Compositions and Methods for Inhibiting Expression of RRM2 Genes

Abstract
The invention relates to a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of a RRM2 gene. The invention also relates to a pharmaceutical composition comprising the dsRNA or nucleic acid molecules or vectors encoding the same together with a pharmaceutically acceptable carrier; methods for treating diseases caused by the expression of a RRM2 gene using said pharmaceutical composition; and methods for inhibiting the expression of RRM2 in a cell.
Description
SEQUENCE LISTING

The present application is being filed along with a Sequence Listing, which is provided in electronic format. The Sequence Listing is provided as a file entitled 27028US4_SequenceListing.txt, created 26 Feb. 2018, and is approximately 294 KB in size. The Sequence Listing is hereby incorporated by reference in its entirety.


BACKGROUND OF THE INVENTION

Cancer remains an important area of high unmet medical need. The majority of current treatments provide small gains in overall survival requiring a delicate balance between efficacy and toxicity. Cancer is a disease characterized by uncontrolled growth and survival driven by improper regulation of the cell cycle. The cell cycle is divided up into four stages culminating in cytokinesis with checkpoint controls ensuring accurate completion of each phase. The cell cycle is designed to duplicate cellular material equally partitioning this material into what will become two new cells. DNA replication occurs during S-phase requiring pools of nucleic acid as the building blocks (dNTP) for new DNA as well as to repair DNA damage. Ribonucleotide reductase (RR) converts ribonucleoside 5′-diphosphates into 2′-deoxyribonucleotides which serve as the dNTP source for DNA synthesis and repair. RR catalyzes the rate-limiting step in the generation of dNTPs and represents an important part of cancer cell growth and repair. (RR) is made up of two subunits called RRM1 and RRM2 both of which are required for catalytic RR activity. RRM2 is overexpressed in a range of tumor types and elevated expression is associated with malignant transformation and metastasis. Overexpression of RRM2 cooperates with other oncogenes to drive the transformation and progression of normal cells. Tumor cells are particularly sensitive to changes in their dNTP pools because they have a high proliferation rate and lack checkpoint controls to monitor and repair DNA damage. Inhibition of RRM2 in tumor cells is expected to enhance DNA damage from a lack of sufficient dNTPs producing an apoptotic response. In nontransformed cells, checkpoint controls monitor the level of dNTPs and have the signaling ability to arrest cells before completing DNA replication and repair protecting cells from incurring incompletely replicated DNA or poorly repaired DNA damage.


Despite significant advances in the field of RNA interference (RNAi) and advances in the treatment of fibrosis and proliferative disorders, like cancers, there remains a need for an agent that can selectively and efficiently silence the RRM2 gene. A specific RRM2 inhibitor is expected to provide an improved therapeutic index over existing inhibitors because it is more selective and tumors cells lacking checkpoint controls are dependent on large dNTP pools to support their rapid proliferation and DNA repair. Also, preclinical data supports the potent tumor cell killing effects following RRM2 inhibition.


RRM2 mRNA overexpression is associated with rapidly proliferating tumor cells. RRM2 expression is cell cycle regulated peaking at S-phase when DNA replication and repair occur followed by rapid degradation during mitosis. RRM2 is overexpressed in Acute myeloid leukemia (AML), bladder cancer, prostate cancer, Non Small Cell Lung Cancer (NSCLC), breast cancer, Hepatocellular Carcinoma (HCC), and colorectal cancers to name a few.


Double-stranded ribonucleic acid (dsRNA) molecules have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi), which is a viable pathway in the development of therapeutically active substances for the treatment of a wide range of proliferating diseases. Accordingly, inhibition of RRM2 expression with the dsRNA molecules of this invention may be used in the treatment of cancer including but not limited to Hepatocellular Carcinoma (HCC) and leukemia as well as other solid tumor types.


SUMMARY OF THE INVENTION

The invention provides double-stranded ribonucleic acid molecules (dsRNAs), as well as compositions and methods for inhibiting the expression of the RRM2 gene, in particular the expression of the RRM2 gene, in a cell, tissue or mammal using such dsRNA. The invention also provides compositions and methods for treating pathological conditions and diseases caused by the expression of the RRM2 gene such as in proliferative disorders like cancer and inflammation.


In one preferred embodiment the described dsRNA molecule is capable of inhibiting the expression of a RRM2 gene by at least 60%, preferably by at least 70%, most preferably by at least 80%. The invention also provides compositions and methods for specifically targeting the liver with RRM2 dsRNA, for treating pathological conditions and diseases caused by the expression of the RRM2 gene including those described above.







DETAILED DESCRIPTION OF THE INVENTION

The invention provides double-stranded ribonucleic acid (dsRNA) molecules able to selectively and efficiently decrease the expression of RRM2. The use of RRM2 RNAi provides a method for the therapeutic and/or prophylactic treatment of diseases/disorders which are associated with inflammation and proliferative disorders, like cancers. Particular disease/disorder states include the therapeutic and/or prophylactic treatment of inflammation and proliferative disorders, like cancers, particularly HCC, leukemia and solid tumors, which method comprises administration of dsRNA targeting RRM2 to a human being or animal.


In one embodiment, the invention provides double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of a RRM2 gene, in particular the expression of the mammalian or human RRM2 gene. The dsRNA comprises at least two sequences that are complementary to each other. The dsRNA comprises a sense strand comprising a first sequence and an antisense strand comprising a second sequence, see sequences provided in the sequence listing and also the specific dsRNA pairs in the appended table 1 and table 2. In one embodiment the sense strand comprises a sequence which has an identity of at least 90% to at least a portion of an mRNA encoding RRM2. Said sequence is located in a region of complementarity of the sense strand to the antisense strand, preferably within nucleotides 2-7 of the 5′ terminus of the antisense strand. In one preferred embodiment the dsRNA specifically targets the human RRM2 gene.


In one embodiment, the antisense strand comprises a nucleotide sequence which is substantially complementary to at least part of an mRNA encoding said RRM2 gene, and the region of complementarity is most preferably less than 30 nucleotides in length. Furthermore, it is preferred that the length of the herein described inventive dsRNA molecules (duplex length) is in the range of about 16 to 30 nucleotides, in particular in the range of about 18 to 28 nucleotides. Particularly useful in context of this invention are duplex lengths of about 19, 20, 21, 22, 23 or 24 nucleotides. Most preferred are duplex stretches of 19, 21 or 23 nucleotides.


The dsRNA, upon delivery to a cell expressing a RRM2 gene, inhibits the expression of a RRM2 gene in vitro by at least 60%, preferably by at least 70%, and most preferably by 80%.


Appended Table 1 relates to preferred molecules to be used as dsRNA in accordance with this invention. Also modified dsRNA molecules are provided herein and are in particular disclosed in appended table 2, providing illustrative examples of modified dsRNA molecules of the present invention. As pointed out herein above, Table 2 provides for illustrative examples of modified dsRNAs of this invention (whereby the corresponding sense strand and antisense strand is provided in this table). The relation of the unmodified preferred molecules shown in Table 1 to the modified dsRNAs of Table 2 is illustrated in Table 5. Yet, the illustrative modifications of these constituents of the inventive dsRNAs are provided herein as examples of modifications.


Tables 3 and 4 provide for selective biological, clinical and pharmaceutical relevant parameters of certain dsRNA molecules of this invention.


Some of the preferred dsRNA molecules are provided in the appended table 1 and, inter alia and preferably, wherein the sense strand is selected from the group consisting of the nucleic acid sequences depicted in SEQ ID NOs: 8, 39, 56, 3, 82, 33, 9, 29, 444, 492 and 442 and the antisense strand is selected from the group consisting of the nucleic acid sequences depicted in SEQ ID NOs: 241, 272, 289, 236, 315, 266, 242, 262, 806, 852 and 851 Accordingly, the inventive dsRNA molecule may, inter alia, comprise the sequence pairs selected from the group consisting of SEQ ID NOs: 8/241, 39/272, 56/289, 3/236, 82/315, 33/266, 9/242, 29/262 444/806, 492/852 and 442/851. In the context of specific dsRNA molecules provided herein, pairs of SEQ ID NOs relate to corresponding sense and antisense strands sequences (5′ to 3′) as also shown in the tables.


In one embodiment the dsRNA molecules comprise an antisense strand with a 3′ overhang of 1-5 nucleotides in length, preferably 1-2 nucleotides in length. Preferably said overhang of the antisense strand comprises uracil or nucleotides which are complementary to the mRNA encoding RRM2. In another preferred embodiment, said dsRNA molecules comprise a sense strand with a 3′ overhang of 1-5 nucleotides in length, preferably 1-2 nucleotides in length. Preferably said overhang of the sense strand comprises uracil or nucleotides which are identical to the mRNA encoding RRM2.


In another preferred embodiment, the dsRNA molecules comprise a sense strand with a 3′ overhang of 1-5 nucleotides in length, preferably 1-2 nucleotides in length, and an antisense strand with a 3′ overhang of 1-5 nucleotides in length, preferably 1-2 nucleotides in length.


Preferably said overhang of the sense strand comprises uracil or nucleotides which are at least 90% identical to the mRNA encoding RRM2 and said overhang of the antisense strand comprises uracil or nucleotides which are at least 90% complementary to the mRNA encoding RRM2.


The dsRNA molecules of the invention may be comprised of naturally occurring nucleotides or may be comprised of at least one modified nucleotide, such as a 2′-O-methyl modified nucleotide, inverted deoxythymidine, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group. 2′ modified nucleotides may have the additional advantage that certain immunostimulatory factors or cytokines are suppressed when the inventive dsRNA molecules are employed in vivo, for example in a medical setting. Alternatively and non-limiting, the modified nucleotide may be chosen from the group of: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide. In one preferred embodiment the dsRNA molecules comprises at least one of the following modified nucleotides: a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group and a deoxythymidine. Preferred dsRNA molecules comprising modified nucleotides are given in table 2. In another preferred embodiment one of those deoxythymidine nucleotides at the 3′ of both strand is a inverted deoxythymidine.


In a preferred embodiment the inventive dsRNA molecules comprise modified nucleotides as detailed in the sequences given in table 2. In one preferred embodiment the inventive dsRNA molecule comprises sequence pairs selected from the group consisting of SEQ ID NOs: 8/241, 39/272, 56/289, 3/236, 82/315, 33/266, 9/242, 29/262, 444/806, 492/852 and 442/851, and comprises overhangs at the antisense and/or sense strand of 1-2 deoxythymidines. In one preferred embodiment the inventive dsRNA molecule comprises sequence pairs selected from the group consisting of SEQ ID NOs: 8/241, 39/272, 56/289, 3/236, 82/315, 33/266, 9/242, 29/262 444/806, 492/852 and 442/851, and comprise modifications as detailed in table 2. Preferred dsRNA molecules comprising modified nucleotides are listed in table 2-4, with the most preferred dsRNA molecules depicted in SEQ ID Nos: 469/742, 475/884, 477/839, 497/711, 501/842, 553/968, 507/841, 525/885, 552/891, 477/963, 526/967 and 476/966.


In another embodiment, the inventive dsRNAs comprise modified nucleotides on positions different from those disclosed in table 2. In one preferred embodiment two deoxythymidine nucleotides are found at the 3′ of both strands of the dsRNA molecule. Preferably said deoxythymidine nucleotides form an overhang.


In one embodiment the dsRNA molecules of the invention comprise a sense and an antisense strand wherein both strands have a half-life of at least 0.9 hours. In one preferred embodiment the dsRNA molecules of the invention comprise a sense and an antisense strand wherein both strands have a half-life of at least 48 hours, preferably in human serum. In another embodiment the dsRNA molecules of the invention are non-immunostimulatory, e.g. do not stimulate INF-alpha (INF-α) and TNF-alpha (TNF-α) in vitro. In another embodiment, the dsRNA molecules of the invention do stimulate INF-α and TNF-α in vitro to a very minor degree.


In another embodiment, a nucleic acid sequence encoding a sense strand and/or an antisense strand comprised in the dsRNAs as defined herein are provided.


The invention also provides for cells comprising at least one of the dsRNAs of the invention. The cell is preferably a mammalian cell, such as a human cell. Furthermore, tissues and/or non-human organisms comprising the herein defined dsRNA molecules are an embodiment of this invention, whereby said non-human organisms are particularly useful for research purposes or as research tools, for example in drug testing.


Furthermore, the invention relates to a method for inhibiting the expression of a RRM2 gene, in particular a mammalian or human RRM2 gene, in a cell, tissue or organism comprising the following steps:

    • a) introducing into the cell, tissue or organism a double-stranded ribonucleic acid (dsRNA) as defined herein; and
    • b) maintaining said cell, tissue or organism produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of a RRM2 gene, thereby inhibiting expression of a RRM2 gene in a given cell.


The invention also relates to pharmaceutical compositions comprising the inventive dsRNAs of the invention. These pharmaceutical compositions are particularly useful in the inhibition of the expression of a RRM2 gene in a cell, a tissue or an organism. The pharmaceutical composition comprising one or more of the dsRNA of the invention may also comprise (a) pharmaceutically acceptable carrier(s), diluent(s) and/or excipient(s).


In another embodiment, the invention provides methods for treating, preventing or managing inflammation and/or proliferative disorders like cancers which are associated with RRM2, said method comprising administering to a subject in need of such treatment, prevention or management a therapeutically or prophylactically effective amount of one or more of the dsRNAs of the invention. Preferably, said subject is a mammal, most preferably a human patient.


In one embodiment, the invention provides a method for treating a subject having a pathological condition mediated by the expression of a RRM2 gene. Such conditions comprise disorders associated with inflammation and proliferative disorders, like cancers, as described above. In this embodiment, the dsRNA acts as a therapeutic agent for controlling the expression of a RRM2 gene. The method comprises administering a pharmaceutical composition of the invention to the patient (e.g., human), such that expression of a RRM2 gene is silenced. Because of their high specificity, the dsRNAs of the invention specifically target mRNAs of a RRM2 gene. In one preferred embodiment the described dsRNAs specifically decrease RRM2 mRNA levels and do not directly affect the expression and/or mRNA levels of off-target genes in the cell.


In one preferred embodiment the described dsRNA decrease RRM2 mRNA levels in the liver by at least 60%, preferably by at least 70%, and most preferably by at least 80% in vivo. In another embodiment the described dsRNAs decrease RRM2 mRNA levels in vivo for at least 4 days. In another preferred embodiment, the dsRNAs of the invention are used for the preparation of a pharmaceutical composition for the treatment of inflammation and proliferative disorders, like cancer. Cancers to be treated with said pharmaceutical composition comprise but are not limited to: HCC, AML, leukemia, bladder cancer, prostate cancer, NSCLC, breast cancer and colorectal cancer.


In another embodiment, the invention provides vectors for inhibiting the expression of a RRM2 gene in a cell, in particular a RRM2 gene comprising a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of the dsRNA molecules of the invention.


In another embodiment, the invention provides a cell comprising a vector for inhibiting the expression of a RRM2 gene in a cell. Said vector comprises a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of the dsRNA molecule of the invention. Yet, it is preferred that said vector comprises, besides said regulatory sequence a sequence that encodes at least one “sense strand” of the inventive dsRNA and at least one “anti-sense strand” of said dsRNA. It is also envisaged that the claimed cell comprises two or more vectors comprising, besides said regulatory sequences, the herein defined sequence(s) that encode(s) at least one strand of the dsRNA molecules of the invention.


In one embodiment, the method comprises administering a composition comprising a dsRNA, wherein the dsRNA comprises a nucleotide sequence which is complementary to at least a part of an RNA transcript of a RRM2 gene of the mammal to be treated. As pointed out above, also vectors and cells comprising nucleic acid molecules that encode for at least one strand of the herein defined dsRNA molecules can be used as pharmaceutical compositions and may, therefore, also be employed in the herein disclosed methods of treating a subject in need of medical intervention. It is also of note that these embodiments relating to pharmaceutical compositions and to corresponding methods of treating a (human) subject also relate to approaches like gene therapy approaches. RRM2 specific dsRNA molecules as provided herein or nucleic acid molecules encoding individual strands of these inventive dsRNA molecules may also be inserted into vectors and used as gene therapy vectors for human patients. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.


In another aspect of the invention, RRM2 specific dsRNA molecules that modulate RRM2 gene expression activity are expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Skillern, A., et al., International PCT Publication No. WO 00/22113). These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be incorporated and inherited as a transgene integrated into the host genome. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).


The individual strands of a dsRNA can be transcribed by promoters on two separate expression vectors and co-transfected into a target cell. Alternatively, each individual strand of the dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In a preferred embodiment, a dsRNA is expressed as an inverted repeat joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.


The recombinant dsRNA expression vectors are preferably DNA plasmids or viral vectors. dsRNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus (for a review, see Muzyczka, et al., Curr. Topics Micro. Immunol. (1992) 158:97-129)); adenovirus (see, for example, Berkner, et al., BioTechniques (1998) 6:616), Rosenfeld et al. (1991, Science 252:431-434), and Rosenfeld et al. (1992), Cell 68:143-155)); or alphavirus as well as others known in the art. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, in vitro and/or in vivo (see, e.g., Danos and Mulligan, Proc. Natl. Acad. Sci. USA (1998) 85:6460-6464). Recombinant retroviral vectors capable of transducing and expressing genes inserted into the genome of a cell can be produced by transfecting the recombinant retroviral genome into suitable packaging cell lines such as PA317 and Psi-CRIP (Comette et al., 1991, Human Gene Therapy 2:5-10; Cone et al., 1984, Proc. Natl. Acad. Sci. USA 81:6349). Recombinant adenoviral vectors can be used to infect a wide variety of cells and tissues in susceptible hosts (e.g., rat, hamster, dog, and chimpanzee) (Hsu et al., 1992, J. Infectious Disease, 166:769), and also have the advantage of not requiring mitotically active cells for infection.


The promoter driving dsRNA expression in either a DNA plasmid or viral vector of the invention may be a eukaryotic RNA polymerase I (e.g. ribosomal RNA promoter), RNA polymerase II (e.g. CMV early promoter or actin promoter or U1 snRNA promoter) or preferably RNA polymerase III promoter (e.g. U6 snRNA or 7SK RNA promoter) or a prokaryotic promoter, for example the T7 promoter, provided the expression plasmid also encodes T7 RNA polymerase required for transcription from a T7 promoter. The promoter can also direct transgene expression to the pancreas (see, e.g. the insulin regulatory sequence for pancreas (Bucchini et al., 1986, Proc. Natl. Acad. Sci. USA 83:2511-2515)).


In addition, expression of the transgene can be precisely regulated, for example, by using an inducible regulatory sequence and expression systems such as a regulatory sequence that is sensitive to certain physiological regulators, e.g., circulating glucose levels, or hormones (Docherty et al., 1994, FASEB 1 8:20-24). Such inducible expression systems, suitable for the control of transgene expression in cells or in mammals include regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-β-D1-thiogalactopyranoside (EPTG). A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the dsRNA transgene.


Preferably, recombinant vectors capable of expressing dsRNA molecules are delivered as described below, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of dsRNA molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the dsRNAs bind to target RNA and modulate its function or expression. Delivery of dsRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.


dsRNA expression DNA plasmids are typically transfected into target cells as a complex with cationic lipid carriers (e.g. Oligofectamine) or non-cationic lipid-based carriers (e.g. Transit-TKO™). Multiple lipid transfections for dsRNA-mediated knockdowns targeting different regions of a single RRM2 gene or multiple RRM2 genes over a period of a week or more are also contemplated by the invention. Successful introduction of the vectors of the invention into host cells can be monitored using various known methods. For example, transient transfection can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Stable transfection of ex vivo cells can be ensured using markers that provide the transfected cell with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.


The following detailed description discloses how to make and use the dsRNA and compositions containing dsRNA to inhibit the expression of a target RRM2 gene, as well as compositions and methods for treating diseases and disorders caused by the expression of said RRM2 gene.


Definitions

For convenience, the meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below. If there is an apparent discrepancy between the usage of a term in other parts of this specification and its definition provided in this section, the definition in this section shall prevail.


“G”, “C”, “A”, “U”, and “T” or “dT” respectively, each generally stand for a nucleotide that contains guanine, cytosine, adenine, uracil and deoxythymidine as a base, respectively. However, the term “ribonucleotide” or “nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety. Sequences comprising such replacement moieties are embodiments of the invention. As detailed below, the herein described dsRNA molecules may also comprise “overhangs”, i.e. unpaired, overhanging nucleotides which are not directly involved in the RNA double helical structure normally formed by the herein defined pair of “sense strand” and “anti-sense strand”. Often, such an overhanging stretch comprises the deoxythymidine nucleotide, in most embodiments, 2 deoxythymidines in the 3′ end. Such overhangs will be described and illustrated below.


The term “RRM2” as used herein relates in particular to the ribonucleotide reductase M2, also known as the ribonucleotide reductase M2 polypeptide, ribonucleoside-diphosphate reductase subunit M2, ribonucleotide reductase small chain, ribonucleotide reductase small subunit, with synonyms R2, RR2, RR2M, and the like and said term relates to the corresponding gene, encoded mRNA, encoded protein/polypeptide as well as functional fragments of the same. Preferred is the human RRM2 gene. In other preferred embodiments the dsRNAs of the invention target the RRM2 gene of human (H. sapiens) and cynomolgous monkey (Macaca fascicularis) RRM2 gene. Also dsRNAs targeting the rat (Rattus norvegicus) and mouse (Mus musculus) RRM2 gene are part of this invention. The term “RRM2 gene/sequence” does not only relate to (the) wild-type sequence(s) but also to mutations and alterations which may be comprised in said gene/sequence. Accordingly, the present invention is not limited to the specific dsRNA molecules provided herein. The invention also relates to dsRNA molecules that comprise an antisense strand that is at least 85% complementary to the corresponding nucleotide stretch of an RNA transcript of a RRM2 gene that comprises such mutations/alterations.


As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a RRM2 gene, including mRNA that is a product of RNA processing of a primary transcription product.


As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature. However, as detailed herein, such a “strand comprising a sequence” may also comprise modifications, like modified nucleotides.


As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence. “Complementary” sequences, as used herein, may also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled.


Sequences referred to as “fully complementary” comprise base-pairing of the oligonucleotide or polynucleotide comprising the first nucleotide sequence to the oligonucleotide or polynucleotide comprising the second nucleotide sequence over the entire length of the first and second nucleotide sequence.


However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they may form one or more, but preferably not more than 13 mismatched base pairs upon hybridization.


The terms “complementary”, “fully complementary” and “substantially complementary” herein may be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, as will be understood from the context of their use.


The term “double-stranded RNA”, “dsRNA molecule”, or “dsRNA”, as used herein, refers to a ribonucleic acid molecule, or complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary nucleic acid strands. The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′ end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a “hairpin loop”. Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′ end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker”. The RNA strands may have the same or a different number of nucleotides. In addition to the duplex structure, a dsRNA may comprise one or more nucleotide overhangs. The nucleotides in said “overhangs” may comprise between 0 and 5 nucleotides, whereby “0” means no additional nucleotide(s) that form(s) an “overhang” and whereas “5” means five additional nucleotides on the individual strands of the dsRNA duplex. These optional “overhangs” are located in the 3′ end of the individual strands. As will be detailed below, also dsRNA molecules which comprise only an “overhang” in one of the two strands may be useful and even advantageous in context of this invention. The “overhang” comprises preferably between 0 and 2 nucleotides. Most preferably 2 “dT” (deoxythymidine) nucleotides are found at the 3′ end of both strands of the dsRNA. Also 2 “U” (uracil) nucleotides can be used as overhangs at the 3′ end of both strands of the dsRNA. Accordingly, a “nucleotide overhang” refers to the unpaired nucleotide or nucleotides that protrude from the duplex structure of a dsRNA when a 3′-end of one strand of the dsRNA extends beyond the 5′-end of the other strand, or vice versa. For example the antisense strand comprises 23 nucleotides and the sense strand comprises 21 nucleotides, forming a 2 nucleotide overhang at the 3′ end of the antisense strand. Preferably, the 2 nucleotide overhang is fully complementary to the mRNA of the target gene. “Blunt” or “blunt end” means that there are no unpaired nucleotides at that end of the dsRNA, i.e., no nucleotide overhang. A “blunt ended” dsRNA is a dsRNA that is double-stranded over its entire length, i.e., no nucleotide overhang at either end of the molecule.


The term “antisense strand” refers to the strand of a dsRNA which includes a region that is substantially complementary to a target sequence. As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence. Where the region of complementarity is not fully complementary to the target sequence, the mismatches are most tolerated outside nucleotides 2-7 of the 5′ terminus of the antisense strand


The term “sense strand,” as used herein, refers to the strand of a dsRNA that includes a region that is substantially complementary to a region of the antisense strand. “Substantially complementary” means preferably at least 85% of the overlapping nucleotides in sense and antisense strand are complementary.


“Introducing into a cell”, when referring to a dsRNA, means facilitating uptake or absorption into the cell, as is understood by those skilled in the art. Absorption or uptake of dsRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. The meaning of this term is not limited to cells in vitro; a dsRNA may also be “introduced into a cell”, wherein the cell is part of a living organism. In such instance, introduction into the cell will include the delivery to the organism. For example, for in vivo delivery, dsRNA can be injected into a tissue site or administered systemically. It is, for example envisaged that the dsRNA molecules of this invention be administered to a subject in need of medical intervention. Such an administration may comprise the injection of the dsRNA, the vector or a cell of this invention into a diseased site in said subject, for example into liver tissue/cells or into cancerous tissues/cells, like liver cancer tissue. In addition, the injection is preferably in close proximity to the diseased tissue envisaged. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection.


As used herein, “proliferating” and “proliferation” refer to cells undergoing mitosis. Throughout this application, the term “proliferative disorder” refers to any disease/disorder marked by unwanted or aberrant proliferation of tissue. As used herein, the term “proliferative disorder” also refers to conditions in which the unregulated and/or abnormal growth of cells can lead to the development of an unwanted condition or disease, which can be cancerous or non-cancerous.


The term “inflammation” as used herein refers to the biologic response of body tissue to injury, irritation, or disease which can be caused by harmful stimuli, for example; pathogens, damaged cells, or irritants. Inflammation is typically characterized by pain and swelling. Inflammation is intended to encompass both acute responses, in which inflammatory processes are active neutrophils and leukocytes), and chronic responses, which are marked by slow progress, a shift in the type of cell present at the site of inflammation, and the formation of connective tissue.


Cancers to be treated comprise, but are again not limited to leukemia, AML, solid tumors, liver cancer, brain cancer, breast cancer, lung cancer, NSCLC, colorectal cancer, bladder cancer and prostate cancer, whereby said liver cancer may, inter alia, be selected from the group consisting of hepatocellular carcinoma (HCC), hepatoblastoma, a mixed liver cancer, a cancer derived from mesenchymal tissue, a liver sarcoma or a cholangiocarcinoma.


The terms “silence”, “inhibit the expression of” and “knock down”, in as far as they refer to a RRM2 gene, herein refer to the at least partial suppression of the expression of a RRM2 gene, as manifested by a reduction of the amount of mRNA transcribed from a RRM2 gene which may be isolated from a first cell or group of cells in which a RRM2 gene is transcribed and which has or have been treated such that the expression of a RRM2 gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells). The degree of inhibition is usually expressed in terms of










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100


%




Alternatively, the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to the RRM2 gene transcription, e.g. the amount of protein encoded by a RRM2 gene which is secreted by a cell, or the number of cells displaying a certain phenotype. As illustrated in the appended examples and in the appended tables provided herein, the inventive dsRNA molecules are capable of inhibiting the expression of a human RRM2 by at least about 60%, preferably by at least 70%, most preferably by at least 80% in vitro assays, i.e. in vitro. The term “in vitro” as used herein includes but is not limited to cell culture assays. In another embodiment the inventive dsRNA molecules are capable of inhibiting the expression of a mouse or rat RRM2 by at least 60% preferably by at least 70%, most preferably by at least 80%. The person skilled in the art can readily determine such an inhibition rate and related effects, in particular in light of the assays provided herein.


The term “off target” as used herein refers to all non-target mRNAs of the transcriptome that are predicted by in silico methods to hybridize to the described dsRNAs based on sequence complementarity. The dsRNAs of the present invention preferably do specifically inhibit the expression of RRM2, i.e. do not inhibit the expression of any off-target.


The term “half-life” as used herein is a measure of stability of a compound or molecule and can be assessed by methods known to a person skilled in the art, especially in light of the assays provided herein.


The term “non-immunostimulatory” as used herein refers to the absence of any induction of a immune response by the invented dsRNA molecules. Methods to determine immune responses are well known to a person skilled in the art, for example by assessing the release of cytokines, as described in the examples section.


The terms “treat”, “treatment”, and the like, mean in context of this invention the relief from or alleviation of a disorder related to RRM2 expression, like inflammation and proliferative disorders, like cancers.


As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of a dsRNA and a pharmaceutically acceptable carrier. However, such a “pharmaceutical composition” may also comprise individual strands of such a dsRNA molecule or the herein described vector(s) comprising a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of a sense or an antisense strand comprised in the dsRNAs of this invention. It is also envisaged that cells, tissues or isolated organs that express or comprise the herein defined dsRNAs may be used as “pharmaceutical compositions”. As used herein, “pharmacologically effective amount,” “therapeutically effective amount” or simply “effective amount” refers to that amount of an RNA effective to produce the intended pharmacological, therapeutic or preventive result.


The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture medium. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives as known to persons skilled in the art.


It is in particular envisaged that the pharmaceutically acceptable carrier allows for the systemic administration of the dsRNAs, vectors or cells of this invention. Whereas also the enteric administration is envisaged the parenteral administration and also transdermal or transmucosal (e.g. insufflation, buccal, vaginal, anal) administration as well as inhalation of the drug are feasible ways of administering to a patient in need of medical intervention the compounds of this invention. When parenteral administration is employed, this can comprise the direct injection of the compounds of this invention into the diseased tissue or at least in close proximity. However, also intravenous, intraarterial, subcutaneous, intramuscular, intraperitoneal, intradermal, intrathecal and other administrations of the compounds of this invention are within the skill of the artisan, for example the attending physician.


For intramuscular, subcutaneous and intravenous use, the pharmaceutical compositions of the invention will generally be provided in sterile aqueous solutions or suspensions, buffered to an appropriate pH and isotonicity. In a preferred embodiment, the carrier consists exclusively of an aqueous buffer. In this context, “exclusively” means no auxiliary agents or encapsulating substances are present which might affect or mediate uptake of dsRNA in the cells that express a RRM2 gene. Aqueous suspensions according to the invention may include suspending agents such as cellulose derivatives, sodium alginate, polyvinyl-pyrrolidone and gum tragacanth, and a wetting agent such as lecithin. Suitable preservatives for aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate. The pharmaceutical compositions useful according to the invention also include encapsulated formulations to protect the dsRNA against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in PCT publication WO 91/06309 which is incorporated by reference herein.


As used herein, a “transformed cell” is a cell into which at least one vector has been introduced from which a dsRNA molecule or at least one strand of such a dsRNA molecule may be expressed. Such a vector is preferably a vector comprising a regulatory sequence operably linked to nucleotide sequence that encodes at least one sense strand or antisense strand of a dsRNA of the present invention.


It can be reasonably expected that shorter dsRNAs comprising one of the sequences in Table 1 and 4 minus only a few nucleotides on one or both ends may be similarly effective as compared to the dsRNAs described above.


In one preferred embodiment the inventive dsRNA molecules comprise nucleotides 1-19 of the sequences given in Table 1.


As pointed out above, in most embodiments of this invention, the dsRNA molecules provided herein comprise a duplex length (i.e. without “overhangs”) of about 16 to about 30 nucleotides.


Particular useful dsRNA duplex lengths are about 19 to about 25 nucleotides. Most preferred are duplex structures with a length of 19 nucleotides. In the inventive dsRNA molecules, the antisense strand is at least partially complementary to the sense strand.


The dsRNA of the invention can contain one or more mismatches to the target sequence. In a preferred embodiment, the dsRNA of the invention contains no more than 13 mismatches. If the antisense strand of the dsRNA contains mismatches to a target sequence, it is preferable that the area of mismatch not be located within nucleotides 2-7 of the 5′ terminus of the antisense strand. In another embodiment it is preferable that the area of mismatch not be located within nucleotides 2-9 of the 5′ terminus of the antisense strand.


As mentioned above, at least one end/strand of the dsRNA may have a single-stranded nucleotide overhang of 1 to 5, preferably 1 or 2 nucleotides. dsRNAs having at least one nucleotide overhang have unexpectedly superior inhibitory properties than their blunt-ended counterparts. Moreover, the present inventors have discovered that the presence of only one nucleotide overhang strengthens the interference activity of the dsRNA, without affecting its overall stability. dsRNA having only one overhang has proven particularly stable and effective in vivo, as well as in a variety of cells, cell culture mediums, blood, and serum. Preferably, the single-stranded overhang is located at the 3′-terminal end of the antisense strand or, alternatively, at the 3′-terminal end of the sense strand. The dsRNA may also have a blunt end, preferably located at the 5′-end of the antisense strand. Preferably, the antisense strand of the dsRNA has a nucleotide overhang at the 3′-end, and the 5′-end is blunt. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.


The dsRNA of the present invention may also be chemically modified to enhance stability. The nucleic acids of the invention may be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry”, Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Chemical modifications may include, but are not limited to 2′ modifications, introduction of non-natural bases, covalent attachment to a ligand, and replacement of phosphate linkages with thiophosphate linkages, inverted deoxythymidines. In this embodiment, the integrity of the duplex structure is strengthened by at least one, and preferably two, chemical linkages. Chemical linking may be achieved by any of a variety of well-known techniques, for example by introducing covalent, ionic or hydrogen bonds; hydrophobic interactions, van der Waals or stacking interactions; by means of metal-ion coordination, or through use of purine analogues. Preferably, the chemical groups that can be used to modify the dsRNA include, without limitation, methylene blue; bifunctional groups, preferably bis-(2-chloroethyl)amine; N-acetyl-N′-(p-glyoxylbenzoyl)cystamine; 4-thiouracil; and psoralen. In one preferred embodiment, the linker is a hexa-ethylene glycol linker. In this case, the dsRNA are produced by solid phase synthesis and the hexa-ethylene glycol linker is incorporated according to standard methods (e.g., Williams, D. J., and K. B. Hall, Biochem. (1996) 35:14665-14670). In a particular embodiment, the 5′-end of the antisense strand and the 3′-end of the sense strand are chemically linked via a hexaethylene glycol linker. In another embodiment, at least one nucleotide of the dsRNA comprises a phosphorothioate or phosphorodithioate groups. The chemical bond at the ends of the dsRNA is preferably formed by triple-helix bonds.


In certain embodiments, a chemical bond may be formed by means of one or several bonding groups, wherein such bonding groups are preferably poly-(oxyphosphinicooxy-1,3-propandiol)- and/or polyethylene glycol chains. In other embodiments, a chemical bond may also be formed by means of purine analogs introduced into the double-stranded structure instead of purines. In further embodiments, a chemical bond may be formed by azabenzene units introduced into the double-stranded structure. In still further embodiments, a chemical bond may be formed by branched nucleotide analogs instead of nucleotides introduced into the double-stranded structure. In certain embodiments, a chemical bond may be induced by ultraviolet light.


In yet another embodiment, the nucleotides at one or both of the two single strands may be modified to prevent or inhibit the activation of cellular enzymes, for example certain nucleases. Techniques for inhibiting the activation of cellular enzymes are known in the art including, but not limited to, 2′-amino modifications, 2′-amino sugar modifications, 2′-F sugar modifications, 2′-F modifications, 2′-alkyl sugar modifications, uncharged backbone modifications, morpholino modifications, 2′-O-methyl modifications, and phosphoramidate (see, e.g., Wagner, Nat. Med. (1995) 1:1116-8). Thus, at least one 2′-hydroxyl group of the nucleotides on a dsRNA is replaced by a chemical group, preferably by a 2′-amino or a 2′-methyl group. Also, at least one nucleotide may be modified to form a locked nucleotide. Such locked nucleotide contains a methylene bridge that connects the 2′-oxygen of ribose with the 4′-carbon of ribose. Introduction of a locked nucleotide into an oligonucleotide improves the affinity for complementary sequences and increases the melting temperature by several degrees.


Modifications of dsRNA molecules provided herein may positively influence their stability in vivo as well as in vitro and also improve their delivery to the (diseased) target side. Furthermore, such structural and chemical modifications may positively influence physiological reactions towards the dsRNA molecules upon administration, e.g. the cytokine release which is preferably suppressed. Such chemical and structural modifications are known in the art and are, inter alia, illustrated in Nawrot (2006) Current Topics in Med Chem, 6, 913-925.


Conjugating a ligand to a dsRNA can enhance its cellular absorption as well as targeting to a particular tissue. In certain instances, a hydrophobic ligand is conjugated to the dsRNA to facilitate direct permeation of the cellular membrane. Alternatively, the ligand conjugated to the dsRNA is a substrate for receptor-mediated endocytosis. These approaches have been used to facilitate cell permeation of antisense oligonucleotides. For example, cholesterol has been conjugated to various antisense oligonucleotides resulting in compounds that are substantially more active compared to their non-conjugated analogs. See M. Manoharan Antisense & Nucleic Acid Drug Development 2002, 12, 103. Other lipophilic compounds that have been conjugated to oligonucleotides include 1-pyrene butyric acid, 1,3-bis-O-(hexadecyl)glycerol, and menthol. One example of a ligand for receptor-mediated endocytosis is folic acid. Folic acid enters the cell by folate-receptor-mediated endocytosis. dsRNA compounds bearing folic acid would be efficiently transported into the cell via the folate-receptor-mediated endocytosis. Attachment of folic acid to the 3′-terminus of an oligonucleotide results in increased cellular uptake of the oligonucleotide (Li, S.; Deshmukh, H. M.; Huang, L. Pharm. Res. 1998, 15, 1540). Other ligands that have been conjugated to oligonucleotides include polyethylene glycols, carbohydrate clusters, cross-linking agents, porphyrin conjugates, and delivery peptides.


In certain instances, conjugation of a cationic ligand to oligonucleotides often results in improved resistance to nucleases. Representative examples of cationic ligands are propylammonium and dimethylpropylammonium. Interestingly, antisense oligonucleotides were reported to retain their high binding affinity to mRNA when the cationic ligand was dispersed throughout the oligonucleotide. See M. Manoharan Antisense & Nucleic Acid Drug Development 2002, 12, 103 and references therein.


The ligand-conjugated dsRNA of the invention may be synthesized by the use of a dsRNA that bears a pendant reactive functionality, such as that derived from the attachment of a linking molecule onto the dsRNA. This reactive oligonucleotide may be reacted directly with commercially-available ligands, ligands that are synthesized bearing any of a variety of protecting groups, or ligands that have a linking moiety attached thereto. The methods of the invention facilitate the synthesis of ligand-conjugated dsRNA by the use of, in some preferred embodiments, nucleoside monomers that have been appropriately conjugated with ligands and that may further be attached to a solid-support material. Such ligand-nucleoside conjugates, optionally attached to a solid-support material, are prepared according to some preferred embodiments of the methods of the invention via reaction of a selected serum-binding ligand with a linking moiety located on the 5′ position of a nucleoside or oligonucleotide. In certain instances, an dsRNA bearing an aralkyl ligand attached to the 3′-terminus of the dsRNA is prepared by first covalently attaching a monomer building block to a controlled-pore-glass support via a long-chain aminoalkyl group. Then, nucleotides are bonded via standard solid-phase synthesis techniques to the monomer building-block bound to the solid support. The monomer building block may be a nucleoside or other organic compound that is compatible with solid-phase synthesis.


The dsRNA used in the conjugates of the invention may be conveniently and routinely made through the well-known technique of solid-phase synthesis. It is also known to use similar techniques to prepare other oligonucleotides, such as the phosphorothioates and alkylated derivatives.


Teachings regarding the synthesis of particular modified oligonucleotides may be found in the following U.S. patents: U.S. Pat. No. 5,218,105, drawn to polyamine conjugated oligonucleotides; U.S. Pat. Nos. 5,541,307, drawn to oligonucleotides having modified backbones; U.S. Pat. No. 5,521,302, drawn to processes for preparing oligonucleotides having chiral phosphorus linkages; U.S. Pat. No. 5,539,082, drawn to peptide nucleic acids; U.S. Pat. No. 5,554,746, drawn to oligonucleotides having β-lactam backbones; U.S. Pat. No. 5,571,902, drawn to methods and materials for the synthesis of oligonucleotides; U.S. Pat. No. 5,578,718, drawn to nucleosides having alkylthio groups, wherein such groups may be used as linkers to other moieties attached at any of a variety of positions of the nucleoside; U.S. Pat. No. 5,587,361 drawn to oligonucleotides having phosphorothioate linkages of high chiral purity; U.S. Pat. No. 5,506,351, drawn to processes for the preparation of 2′-O-alkyl guanosine and related compounds, including 2,6-diaminopurine compounds; U.S. Pat. No. 5,587,469, drawn to oligonucleotides having N-2 substituted purines; U.S. Pat. No. 5,587,470, drawn to oligonucleotides having 3-deazapurines; U.S. Pat. No. 5,608,046, both drawn to conjugated 4′-desmethyl nucleoside analogs; U.S. Pat. No. 5,610,289, drawn to backbone-modified oligonucleotide analogs; U.S. Pat. No. 6,262,241 drawn to, inter alia, methods of synthesizing 2′-fluoro-oligonucleotides.


In the ligand-conjugated dsRNA and ligand-molecule bearing sequence-specific linked nucleosides of the invention, the oligonucleotides and oligonucleosides may be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors that already bear the linking moiety, ligand-nucleotide or nucleoside-conjugate precursors that already bear the ligand molecule, or non-nucleoside ligand-bearing building blocks.


When using nucleotide-conjugate precursors that already bear a linking moiety, the synthesis of the sequence-specific linked nucleosides is typically completed, and the ligand molecule is then reacted with the linking moiety to form the ligand-conjugated oligonucleotide. Oligonucleotide conjugates bearing a variety of molecules such as steroids, vitamins, lipids and reporter molecules, has previously been described (see Manoharan et al., PCT Application WO 93/07883). In a preferred embodiment, the oligonucleotides or linked nucleosides of the invention are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside conjugates in addition to commercially available phosphoramidites.


The incorporation of a 2′-O-methyl, 2′-O-ethyl, 2′-O-propyl, 2′-O-allyl, 2′-O-aminoalkyl or 2′-deoxy-2′-fluoro group in nucleosides of an oligonucleotide confers enhanced hybridization properties to the oligonucleotide. Further, oligonucleotides containing phosphorothioate backbones have enhanced nuclease stability. Thus, functionalized, linked nucleosides of the invention can be augmented to include either or both a phosphorothioate backbone or a 2′-O-methyl, 2′-O-ethyl, 2′-O-propyl, 2′-O-aminoalkyl, 2′-O-allyl or 2′-deoxy-2′-fluoro group.


In some preferred embodiments, functionalized nucleoside sequences of the invention possessing an amino group at the 5′-terminus are prepared using a DNA synthesizer, and then reacted with an active ester derivative of a selected ligand. Active ester derivatives are well known to those skilled in the art. Representative active esters include N-hydrosuccinimide esters, tetrafluorophenolic esters, pentafluorophenolic esters and pentachlorophenolic esters. The reaction of the amino group and the active ester produces an oligonucleotide in which the selected ligand is attached to the 5′-position through a linking group. The amino group at the 5′-terminus can be prepared utilizing a 5′-Amino-Modifier C6 reagent. In a preferred embodiment, ligand molecules may be conjugated to oligonucleotides at the 5′-position by the use of a ligand-nucleoside phosphoramidite wherein the ligand is linked to the 5′-hydroxy group directly or indirectly via a linker. Such ligand-nucleoside phosphoramidites are typically used at the end of an automated synthesis procedure to provide a ligand-conjugated oligonucleotide bearing the ligand at the 5′-terminus.


In one preferred embodiment of the methods of the invention, the preparation of ligand conjugated oligonucleotides commences with the selection of appropriate precursor molecules upon which to construct the ligand molecule. Typically, the precursor is an appropriately-protected derivative of the commonly-used nucleosides. For example, the synthetic precursors for the synthesis of the ligand-conjugated oligonucleotides of the invention include, but are not limited to, 2′-aminoalkoxy-5′-ODMT-nucleosides, 2′-6-aminoalkylamino-5′-ODMT-nucleosides, 5′-6-aminoalkoxy-2′-deoxy-nucleosides, 5′-6-aminoalkoxy-2-protected-nucleosides, 3′-6-aminoalkoxy-5′-ODMT-nucleosides, and 3′-aminoalkylamino-5′-ODMT-nucleosides that may be protected in the nucleobase portion of the molecule. Methods for the synthesis of such amino-linked protected nucleoside precursors are known to those of ordinary skill in the art.


In many cases, protecting groups are used during the preparation of the compounds of the invention. As used herein, the term “protected” means that the indicated moiety has a protecting group appended thereon. In some preferred embodiments of the invention, compounds contain one or more protecting groups. A wide variety of protecting groups can be employed in the methods of the invention. In general, protecting groups render chemical functionalities inert to specific reaction conditions, and can be appended to and removed from such functionalities in a molecule without substantially damaging the remainder of the molecule.


Representative hydroxyl protecting groups, as well as other representative protecting groups, are disclosed in Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2d ed., John Wiley & Sons, New York, 1991, and Oligonucleotides And Analogues A Practical Approach, Ekstein, F. Ed., IRL Press, N.Y., 1991.


Amino-protecting groups stable to acid treatment are selectively removed with base treatment, and are used to make reactive amino groups selectively available for substitution. Examples of such groups are the Fmoc (E. Atherton and R. C. Sheppard in The Peptides, S. Udenfriend, J. Meienhofer, Eds., Academic Press, Orlando, 1987, volume 9, p. 1) and various substituted sulfonylethyl carbamates exemplified by the Nsc group (Samukov et al., Tetrahedron Lett., 1994, 35:7821.


Additional amino-protecting groups include, but are not limited to, carbamate protecting groups, such as 2-trimethylsilylethoxycarbonyl (Teoc), 1-methyl-1-(4-biphenylyl)-ethoxycarbonyl (Bpoc), t-butoxycarbonyl (BOC), allyloxycarbonyl (Alloc), 9-fluorenyl-methyloxycarbonyl (Fmoc), and benzyloxycarbonyl (Cbz); amide protecting groups, such as formyl, acetyl, trihaloacetyl, benzoyl, and nitrophenylacetyl; sulfonamide protecting groups, such as 2-nitrobenzenesulfonyl; and imine and cyclic imide protecting groups, such as phthalimido and dithiasuccinoyl. Equivalents of these amino-protecting groups are also encompassed by the compounds and methods of the invention.


Many solid supports are commercially available and one of ordinary skill in the art can readily select a solid support to be used in the solid-phase synthesis steps. In certain embodiments, a universal support is used. A universal support allows for the preparation of oligonucleotides having unusual or modified nucleotides located at the 3′-terminus of the oligonucleotide. For further details about universal supports see Scott et al., Innovations and Perspectives in solid-phase Synthesis, 3rd International Symposium, 1994, Ed. Roger Epton, Mayflower Worldwide, 115-124]. In addition, it has been reported that the oligonucleotide can be cleaved from the universal support under milder reaction conditions when the oligonucleotide is bonded to the solid support via a syn-1,2-acetoxyphosphate group which more readily undergoes basic hydrolysis. See Guzaev, A. I.; Manoharan, M. J. Am. Chem. Soc. 2003, 125, 2380.


The nucleosides are linked by phosphorus-containing or non-phosphorus-containing covalent internucleoside linkages. For the purposes of identification, such conjugated nucleosides can be characterized as ligand-bearing nucleosides or ligand-nucleoside conjugates. The linked nucleosides having an aralkyl ligand conjugated to a nucleoside within their sequence will demonstrate enhanced dsRNA activity when compared to like dsRNA compounds that are not conjugated.


The aralkyl-ligand-conjugated oligonucleotides of the invention also include conjugates of oligonucleotides and linked nucleosides wherein the ligand is attached directly to the nucleoside or nucleotide without the intermediacy of a linker group. The ligand may preferably be attached, via linking groups, at a carboxyl, amino or oxo group of the ligand. Typical linking groups may be ester, amide or carbamate groups.


Specific examples of preferred modified oligonucleotides envisioned for use in the ligand-conjugated oligonucleotides of the invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined here, oligonucleotides having modified backbones or internucleoside linkages include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of the invention, modified oligonucleotides that do not have a phosphorus atom in their intersugar backbone can also be considered to be oligonucleosides.


Specific oligonucleotide chemical modifications are described below. It is not necessary for all positions in a given compound to be uniformly modified. Conversely, more than one modifications may be incorporated in a single dsRNA compound or even in a single nucleotide thereof.


Preferred modified internucleoside linkages or backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free-acid forms are also included.


Representative United States Patents relating to the preparation of the above phosphorus-atom-containing linkages include, but are not limited to, U.S. Pat. Nos. 4,469,863; 5,023,243; 5,264,423; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233 and 5,466,677, each of which is herein incorporated by reference in their entirety.


Preferred modified internucleoside linkages or backbones that do not include a phosphorus atom therein (i.e., oligonucleosides) have backbones that are formed by short chain alkyl or cycloalkyl intersugar linkages, mixed heteroatom and alkyl or cycloalkyl intersugar linkages, or one or more short chain heteroatomic or heterocyclic intersugar linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.


Representative United States patents relating to the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,214,134; 5,216,141; 5,264,562; 5,466,677; 5,470,967; 5,489,677; 5,602,240 and 5,663,312, each of which is herein incorporated by reference.


In other preferred oligonucleotide mimetics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleoside units are replaced with novel groups. The nucleobase units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligonucleotide, an oligonucleotide mimetic, that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide-containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to atoms of the amide portion of the backbone. Teaching of PNA compounds can be found for example in U.S. Pat. No. 5,539,082.


Some preferred embodiments of the invention employ oligonucleotides with phosphorothioate linkages and oligonucleosides with heteroatom backbones, and in particular —CH2NH—O—CH2, —CH2—N(CH3)—O—CH2—[known as a methylene (methylimino) or MMI backbone], —CH2—O—N(CH3)—CH2—, —CH2—N(CH3)—N(CH3)—CH2—, and —O—N(CH3)—CH2—CH2—[wherein the native phosphodiester backbone is represented as —O—P—O—CH2] of the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.


The oligonucleotides employed in the ligand-conjugated oligonucleotides of the invention may additionally or alternatively comprise nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.


Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in the Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligonucleotides of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Id., pages 276-278) and are presently preferred base substitutions, even more particularly when combined with 2′-methoxyethyl sugar modifications.


Representative United States patents relating to the preparation of certain of the above-noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 5,134,066; 5,459,255; 5,552,540; 5,594,121 and 5,596,091 all of which are hereby incorporated by reference.


In certain embodiments, the oligonucleotides employed in the ligand-conjugated oligonucleotides of the invention may additionally or alternatively comprise one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2′ position: OH; F; O-, S-, or N-alkyl, O-, S-, or N-alkenyl, or O, S- or N-alkynyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Particularly preferred are O[(CH2)nO]mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2′ position: C1 to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy [2′-O—CH2CH2OCH3, also known as 2′-O-(2-methoxyethyl) or 2′-MOE], i.e., an alkoxyalkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2′-DMAOE, as described in U.S. Pat. No. 6,127,533, filed on Jan. 30, 1998, the contents of which are incorporated by reference.


Other preferred modifications include 2′-methoxy (2′-O—CH3), 2′-aminopropoxy (2′-OCH2CH2CH2NH2) and 2′-fluoro (2′-F). Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides.


As used herein, the term “sugar substituent group” or “2′-substituent group” includes groups attached to the 2′-position of the ribofuranosyl moiety with or without an oxygen atom. Sugar substituent groups include, but are not limited to, fluoro, O-alkyl, O-alkylamino, O-alkylalkoxy, protected O-alkylamino, O-alkylaminoalkyl, O-alkyl imidazole and polyethers of the formula (O-alkyl)m, wherein m is 1 to about 10. Preferred among these polyethers are linear and cyclic polyethylene glycols (PEGs), and (PEG)-containing groups, such as crown ethers and, inter alia, those which are disclosed by Delgardo et. al. (Critical Reviews in Therapeutic Drug Carrier Systems 1992, 9:249), which is hereby incorporated by reference in its entirety. Further sugar modifications are disclosed by Cook (Anti-fibrosis Drug Design, 1991, 6:585-607). Fluoro, O-alkyl, O-alkylamino, O-alkyl imidazole, O-alkylaminoalkyl, and alkyl amino substitution is described in U.S. Pat. No. 6,166,197, entitled “Oligomeric Compounds having Pyrimidine Nucleotide(s) with 2′ and 5′ Substitutions,” hereby incorporated by reference in its entirety.


Additional sugar substituent groups amenable to the invention include 2′-SR and 2′-NR2 groups, wherein each R is, independently, hydrogen, a protecting group or substituted or unsubstituted alkyl, alkenyl, or alkynyl. 2′-SR Nucleosides are disclosed in U.S. Pat. No. 5,670,633, hereby incorporated by reference in its entirety. The incorporation of 2′-SR monomer synthons is disclosed by Hamm et al. (J. Org. Chem., 1997, 62:3415-3420). 2′-NR nucleosides are disclosed by Goettingen, M., J. Org. Chem., 1996, 61, 6273-6281; and Polushin et al., Tetrahedron Lett., 1996, 37, 3227-3230. Further representative 2′-substituent groups amenable to the invention include those having one of formula I or II:




embedded image


wherein,

    • E is C1-C10 alkyl, N(Q3)(Q4) or N═C (Q3)(Q4); each Q3 and Q4 is, independently, H, C1-C10 alkyl, dialkylaminoalkyl, a nitrogen protecting group, a tethered or untethered conjugate group, a linker to a solid support; or Q3 and Q4, together, form a nitrogen protecting group or a ring structure optionally including at least one additional heteroatom selected from N and 0;
    • q1 is an integer from 1 to 10;
    • q2 is an integer from 1 to 10;
    • q3 is 0 or 1;
    • q4 is 0, 1 or 2;
    • each Z1, Z2 and Z3 is, independently, C4-C7 cycloalkyl, C5-C14 aryl or C3-C15 heterocyclyl, wherein the heteroatom in said heterocyclyl group is selected from oxygen, nitrogen and sulfur;
    • Z4 is OM1, SM1, or N(M1)2; each M1 is, independently, H, C1-C8 alkyl, C1-C8 haloalkyl, C(═NH)N(H)M2, C(═O)N(H)M2 or OC(═O)N(H)M2; M2 is H or C1-C8 alkyl; and
    • Z5 is C1-C10 alkyl, C1-C10 haloalkyl, C2-C10 alkenyl, C2-C10 alkynyl, C6-C14 aryl, N(Q3)(Q4), OQ3, halo, SQ3 or CN.


Representative 2′-O-sugar substituent groups of formula I are disclosed in U.S. Pat. No. 6,172,209, entitled “Capped 2′-Oxyethoxy Oligonucleotides,” hereby incorporated by reference in its entirety. Representative cyclic 2′-O-sugar substituent groups of formula II are disclosed in U.S. Pat. No. 6,271,358, entitled “RNA Targeted 2′-Modified Oligonucleotides that are Conformationally Preorganized,” hereby incorporated by reference in its entirety.


Sugars having O-substitutions on the ribosyl ring are also amenable to the invention. Representative substitutions for ring O include, but are not limited to, S, CH2, CHF, and CF2.


Oligonucleotides may also have sugar mimetics, such as cyclobutyl moieties, in place of the pentofuranosyl sugar. Representative United States patents relating to the preparation of such modified sugars include, but are not limited to, U.S. Pat. Nos. 5,359,044; 5,466,786; 5,519,134; 5,591,722; 5,597,909; 5,646,265 and 5,700,920, all of which are hereby incorporated by reference.


Additional modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide. For example, one additional modification of the ligand-conjugated oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more additional non-ligand moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties, such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 111; Kabanov et al., FEBS Lett., 1990, 259, 327; Svinarchuk et al., Biochimie, 1993, 75, 49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990, 18, 3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923).


The invention also includes compositions employing oligonucleotides that are substantially chirally pure with regard to particular positions within the oligonucleotides. Examples of substantially chirally pure oligonucleotides include, but are not limited to, those having phosphorothioate linkages that are at least 75% Sp or Rp (Cook et al., U.S. Pat. No. 5,587,361) and those having substantially chirally pure (Sp or Rp) alkylphosphonate, phosphoramidate or phosphotriester linkages (Cook, U.S. Pat. Nos. 5,212,295 and 5,521,302).


In certain instances, the oligonucleotide may be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to oligonucleotides in order to enhance the activity, cellular distribution or cellular uptake of the oligonucleotide, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Typical conjugation protocols involve the synthesis of oligonucleotides bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction may be performed either with the oligonucleotide still bound to the solid support or following cleavage of the oligonucleotide in solution phase. Purification of the oligonucleotide conjugate by HPLC typically affords the pure conjugate.


Alternatively, the molecule being conjugated may be converted into a building block, such as a phosphoramidite, via an alcohol group present in the molecule or by attachment of a linker bearing an alcohol group that may be phosphorylated.


Importantly, each of these approaches may be used for the synthesis of ligand conjugated oligonucleotides. Amino linked oligonucleotides may be coupled directly with ligand via the use of coupling reagents or following activation of the ligand as an NHS or pentfluorophenolate ester. Ligand phosphoramidites may be synthesized via the attachment of an aminohexanol linker to one of the carboxyl groups followed by phosphitylation of the terminal alcohol functionality. Other linkers, such as cysteamine, may also be utilized for conjugation to a chloroacetyl linker present on a synthesized oligonucleotide.


The person skilled in the art is readily aware of methods to introduce the molecules of this invention into cells, tissues or organisms. Corresponding examples have also been provided in the detailed description of the invention above. For example, the nucleic acid molecules or the vectors of this invention, encoding for at least one strand of the inventive dsRNAs may be introduced into cells or tissues by methods known in the art, like transfections etc.


Also for the introduction of dsRNA molecules, means and methods have been provided. For example, targeted delivery by glycosylated and folate-modified molecules, including the use of polymeric carriers with ligands, such as galactose and lactose or the attachment of folic acid to various macromolecules allows the binding of molecules to be delivered to folate receptors. Targeted delivery by peptides and proteins other than antibodies, for example, including RGD-modified nanoparticles to deliver siRNA in vivo or multicomponent (nonviral) delivery systems including short cyclodextrins, adamantine-PEG are known. Yet, also the targeted delivery using antibodies or antibody fragments, including (monovalent) Fab-fragments of an antibody (or other fragments of such an antibody) or single-chain antibodies are envisaged.


Injection approaches for target directed delivery comprise, inter alia, hydrodynamic i.v. injection. Also cholesterol conjugates of dsRNA may be used for targeted delivery, whereby the conjugation to lipohilic groups enhances cell uptake and improve pharmacokinetics and tissue biodistribution of oligonucleotides. Also cationic delivery systems are known, whereby synthetic vectors with net positive (cationic) charge to facilitate the complex formation with the polyanionic nucleic acid and interaction with the negatively charged cell membrane. Such cationic delivery systems comprise also cationic liposomal delivery systems, cationic polymer and peptide delivery systems. Other delivery systems for the cellular uptake of dsRNA/siRNA are aptamer-ds/siRNA. Also gene therapy approaches can be used to deliver the inventive dsRNA molecules or nucleic acid molecules encoding the same. Such systems comprise the use of non-pathogenic virus, modified viral vectors, as well as deliveries with nanoparticles or liposomes. Other delivery methods for the cellular uptake of dsRNA are extracorporeal, for example ex vivo treatments of cells, organs or tissues. Certain of these technologies are described and summarized in publications, like Akhtar (2007), Journal of Clinical Investigation 117, 3623-3632, Nguyen et al. (2008), Current Opinion in Moleculare Therapeutics 10, 158-167, Zamboni (2005), Clin. Cancer Res. 11, 8230-8234 or Ikeda et al. (2006), Pharmaceutical Research 23, 1631-1640


Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.


The above provided embodiments and items of the present invention are now illustrated with the following, non-limiting examples.


DESCRIPTION OF FIGURES AND APPENDED TABLES



  • Table 1. Core sequences of dsRNAs targeting human RRM2 gene Letters in capitals represent RNA nucleotides.

  • Table 2. Characterization of dsRNAs targeting human RRM2: Activity testing with single dose in HeLa-S3 cells. Letters in capitals represent RNA nucleotides, lower case letters “c”, “g”, “a” and “u” represent 2′-O-methyl-modified nucleotides, “s” represents phosphorothioate, “dT” deoxythymidine, and “p” represents 5′-phosphate group (sequences without a “p” are lacking a 5′-phosphate group). S.d.=standard deviation, % mRNA=mean mRNA knockdown.

  • Table 3. Characterization of dsRNAs targeting human RRM2: Activity testing for dose response in HeLa-S3 cells. IC 50: 50% inhibitory concentration, IC 80: 80% inhibitory concentration, IC 20: 20% inhibitory concentration.

  • Table 4. Characterization of dsRNAs targeting human RRM2: Stability and Cytokine Induction. t 1/2: half-life of a strand as defined in examples, PBMC: Human peripheral blood mononuclear cells.

  • Table 5. Core sequences of dsRNAs targeting human RRM2 gene and their modified counterparts. Letters in capitals represent RNA nucleotides, lower case letters “c”, “g”, “a” and “u” represent 2′-O-methyl-modified nucleotides, “s” represents phosphorothioate, “dT” deoxythymidine, and “p” represents 5′-phosphate group (sequences without a “p” in columns 6 and 8 are lacking a 5′-phosphate group).

  • Table 6. Sequences of bDNA probes for determination of human RRM2. LE=label extender, CE=capture extender, BL=blocking probe.

  • Table 7. Sequences of bDNA probes for determination of human GAPDH. LE=label extender, CE=capture extender, BL=blocking probe.

  • Table 8. mRNA knockdown and cell viability dose-response curves, IC50 summary.

  • Table 9. Time course of mRNA knockdown: >80% mRNA knockdown observed at 24 hr, Percent mRNA knockdown with 5 nM siRNA, relative to mock transfection.


  • FIG. 1. RRM2 protein knockdown dose-response 24 h post-transfection. A: Western blot B: Quantitation of RRM2 protein in Western blot.


  • FIG. 2. Cell cycle analysis of HepG2 cells with dsRNA 477/839: FACS analysis following propidium iodide staining indicates that following siRNA treatment cells accumulate in S-phase.


  • FIG. 3. Cell cycle analysis of HepG2 cells with dsRNA 477/839: FACS analysis following propidium iodide staining indicates that following siRNA treatment cells accumulate in S-phase.


  • FIG. 4. Cell cycle analysis of HLF cells with dsRNA 477/839: FACS analysis following propidium iodide staining indicates that following siRNA treatment cells accumulate in S-phase.


  • FIG. 5. Cell cycle analysis of HLF cells with dsRNA 477/839: FACS analysis following propidium iodide staining indicates that following siRNA treatment cells accumulate in S-phase.


  • FIG. 6. Apoptosis assay in HepG2 cells. dsRNA 477/839 activates caspase-3/7.


  • FIG. 7. Apoptosis assay in HLF cells. dsRNA 477/839 activates caspase-3/7.


  • FIG. 8. Effects on components of DNA damage pathway 48 hr post-transfection with RRM2 siRNA (5 nM). RRM2 knockdown activates the pathway (pChk1 and γ-H2AX) even in the absence of phleomycin-induced DNA damage.



EXAMPLES

Identification of dsRNAs for Therapeutic Use


dsRNA design was carried out to identify dsRNAs specifically targeting human RRM2 for therapeutic use. First, the known mRNA sequences of human (Homo sapiens) RRM2 (NM_001034.3 and NM_001165931.1 listed as SEQ ID NO. 1013 and 1014) were downloaded from NCBI Genbank.


The cynomolgous monkey (Macaca fascicularis) RRM2 gene was sequenced (see SEQ ID NO. 1015)


The cynomolgus monkey sequence (SEQ ID NO. 1015) was examined together with the human RRM2 mRNA sequences (SEQ ID NO. 1013 and 1014) by computer analysis to identify homologous sequences of 19 nucleotides that yield RNA interference (RNAi) agents cross-reactive to both sequences.


In identifying RNAi agents, the selection was limited to 19mer sequences having at least 2 mismatches to any other sequence in the human RefSeq database (release 38), which we assumed to represent the comprehensive human transcriptome, by using a proprietary algorithm.


All sequences containing 4 or more consecutive U's (poly-U sequences) or G's (poly-G sequences) were excluded from the synthesis.


The sequences thus identified formed the basis for the synthesis of the RNAi agents in appended Tables 1, 2 and 5. dsRNAs cross-reactive to human as well as cynomolgous monkey were defined as most preferable for therapeutic use.


dsRNA Synthesis


Where the source of a reagent is not specifically given herein, such reagent may be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.


Single-stranded RNAs were produced by solid phase synthesis on a scale of 1 mole using an Expedite 8909 synthesizer (Applied Biosystems, Applera Deutschland GmbH, Darmstadt, Germany) and controlled pore glass (CPG, 500 Å, Proligo Biochemie GmbH, Hamburg, Germany) as solid support. RNA and RNA containing 2′-O-methyl nucleotides were generated by solid phase synthesis employing the corresponding phosphoramidites and 2′-O-methyl phosphoramidites, respectively (Proligo Biochemie GmbH, Hamburg, Germany). These building blocks were incorporated at selected sites within the sequence of the oligoribonucleotide chain using standard nucleoside phosphoramidite chemistry such as described in Current protocols in nucleic acid chemistry, Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA. Phosphorothioate linkages were introduced by replacement of the iodine oxidizer solution with a solution of the Beaucage reagent (Chruachem Ltd, Glasgow, UK) in acetonitrile (1%). Further ancillary reagents were obtained from Mallinckrodt Baker (Griesheim, Germany).


Deprotection and purification of the crude oligoribonucleotides by anion exchange HPLC were carried out according to established procedures. Yields and concentrations were determined by UV absorption of a solution of the respective RNA at a wavelength of 260 nm using a spectral photometer (DU 640B, Beckman Coulter GmbH, Unterschleißheim, Germany). Double stranded RNA was generated by mixing an equimolar solution of complementary strands in annealing buffer (20 mM sodium phosphate, pH 6.8; 100 mM sodium chloride), heated in a water bath at 85-90° C. for 3 minutes and cooled to room temperature over a period of 3-4 hours. The annealed RNA solution was stored at −20° C. until use.


Activity Testing


The activity of the RRM2 dsRNAs for therapeutic use described above was tested in HeLa-S3 cells. Cells in culture were used for quantitation of RRM2 mRNA by branched DNA in total mRNA derived from cells incubated with RRM2-targeting dsRNAs.


HeLa-S3 cells were obtained from American Type Culture Collection (Rockville, Md., cat. No. CCL-2.2) and cultured in Ham's F12 (Biochrom AG, Berlin, Germany, cat. No. FG 0815) supplemented to contain 10% fetal calf serum (FCS) (Biochrom AG, Berlin, Germany, cat. No. S0115), Penicillin 100 U/ml, Streptomycin 100 mg/ml (Biochrom AG, Berlin, Germany, cat. No. A2213) at 37° C. in an atmosphere with 5% CO2 in a humidified incubator (Heraeus HERAcell, Kendro Laboratory Products, Langenselbold, Germany).


Cell seeding and transfection of dsRNA were performed at the same time. For transfection with dsRNA, cells were seeded at a density of 2.0 times.10.sup.4 cells/well in 96-well plates. Transfection with dsRNA was carried out with lipofectamine 2000 (Invitrogen GmbH, Karlsruhe, Germany, cat.No. 11668-019) as described by the manufacturer. In a first single dose experiment dsRNAs were transfected at a concentration of 50 nM. In a second single dose experiment most active dsRNAs were reanalyzed at 500 pM. Most potent dsRNAs and modification variants thereof were tested for improved silencing of RRM2 in single dose at 30 pM. Very effective dsRNAs from single dose screens were further characterized by dose response curves. For this, transfections were performed as described for the single dose screen above, but with the following concentrations of dsRNA (nM): 24, 6, 1.5, 0.375, 0.0938, 0.0234, 0.0059, 0.0015, 0.0004 and 0.0001 nM. After transfection cells were incubated for 24 h at 37° C. and 5% CO2 in a humidified incubator (Heraeus HERAcell, Kendro Laboratory Products, Langenselbold, Germany). bDNA Assay Kit QuantiGene 2.0 (Panomics/Affymetrix, Fremont, USA, Cat-No: 15735) was used for quantification of RRM2 mRNA, while QuantiGene Assay 1.0 (Panomics/Affymetrix, Fremont, USA, Cat-No: QG0004) was used for quantification of GAPDH mRNA. 24 hours after transfection cells were harvested and lysed at 53° C. following procedures recommended by the manufacturer Panomics/Affymetrix for bDNA quantitation of mRNA. Afterwards, 50 μl of the lysates were incubated with probesets specific to human RRM2 and human GAPDH (sequence of probesets see appended tables 7 and 8) and processed according to the manufacturer's protocol for QuantiGene Assay Kit 1 or 2, respectively. Chemoluminescence was measured in a Victor2-Light (Perkin Elmer, Wiesbaden, Germany) as RLUs (relative light units) and values obtained with the human RRM2 probeset were normalized to the respective human GAPDH values for each well. Unrelated control dsRNAs were used as a negative control.


Inhibition data are given in appended tables 2 and 3.


Stability of dsRNAs


Stability of dsRNAs targeting human RRM2 was determined in in vitro assays with either human or mouse serum by measuring the half-life of each single strand.


Measurements were carried out in triplicates for each time point, using 3 μl 50 μM dsRNA sample mixed with 30 μl human serum (Sigma) or mouse serum (Sigma). Mixtures were incubated for either 0 min, 30 min, 1 h, 3 h, 6 h, 24 h, or 48 h at 37° C. As control for unspecific degradation dsRNA was incubated with 30 μl 1×PBS pH 6.8 for 48 h. Reactions were stopped by the addition of 4 μl proteinase K (20 mg/ml), 25 μl of—“Tissue and Cell Lysis Solution” (Epicentre) and 38 μl Millipore water for 30 min at 65° C. Samples were afterwards spin filtered through a 0.2 m 96 well filter plate at 1400 rpm for 8 min, washed with 55 μl Millipore water twice and spin filtered again.


For separation of single strands and analysis of remaining full length product (FLP), samples were run through an ion exchange Dionex Summit HPLC under denaturing conditions using as eluent A 20 mM Na3PO4 in 10% ACN pH=1 and for eluent B 1 M NaBr in eluent A.


The following gradient was applied:

















Time
% A
% B




















−1.0 min
75
25



1.00 min
75
25



19.0 min
38
62



19.5 min
0
100



21.5 min
0
100



22.0 min
75
25



24.0 min
75
25










For every injection, the chromatograms were integrated automatically by the Dionex Chromeleon 6.60 HPLC software, and were adjusted manually if necessary. All peak areas were corrected to the internal standard (IS) peak and normalized to the incubation at t=0 min. The area under the peak and resulting remaining FLP was calculated for each single strand and triplicate separately. Half-life (t1/2) of a strand was defined by the average time point [h] for triplicates at which half of the FLP was degraded. Results are given in appended table 4.


Cytokine Induction


Potential cytokine induction of dsRNAs was determined by measuring the release of INF-α and TNF-α in an in vitro PBMC assay.


Human peripheral blood mononuclear cells (PBMC) were isolated from buffy coat blood of two donors by Ficoll centrifugation at the day of transfection. Cells were transfected in quadruplicates with dsRNA and cultured for 24 h at 37° C. at a final concentration of 130 nM in Opti-MEM, using either Gene Porter 2 (GP2) or DOTAP. dsRNA sequences that were known to induce INF-α and TNF-α in this assay, as well as a CpG oligo, were used as positive controls. Chemical conjugated dsRNA or CpG oligonucleotides that did not need a transfection reagent for cytokine induction, were incubated at a concentration of 500 nM in culture medium. At the end of incubation, the quadruplicate culture supernatant were pooled.


INF-α and TNF-α was then measured in these pooled supernatants by standard sandwich ELISA with two data points per pool. The degree of cytokine induction was expressed relative to positive controls using a score from 0 to 5, with 5 indicating maximum induction. Results are given in appended table 4.


Cell Culture and siRNA Transfections.


HepG2, HLF and A549 cells were obtained from ATCC and maintained in the recommended media, supplemented with 10% fetal bovine serum and 2 mM 1-Glutamine (HepG2: MEME; HLF and A549: DMEM). Cells were transfected using 0.1 μl DharmaFect 1 (Thermo Fisher) per well of a 96-well plate, with each well containing a final volume of 100 μl growth media. Transfections in 6-well plates were carried out in a similar manner, with volumes adjusted for the larger well size. Transfections were performed using a “reverse transfection” protocol, in which cells (HepG2: 5,000 cells; HLF: 2,000 cells; A540: 4,000 cells) were mixed with transfection mix immediately prior to plating.


bDNA Assay.


QuantiGene branched DNA assays (Affymetrix) for mRNA quantitation were run according to the manufacturer's directions using probe sets designed and synthesized by Affymetrix. 40 μl of lysate were used, and signal was normalized to expression of cyclophin B. Results are summarized in table 8 and 9.


Cell Growth Assays.


Cell-Titer Glo assays (Promega) were performed according to the manufacturer's directions. Real-time growth assays were performed on the xCELLigence instrument (Roche) using E-Plate 96 plates. Cell index was measured every hour, and area under the curve calculated at particular time points. Results are summarized in table 8.


Western Blots.


Antibodies for Western blots were obtained from the following sources: Santa Cruz Biotechnology, Inc. (goat polyclonal anti-RRM2, catalog number sc-10844; goat anti-mouse-HRP, catalog number sc-2005); Cell Signaling Technology (rabbit polyclonal anti-Chk1, catalog number 2345; rabbit polyclonal anti-pChk1 (S317), catalog number 2344; goat anti-rabbit-HRP, catalog number 7074; loading control antibody sampler kit (HRP conjugate), catalog number 4670); R&D Systems (mouse monoclonal anti-H2AX, catalog number MAB3406; rabbit polyclonal anti-gamma-H2AX, catalog number AF2288); Promega (donkey anti-goat-HRP, catalog number V8051).


Cells were transfected in 6-well plates and lysed with Pierce M-PER Mammalian Protein Extraction Reagent containing Pierce Halt Protease and Phosphatase Inhibitor Cocktail, according to the manufacturer's recommendations. Protein concentrations were determined using the Pierce Micro BCA Protein Assay Kit. Lysates were run on Novex NuPAGE 4-12% Bis-Tris gels. Protein was transferred to nitrocellulose using the Invitrogen iBlot Dry Blotting System. Western blots were probed using dilutions of antibodies recommended by the manufacturers, and detected using Amersham ECL Plus Western Blotting Detection Reagents. Gel images were collected on a FujiFilm LAS-4000 instrument and quantitated using Multi Gauge v3.1 software. RRM2 protein knock-down dose-response results are shown in FIG. 1. Effects on components of DNA damage pathway (in vivo pharmacodynamic markers) are shown in FIG. 8.


Cell Cycle Assay.


Cells were transfected in 6-well plates and harvested at 24, 48, 72 and 96 hours after transfection. Media was removed and collected in a 50-ml conical tube. Wells were washed with 2 ml PBS and added to the corresponding tubes. Cells were trypsinized, and, once displaced, added to the appropriate tubes. Tubes were centrifuged at 2,000 rpm for 10 min, washed once with 2 ml PBS, and then centrifuged again at 2,000 rpm for 5 min. Supernatants were carefully removed. To fix cells, tubes were tapped to loosen the pellets, and 1.2 ml cold 70% ETOH was added. After vortexing, samples were stored at −20° C. overnight. After thawing cells at room temperature for 20-30 min, 1.2 ml cold PBS was added, and tubes were centrifuged at 2,000 rpm for 10 min. Pellets were washed with 2 ml PBS and centrifuged again at 2,000 rpm. To the pellets was added 0.5 ml propidium iodide/RNase staining buffer (BD Pharmingen, catalog number 550825). Following a 15-min incubation at 37° C., data was collected on FACS LSRII instrument (BD) using DIVA software (10,000 events per sample) and analyzed using FlowJo software. Results are shown in FIGS. 2-5.


Caspase Assay.


Activation of caspase 3/7 was determined using the Apo-ONE Homogeneous Caspase-3/7 assay (Promega), according to the manufacturer's recommendations. Results are shown in FIGS. 6 and 7.


All ranges recited herein encompass all combinations and subcombinations included within that range limit. All patents and publications cited herein are hereby incorporated by reference in their entirety.












TABLE 1





SEQ 

SEQ



ID
Sense strand 
ID
Antisense strand


NO
sequence (5′-3′)
NO
sequence (5′-3′)







  1
UUGUGGCAGACAGACUUAU
234
AUAAGUCUGUCUGCCACAA





  2
CCUGAUGUUCAAACACCUG
235
CAGGUGUUUGAACAUCAGG





  3
AGUCCAACAGAGAAUUCUU
236
AAGAAUUCUCUGUUGGACU





  4
UAGGCGAGUAUCAGAGGAU
237
AUCCUCUGAUACUCGCCUA





  5
GGCGAGUAUCAGAGGAUGG
238
CCAUCCUCUGAUACUCGCC





  6
UGUUCAAACACCUGGUACA
239
UGUACCAGGUGUUUGAACA





  7
GGGUGACCCUUUAGUGAGC
240
GCUCACUAAAGGGUCACCC





  8
GAAGGAAAGACUAACUUCU
241
AGAAGUUAGUCUUUCCUUC





  9
UUCUGAAAUGUAUAGUCUU
242
AAGACUAUACAUUUCAGAA





 10
CUGUGUAGCUACCUCACAA
243
UUGUGAGGUAGCUACACAG





 11
UGCACUCUAAUGAAGCAAU
244
AUUGCUUCAUUAGAGUGCA





 12
CCCAUUUGACUUUAUGGAG
245
CUCCAUAAAGUCAAAUGGG





 13
AAAUGUAUAGUCUUCUUAU
246
AUAAGAAGACUAUACAUUU





 14
UACAUUGAGUUUGUGGCAG
247
CUGCCACAAACUCAAUGUA





 15
CAAUACAUUGAGUUUGUGG
248
CCACAAACUCAAUGUAUUG





 16
GAACAGGAGUUCCUCACUG
249
CAGUGAGGAACUCCUGUUC





 17
AUCCCAUGUUCUGGCUUUC
250
GAAAGCCAGAACAUGGGAU





 18
GUAGGUUGUGUGAGUUAAU
251
AUUAACUCACACAACCUAC





 19
AUAGUCUUCUUAUUGACAC
252
GUGUCAAUAAGAAGACUAU





 20
AUUGCACUCUAAUGAAGCA
253
UGCUUCAUUAGAGUGCAAU





 21
UUAUCAAUGCUGUUCGGAU
254
AUCCGAACAGCAUUGAUAA





 22
AGAAACGAGGACUGAUGCC
255
GGCAUCAGUCCUCGUUUCU





 23
CAUUGAGUUUGUGGCAGAC
256
GUCUGCCACAAACUCAAUG





 24
ACAUUCAGCACUGGGAAUC
257
GAUUCCCAGUGCUGAAUGU





 25
UGAUGUUCAAACACCUGGU
258
ACCAGGUGUUUGAACAUCA





 26
GGAUAGAACAGGAGUUCCU
259
AGGAACUCCUGUUCUAUCC





 27
AAUAUUUCACUGGAAGGAA
260
UUCCUUCCAGUGAAAUAUU





 28
AAUAAACAUUGUUUGUACU
261
AGUACAAACAAUGUUUAUU





 29
UCCCAUGUUCUGGCUUUCU
262
AGAAAGCCAGAACAUGGGA





 30
UUCGGAUAGAACAGGAGUU
263
AACUCCUGUUCUAUCCGAA





 31
AAGUAGGUUGUGUGAGUUA
264
UAACUCACACAACCUACUU





 32
UUAUAGUGCUGGUAGUAUC
265
GAUACUACCAGCACUAUAA





 33
CUUCUUAUUGACACUUACA
266
UGUAAGUGUCAAUAAGAAG





 34
UACAGAAGCCCGCUGUUUC
267
GAAACAGCGGGCUUCUGUA





 35
GUGACCCUUUAGUGAGCUU
268
AAGCUCACUAAAGGGUCAC





 36
AUAGAACAGGAGUUCCUCA
269
UGAGGAACUCCUGUUCUAU





 37
CUGGCACUUUACAAACAAA
270
UUUGUUUGUAAAGUGCCAG





 38
UCUAAUGAAGCAAUACAUU
271
AAUGUAUUGCUUCAUUAGA





 39
UCUUCUUAUUGACACUUAC
272
GUAAGUGUCAAUAAGAAGA





 40
UGUUCGGAUAGAACAGGAG
273
CUCCUGUUCUAUCCGAACA





 41
AGUACCAUGAUAUCUGGCA
274
UGCCAGAUAUCAUGGUACU





 42
CAGAGAUGAGGGUUUACAC
275
GUGUAAACCCUCAUCUCUG





 43
GAAACGAGGACUGAUGCCU
276
AGGCAUCAGUCCUCGUUUC





 44
AAGAGAGUAGGCGAGUAUC
277
GAUACUCGCCUACUCUCUU





 45
CAUUAGCUGAAUAAUGUGA
278
UCACAUUAUUCAGCUAAUG





 46
AGUAGAGAACCCAUUUGAC
279
GUCAAAUGGGUUCUCUACU





 47
AGGCGAGUAUCAGAGGAUG
280
CAUCCUCUGAUACUCGCCU





 48
UAGACUAAGCAUGUAAUUU
281
AAAUUACAUGCUUAGUCUA





 49
AACAUUGUUUGUACUCACA
282
UGUGAGUACAAACAAUGUU





 50
GAUGGGAGUGAUGUCAAGU
283
ACUUGACAUCACUCCCAUC





 51
CAGACCAUUUCCUAAUCAG
284
CUGAUUAGGAAAUGGUCUG





 52
GAUUACAGAAGCCCGCUGU
285
ACAGCGGGCUUCUGUAAUC





 53
CAUUGAAACGAUGCCUUGU
286
ACAAGGCAUCGUUUCAAUG





 54
ACUUAUGCUGGAACUGGGU
287
ACCCAGUUCCAGCAUAAGU





 55
GUCGACAAGGAGAACACGC
288
GCGUGUUCUCCUUGUCGAC





 56
AGGAAAGACUAACUUCUUU
289
AAAGAAGUUAGUCUUUCCU





 57
CAAGACCGCGAGGAGGAUC
290
GAUCCUCCUCGCGGUCUUG





 58
GACAAUGGCAGUCUUGGCU
291
AGCCAAGACUGCCAUUGUC





 59
AUGCCUUGUGUCAAGAAGA
292
UCUUCUUGACACAAGGCAU





 60
GCCUCACUGCUUCAACGCA
293
UGCGUUGAAGCAGUGAGGC





 61
UACCUCACAACCAGUCCUG
294
CAGGACUGGUUGUGAGGUA





 62
GAGAAGAGAGUAGGCGAGU
295
ACUCGCCUACUCUCUUCUC





 63
AGACUUAUGCUGGAACUGG
296
CCAGUUCCAGCAUAAGUCU





 64
UUACAGAAGCCCGCUGUUU
297
AAACAGCGGGCUUCUGUAA





 65
UUAUGCUGGAACUGGGUUU
298
AAACCCAGUUCCAGCAUAA





 66
AUAAACAUUGUUUGUACUC
299
GAGUACAAACAAUGUUUAU





 67
UCAAUGCCAUUGAAACGAU
300
AUCGUUUCAAUGGCAUUGA





 68
AUAGUGCUGGUAGUAUCAC
301
GUGAUACUACCAGCACUAU





 69
CAGCCUCACUGCUUCAACG
302
CGUUGAAGCAGUGAGGCUG





 70
UCUUGGCUUUAAAGUGAGG
303
CCUCACUUUAAAGCCAAGA





 71
GGCUGUGACUUACCAUAGC
304
GCUAUGGUAAGUCACAGCC





 72
GGCUACCUAUGGUGAACGU
305
ACGUUCACCAUAGGUAGCC





 73
CGCGAGGAGGAUCUUCCAG
306
CUGGAAGAUCCUCCUCGCG





 74
GCCAUUGAAACGAUGCCUU
307
AAGGCAUCGUUUCAAUGGC





 75
AGCCUCACUGCUUCAACGC
308
GCGUUGAAGCAGUGAGGCU





 76
GGCAGACAGACUUAUGCUG
309
CAGCAUAAGUCUGUCUGCC





 77
GUGACUAAAGUAAGUUAAA
310
UUUAACUUACUUUAGUCAC





 78
AGUUAUUGUUACCUAAAGU
311
ACUUUAGGUAACAAUAACU





 79
GCCUUUAUGUUUGGGAGAA
312
UUCUCCCAAACAUAAAGGC





 80
UUCAGAGUAGAGAACCCAU
313
AUGGGUUCUCUACUCUGAA





 81
AAACGAGGACUGAUGCCUG
314
CAGGCAUCAGUCCUCGUUU





 82
GUAGGCGAGUAUCAGAGGA
315
UCCUCUGAUACUCGCCUAC





 83
AAGCCCGCUGUUUCUAUGG
316
CCAUAGAAACAGCGGGCUU





 84
UCAGCACUGGGAAUCCCUG
317
CAGGGAUUCCCAGUGCUGA





 85
GAAUAAUGUGAGGAUUAAC
318
GUUAAUCCUCACAUUAUUC





 86
UGUGGCAGACAGACUUAUG
319
CAUAAGUCUGUCUGCCACA





 87
AGAGAUAAAUGUUGAUCUU
320
AAGAUCAACAUUUAUCUCU





 88
UACCAUGAUAUCUGGCAGA
321
UCUGCCAGAUAUCAUGGUA





 89
CUUCCAAAUUGCCAUGGAA
322
UUCCAUGGCAAUUUGGAAG





 90
ACCGCGAGGAGGAUCUUCC
323
GGAAGAUCCUCCUCGCGGU





 91
GAAAUGUAUAGUCUUCUUA
324
UAAGAAGACUAUACAUUUC





 92
AUGUUCAAACACCUGGUAC
325
GUACCAGGUGUUUGAACAU





 93
AGGGAAUUUCUCUUCAAUG
326
CAUUGAAGAGAAAUUCCCU





 94
CCCUGUUAAGUGGUGAAAU
327
AUUUCACCACUUAACAGGG





 95
GAUGAGGGUUUACACUGUG
328
CACAGUGUAAACCCUCAUC





 96
UGUGUGAGUUAAUUCAUUU
329
AAAUGAAUUAACUCACACA





 97
UUGCCUGAUGUUCAAACAC
330
GUGUUUGAACAUCAGGCAA





 98
AAACUUGUGUAGACUAAGC
331
GCUUAGUCUACACAAGUUU





 99
UAUAUCCCAUGUUCUGGCU
332
AGCCAGAACAUGGGAUAUA





100
UUGUGUAGACUAAGCAUGU
333
ACAUGCUUAGUCUACACAA





101
AUGCUGUUCGGAUAGAACA
334
UGUUCUAUCCGAACAGCAU





102
AAUUAUCAAUGCUGUUCGG
335
CCGAACAGCAUUGAUAAUU





103
GCCUGAUGUUCAAACACCU
336
AGGUGUUUGAACAUCAGGC





104
CAUAGCAGUGACAAUGGCA
337
UGCCAUUGUCACUGCUAUG





105
UGUGAGUUAAUUCAUUUAU
338
AUAAAUGAAUUAACUCACA





106
AGUGCUGGUAGUAUCACCU
339
AGGUGAUACUACCAGCACU





107
UAUCAAUGCUGUUCGGAUA
340
UAUCCGAACAGCAUUGAUA





108
GACUAAAGUAAGUUAAACU
341
AGUUUAACUUACUUUAGUC





109
AAUGCUGUUCGGAUAGAAC
342
GUUCUAUCCGAACAGCAUU





110
AGAAUAUUUCACUGGAAGG
343
CCUUCCAGUGAAAUAUUCU





111
AUCUGGCAGAUGUAUAAGA
344
UCUUAUACAUCUGCCAGAU





112
UAUAGUGCUGGUAGUAUCA
345
UGAUACUACCAGCACUAUA





113
GGCCAGCAAGACCGCGAGG
346
CCUCGCGGUCUUGCUGGCC





114
CCAUGAUAUCUGGCAGAUG
347
CAUCUGCCAGAUAUCAUGG





115
UUAAACUUGUGUAGACUAA
348
UUAGUCUACACAAGUUUAA





116
UUCAAUGCCAUUGAAACGA
349
UCGUUUCAAUGGCAUUGAA





117
AGAAAGCUGAGACAUUGCA
350
UGCAAUGUCUCAGCUUUCU





118
CUAUGGCUUCCAAAUUGCC
351
GGCAAUUUGGAAGCCAUAG





119
AAGUGACUAAAGUAAGUUA
352
UAACUUACUUUAGUCACUU





120
UGACUAAAGUAAGUUAAAC
353
GUUUAACUUACUUUAGUCA





121
UGCUGUUCGGAUAGAACAG
354
CUGUUCUAUCCGAACAGCA





122
GCGAGUAUCAGAGGAUGGG
355
CCCAUCCUCUGAUACUCGC





123
GGGCCUUGCGCUGGAUUGG
356
CCAAUCCAGCGCAAGGCCC





124
ACCUCACAACCAGUCCUGU
357
ACAGGACUGGUUGUGAGGU





125
ACUAAGUGACUAAAGUAAG
358
CUUACUUUAGUCACUUAGU





126
AUUACAGAAGCCCGCUGUU
359
AACAGCGGGCUUCUGUAAU





127
GAGUAGGCGAGUAUCAGAG
360
CUCUGAUACUCGCCUACUC





128
CAGUGACAAUGGCAGUCUU
361
AAGACUGCCAUUGUCACUG





129
GGCCUUGCGCUGGAUUGGG
362
CCCAAUCCAGCGCAAGGCC





130
UUCUUAUUGACACUUACAU
363
AUGUAAGUGUCAAUAAGAA





131
UUCACUAAGUGACUAAAGU
364
ACUUUAGUCACUUAGUGAA





132
GUGUGAGUUAAUUCAUUUA
365
UAAAUGAAUUAACUCACAC





133
CCCGCUCGCGCCCAUCACG
366
CGUGAUGGGCGCGAGCGGG





134
GUAAGUUAAACUUGUGUAG
367
CUACACAAGUUUAACUUAC





135
CGGAAGUUGGAAUCAGGUU
368
AACCUGAUUCCAACUUCCG





136
AUGUGAGGAUUAACUUCUG
369
CAGAAGUUAAUCCUCACAU





137
UUAAGUGGUGAAAUCAACU
370
AGUUGAUUUCACCACUUAA





138
UGUAGACUAAGCAUGUAAU
371
AUUACAUGCUUAGUCUACA





139
AUAAUGUGAGGAUUAACUU
372
AAGUUAAUCCUCACAUUAU





140
GGCUGGCUGUGACUUACCA
373
UGGUAAGUCACAGCCAGCC





141
AAGAGGCUACCUAUGGUGA
374
UCACCAUAGGUAGCCUCUU





142
CAGAUUACAGAAGCCCGCU
375
AGCGGGCUUCUGUAAUCUG





143
UGAGGCCUUGCCUGUGAAG
376
CUUCACAGGCAAGGCCUCA





144
AUAAUUAUCAAUGCUGUUC
377
GAACAGCAUUGAUAAUUAU





145
GUGACUUACCAUAGCAGUG
466
CACUGCUAUGGUAAGUCAC





146
UAGGGCUACUUUGAAUUAA
378
UUAAUUCAAAGUAGCCCUA





147
UGGCAGAUGUAUAAGAAGG
379
CCUUCUUAUACAUCUGCCA





148
AUAGCUUGAUUUAUUUGGU
380
ACCAAAUAAAUCAAGCUAU





149
CAGCAAGACCGCGAGGAGG
381
CCUCCUCGCGGUCUUGCUG





150
GACUGAUGCCUGGCCUCAC
382
GUGAGGCCAGGCAUCAGUC





151
UUACCUUGGAUGCUGACUU
383
AAGUCAGCAUCCAAGGUAA





152
AUUCAGCACUGGGAAUCCC
384
GGGAUUCCCAGUGCUGAAU





153
AGCAAGACCGCGAGGAGGA
385
UCCUCCUCGCGGUCUUGCU





154
AGGGCUACUUUGAAUUAAU
386
AUUAAUUCAAAGUAGCCCU





155
UAAGUUAUUGUUACCUAAA
387
UUUAGGUAACAAUAACUUA





156
UUUAUAGUGCUGGUAGUAU
388
AUACUACCAGCACUAUAAA





157
GCAAGACCGCGAGGAGGAU
389
AUCCUCCUCGCGGUCUUGC





158
UCUAUGGCUUCCAAAUUGC
390
GCAAUUUGGAAGCCAUAGA





159
AAAGACUAACUUCUUUGAG
391
CUCAAAGAAGUUAGUCUUU





160
ACCAUGAUAUCUGGCAGAU
392
AUCUGCCAGAUAUCAUGGU





161
GACCAUUUCCUAAUCAGUU
393
AACUGAUUAGGAAAUGGUC





162
UUACCAUAGCAGUGACAAU
394
AUUGUCACUGCUAUGGUAA





163
AAUGUGAGGAUUAACUUCU
395
AGAAGUUAAUCCUCACAUU





164
UAGUGUCCUGGGAUUCUCU
396
AGAGAAUCCCAGGACACUA





165
UGUUAAGUGGUGAAAUCAA
397
UUGAUUUCACCACUUAACA





166
ACAAAUAUUCUUAAUAGGG
398
CCCUAUUAAGAAUAUUUGU





167
GCGGAAGUUGGAAUCAGGU
399
ACCUGAUUCCAACUUCCGC





168
AACUUGUGUAGACUAAGCA
400
UGCUUAGUCUACACAAGUU





169
AUUCUUAAUAGGGCUACUU
401
AAGUAGCCCUAUUAAGAAU





170
CCUAAAGUUAAUCCAGAUU
402
AAUCUGGAUUAACUUUAGG





171
UAUUGUUACCUAAAGUUAA
403
UUAACUUUAGGUAACAAUA





172
GUGCUGGUAGUAUCACCUU
404
AAGGUGAUACUACCAGCAC





173
CUGUGACUUACCAUAGCAG
405
CUGCUAUGGUAAGUCACAG





174
GAGCUUCUUAAGUUAAAUC
406
GAUUUAACUUAAGAAGCUC





175
CUGUUCGGAUAGAACAGGA
407
UCCUGUUCUAUCCGAACAG





176
GUUAUUGUUACCUAAAGUU
408
AACUUUAGGUAACAAUAAC





177
UAAUGUGAGGAUUAACUUC
409
GAAGUUAAUCCUCACAUUA





178
ACCACUAAUGGGAGCCAAU
410
AUUGGCUCCCAUUAGUGGU





179
UGUGUAGACUAAGCAUGUA
411
UACAUGCUUAGUCUACACA





180
UGGGCCUUGCGCUGGAUUG
412
CAAUCCAGCGCAAGGCCCA





181
AGGAGCUUCUUAAGUUAAA
413
UUUAACUUAAGAAGCUCCU





182
GGUGACCCUUUAGUGAGCU
414
AGCUCACUAAAGGGUCACC





183
AGAGUAGGCGAGUAUCAGA
415
UCUGAUACUCGCCUACUCU





184
GCAGUGACAAUGGCAGUCU
416
AGACUGCCAUUGUCACUGC





185
AAACGAUGCCUUGUGUCAA
417
UUGACACAAGGCAUCGUUU





186
GGACUGAUGCCUGGCCUCA
418
UGAGGCCAGGCAUCAGUCC





187
UGAGAGAUAAAUGUUGAUC
419
GAUCAACAUUUAUCUCUCA





188
UGGUUUCUACACCAAAUAC
420
GUAUUUGGUGUAGAAACCA





189
UCUCUGUAAUAUGAUACAU
421
AUGUAUCAUAUUACAGAGA





190
GAGAGAUAAAUGUUGAUCU
422
AGAUCAACAUUUAUCUCUC





191
ACUCUAAUGAAGCAAUACA
423
UGUAUUGCUUCAUUAGAGU





192
UGAAGUGUUACCAACUAGC
424
GCUAGUUGGUAACACUUCA





193
AAUGAAGCAAUACAUUGAG
425
CUCAAUGUAUUGCUUCAUU





194
ACGAUGCCUUGUGUCAAGA
426
UCUUGACACAAGGCAUCGU





195
AGACCGCGAGGAGGAUCUU
427
AAGAUCCUCCUCGCGGUCU





196
UUGUUACCUAAAGUUAAUC
428
GAUUAACUUUAGGUAACAA





197
CAGAAGCCCGCUGUUUCUA
429
UAGAAACAGCGGGCUUCUG





198
UUUGACUUUAUGGAGAAUA
430
UAUUCUCCAUAAAGUCAAA





199
UACCUAAAGUUAAUCCAGA
431
UCUGGAUUAACUUUAGGUA





200
UUCAAACACCUGGUACACA
432
UGUGUACCAGGUGUUUGAA





201
UUGCACUCUAAUGAAGCAA
433
UUGCUUCAUUAGAGUGCAA





202
UGUUACCUAAAGUUAAUCC
434
GGAUUAACUUUAGGUAACA





203
CACUAAGUGACUAAAGUAA
435
UUACUUUAGUCACUUAGUG





204
UGCCAGAUAGAAGACAGGU
436
ACCUGUCUUCUAUCUGGCA





205
AAUGUAUAGUCUUCUUAUU
437
AAUAAGAAGACUAUACAUU





206
GACCACUAAUGGGAGCCAA
438
UUGGCUCCCAUUAGUGGUC





207
GUUACCUAAAGUUAAUCCA
439
UGGAUUAACUUUAGGUAAC





208
UGAUGCCUGGCCUCACAUU
440
AAUGUGAGGCCAGGCAUCA





209
CCAACUUUAAAGUCAGUCC
441
GGACUGACUUUAAAGUUGG





210
UAAACUUGUGUAGACUAAG
442
CUUAGUCUACACAAGUUUA





211
AGUAGGUUGUGUGAGUUAA
443
UUAACUCACACAACCUACU





212
GUUAAACUUGUGUAGACUA
444
UAGUCUACACAAGUUUAAC





213
CUGACCACUAAUGGGAGCC
445
GGCUCCCAUUAGUGGUCAG





214
UAUUCUUAAUAGGGCUACU
446
AGUAGCCCUAUUAAGAAUA





215
GUAGUGUCCUGGGAUUCUC
447
GAGAAUCCCAGGACACUAC





216
UAUCUGGCAGAUGUAUAAG
448
CUUAUACAUCUGCCAGAUA





217
AGGCUACCUAUGGUGAACG
449
CGUUCACCAUAGGUAGCCU





218
UCAGACCAUUUCCUAAUCA
450
UGAUUAGGAAAUGGUCUGA





219
UUACCUAAAGUUAAUCCAG
451
CUGGAUUAACUUUAGGUAA





220
GGUUUCUACACCAAAUACA
452
UGUAUUUGGUGUAGAAACC





221
GUUGGUGCCAGAUAGAAGA
453
UCUUCUAUCUGGCACCAAC





222
GCUACCUAUGGUGAACGUG
454
CACGUUCACCAUAGGUAGC





223
UCACUAAGUGACUAAAGUA
455
UACUUUAGUCACUUAGUGA





224
UUAUUGUUACCUAAAGUUA
456
UAACUUUAGGUAACAAUAA





225
UAGCUGAAUAAUGUGAGGA
457
UCCUCACAUUAUUCAGCUA





226
UGACCACUAAUGGGAGCCA
458
UGGCUCCCAUUAGUGGUCA





227
GUAGCUACCUCACAACCAG
459
CUGGUUGUGAGGUAGCUAC





228
UCCCGCUCGCGCCCAUCAC
460
GUGAUGGGCGCGAGCGGGA





229
CUUGGCUUUAAAGUGAGGG
461
CCCUCACUUUAAAGCCAAG





230
AGAAGCCCGCUGUUUCUAU
462
AUAGAAACAGCGGGCUUCU





231
ACUAAAGUAAGUUAAACUU
463
AAGUUUAACUUACUUUAGU





232
AGUAAGUUAAACUUGUGUA
464
UACACAAGUUUAACUUACU





233
AAUAAUUAUCAAUGCUGUU
465
AACAGCAUUGAUAAUUAUU

























TABLE 2











Activity


Activity




















testing with
Activity
testing with
Activity






50 nM siRNA in
testing with
50 nM siRNA in
testing with






HeLaS3 cells,
500 pM siRNA
HeLaS3 cells,
30 pM siRNA


SEQ

SEQ

transfection 1
in HeLaS3 cells
transfection 2
in HeLaS3 cells


















ID
sense strand sequence
ID
antisense strand sequence
mRNA
s.d.
mRNA
s.d.
mRNA
s.d.
mRNA
s.d.


NO
(5'-3')
NO
(5'-3')
[%]
[%]
[%]
[%]
[%]
[%]
[%]
[%]





526
agGAAAGAcuAAcuucuuudTsdT
967
AAAGAAGUuAGUCUUUCCUdTsdT
n.d.
n.d.
n.d.
n.d.
10
 2
 42
 1





477
uucuGAAAuGuAuAGucuudTsdT
963
AAGACuAuAcAUUUcAGAAdTsdT
n.d.
n.d.
n.d.
n.d.
 8
 1
 48
 2





470
aguccAAcAGAGAAuucuudTsdT
962
AAGAAUUCUCUGUUGGACUdTsdT
n.d.
n.d.
n.d.
n.d.
 7
 0
 49
 2





476
gaAGGAAAGAcuAAcuucudTsdT
966
AGAAGUuAGUCUUUCCUUCdTsdT
n.d.
n.d.
n.d.
n.d.
 8
 2
 50
 2





507
ucuucuuAuuGAcAcuuAcdTsdT
964
GUAAGUGUcAAuAAGAAGAdTsdT
n.d.
n.d.
n.d.
n.d.
 3
 1
 53
 3





553
guAGGcGAGuAucAGAGGAdTsdT
968
UCCUCUGAuACUCGCCuACdTsdT
n.d.
n.d.
n.d.
n.d.
12
 2
 54
 4





501
cuucuuAuuGAcAcuuAcAdTsdT
965
UGuAAGUGUcAAuAAGAAGdTsdT
n.d.
n.d.
n.d.
n.d.
 9
 1
 58
 7





497
ucccAuGuucuGGcuuucudTsdT
961
AGAAAGCcAGAAcAUGGGAdTsdT
n.d.
n.d.
n.d.
n.d.
 5
 1
 70
 7





508
ucuucuUAuuGAcAcuuAcdTsdT
972
GUAAGUGUcAAuAaGAAGAdTsdT
n.d.
n.d.
n.d.
n.d.
14
 1
 85
 8





470
aguccAAcAGAGAAuucuudTsdT
970
AAGAAUUCUCUGUUGGACUdTsdT
n.d.
n.d.
n.d.
n.d.
15
 1
 89
 2





477
uucuGAAAuGuAuAGucuudTsdT
971
AAGACuAuAcAUUucAGAAdTsdT
n.d.
n.d.
n.d.
n.d.
18
 3
 89
 9





508
ucuucuuAuuGAcAcuuAcdTsdT
985
GuAAGUGUcAAuAAGAAGAdTsdT
n.d.
n.d.
n.d.
n.d.
11
 2
 91
 5





507
ucuucuuAuuGAcAcuuAcdTsdT
972
GUAAGUGUcAAuAaGAAGAdTsdT
n.d.
n.d.
n.d.
n.d.
14
 2
 91
10





470
aguccAAcAGAGAAuucuudTsdT
978
AaGAAUUCUCUGUuGGACUdTsdT
n.d.
n.d.
n.d.
n.d.
24
 3
 92
 7





501
cuucuuAuuGAcAcuuAcAdTsdT
973
UGuAAGUGUcAAuaAGAAGdTsdT
n.d.
n.d.
n.d.
n.d.
34
 4
 95
 5





497
ucccAuGuucuGGcuuucudTsdT
969
AGAAAGCcAGAAcaUGGGAdTsdT
n.d.
n.d.
n.d.
n.d.
39
 5
 96
 9





476
gaAGGAAAGAcuAAcuucudTsdT
974
AGAAGUuAGUCUUUCCUUCdTsdT
n.d.
n.d.
n.d.
n.d.
14
 1
 97
 8





553
guAGGcGAGuAucAGAGGAdTsdT
984
UcCUCUGAuACUCgCCuACdTsdT
n.d.
n.d.
n.d.
n.d.
35
 3
 98
 9





526
agGAAAGAcuAAcuucuuudTsdT
983
AaAGAAGUuAGUCuUUCCUdTsdT
n.d.
n.d.
n.d.
n.d.
26
 3
 98
 8





553
guAGGcGAGuAucAGAGGAdTsdT
976
UCCUCUGAuACUCgCCuACdTsdT
n.d.
n.d.
n.d.
n.d.
23
 1
 99
11





526
agGAAAGAcuAAcuucuuudTsdT
975
AAAGAAGUuAGUCuUUCCUdTsdT
n.d.
n.d.
n.d.
n.d.
20
 1
102
 6





508
ucuucuUAuuGAcAcuuAcdTsdT
980
GuAAGUGUcAAuAaGAAGAdTsdT
n.d.
n.d.
n.d.
n.d.
21
 1
102
13





477
uucuGAAAuGuAuAGucuudTsdT
979
AaGACuAuAcAUUucAGAAdTsdT
n.d.
n.d.
n.d.
n.d.
25
 3
104
16





497
ucccAuGuucuGGcuuucudTsdT
977
AgAAAGCcAGAAcaUGGGAdTsdT
n.d.
n.d.
n.d.
n.d.
43
 2
106
 8





501
cuucuuAuuGAcAcuuAcAdTsdT
981
UguAAGUGUcAAuaAGAAGdTsdT
n.d.
n.d.
n.d.
n.d.
66
14
111
14





476
gaAGGAAAGAcuAAcuucudTSdT
982
AgAAGUuAGUCUUuCCUUCdTsdT
n.d.
n.d.
n.d.
n.d.
23
 4
111
13





507
ucuucuuAuuGAcAcuuAcdTsdT
980
GuAAGUGUcAAuAaGAAGAdTsdT
n.d.
n.d.
n.d.
n.d.
21
 5
115
15





507
ucuucuuAuuGAcAcuuAcdTsdT
841
pGUAAGUGUcAAuAAGAAGAdTsdT
  7
 2
 11
 2
 2
 0
 45
 2





477
uucuGAAAuGuAuAGucuudTsdT
839
pAAGACuAuAcAUUUcAGAAdTsdT
 10
 1
 12
 2
 8
 1
 43
 4





501
cuucuuAuuGAcAcuuAcAdTsdT
842
pUGuAAGUGUcAAuAAGAAGdTsdT
 12
 1
 14
 0
 8
 2
 52
 6





469
AguccAAcAGAGAAuucuudTsdT
742
pAAGAAUUCUCUGUUGGACUdTsdT
 11
 1
 14
 0
 8
 0
 38
 4





475
GaAGGAAAGAcuAAcuucudTsdT
884
pAGAAGUuAGUCUUUCCUUCdTsdT
 10
 1
 14
 0
 7
 1
 48
 5





522
cauuGAAAcGAuGccuuGudTsdT
845
pACAAGGcAUCGUUUcAAUGdTsdT
 11
 2
 14
 2
n.d.
n.d.
n.d.
n.d.





525
AgGAAAGAcuAAcuucuuudTsdT
885
pAAAGAAGUuAGUCUUUCCUdTsdT
 12
 2
 15
 1
 8
 1
 44
 6





497
ucccAuGuucuGGcuuucudTsdT
711
pAGAAAGCcAGAAcAUGGGAdTsdT
 12
 1
 15
 2
 6
 1
 71
 5





471
uaGGcGAGuAucAGAGGAudTsdT
892
pAUCCUCUGAuACUCGCCuAdTsdT
 14
 1
 16
 1
n.d.
n.d.
n.d.
n.d.





552
GuAGGcGAGuAucAGAGGAdTsdT
891
pUCCUCUGAuACUCGCCuACdTsdT
 14
 2
 16
 1
10
 0
 51
 5





494
GgAuAGAAcAGGAGuuccudTsdT
866
pAGGAACUCCUGUUCuAUCCdTsdT
 17
 1
 16
 1
n.d.
n.d.
n.d.
n.d.





473
uguucAAAcAccuGGuAcAdTsdT
863
pUGuACcAGGUGUUUGAAcAdTsdT
 17
 1
 16
 0
n.d.
n.d.
n.d.
n.d.





560
cuuccAAAuuGccAuGGAAdTsdT
838
pUUCcAUGGcAAUUUGGAAGdTsdT
 16
 2
 16
 1
n.d.
n.d.
n.d.
n.d.





544
GccAuuGAAAcGAuGccuudTsdT
718
pAAGGcAUCGUUUcAAUGGCdTsdT
 13
 2
 17
 1
n.d.
n.d.
n.d.
n.d.





493
ugAuGuucAAAcAccuGGudTsdT
861
pACcAGGUGUUUGAAcAUcAdTsdT
 21
 1
 17
 1
n.d.
n.d.
n.d.
n.d.





481
AaAuGuAuAGucuucuuAudTsdT
714
pAUAAGAAGACuAuAcAUUUdTsdT
 14
 1
 17
 1
n.d.
n.d.
n.d.
n.d.





488
AuuGcAcucuAAuGAAGcAdTsdT
735
pUGCUUcAUuAGAGUGcAAUdTsdT
 13
 1
 18
 2
n.d.
n.d.
n.d.
n.d.





532
GaGAAGAGAGuAGGcGAGudTsdT
887
pACUCGCCuACUCUCUUCUCdTsdT
 18
 2
 18
 1
n.d.
n.d.
n.d.
n.d.





535
uuAuGcuGGAAcuGGGuuudTsdT
881
pAAACCcAGUUCcAGcAuAAdTsdT
 19
 2
 18
 1
n.d.
n.d.
n.d.
n.d.





467
uuGuGGcAGAcAGAcuuAudTsdT
737
pAUAAGUCUGUCUGCcAcAAdTsdT
 22
 0
 19
 1
n.d.
n.d.
n.d.
n.d.





489
uuAucAAuGcuGuucGGAudTsdT
728
pAUCCGAAcAGcAUUGAuAAdTsdT
 15
 1
 19
 1
n.d.
n.d.
n.d.
n.d.





480
cccAuuuGAcuuuAuGGAGdTsdT
739
pCUCcAuAAAGUcAAAUGGGdTsdT
 13
 1
 19
 1
n.d.
n.d.
n.d.
n.d.





533
AgAcuuAuGcuGGAAcuGGdTsdT
879
pCCAGUUCcAGcAuAAGUCUdTsdT
 14
 2
 19
 2
n.d.
n.d.
n.d.
n.d.





582
AucuGGcAGAuGuAuAAGAdTsdT
825
pUCUuAuAcAUCUGCcAGAUdTsdT
 19
 3
 19
 1
n.d.
n.d.
n.d.
n.d.





495
AauAuuucAcuGGAAGGAAdTsdT
741
pUUCCUUCcAGUGAAAuAUUdTsdT
 16
 1
 19
 2
n.d.
n.d.
n.d.
n.d.





534
uuAcAGAAGcccGcuGuuudTsdT
831
pAAAcAGCGGGCUUCUGuAAdTsdT
 23
 2
 20
 3
n.d.
n.d.
n.d.
n.d.





487
AuAGucuucuuAuuGAcAcdTsdT
715
pGUGUcAAuAAGAAGACuAUdTsdT
 19
 1
 20
 3
n.d.
n.d.
n.d.
n.d.





521
GauuAcAGAAGcccGcuGudTsdT
712
pACAGCGGGCUUCUGuAAUCdTsdT
 19
 2
 20
 1
n.d.
n.d.
n.d.
n.d.





663
ugAAGuGuuAccAAcuAGcdTsdT
743
pGCuAGUUGGuAAcACUUcAdTsdT
 29
 5
 20
 1
n.d.
n.d.
n.d.
n.d.





578
uaucAAuGcuGuucGGAuAdTsdT
865
puAUCCGAAcAGcAUUGAuAdTsdT
 19
 3
 20
 2
n.d.
n.d.
n.d.
n.d.





482
uacAuuGAGuuuGuGGcAGdTsdT
875
pCUGCcAcAAACUcAAUGuAdTsdT
 15
 1
 20
 2
n.d.
n.d.
n.d.
n.d.





528
GacAAuGGcAGucuuGGcudTsdT
755
pAGCcAAGACUGCcAUUGUCdTsdT
 20
 2
 21
 3
n.d.
n.d.
n.d.
n.d.





479
ugcAcucuAAuGAAGcAAudTsdT
871
pAUUGCUUcAUuAGAGUGcAdTsdT
 15
 1
 21
 1
n.d.
n.d.
n.d.
n.d.





559
uaccAuGAuAucuGGcAGAdTsdT
821
pUCUGCcAGAuAUcAUGGuAdTsdT
 23
 2
 21
 2
n.d.
n.d.
n.d.
n.d.





481
AaAuGuAuAGucuucuuAudTsdT
939
pAuAAGAAGACuAuAcAUUUdTsdT
 18
 2
 21
 1
n.d.
n.d.
n.d.
n.d.





484
GaAcAGGAGuuccucAcuGdTsdT
868
pcAGUGAGGAACUCCUGUUCdTsdT
 28
 1
 21
 1
n.d.
n.d.
n.d.
n.d.





498
uucGGAuAGAAcAGGAGuudTsdT
734
pAACUCCUGUUCuAUCCGAAdTsdT
 18
 1
 21
 1
n.d.
n.d.
n.d.
n.d.





512
GaAAcGAGGAcuGAuGccudTsdT
852
pAGGcAUcAGUCCUCGUUUCdTsdT
 17
 2
 21
 2
n.d.
n.d.
n.d.
n.d.





491
cauuGAGuuuGuGGcAGAcdTsdT
876
pGUCUGCcAcAAACUcAAUGdTsdT
 17
 1
 21
 2
n.d.
n.d.
n.d.
n.d.





485
AucccAuGuucuGGcuuucdTsdT
710
pGAAAGCcAGAAcAUGGGAUdTsdT
 12
 1
 22
 2
n.d.
n.d.
n.d.
n.d.





516
AgGcGAGuAucAGAGGAuGdTsdT
893
pcAUCCUCUGAuACUCGCCUdTsdT
 21
 2
 22
 2
n.d.
n.d.
n.d.
n.d.





518
AacAuuGuuuGuAcucAcAdTsdT
913
pUGUGAGuAcAAAcAAUGUUdTsdT
 27
 2
 22
 2
n.d.
n.d.
n.d.
n.d.





502
uacAGAAGcccGcuGuuucdTsdT
832
pGAAAcAGCGGGCUUCUGuAdTsdT
 23
 1
 22
 1
n.d.
n.d.
n.d.
n.d.





504
AuAGAAcAGGAGuuccucAdTsdT
867
pUGAGGAACUCCUGUUCuAUdTsdT
 23
 1
 22
 2
n.d.
n.d.
n.d.
n.d.





561
AccGcGAGGAGGAucuuccdTsdT
818
pGGAAGAUCCUCCUCGCGGUdTsdT
 21
 2
 22
 5
n.d.
n.d.
n.d.
n.d.





529
AuGccuuGuGucAAGAAGAdTsdT
846
pUCUUCUUGAcAcAAGGcAUdTsdT
 19
 2
 22
 0
n.d.
n.d.
n.d.
n.d.





568
uuGccuGAuGuucAAAcAcdTsdT
858
pGUGUUUGAAcAUcAGGcAAdTsdT
 31
 3
 22
 1
n.d.
n.d.
n.d.
n.d.





597
AuuAcAGAAGcccGcuGuudTsdT
830
pAAcAGCGGGCUUCUGuAAUdTsdT
 23
 3
 23
 2
n.d.
n.d.
n.d.
n.d.





510
AguAccAuGAuAucuGGcAdTsdT
820
pUGCcAGAuAUcAUGGuACUdTsdT
 26
 2
 23
 2
n.d.
n.d.
n.d.
n.d.





633
uuAccAuAGcAGuGAcAAudTsdT
754
pAUUGUcACUGCuAUGGuAAdTsdT
 20
 4
 23
 1
n.d.
n.d.
n.d.
n.d.





564
AgGGAAuuucucuucAAuGdTsdT
716
pcAUUGAAGAGAAAUUCCCUdTsdT
 22
 2
 23
 1
n.d.
n.d.
n.d.
n.d.





515
AguAGAGAAcccAuuuGAcdTsdT
738
pGUcAAAUGGGUUCUCuACUdTsdT
 20
 2
 23
 3
n.d.
n.d.
n.d.
n.d.





523
AcuuAuGcuGGAAcuGGGudTsdT
880
pACCcAGUUCcAGcAuAAGUdTsdT
 20
 2
 23
 2
n.d.
n.d.
n.d.
n.d.





490
AgAAAcGAGGAcuGAuGccdTsdT
851
pGGcAUcAGUCCUCGUUUCUdTsdT
 20
 1
 23
 2
n.d.
n.d.
n.d.
n.d.





545
AgccucAcuGcuucAAcGcdTsdT
758
pGCGUUGAAGcAGUGAGGCUdTsdT
 23
 2
 23
 1
n.d.
n.d.
n.d.
n.d.





599
caGuGAcAAuGGcAGucuudTsdT
904
pAAGACUGCcAUUGUcACUGdTsdT
 26
 3
 23
 2
n.d.
n.d.
n.d.
n.d.





539
caGccucAcuGcuucAAcGdTsdT
908
pCGUUGAAGcAGUGAGGCUGdTsdT
 25
 2
 23
 2
n.d.
n.d.
n.d.
n.d.





521
GauuAcAGAAGcccGcuGudTsdT
938
pAcAGCGGGCUUCUGuAAUCdTsdT
 22
 1
 23
 1
n.d.
n.d.
n.d.
n.d.





573
AauuAucAAuGcuGuucGGdTsdT
727
pCCGAAcAGcAUUGAuAAUUdTsdT
 20
 3
 24
 2
n.d.
n.d.
n.d.
n.d.





505
CuGGcAcuuuAcAAAcAAAdTsdT
910
pUUUGUUUGuAAAGUGCcAGdTsdT
 32
 1
 24
 2
n.d.
n.d.
n.d.
n.d.





585
ccAuGAuAucuGGcAGAuGdTsdT
823
pcAUCUGCcAGAuAUcAUGGdTsdT
 23
 3
 25
 1
n.d.
n.d.
n.d.
n.d.





531
uaccucAcAAccAGuccuGdTsdT
745
pcAGGACUGGUUGUGAGGuAdTsdT
 25
 2
 25
 3
n.d.
n.d.
n.d.
n.d.





574
GccuGAuGuucAAAcAccudTsdT
859
pAGGUGUUUGAAcAUcAGGCdTsdT
 21
 3
 25
 1
n.d.
n.d.
n.d.
n.d.





492
AcAuucAGcAcuGGGAAucdTsdT
708
pGAUUCCcAGUGCUGAAUGUdTsdT
 17
 1
 25
 2
n.d.
n.d.
n.d.
n.d.





513
AaGAGAGuAGGcGAGuAucdTsdT
888
pGAuACUCGCCuACUCUCUUdTsdT
 21
 2
 25
 4
n.d.
n.d.
n.d.
n.d.





474
GgGuGAcccuuuAGuGAGcdTsdT
756
pGCUcACuAAAGGGUcACCCdTsdT
 24
 1
 25
 2
n.d.
n.d.
n.d.
n.d.





598
GaGuAGGcGAGuAucAGAGdTsdT
890
pCUCUGAuACUCGCCuACUCdTsdT
 23
 3
 25
 1
n.d.
n.d.
n.d.
n.d.





496
AauAAAcAuuGuuuGuAcudTsdT
911
pAGuAcAAAcAAUGUUuAUUdTsdT
 25
 1
 25
 2
n.d.
n.d.
n.d.
n.d.





506
ucuAAuGAAGcAAuAcAuudTsdT
872
pAAUGuAUUGCUUcAUuAGAdTsdT
 22
 1
 26
 1
n.d.
n.d.
n.d.
n.d.





500
uuAuAGuGcuGGuAGuAucdTsdT
746
pGAuACuACcAGcACuAuAAdTsdT
 28
 1
 26
 1
n.d.
n.d.
n.d.
n.d.





583
uauAGuGcuGGuAGuAucAdTsdT
747
pUGAuACuACcAGcACuAuAdTsdT
 26
 3
 26
 1
n.d.
n.d.
n.d.
n.d.





563
AuGuucAAAcAccuGGuAcdTsdT
862
pGUACcAGGUGUUUGAAcAUdTsdT
 20
 2
 26
 2
n.d.
n.d.
n.d.
n.d.





468
ccuGAuGuucAAAcAccuGdTsdT
860
pcAGGUGUUUGAAcAUcAGGdTsdT
 18
 0
 27
 2
n.d.
n.d.
n.d.
n.d.





587
uucAAuGccAuuGAAAcGAdTsdT
844
pUCGUUUcAAUGGcAUUGAAdTsdT
 27
 3
 27
 2
n.d.
n.d.
n.d.
n.d.





550
uucAGAGuAGAGAAcccAudTsdT
882
pAUGGGUUCUCuACUCUGAAdTsdT
 25
 2
 27
 2
n.d.
n.d.
n.d.
n.d.





575
cauAGcAGuGAcAAuGGcAdTsdT
902
pUGCcAUUGUcACUGCuAUGdTsdT
 33
 3
 28
 1
n.d.
n.d.
n.d.
n.d.





624
AgcAAGAccGcGAGGAGGAdTsdT
814
pUCCUCCUCGCGGUCUUGCUdTsdT
 29
 4
 28
 4
n.d.
n.d.
n.d.
n.d.





627
uuuAuAGuGcuGGuAGuAudTsdT
900
pAUACuACcAGcACuAuAAAdTsdT
 30
 4
 28
 3
n.d.
n.d.
n.d.
n.d.





613
caGAuuAcAGAAGcccGcudTsdT
829
pAGCGGGCUUCUGuAAUCUGdTsdT
 27
 3
 29
 2
n.d.
n.d.
n.d.
n.d.





507
ucuucuuAuuGAcAcuuAcdTsdT
949
pGuAAGUGUcAAuAAGAAGAdTsdT
 18
 2
 29
 2
n.d.
n.d.
n.d.
n.d.





509
uguucGGAuAGAAcAGGAGdTsdT
733
pCUCCUGUUCuAUCCGAAcAdTsdT
 18
 2
 30
 4
n.d.
n.d.
n.d.
n.d.





530
GccucAcuGcuucAAcGcAdTsdT
909
pUGCGUUGAAGcAGUGAGGCdTsdT
 28
 2
 30
 5
n.d.
n.d.
n.d.
n.d.





483
caAuAcAuuGAGuuuGuGGdTsdT
874
pCCAcAAACUcAAUGuAUUGdTsdT
 19
 1
 30
 1
n.d.
n.d.
n.d.
n.d.





519
GauGGGAGuGAuGucAAGudTsdT
896
pACUUGAcAUcACUCCcAUCdTsdT
 26
 2
 30
 2
n.d.
n.d.
n.d.
n.d.





537
ucAAuGccAuuGAAAcGAudTsdT
717
pAUCGUUUcAAUGGcAUUGAdTsdT
 31
 2
 30
 2
n.d.
n.d.
n.d.
n.d.





511
caGAGAuGAGGGuuuAcAcdTsdT
856
pGUGuAAACCCUcAUCUCUGdTsdT
 23
 2
 30
 3
n.d.
n.d.
n.d.
n.d.





620
caGcAAGAccGcGAGGAGGdTsdT
707
pCCUCCUCGCGGUCUUGCUGdTsdT
 30
 4
 31
 4
n.d.
n.d.
n.d.
n.d.





595
AccucAcAAccAGuccuGudTsdT
899
pACAGGACUGGUUGUGAGGUdTsdT
 31
 3
 31
 1
n.d.
n.d.
n.d.
n.d.





668
caGAAGcccGcuGuuucuAdTsdT
833
puAGAAAcAGCGGGCUUCUGdTsdT
 32
 6
 32
 3
n.d.
n.d.
n.d.
n.d.





577
AguGcuGGuAGuAucAccudTsdT
749
pAGGUGAuACuACcAGcACUdTsdT
 28
 3
 32
 2
n.d.
n.d.
n.d.
n.d.





654
AgAGuAGGcGAGuAucAGAdTsdT
889
pUCUGAuACUCGCCuACUCUdTsdT
 48
 5
 33
 1
n.d.
n.d.
n.d.
n.d.





546
GgcAGAcAGAcuuAuGcuGdTsdT
878
pcAGcAuAAGUCUGUCUGCCdTsdT
 21
 2
 33
 3
n.d.
n.d.
n.d.
n.d.





622
uuAccuuGGAuGcuGAcuudTsdT
897
pAAGUcAGcAUCcAAGGuAAdTsdT
 50
 4
 33
 2
n.d.
n.d.
n.d.
n.d.





541
GgcuGuGAcuuAccAuAGcdTsdT
751
pGCuAUGGuAAGUcAcAGCCdTsdT
 21
 2
 33
 2
n.d.
n.d.
n.d.
n.d.





566
GauGAGGGuuuAcAcuGuGdTsdT
857
pcAcAGUGuAAACCCUcAUCdTsdT
 33
 2
 34
 2
n.d.
n.d.
n.d.
n.d.





503
GuGAcccuuuAGuGAGcuudTsdT
907
pAAGCUcACuAAAGGGUcACdTsdT
 25
 1
 34
 2
n.d.
n.d.
n.d.
n.d.





527
caAGAccGcGAGGAGGAucdTsdT
816
pGAUCCUCCUCGCGGUCUUGdTsdT
 29
 2
 35
 8
n.d.
n.d.
n.d.
n.d.





589
cuAuGGcuuccAAAuuGccdTsdT
837
pGGcAAUUUGGAAGCcAuAGdTsdT
 23
 3
 36
 3
n.d.
n.d.
n.d.
n.d.





643
GuGcuGGuAGuAucAccuudTsdT
750
pAAGGUGAuACuACcAGcACdTsdT
 37
 4
 37
 2
n.d.
n.d.
n.d.
n.d.





600
GgccuuGcGcuGGAuuGGGdTsdT
849
pCCcAAUCcAGCGcAAGGCCdTsdT
 27
 3
 38
 2
n.d.
n.d.
n.d.
n.d.





572
AuGcuGuucGGAuAGAAcAdTsdT
730
pUGUUCuAUCCGAAcAGcAUdTsdT
 32
 3
 38
 1
n.d.
n.d.
n.d.
n.d.





517
uaGAcuAAGcAuGuAAuuudTsdT
784
pAAAUuAcAUGCUuAGUCuAdTsdT
 32
 2
 39
 3
n.d.
n.d.
n.d.
n.d.





611
GgcuGGcuGuGAcuuAccAdTsdT
901
pUGGuAAGUcAcAGCcAGCCdTsdT
 38
 3
 40
 2
n.d.
n.d.
n.d.
n.d.





540
ucuuGGcuuuAAAGuGAGGdTsdT
905
pCCUcACUUuAAAGCcAAGAdTsdT
 23
 2
 40
 2
n.d.
n.d.
n.d.
n.d.





536
AuAAAcAuuGuuuGuAcucdTsdT
912
pGAGuAcAAAcAAUGUUuAUdTsdT
 50
 2
 40
 4
n.d.
n.d.
n.d.
n.d.





524
GucGAcAAGGAGAAcAcGcdTsdT
705
pGCGUGUUCUCCUUGUCGACdTsdT
 32
 2
 41
 3
n.d.
n.d.
n.d.
n.d.





551
AaAcGAGGAcuGAuGccuGdTsdT
724
pcAGGcAUcAGUCCUCGUUUdTsdT
 26
 2
 42
 4
n.d.
n.d.
n.d.
n.d.





601
uucuuAuuGAcAcuuAcAudTsdT
843
pAUGuAAGUGUcAAuAAGAAdTsdT
 31
 3
 43
 3
n.d.
n.d.
n.d.
n.d.





586
uuAAAcuuGuGuAGAcuAAdTsdT
777
pUUAGUCuAcAcAAGUUuAAdTsdT
 37
 3
 43
 4
n.d.
n.d.
n.d.
n.d.





616
GuGAcuuAccAuAGcAGuGdTsdT
753
pcACUGCuAUGGuAAGUcACdTsdT
 24
 4
 43
 4
n.d.
n.d.
n.d.
n.d.





472
GgcGAGuAucAGAGGAuGGdTsdT
894
pCCAUCCUCUGAuACUCGCCdTsdT
 26
 1
 44
 2
n.d.
n.d.
n.d.
n.d.





570
uauAucccAuGuucuGGcudTsdT
828
pAGCcAGAAcAUGGGAuAuAdTsdT
 28
 3
 44
 8
n.d.
n.d.
n.d.
n.d.





655
GcAGuGAcAAuGGcAGucudTsdT
903
pAGACUGCcAUUGUcACUGCdTsdT
 34
 5
 44
 2
n.d.
n.d.
n.d.
n.d.





671
uucAAAcAccuGGuAcAcAdTsdT
864
pUGUGuACcAGGUGUUUGAAdTsdT
 59
 6
 44
 4
n.d.
n.d.
n.d.
n.d.





580
AauGcuGuucGGAuAGAAcdTsdT
729
pGUUCuAUCCGAAcAGcAUUdTsdT
 31
 3
 44
 1
n.d.
n.d.
n.d.
n.d.





618
ugGcAGAuGuAuAAGAAGGdTsdT
826
pCCUUCUuAuAcAUCUGCcAdTsdT
 36
 4
 44
 7
n.d.
n.d.
n.d.
n.d.





478
cuGuGuAGcuAccucAcAAdTsdT
744
pUUGUGAGGuAGCuAcAcAGdTsdT
 24
 1
 46
 3
n.d.
n.d.
n.d.
n.d.





533
AgAcuuAuGcuGGAAcuGGdTsdT
954
pCcAGUUCcAGcAuAAGUCUdTsdT
 26
 1
 46
 3
n.d.
n.d.
n.d.
n.d.





605
GuAAGuuAAAcuuGuGuAGdTsdT
775
pCUAcAcAAGUUuAACUuACdTsdT
 44
 3
 46
 3
n.d.
n.d.
n.d.
n.d.





571
uuGuGuAGAcuAAGcAuGudTsdT
781
pACAUGCUuAGUCuAcAcAAdTsdT
 47
 3
 47
 2
n.d.
n.d.
n.d.
n.d.





557
uguGGcAGAcAGAcuuAuGdTsdT
877
pcAuAAGUCUGUCUGCcAcAdTsdT
 31
 2
 47
 7
n.d.
n.d.
n.d.
n.d.





467
uuGuGGcAGAcAGAcuuAudTsdT
940
pAuAAGUCUGUCUGCcAcAAdTsdT
 86
15
 47
 5
n.d.
n.d.
n.d.
n.d.





579
GacuAAAGuAAGuuAAAcudTsdT
772
pAGUUuAACUuACUUuAGUCdTsdT
 32
 3
 47
 2
n.d.
n.d.
n.d.
n.d.





543
cgcGAGGAGGAucuuccAGdTsdT
819
pCUGGAAGAUCCUCCUCGCGdTsdT
 52
 2
 48
 7
n.d.
n.d.
n.d.
n.d.





547
GuGAcuAAAGuAAGuuAAAdTsdT
770
pUUuAACUuACUUuAGUcACdTsdT
 39
 2
 48
 2
n.d.
n.d.
n.d.
n.d.





602
uucAcuAAGuGAcuAAAGudTsdT
765
pACUUuAGUcACUuAGUGAAdTsdT
 39
 3
 49
 4
n.d.
n.d.
n.d.
n.d.





628
GcAAGAccGcGAGGAGGAudTsdT
815
pAUCCUCCUCGCGGUCUUGCdTsdT
 36
 4
 49
 3
n.d.
n.d.
n.d.
n.d.





571
uuGuGuAGAcuAAGcAuGudTsdT
945
pAcAUGCUuAGUCuAcAcAAdTsdT
 59
 3
 50
 4
n.d.
n.d.
n.d.
n.d.





562
GaAAuGuAuAGucuucuuAdTsdT
713
puAAGAAGACuAuAcAUUUCdTsdT
 49
 2
 51
 6
n.d.
n.d.
n.d.
n.d.





593
GcGAGuAucAGAGGAuGGGdTsdT
895
pCCcAUCCUCUGAuACUCGCdTsdT
 27
 3
 52
 5
n.d.
n.d.
n.d.
n.d.





538
AuAGuGcuGGuAGuAucAcdTsdT
748
pGUGAuACuACcAGcACuAUdTsdT
 57
 2
 52
 3
n.d.
n.d.
n.d.
n.d.





666
AgAccGcGAGGAGGAucuudTsdT
817
pAAGAUCCUCCUCGCGGUCUdTsdT
 52
 6
 52
15
n.d.
n.d.
n.d.
n.d.





567
uguGuGAGuuAAuucAuuudTsdT
922
pAAAUGAAUuAACUcAcAcAdTsdT
 48
 2
 53
 4
n.d.
n.d.
n.d.
n.d.





629
ucuAuGGcuuccAAAuuGcdTsdT
836
pGCAAUUUGGAAGCcAuAGAdTsdT
 63
 4
 53
 3
n.d.
n.d.
n.d.
n.d.





590
AaGuGAcuAAAGuAAGuuAdTsdT
769
puAACUuACUUuAGUcACUUdTsdT
 62
 3
 53
 7
n.d.
n.d.
n.d.
n.d.





681
uaAAcuuGuGuAGAcuAAGdTsdT
778
pCUuAGUCuAcAcAAGUUuAdTsdT
 52
 7
 53
 3
n.d.
n.d.
n.d.
n.d.





703
AguAAGuuAAAcuuGuGuAdTsdT
774
puAcAcAAGUUuAACUuACUdTsdT
 50
13
 54
 2
n.d.
n.d.
n.d.
n.d.





603
GuGuGAGuuAAuucAuuuAdTsdT
791
puAAAUGAAUuAACUcAcACdTsdT
 46
 3
 54
 8
n.d.
n.d.
n.d.
n.d.





642
uauuGuuAccuAAAGuuAAdTsdT
803
pUUAACUUuAGGuAAcAAuAdTsdT
 44
 4
 55
 6
n.d.
n.d.
n.d.
n.d.





674
cacuAAGuGAcuAAAGuAAdTsdT
942
pUuACUUuAGUcACUuAGUGdTsdT
 51
 3
 55
 2
n.d.
n.d.
n.d.
n.d.





569
AaAcuuGuGuAGAcuAAGcdTsdT
779
pGCUuAGUCuAcAcAAGUUUdTsdT
 60
 3
 56
 3
n.d.
n.d.
n.d.
n.d.





636
uguuAAGuGGuGAAAucAAdTsdT
918
pUUGAUUUcACcACUuAAcAdTsdT
 38
 4
 56
 3
n.d.
n.d.
n.d.
n.d.





659
ugGuuucuAcAccAAAuAcdTsdT
941
pGuAUUUGGUGuAGAAACcAdTsdT
 51
 4
 56
 8
n.d.
n.d.
n.d.
n.d.





588
AgAAAGcuGAGAcAuuGcAdTsdT
925
pUGcAAUGUCUcAGCUUUCUdTsdT
 43
 3
 57
 1
n.d.
n.d.
n.d.
n.d.





596
AcuAAGuGAcuAAAGuAAGdTsdT
768
pCUuACUUuAGUcACUuAGUdTsdT
 52
 3
 57
 5
n.d.
n.d.
n.d.
n.d.





609
uguAGAcuAAGcAuGuAAudTsdT
783
pAUuAcAUGCUuAGUCuAcAdTsdT
 60
 3
 58
 5
n.d.
n.d.
n.d.
n.d.





650
uguGuAGAcuAAGcAuGuAdTsdT
782
puAcAUGCUuAGUCuAcAcAdTsdT
 54
 5
 58
 4
n.d.
n.d.
n.d.
n.d.





591
ugAcuAAAGuAAGuuAAAcdTsdT
771
pGUUuAACUuACUUuAGUcAdTsdT
 54
 3
 58
 1
n.d.
n.d.
n.d.
n.d.





639
AacuuGuGuAGAcuAAGcAdTsdT
780
pUGCUuAGUCuAcAcAAGUUdTsdT
 57
 4
 58
 2
n.d.
n.d.
n.d.
n.d.





683
GuuAAAcuuGuGuAGAcuAdTsdT
776
puAGUCuAcAcAAGUUuAACdTsdT
 55
 7
 58
 3
n.d.
n.d.
n.d.
n.d.





586
uuAAAcuuGuGuAGAcuAAdTsdT
944
pUuAGUCuAcAcAAGUUuAAdTsdT
 62
 6
 59
 2
n.d.
n.d.
n.d.
n.d.





653
GguGAcccuuuAGuGAGcudTsdT
757
pAGCUcACuAAAGGGUcACCdTsdT
 60
 5
 60
 7
n.d.
n.d.
n.d.
n.d.





542
GgcuAccuAuGGuGAAcGudTsdT
722
pACGUUcACcAuAGGuAGCCdTsdT
 30
 2
 60
 6
n.d.
n.d.
n.d.
n.d.





631
AccAuGAuAucuGGcAGAudTsdT
822
pAUCUGCcAGAuAUcAUGGUdTsdT
 65
 4
 60
 4
n.d.
n.d.
n.d.
n.d.





605
GuAAGuuAAAcuuGuGuAGdTsdT
943
pCuAcAcAAGUUuAACUuACdTsdT
 51
 3
 60
 6
n.d.
n.d.
n.d.
n.d.





676
AauGuAuAGucuucuuAuudTsdT
840
pAAuAAGAAGACuAuAcAUUdTsdT
 62
 6
 60
 8
n.d.
n.d.
n.d.
n.d.





499
AaGuAGGuuGuGuGAGuuAdTsdT
790
puAACUcAcAcAACCuACUUdTsdT
 56
 1
 60
 2
n.d.
n.d.
n.d.
n.d.





669
uuuGAcuuuAuGGAGAAuAdTsdT
883
puAUUCUCcAuAAAGUcAAAdTsdT
 61
 6
 60
 2
n.d.
n.d.
n.d.
n.d.





677
GaccAcuAAuGGGAGccAAdTsdT
914
pUUGGCUCCcAUuAGUGGUCdTsdT
 56
 6
 60
 6
n.d.
n.d.
n.d.
n.d.





627
uuuAuAGuGcuGGuAGuAudTsdT
957
pAuACuACcAGcACuAuAAAdTsdT
 74
 9
 61
 1
n.d.
n.d.
n.d.
n.d.





656
AaAcGAuGccuuGuGucAAdTsdT
719
pUUGAcAcAAGGcAUCGUUUdTsdT
 40
 5
 62
 8
n.d.
n.d.
n.d.
n.d.





647
GuuAuuGuuAccuAAAGuudTsdT
801
pAACUUuAGGuAAcAAuAACdTsdT
 63
 5
 62
14
n.d.
n.d.
n.d.
n.d.





644
cuGuGAcuuAccAuAGcAGdTsdT
752
pCUGCuAUGGuAAGUcAcAGdTsdT
 30
 4
 62
 6
n.d.
n.d.
n.d.
n.d.





652
AgGAGcuucuuAAGuuAAAdTsdT
788
pUUuAACUuAAGAAGCUCCUdTsdT
 61
 5
 62
 4
n.d.
n.d.
n.d.
n.d.





672
uuGcAcucuAAuGAAGcAAdTsdT
870
pUUGCUUcAUuAGAGUGcAAdTsdT
 88
 6
 63
 7
n.d.
n.d.
n.d.
n.d.





682
AguAGGuuGuGuGAGuuAAdTsdT
920
pUUAACUcAcAcAACCuACUdTsdT
 60
 7
 63
 5
n.d.
n.d.
n.d.
n.d.





594
GgGccuuGcGcuGGAuuGGdTsdT
848
pCCAAUCcAGCGcAAGGCCCdTsdT
 32
 3
 63
 5
n.d.
n.d.
n.d.
n.d.





701
AgAAGcccGcuGuuucuAudTsdT
834
pAUAGAAAcAGCGGGCUUCUdTsdT
 79
11
 63
 5
n.d.
n.d.
n.d.
n.d.





697
ugAccAcuAAuGGGAGccAdTsdT
763
pUGGCUCCcAUuAGUGGUcAdTsdT
 68
 9
 64
 8
n.d.
n.d.
n.d.
n.d.





664
AauGAAGcAAuAcAuuGAGdTsdT
873
pCUcAAUGuAUUGCUUcAUUdTsdT
 63
 5
 64
 4
n.d.
n.d.
n.d.
n.d.





662
AcucuAAuGAAGcAAuAcAdTsdT
736
pUGuAUUGCUUcAUuAGAGUdTsdT
 71
 5
 64
 6
n.d.
n.d.
n.d.
n.d.





659
ugGuuucuAcAccAAAuAcdTsdT
760
pGUAUUUGGUGuAGAAACcAdTsdT
 54
 5
 64
 3
n.d.
n.d.
n.d.
n.d.





615
AuAAuuAucAAuGcuGuucdTsdT
726
pGAAcAGcAUUGAuAAUuAUdTsdT
 45
 4
 65
 4
n.d.
n.d.
n.d.
n.d.





558
AgAGAuAAAuGuuGAucuudTsdT
798
pAAGAUcAAcAUUuAUCUCUdTsdT
 67
 2
 65
 7
n.d.
n.d.
n.d.
n.d.





651
ugGGccuuGcGcuGGAuuGdTsdT
847
pcAAUCcAGCGcAAGGCCcAdTsdT
 67
 5
 65
 3
n.d.
n.d.
n.d.
n.d.





698
GuAGcuAccucAcAAccAGdTsdT
898
pCUGGUUGUGAGGuAGCuACdTsdT
 61
 9
 65
 2
n.d.
n.d.
n.d.
n.d.





680
ccAAcuuuAAAGucAGuccdTsdT
915
pGGACUGACUUuAAAGUUGGdTsdT
 59
 6
 65
 2
n.d.
n.d.
n.d.
n.d.





645
GaGcuucuuAAGuuAAAucdTsdT
917
pGAUUuAACUuAAGAAGCUCdTsdT
 59
 5
 65
 2
n.d.
n.d.
n.d.
n.d.





642
uauuGuuAccuAAAGuuAAdTsdT
947
pUuAACUUuAGGuAAcAAuAdTsdT
 69
 4
 65
 3
n.d.
n.d.
n.d.
n.d.





486
GuAGGuuGuGuGAGuuAAudTsdT
921
pAUuAACUcAcAcAACCuACdTsdT
 61
 1
 65
 4
n.d.
n.d.
n.d.
n.d.





692
GuuGGuGccAGAuAGAAGAdTsdT
785
pUCUUCuAUCUGGcACcAACdTsdT
 65
 8
 65
 4
n.d.
n.d.
n.d.
n.d.





691
GguuucuAcAccAAAuAcAdTsdT
761
pUGuAUUUGGUGuAGAAACCdTsdT
 63
 7
 66
 6
n.d.
n.d.
n.d.
n.d.





674
cacuAAGuGAcuAAAGuAAdTsdT
767
pUUACUUuAGUcACUuAGUGdTsdT
 61
 6
 66
 7
n.d.
n.d.
n.d.
n.d.





623
AuucAGcAcuGGGAAucccdTsdT
709
pGGGAUUCCcAGUGCUGAAUdTsdT
 68
 4
 66
 9
n.d.
n.d.
n.d.
n.d.





581
AgAAuAuuucAcuGGAAGGdTsdT
740
pCCUUCcAGUGAAAuAUUCUdTsdT
 43
 3
 67
 4
n.d.
n.d.
n.d.
n.d.





673
uguuAccuAAAGuuAAuccdTsdT
805
pGGAUuAACUUuAGGuAAcAdTsdT
 70
 6
 67
 9
n.d.
n.d.
n.d.
n.d.





607
AuGuGAGGAuuAAcuucuGdTsdT
810
pcAGAAGUuAAUCCUcAcAUdTsdT
 69
 3
 67
 6
n.d.
n.d.
n.d.
n.d.





686
GuAGuGucouGGGAuucucdTsdT
787
pGAGAAUCCcAGGAcACuACdTsdT
 72
 7
 68
 4
n.d.
n.d.
n.d.
n.d.





576
uguGAGuuAAuucAuuuAudTsdT
923
pAUAAAUGAAUuAACUcAcAdTsdT
 64
 3
 68
 5
n.d.
n.d.
n.d.
n.d.





619
AuAGcuuGAuuuAuuuGGudTsdT
759
pACcAAAuAAAUcAAGCuAUdTsdT
 59
 4
 68
 5
n.d.
n.d.
n.d.
n.d.





608
uuAAGuGGuGAAAucAAcudTsdT
919
pAGUUGAUUUcACcACUuAAdTsdT
 62
 3
 68
 9
n.d.
n.d.
n.d.
n.d.





682
AguAGGuuGuGuGAGuuAAdTsdT
958
pUuAACUcAcAcAACCuACUdTsdT
 75
 5
 69
 5
n.d.
n.d.
n.d.
n.d.





549
GccuuuAuGuuuGGGAGAAdTsdT
924
pUUCUCCcAAAcAuAAAGGCdTsdT
 48
 2
 69
 4
n.d.
n.d.
n.d.
n.d.





520
caGAccAuuuccuAAucAGdTsdT
937
pCUGAUuAGGAAAUGGUCUGdTsdT
 70
 2
 69
 3
n.d.
n.d.
n.d.
n.d.





556
GaAuAAuGuGAGGAuuAAcdTsdT
933
pGUuAAUCCUcAcAUuAUUCdTsdT
 64
 2
 70
 7
n.d.
n.d.
n.d.
n.d.





679
ugAuGccuGGccucAcAuudTsdT
855
pAAUGUGAGGCcAGGcAUcAdTsdT
 72
 6
 70
 8
n.d.
n.d.
n.d.
n.d.





660
ucucuGuAAuAuGAuAcAudTsdT
927
pAUGuAUcAuAUuAcAGAGAdTsdT
 62
 5
 70
 3
n.d.
n.d.
n.d.
n.d.





694
ucAcuAAGuGAcuAAAGuAdTsdT
766
puACUUuAGUcACUuAGUGAdTsdT
 68
 8
 70
 5
n.d.
n.d.
n.d.
n.d.





675
ugccAGAuAGAAGAcAGGudTsdT
786
pACCUGUCUUCuAUCUGGcAdTsdT
 64
 6
 70
 3
n.d.
n.d.
n.d.
n.d.





658
ugAGAGAuAAAuGuuGAucdTsdT
797
pGAUcAAcAUUuAUCUCUcAdTsdT
 70
 5
 71
 9
n.d.
n.d.
n.d.
n.d.





684
cuGAccAcuAAuGGGAGccdTsdT
762
pGGCUCCcAUuAGUGGUcAGdTsdT
 68
 7
 72
 9
n.d.
n.d.
n.d.
n.d.





625
AgGGcuAcuuuGAAuuAAudTsdT
796
pAUuAAUUcAAAGuAGCCCUdTsdT
 70
 4
 72
 9
n.d.
n.d.
n.d.
n.d.





649
AccAcuAAuGGGAGccAAudTsdT
764
pAUUGGCUCCcAUuAGUGGUdTsdT
 61
 5
 72
 4
n.d.
n.d.
n.d.
n.d.





617
uaGGGcuAcuuuGAAuuAAdTsdT
946
pUuAAUUcAAAGuAGCCCuAdTsdT
 73
 3
 72
 6
n.d.
n.d.
n.d.
n.d.





606
cgGAAGuuGGAAucAGGuudTsdT
931
pAACCUGAUUCcAACUUCCGdTsdT
 57
 3
 73
 2
n.d.
n.d.
n.d.
n.d.





661
GaGAGAuAAAuGuuGAucudTsdT
926
pAGAUcAAcAUUuAUCUCUCdTsdT
 60
 5
 73
 6
n.d.
n.d.
n.d.
n.d.





584
GgccAGcAAGAccGcGAGGdTsdT
706
pCCUCGCGGUCUUGCUGGCCdTsdT
 49
 3
 74
 5
n.d.
n.d.
n.d.
n.d.





695
uuAuuGuuAccuAAAGuuAdTsdT
802
puAACUUuAGGuAAcAAuAAdTsdT
 74
 9
 74
13
n.d.
n.d.
n.d.
n.d.





576
uguGAGuuAAuucAuuuAudTsdT
959
pAuAAAUGAAUuAACUcAcAdTsdT
 67
 4
 74
 5
n.d.
n.d.
n.d.
n.d.





617
uaGGGcuAcuuuGAAuuAAdTsdT
795
pUUAAUUcAAAGuAGCCCuAdTsdT
 51
 4
 74
13
n.d.
n.d.
n.d.
n.d.





635
uaGuGuccuGGGAuucucudTsdT
916
pAGAGAAUCCcAGGAcACuAdTsdT
 72
 4
 74
 6
n.d.
n.d.
n.d.
n.d.





687
uaucuGGcAGAuGuAuAAGdTsdT
824
pCUuAuAcAUCUGCcAGAuAdTsdT
 86
 7
 74
 5
n.d.
n.d.
n.d.
n.d.





632
GaccAuuuccuAAucAGuudTsdT
811
pAACUGAUuAGGAAAUGGUCdTsdT
 61
 4
 75
10
n.d.
n.d.
n.d.
n.d.





626
uaAGuuAuuGuuAccuAAAdTsdT
799
pUUuAGGuAAcAAuAACUuAdTsdT
 84
 4
 75
 9
n.d.
n.d.
n.d.
n.d.





667
uuGuuAccuAAAGuuAAucdTsdT
804
pGAUuAACUUuAGGuAAcAAdTsdT
 76
 6
 76
 9
n.d.
n.d.
n.d.
n.d.





630
AaAGAcuAAcuucuuuGAGdTsdT
886
pCUcAAAGAAGUuAGUCUUUdTsdT
 49
 4
 76
 3
n.d.
n.d.
n.d.
n.d.





621
GacuGAuGccuGGccucAcdTsdT
854
pGUGAGGCcAGGcAUcAGUCdTsdT
 67
 4
 76
 4
n.d.
n.d.
n.d.
n.d.





670
uaccuAAAGuuAAuccAGAdTsdT
807
pUCUGGAUuAACUUuAGGuAdTsdT
 80
 6
 76
10
n.d.
n.d.
n.d.
n.d.





634
AauGuGAGGAuuAAcuucudTsdT
809
pAGAAGUuAAUCCUcAcAUUdTsdT
 69
 4
 77
 2
n.d.
n.d.
n.d.
n.d.





612
AaGAGGcuAccuAuGGuGAdTsdT
850
pUCACcAuAGGuAGCCUCUUdTsdT
 65
 3
 78
 2
n.d.
n.d.
n.d.
n.d.





678
GuuAccuAAAGuuAAuccAdTsdT
928
pUGGAUuAACUUuAGGuAACdTsdT
 73
 6
 79
 5
n.d.
n.d.
n.d.
n.d.





648
uaAuGuGAGGAuuAAcuucdTsdT
935
pGAAGUuAAUCCUcAcAUuAdTsdT
 75
 5
 80
 2
n.d.
n.d.
n.d.
n.d.





689
ucAGAccAuuuccuAAucAdTsdT
936
pUGAUuAGGAAAUGGUCUGAdTsdT
 75
 7
 81
 6
n.d.
n.d.
n.d.
n.d.





641
ccuAAAGuuAAuccAGAuudTsdT
929
pAAUCUGGAUuAACUUuAGGdTsdT
 72
 4
 81
 4
n.d.
n.d.
n.d.
n.d.





555
ucAGcAcuGGGAAucccuGdTsdT
827
pcAGGGAUUCCcAGUGCUGAdTsdT
 81
 2
 82
12
n.d.
n.d.
n.d.
n.d.





565
cccuGuuAAGuGGuGAAAudTsdT
789
pAUUUcACcACUuAAcAGGGdTsdT
 76
 2
 83
 4
n.d.
n.d.
n.d.
n.d.





554
AaGcccGcuGuuucuAuGGdTsdT
835
pCCAuAGAAAcAGCGGGCUUdTsdT
 72
 2
 84
 5
n.d.
n.d.
n.d.
n.d.





696
uaGcuGAAuAAuGuGAGGAdTsdT
808
pUCCUcAcAUuAUUcAGCuAdTsdT
 84
 9
 84
11
n.d.
n.d.
n.d.
n.d.





483
caAuAcAuuGAGuuuGuGGdTsdT
953
pCcAcAAACUcAAUGuAUUGdTsdT
 31
 2
 84
 6
n.d.
n.d.
n.d.
n.d.





638
GcGGAAGuuGGAAucAGGudTsdT
930
pACCUGAUUCcAACUUCCGCdTsdT
 72
 4
 84
 7
n.d.
n.d.
n.d.
n.d.





514
cauuAGcuGAAuAAuGuGAdTsdT
932
pUCAcAUuAUUcAGCuAAUGdTsdT
 72
 2
 85
 6
n.d.
n.d.
n.d.
n.d.





702
AcuAAAGuAAGuuAAAcuudTsdT
773
pAAGUUuAACUuACUUuAGUdTsdT
 87
12
 86
 6
n.d.
n.d.
n.d.
n.d.





595
AccucAcAAccAGuccuGudTsdT
956
pAcAGGACUGGUUGUGAGGUdTsdT
 35
 1
 86
 5
n.d.
n.d.
n.d.
n.d.





472
GgcGAGuAucAGAGGAuGGdTsdT
955
pCcAUCCUCUGAuACUCGCCdTsdT
 23
 1
 87
 5
n.d.
n.d.
n.d.
n.d.





690
uuAccuAAAGuuAAuccAGdTsdT
806
pCUGGAUuAACUUuAGGuAAdTsdT
 89
 7
 88
12
n.d.
n.d.
n.d.
n.d.





592
ugcuGuucGGAuAGAAcAGdTsdT
731
pCUGUUCuAUCCGAAcAGcAdTsdT
 76
 3
 89
 3
n.d.
n.d.
n.d.
n.d.





693
GcuAccuAuGGuGAAcGuGdTsdT
723
pcACGUUcACcAuAGGuAGCdTsdT
 77
 8
 89
 7
n.d.
n.d.
n.d.
n.d.





665
AcGAuGccuuGuGucAAGAdTsdT
720
pUCUUGAcAcAAGGcAUCGUdTsdT
 76
 6
 90
 4
n.d.
n.d.
n.d.
n.d.





688
AgGcuAccuAuGGuGAAcGdTsdT
721
pCGUUcACcAuAGGuAGCCUdTsdT
 90
 7
 90
 8
n.d.
n.d.
n.d.
n.d.





685
uauucuuAAuAGGGcuAcudTsdT
793
pAGuAGCCCuAUuAAGAAuAdTsdT
 93
 7
 90
17
n.d.
n.d.
n.d.
n.d.





701
AgAAGcccGcuGuuucuAudTsdT
948
pAuAGAAAcAGCGGGCUUCUdTsdT
 79
 3
 90
 4
n.d.
n.d.
n.d.
n.d.





646
CuGuucGGAuAGAAcAGGAdTsdT
732
pUCCUGUUCuAUCCGAAcAGdTsdT
 67
 5
 91
 9
n.d.
n.d.
n.d.
n.d.





640
AuucuuAAuAGGGcuAcuudTsdT
794
pAAGuAGCCCuAUuAAGAAUdTsdT
114
 4
 92
15
n.d.
n.d.
n.d.
n.d.





548
AguuAuuGuuAccuAAAGudTsdT
800
pACUUuAGGuAAcAAuAACUdTsdT
 67
 2
 92
10
n.d.
n.d.
n.d.
n.d.





610
AuAAuGuGAGGAuuAAcuudTsdT
934
pAAGUuAAUCCUcAcAUuAUdTsdT
 90
 3
 93
 6
n.d.
n.d.
n.d.
n.d.





637
AcAAAuAuucuuAAuAGGGdTsdT
792
pCCCuAUuAAGAAuAUUUGUdTsdT
 79
 4
 94
18
n.d.
n.d.
n.d.
n.d.





514
cauuAGcuGAAuAAuGuGAdTsdT
960
pUcAcAUuAUUcAGCuAAUGdTsdT
 85
 4
 94
 2
n.d.
n.d.
n.d.
n.d.





614
ugAGGccuuGccuGuGAAGdTsdT
869
pCUUcAcAGGcAAGGCCUcAdTsdT
100
 3
 95
 6
n.d.
n.d.
n.d.
n.d.





563
AuGuucAAAcAccuGGuAcdTsdT
952
pGuACcAGGUGUUUGAAcAUdTsdT
 96
 9
 95
14
n.d.
n.d.
n.d.
n.d.





699
ucccGcucGcGcccAucAcdTsdT
812
pGUGAUGGGCGCGAGCGGGAdTsdT
 94
10
 95
 8
n.d.
n.d.
n.d.
n.d.





604
cccGcucGcGcccAucAcGdTsdT
813
pCGUGAUGGGCGCGAGCGGGdTsdT
 90
 3
 96
 4
n.d.
n.d.
n.d.
n.d.





657
GgAcuGAuGccuGGccucAdTsdT
853
pUGAGGCcAGGcAUcAGUCCdTsdT
 87
 5
 97
 8
n.d.
n.d.
n.d.
n.d.





612
AaGAGGcuAccuAuGGuGAdTsdT
951
pUcACcAuAGGuAGCCUCUUdTsdT
 79
 9
 97
 7
n.d.
n.d.
n.d.
n.d.





522
cauuGAAAcGAuGccuuGudTsdT
950
pAcAAGGcAUCGUUUcAAUGdTsdT
 93
11
100
 6
n.d.
n.d.
n.d.
n.d.





700
cuuGGcuuuAAAGuGAGGGdTsdT
906
pCCCUcACUUuAAAGCcAAGdTsdT
 90
10
100
 7
n.d.
n.d.
n.d.
n.d.





704
AauAAuuAucAAuGcuGuudTsdT
725
pAAcAGcAUUGAuAAUuAUUdTsdT
134
14
101
 2
n.d.
n.d.
n.d.
n.d.




















TABLE 3









Activity testing for dose
Activity testing for
Activity testing for



response in HeLaS3 cells,
dose response in HeLaS3
dose response in HeLaS3



means of two transfections
cells, transfection 3
cells, transfection 4























mean



mean



mean



mean
mean
mean
max.
mean
mean
mean
max.
mean
mean
mean
max.


SEQ ID NO
IC50
IC80
IC20
inh
IC50
IC80
IC20
inh
IC50
IC80
IC20
inh


pair
[nM]
[nM]
[nM]
[%]
[nM]
[nM]
[nM]
[%]
[nM]
[nM]
[nM]
[%]






















469/742
0.004
0.059
0.001
91
0.013
0.124
0.002
95
0.014
0.158
0.002
91


525/885
0.004
0.074
0.001
90
0.01
0.10
0.00
92
n.d.
n.d.
n.d.
n.d.


477/839
0.006
0.066
0.001
91
0.016
0.143
0.003
93
0.013
0.198
0.002
90


552/891
0.006
0.107
0.001
88
0.02
0.14
0.00
90
n.d.
n.d.
n.d.
n.d.


475/884
0.006
0.073
0.001
91
0.02
0.16
0.00
93
n.d.
n.d.
n.d.
n.d.


501/842
0.007
0.091
0.001
90
0.02
0.18
0.00
91
n.d.
n.d.
n.d.
n.d.


473/863
0.009
0.330
0.002
84
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.


507/841
0.010
0.081
0.002
97
0.03
0.21
0.01
97
n.d.
n.d.
n.d.
n.d.


494/866
0.011
0.810
0.003
84
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.


544/718
0.014
0.145
0.003
92
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.


497/711
0.015
0.116
0.003
89
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.


560/838
0.021
0.265
0.004
86
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.


522/845
0.029
0.295
0.006
90
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.


471/892
0.029
0.361
0.005
88
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.


488/735
0.030
0.262
0.006
89
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.


507/949
0.204
2.588
0.053
82
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.


526/967
n.d.
n.d.
n.d.
n.d.
0.015
0.120
0.0016
92
n.d.
n.d.
n.d.
n.d.


470/962
n.d.
n.d.
n.d.
n.d.
0.022
0.219
0.004
94
0.020
0.314
0.002
90


553/968
n.d.
n.d.
n.d.
n.d.
0.024
0.152
0.0041
89
n.d.
n.d.
n.d.
n.d.


477/963
n.d.
n.d.
n.d.
n.d.
0.024
0.189
0.005
93
0.016
0.251
0.002
90


501/965
n.d.
n.d.
n.d.
n.d.
0.036
0.281
0.0053
91
n.d.
n.d.
n.d.
n.d.


476/966
n.d.
n.d.
n.d.
n.d.
0.038
0.313
0.0062
93
n.d.
n.d.
n.d.
n.d.


507/964
n.d.
n.d.
n.d.
n.d.
0.045
0.310
0.0077
97
n.d.
n.d.
n.d.
n.d.


477/971
n.d.
n.d.
n.d.
n.d.
0.154
2.062
0.034
85
n.d.
n.d.
n.d.
n.d.


470/970
n.d.
n.d.
n.d.
n.d.
0.184
2.134
0.040
87
n.d.
n.d.
n.d.
n.d.


470/978
n.d.
n.d.
n.d.
n.d.
0.479
#N/A
0.073
72
n.d.
n.d.
n.d.
n.d.


477/979
n.d.
n.d.
n.d.
n.d.
0.906
#N/A
0.142
78
n.d.
n.d.
n.d.
n.d.





















TABLE 4









Stability Cyno
Stability Human
Stability Mouse




Serum
Serum
Serum















sense
antisense
sense
antisense
sense
antisense
Human PBMC


SEQ ID NO






assay















pair
[hr]
[hr]
[hr]
[hr]
[hr]
[hr]
IFN-a
TNF-a


















475/884
15.8
2.2
>48
0.9
15.5
9.2
0
0


507/841
16.2
0.7
>48
1.7
12.1
0.6
0
0


525/885
41.1
4.2
>48
2.0
13.1
7.7
0
0


469/742
36.4
4.7
>48
2.5
14.2
7.5
0
0


552/891
13.6
9.8
>48
7.1
10.3
11.6
0
0


501/842
17.4
2.2
>48
11.4
13.5
3.2
0
0


477/839
40.8
16.8
>48
>48
27.4
10.7
0
0


497/711
>48
11.8
>48
>48
>48
14.2
0
0























TABLE 5






sense

antisense



antisense


SEQ ID
strand sequence
SEQ ID
strand sequence
SEQ ID
sense strand sequence
SEQ ID
strand sequence


NO
(5′-3′)
NO
(5′-3′)
NO
(5′-3′)
NO
(5′-3′)







  1
UUGUGGCAGACAGACUUAU
234
AUAAGUCUGUCUGCCACAA
467
uuGuGGcAGAcAGAcuuAudTsdT
737
pAUAAGUCUGUCUGCcAcAAdTsdT





  1
UUGUGGCAGACAGACUUAU
234
AUAAGUCUGUCUGCCACAA
467
uuGuGGcAGAcAGAcuuAudTsdT
940
pAuAAGUCUGUCUGCcAcAAdTsdT





  2
CCUGAUGUUCAAACACCUG
235
CAGGUGUUUGAACAUCAGG
468
ccuGAuGuucAAAcAccuGdTsdT
860
pcAGGUGUUUGAAcAUcAGGdTsdT





  3
AGUCCAACAGAGAAUUCUU
236
AAGAAUUCUCUGUUGGACU
469
AguccAAcAGAGAAuucuudTsdT
742
pAAGAAUUCUCUGUUGGACUdTsdT





  3
AGUCCAACAGAGAAUUCUU
236
AAGAAUUCUCUGUUGGACU
470
aguccAAcAGAGAAuucuudTsdT
962
AAGAAUUCUCUGUUGGACUdTsdT





  3
AGUCCAACAGAGAAUUCUU
236
AAGAAUUCUCUGUUGGACU
470
aguccAAcAGAGAAuucuudTsdT
970
AAGAAUUCUCUGUuGGACUdTsdT





  3
AGUCCAACAGAGAAUUCUU
236
AAGAAUUCUCUGUUGGACU
470
aguccAAcAGAGAAuucuudTsdT
978
AaGAAUUCUCUGUuGGACUdTsdT





  4
UAGGCGAGUAUCAGAGGAU
237
AUCCUCUGAUACUCGCCUA
471
uaGGcGAGuAucAGAGGAudTsdT
892
pAUCCUCUGAuACUCGCCuAdTsdT





  5
GGCGAGUAUCAGAGGAUGG
238
CCAUCCUCUGAUACUCGCC
472
GgcGAGuAucAGAGGAuGGdTsdT
894
pCCAUCCUCUGAuACUCGCCdTsdT





  5
GGCGAGUAUCAGAGGAUGG
238
CCAUCCUCUGAUACUCGCC
472
GgcGAGuAucAGAGGAuGGdTsdT
955
pCcAUCCUCUGAuACUCGCCdTsdT





  6
UGUUCAAACACCUGGUACA
239
UGUACCAGGUGUUUGAACA
473
uguucAAAcAccuGGuAcAdTsdT
863
pUGuACcAGGUGUUUGAAcAdTsdT





  7
GGGUGACCCUUUAGUGAGC
240
GCUCACUAAAGGGUCACCC
474
GgGuGAcccuuuAGuGAGcdTsdT
756
pGCUcACuAAAGGGUcACCCdTsdT





  8
GAAGGAAAGACUAACUUCU
241
AGAAGUUAGUCUUUCCUUC
475
GaAGGAAAGAcuAAcuucudTsdT
884
pAGAAGUuAGUCUUUCCUUCdTsdT





  8
GAAGGAAAGACUAACUUCU
241
AGAAGUUAGUCUUUCCUUC
476
gaAGGAAAGAcuAAcuucudTsdT
966
AGAAGUuAGUCUUUCCUUCdTsdT





  8
GAAGGAAAGACUAACUUCU
241
AGAAGUUAGUCUUUCCUUC
476
gaAGGAAAGAcuAAcuucudTsdT
974
AGAAGUuAGUCUUuCCUUCdTsdT





  8
GAAGGAAAGACUAACUUCU
241
AGAAGUUAGUCUUUCCUUC
476
gaAGGAAAGAcuAAcuucudTsdT
982
AgAAGUuAGUCUUuCCUUCdTsdT





  9
UUCUGAAAUGUAUAGUCUU
242
AAGACUAUACAUUUCAGAA
477
uucuGAAAuGuAuAGucuudTsdT
839
pAAGACuAuAcAUUUcAGAAdTsdT





  9
UUCUGAAAUGUAUAGUCUU
242
AAGACUAUACAUUUCAGAA
477
uucuGAAAuGuAuAGucuudTsdT
963
AAGACuAuAcAUUUcAGAAdTsdT





  9
UUCUGAAAUGUAUAGUCUU
242
AAGACUAUACAUUUCAGAA
477
uucuGAAAuGuAuAGucuudTsdT
971
AAGACuAuAcAUUucAGAAdTsdT





  9
UUCUGAAAUGUAUAGUCUU
242
AAGACUAUACAUUUCAGAA
477
uucuGAAAuGuAuAGucuudTsdT
979
AaGACuAuAcAUUucAGAAdTsdT





 10
CUGUGUAGCUACCUCACAA
243
UUGUGAGGUAGCUACACAG
478
cuGuGuAGcuAccucAcAAdTsdT
744
pUUGUGAGGuAGCuAcAcAGdTsdT





 11
UGCACUCUAAUGAAGCAAU
244
AUUGCUUCAUUAGAGUGCA
479
ugcAcucuAAuGAAGcAAudTsdT
871
pAUUGCUUcAUuAGAGUGcAdTsdT





 12
CCCAUUUGACUUUAUGGAG
245
CUCCAUAAAGUCAAAUGGG
480
cccAuuuGAcuuuAuGGAGdTsdT
739
pCUCcAuAAAGUcAAAUGGGdTsdT





 13
AAAUGUAUAGUCUUCUUAU
246
AUAAGAAGACUAUACAUUU
481
AaAuGuAuAGucuucuuAudTsdT
714
pAUAAGAAGACuAuAcAUUUdTsdT





 13
AAAUGUAUAGUCUUCUUAU
246
AUAAGAAGACUAUACAUUU
481
AaAuGuAuAGucuucuuAudTsdT
939
pAuAAGAAGACuAuAcAUUUdTsdT





 14
UACAUUGAGUUUGUGGCAG
247
CUGCCACAAACUCAAUGUA
482
uacAuuGAGuuuGuGGcAGdTsdT
875
pCUGCcAcAAACUcAAUGuAdTsdT





 15
CAAUACAUUGAGUUUGUGG
248
CCACAAACUCAAUGUAUUG
483
caAuAcAuuGAGuuuGuGGdTsdT
874
pCCAcAAACUcAAUGuAUUGdTsdT





 15
CAAUACAUUGAGUUUGUGG
248
CCACAAACUCAAUGUAUUG
483
caAuAcAuuGAGuuuGuGGdTsdT
953
pCcAcAAACUcAAUGuAUUGdTsdT





 16
GAACAGGAGUUCCUCACUG
249
CAGUGAGGAACUCCUGUUC
484
GaAcAGGAGuuccucAcuGdTsdT
868
pcAGUGAGGAACUCCUGUUCdTsdT





 17
AUCCCAUGUUCUGGCUUUC
250
GAAAGCCAGAACAUGGGAU
485
AucccAuGuucuGGcuuucdTsdT
710
pGAAAGCcAGAAcAUGGGAUdTsdT





 18
GUAGGUUGUGUGAGUUAAU
251
AUUAACUCACACAACCUAC
486
GuAGGuuGuGuGAGuuAAudTsdT
921
pAUuAACUcAcAcAACCuACdTsdT





 19
AUAGUCUUCUUAUUGACAC
252
GUGUCAAUAAGAAGACUAU
487
AuAGucuucuuAuuGAcAcdTsdT
715
pGUGUcAAuAAGAAGACuAUdTsdT





 20
AUUGCACUCUAAUGAAGCA
253
UGCUUCAUUAGAGUGCAAU
488
AuuGcAcucuAAuGAAGcAdTsdT
735
pUGCUUcAUuAGAGUGcAAUdTsdT





 21
UUAUCAAUGCUGUUCGGAU
254
AUCCGAACAGCAUUGAUAA
489
uuAucAAuGcuGuucGGAudTsdT
728
pAUCCGAAcAGcAUUGAuAAdTsdT





 22
AGAAACGAGGACUGAUGCC
255
GGCAUCAGUCCUCGUUUCU
490
AgAAAcGAGGAcuGAuGccdTsdT
851
pGGcAUcAGUCCUCGUUUCUdTsdT





 23
CAUUGAGUUUGUGGCAGAC
256
GUCUGCCACAAACUCAAUG
491
cauuGAGuuuGuGGcAGAcdTsdT
876
pGUCUGCcAcAAACUcAAUGdTsdT





 24
ACAUUCAGCACUGGGAAUC
257
GAUUCCCAGUGCUGAAUGU
492
AcAuucAGcAcuGGGAAucdTsdT
708
pGAUUCCcAGUGCUGAAUGUdTsdT





 25
UGAUGUUCAAACACCUGGU
258
ACCAGGUGUUUGAACAUCA
493
ugAuGuucAAAcAccuGGudTsdT
861
pACcAGGUGUUUGAAcAUcAdTsdT





 26
GGAUAGAACAGGAGUUCCU
259
AGGAACUCCUGUUCUAUCC
494
GgAuAGAAcAGGAGuuccudTsdT
866
pAGGAACUCCUGUUCuAUCCdTsdT





 27
AAUAUUUCACUGGAAGGAA
260
UUCCUUCCAGUGAAAUAUU
495
AauAuuucAcuGGAAGGAAdTsdT
741
pUUCCUUCcAGUGAAAuAUUdTsdT





 28
AAUAAACAUUGUUUGUACU
261
AGUACAAACAAUGUUUAUU
496
AauAAAcAuuGuuuGuAcudTsdT
911
pAGuAcAAAcAAUGUUuAUUdTsdT





 29
UCCCAUGUUCUGGCUUUCU
262
AGAAAGCCAGAACAUGGGA
497
ucccAuGuucuGGcuuucudTsdT
711
pAGAAAGCcAGAAcAUGGGAdTsdT





 29
UCCCAUGUUCUGGCUUUCU
262
AGAAAGCCAGAACAUGGGA
497
ucccAuGuucuGGcuuucudTsdT
961
AGAAAGCcAGAAcAUGGGAdTsdT





 29
UCCCAUGUUCUGGCUUUCU
262
AGAAAGCCAGAACAUGGGA
497
ucccAuGuucuGGcuuucudTsdT
969
AGAAAGCcAGAAcaUGGGAdTsdT





 29
UCCCAUGUUCUGGCUUUCU
262
AGAAAGCCAGAACAUGGGA
497
ucccAuGuucuGGcuuucudTsdT
977
AgAAAGCcAGAAcaUGGGAdTsdT





 30
UUCGGAUAGAACAGGAGUU
263
AACUCCUGUUCUAUCCGAA
498
uucGGAuAGAAcAGGAGuudTsdT
734
pAACUCCUGUUCuAUCCGAAdTsdT





 31
AAGUAGGUUGUGUGAGUUA
264
UAACUCACACAACCUACUU
499
AaGuAGGuuGuGuGAGuuAdTsdT
790
puAACUcAcAcAACCuACUUdTsdT





 32
UUAUAGUGCUGGUAGUAUC
265
GAUACUACCAGCACUAUAA
500
uuAuAGuGcuGGuAGuAucdTsdT
746
pGAuACuACcAGcACuAuAAdTsdT





 33
CUUCUUAUUGACACUUACA
266
UGUAAGUGUCAAUAAGAAG
501
cuucuuAuuGAcAcuuAcAdTsdT
842
pUGuAAGUGUcAAuAAGAAGdTsdT





 33
CUUCUUAUUGACACUUACA
266
UGUAAGUGUCAAUAAGAAG
501
cuucuuAuuGAcAcuuAcAdTsdT
965
UGuAAGUGUcAAuAAGAAGdTsdT





 33
CUUCUUAUUGACACUUACA
266
UGUAAGUGUCAAUAAGAAG
501
cuucuuAuuGAcAcuuAcAdTsdT
973
UGuAAGUGUcAAuaAGAAGdTsdT





 33
CUUCUUAUUGACACUUACA
266
UGUAAGUGUCAAUAAGAAG
501
cuucuuAuuGAcAcuuAcAdTsdT
981
UguAAGUGUcAAuaAGAAGdTsdT





 34
UACAGAAGCCCGCUGUUUC
267
GAAACAGCGGGCUUCUGUA
502
uacAGAAGcccGcuGuuucdTsdT
832
pGAAAcAGCGGGCUUCUGuAdTsdT





 35
GUGACCCUUUAGUGAGCUU
268
AAGCUCACUAAAGGGUCAC
503
GuGAcccuuuAGuGAGcuudTsdT
907
pAAGCUcACuAAAGGGUcACdTsdT





 36
AUAGAACAGGAGUUCCUCA
269
UGAGGAACUCCUGUUCUAU
504
AuAGAAcAGGAGuuccucAdTsdT
867
pUGAGGAACUCCUGUUCuAUdTsdT





 37
CUGGCACUUUACAAACAAA
270
UUUGUUUGUAAAGUGCCAG
505
cuGGcAcuuuAcAAAcAAAdTsdT
910
pUUUGUUUGuAAAGUGCcAGdTsdT





 38
UCUAAUGAAGCAAUACAUU
271
AAUGUAUUGCUUCAUUAGA
506
ucuAAuGAAGcAAuAcAuudTsdT
872
pAAUGuAUUGCUUcAUuAGAdTsdT





 39
UCUUCUUAUUGACACUUAC
272
GUAAGUGUCAAUAAGAAGA
507
ucuucuuAuuGAcAcuuAcdTsdT
841
pGUAAGUGUcAAuAAGAAGAdTsdT





 39
UCUUCUUAUUGACACUUAC
272
GUAAGUGUCAAUAAGAAGA
507
ucuucuuAuuGAcAcuuAcdTsdT
949
pGuAAGUGUcAAuAAGAAGAdTsdT





 39
UCUUCUUAUUGACACUUAC
272
GUAAGUGUCAAUAAGAAGA
507
ucuucuuAuuGAcAcuuAcdTsdT
964
GUAAGUGUcAAuAAGAAGAdTsdT





 39
UCUUCUUAUUGACACUUAC
272
GUAAGUGUCAAUAAGAAGA
507
ucuucuuAuuGAcAcuuAcdTsdT
972
GUAAGUGUcAAuAaGAAGAdTsdT





 39
UCUUCUUAUUGACACUUAC
272
GUAAGUGUCAAUAAGAAGA
508
ucuucuUAuuGAcAcuuAcdTsdT
972
GUAAGUGUcAAuAaGAAGAdTsdT





 39
UCUUCUUAUUGACACUUAC
272
GUAAGUGUCAAUAAGAAGA
507
ucuucuuAuuGAcAcuuAcdTsdT
980
GuAAGUGUcAAuAaGAAGAdTsdT





 39
UCUUCUUAUUGACACUUAC
272
GUAAGUGUCAAUAAGAAGA
508
ucuucuUAuuGAcAcuuAcdTsdT
980
GuAAGUGUcAAuAaGAAGAdTsdT





 39
UCUUCUUAUUGACACUUAC
272
GUAAGUGUCAAUAAGAAGA
508
ucuucuUAuuGAcAcuuAcdTsdT
985
GuAAGUGUcAAuAAGAAGAdTsdT





 40
UGUUCGGAUAGAACAGGAG
273
CUCCUGUUCUAUCCGAACA
509
uguucGGAuAGAAcAGGAGdTsdT
733
pCUCCUGUUCuAUCCGAAcAdTsdT





 41
AGUACCAUGAUAUCUGGCA
274
UGCCAGAUAUCAUGGUACU
510
AguAccAuGAuAucuGGcAdTsdT
820
pUGCcAGAuAUcAUGGuACUdTsdT





 42
CAGAGAUGAGGGUUUACAC
275
GUGUAAACCCUCAUCUCUG
511
caGAGAuGAGGGuuuAcAcdTsdT
856
pGUGuAAACCCUcAUCUCUGdTsdT





 43
GAAACGAGGACUGAUGCCU
276
AGGCAUCAGUCCUCGUUUC
512
GaAAcGAGGAcuGAuGccudTsdT
852
pAGGcAUcAGUCCUCGUUUCdTsdT





 44
AAGAGAGUAGGCGAGUAUC
277
GAUACUCGCCUACUCUCUU
513
AaGAGAGuAGGcGAGuAucdTsdT
888
pGAuACUCGCCuACUCUCUUdTsdT





 45
CAUUAGCUGAAUAAUGUGA
278
UCACAUUAUUCAGCUAAUG
514
cauuAGcuGAAuAAuGuGAdTsdT
932
pUCAcAUuAUUcAGCuAAUGdTsdT





 45
CAUUAGCUGAAUAAUGUGA
278
UCACAUUAUUCAGCUAAUG
514
cauuAGcuGAAuAAuGuGAdTsdT
960
pUcAcAUuAUUcAGCuAAUGdTsdT





 46
AGUAGAGAACCCAUUUGAC
279
GUCAAAUGGGUUCUCUACU
515
AguAGAGAAcccAuuuGAcdTsdT
738
pGUcAAAUGGGUUCUCuACUdTsdT





 47
AGGCGAGUAUCAGAGGAUG
280
CAUCCUCUGAUACUCGCCU
516
AgGcGAGuAucAGAGGAuGdTsdT
893
pcAUCCUCUGAuACUCGCCUdTsdT





 48
UAGACUAAGCAUGUAAUUU
281
AAAUUACAUGCUUAGUCUA
517
uaGAcuAAGcAuGuAAuuudTsdT
784
pAAAUuAcAUGCUuAGUCuAdTsdT





 49
AACAUUGUUUGUACUCACA
282
UGUGAGUACAAACAAUGUU
518
AacAuuGuuuGuAcucAcAdTsdT
913
pUGUGAGuAcAAAcAAUGUUdTsdT





 50
GAUGGGAGUGAUGUCAAGU
283
ACUUGACAUCACUCCCAUC
519
GauGGGAGuGAuGucAAGudTsdT
896
pACUUGAcAUcACUCCcAUCdTsdT





 51
CAGACCAUUUCCUAAUCAG
284
CUGAUUAGGAAAUGGUCUG
520
caGAccAuuuccuAAucAGdTsdT
937
pCUGAUuAGGAAAUGGUCUGdTsdT





 52
GAUUACAGAAGCCCGCUGU
285
ACAGCGGGCUUCUGUAAUC
521
GauuAcAGAAGcccGcuGudTsdT
712
pACAGCGGGCUUCUGuAAUCdTsdT





 52
GAUUACAGAAGCCCGCUGU
285
ACAGCGGGCUUCUGUAAUC
521
GauuAcAGAAGcccGcuGudTsdT
938
pAcAGCGGGCUUCUGuAAUCdTsdT





 53
CAUUGAAACGAUGCCUUGU
286
ACAAGGCAUCGUUUCAAUG
522
cauuGAAAcGAuGccuuGudTsdT
845
pACAAGGcAUCGUUUcAAUGdTsdT





 53
CAUUGAAACGAUGCCUUGU
286
ACAAGGCAUCGUUUCAAUG
522
cauuGAAAcGAuGccuuGudTsdT
950
pAcAAGGcAUCGUUUcAAUGdTsdT





 54
ACUUAUGCUGGAACUGGGU
287
ACCCAGUUCCAGCAUAAGU
523
AcuuAuGcuGGAAcuGGGudTsdT
880
pACCcAGUUCcAGcAuAAGUdTsdT





 55
GUCGACAAGGAGAACACGC
288
GCGUGUUCUCCUUGUCGAC
524
GucGAcAAGGAGAAcAcGcdTsdT
705
pGCGUGUUCUCCUUGUCGACdTsdT





 56
AGGAAAGACUAACUUCUUU
289
AAAGAAGUUAGUCUUUCCU
525
AgGAAAGAcuAAcuucuuudTsdT
885
pAAAGAAGUuAGUCUUUCCUdTsdT





 56
AGGAAAGACUAACUUCUUU
289
AAAGAAGUUAGUCUUUCCU
526
agGAAAGAcuAAcuucuuudTsdT
967
AAAGAAGUuAGUCUUUCCUdTsdT





 56
AGGAAAGACUAACUUCUUU
289
AAAGAAGUUAGUCUUUCCU
526
agGAAAGAcuAAcuucuuudTsdT
975
AAAGAAGUuAGUCuUUCCUdTsdT





 56
AGGAAAGACUAACUUCUUU
289
AAAGAAGUUAGUCUUUCCU
526
agGAAAGAcuAAcuucuuudTsdT
983
AaAGAAGUuAGUCuUUCCUdTsdT





 57
CAAGACCGCGAGGAGGAUC
290
GAUCCUCCUCGCGGUCUUG
527
caAGAccGcGAGGAGGAucdTsdT
816
pGAUCCUCCUCGCGGUCUUGdTsdT





 58
GACAAUGGCAGUCUUGGCU
291
AGCCAAGACUGCCAUUGUC
528
GacAAuGGcAGucuuGGcudTsdT
755
pAGCcAAGACUGCcAUUGUCdTsdT





 59
AUGCCUUGUGUCAAGAAGA
292
UCUUCUUGACACAAGGCAU
529
AuGccuuGuGucAAGAAGAdTsdT
846
pUCUUCUUGAcAcAAGGcAUdTsdT





 60
GCCUCACUGCUUCAACGCA
293
UGCGUUGAAGCAGUGAGGC
530
GccucAcuGcuucAAcGcAdTsdT
909
pUGCGUUGAAGcAGUGAGGCdTsdT





 61
UACCUCACAACCAGUCCUG
294
CAGGACUGGUUGUGAGGUA
531
uaccucAcAAccAGuccuGdTsdT
745
pcAGGACUGGUUGUGAGGuAdTsdT





 62
GAGAAGAGAGUAGGCGAGU
295
ACUCGCCUACUCUCUUCUC
532
GaGAAGAGAGuAGGcGAGudTsdT
887
pACUCGCCuACUCUCUUCUCdTsdT





 63
AGACUUAUGCUGGAACUGG
296
CCAGUUCCAGCAUAAGUCU
533
AgAcuuAuGcuGGAAcuGGdTsdT
879
pCCAGUUCcAGcAuAAGUCUdTsdT





 63
AGACUUAUGCUGGAACUGG
296
CCAGUUCCAGCAUAAGUCU
533
AgAcuuAuGcuGGAAcuGGdTsdT
954
pCcAGUUCcAGcAuAAGUCUdTsdT





 64
UUACAGAAGCCCGCUGUUU
297
AAACAGCGGGCUUCUGUAA
534
uuAcAGAAGcccGcuGuuudTsdT
831
pAAAcAGCGGGCUUCUGuAAdTsdT





 65
UUAUGCUGGAACUGGGUUU
298
AAACCCAGUUCCAGCAUAA
535
uuAuGcuGGAAcuGGGuuudTsdT
881
pAAACCcAGUUCcAGcAuAAdTsdT





 66
AUAAACAUUGUUUGUACUC
299
GAGUACAAACAAUGUUUAU
536
AuAAAcAuuGuuuGuAcucdTsdT
912
pGAGuAcAAAcAAUGUUuAUdTsdT





 67
UCAAUGCCAUUGAAACGAU
300
AUCGUUUCAAUGGCAUUGA
537
ucAAuGccAuuGAAAcGAudTsdT
717
pAUCGUUUcAAUGGcAUUGAdTsdT





 68
AUAGUGCUGGUAGUAUCAC
301
GUGAUACUACCAGCACUAU
538
AuAGuGcuGGuAGuAucAcdTsdT
748
pGUGAuACuACcAGcACuAUdTsdT





 69
CAGCCUCACUGCUUCAACG
302
CGUUGAAGCAGUGAGGCUG
539
caGccucAcuGcuucAAcGdTsdT
908
pCGUUGAAGcAGUGAGGCUGdTsdT





 70
UCUUGGCUUUAAAGUGAGG
303
CCUCACUUUAAAGCCAAGA
540
ucuuGGcuuuAAAGuGAGGdTsdT
905
pCCUcACUUuAAAGCcAAGAdTsdT





 71
GGCUGUGACUUACCAUAGC
304
GCUAUGGUAAGUCACAGCC
541
GgcuGuGAcuuAccAuAGcdTsdT
751
pGCuAUGGuAAGUcAcAGCCdTsdT





 72
GGCUACCUAUGGUGAACGU
305
ACGUUCACCAUAGGUAGCC
542
GgcuAccuAuGGuGAAcGudTsdT
722
pACGUUcACcAuAGGuAGCCdTsdT





 73
CGCGAGGAGGAUCUUCCAG
306
CUGGAAGAUCCUCCUCGCG
543
cgcGAGGAGGAucuuccAGdTsdT
819
pCUGGAAGAUCCUCCUCGCGdTsdT





 74
GCCAUUGAAACGAUGCCUU
307
AAGGCAUCGUUUCAAUGGC
544
GccAuuGAAAcGAuGccuudTsdT
718
pAAGGcAUCGUUUcAAUGGCdTsdT





 75
AGCCUCACUGCUUCAACGC
308
GCGUUGAAGCAGUGAGGCU
545
AgccucAcuGcuucAAcGcdTsdT
758
pGCGUUGAAGcAGUGAGGCUdTsdT





 76
GGCAGACAGACUUAUGCUG
309
CAGCAUAAGUCUGUCUGCC
546
GgcAGAcAGAcuuAuGcuGdTsdT
878
pcAGcAuAAGUCUGUCUGCCdTsdT





 77
GUGACUAAAGUAAGUUAAA
310
UUUAACUUACUUUAGUCAC
547
GuGAcuAAAGuAAGuuAAAdTsdT
770
pUUuAACUuACUUuAGUcACdTsdT





 78
AGUUAUUGUUACCUAAAGU
311
ACUUUAGGUAACAAUAACU
548
AguuAuuGuuAccuAAAGudTsdT
800
pACUUuAGGuAAcAAuAACUdTsdT





 79
GCCUUUAUGUUUGGGAGAA
312
UUCUCCCAAACAUAAAGGC
549
GccuuuAuGuuuGGGAGAAdTsdT
924
pUUCUCCcAAAcAuAAAGGCdTsdT





 80
UUCAGAGUAGAGAACCCAU
313
AUGGGUUCUCUACUCUGAA
550
uucAGAGuAGAGAAcccAudTsdT
882
pAUGGGUUCUCuACUCUGAAdTsdT





 81
AAACGAGGACUGAUGCCUG
314
CAGGCAUCAGUCCUCGUUU
551
AaAcGAGGAcuGAuGccuGdTsdT
724
pcAGGcAUcAGUCCUCGUUUdTsdT





 82
GUAGGCGAGUAUCAGAGGA
315
UCCUCUGAUACUCGCCUAC
552
GuAGGcGAGuAucAGAGGAdTsdT
891
pUCCUCUGAuACUCGCCuACdTsdT





 82
GUAGGCGAGUAUCAGAGGA
315
UCCUCUGAUACUCGCCUAC
553
guAGGcGAGuAucAGAGGAdTsdT
968
UCCUCUGAuACUCGCCuACdTsdT





 82
GUAGGCGAGUAUCAGAGGA
315
UCCUCUGAUACUCGCCUAC
553
guAGGcGAGuAucAGAGGAdTsdT
976
UCCUCUGAuACUCgCCuACdTsdT





 82
GUAGGCGAGUAUCAGAGGA
315
UCCUCUGAUACUCGCCUAC
553
guAGGcGAGuAucAGAGGAdTsdT
984
UcCUCUGAuACUCgCCuACdTsdT





 83
AAGCCCGCUGUUUCUAUGG
316
CCAUAGAAACAGCGGGCUU
554
AaGcccGcuGuuucuAuGGdTsdT
835
pCCAuAGAAAcAGCGGGCUUdTsdT





 84
UCAGCACUGGGAAUCCCUG
317
CAGGGAUUCCCAGUGCUGA
555
ucAGcAcuGGGAAucccuGdTsdT
827
pcAGGGAUUCCcAGUGCUGAdTsdT





 85
GAAUAAUGUGAGGAUUAAC
318
GUUAAUCCUCACAUUAUUC
556
GaAuAAuGuGAGGAuuAAcdTsdT
933
pGUuAAUCCUcAcAUuAUUCdTsdT





 86
UGUGGCAGACAGACUUAUG
319
CAUAAGUCUGUCUGCCACA
557
uguGGcAGAcAGAcuuAuGdTsdT
877
pcAuAAGUCUGUCUGCcAcAdTsdT





 87
AGAGAUAAAUGUUGAUCUU
320
AAGAUCAACAUUUAUCUCU
558
AgAGAuAAAuGuuGAucuudTsdT
798
pAAGAUcAAcAUUuAUCUCUdTsdT





 88
UACCAUGAUAUCUGGCAGA
321
UCUGCCAGAUAUCAUGGUA
559
uaccAuGAuAucuGGcAGAdTsdT
821
pUCUGCcAGAuAUcAUGGuAdTsdT





 89
CUUCCAAAUUGCCAUGGAA
322
UUCCAUGGCAAUUUGGAAG
560
cuuccAAAuuGccAuGGAAdTsdT
838
pUUCcAUGGcAAUUUGGAAGdTsdT





 90
ACCGCGAGGAGGAUCUUCC
323
GGAAGAUCCUCCUCGCGGU
561
AccGcGAGGAGGAucuuccdTsdT
818
pGGAAGAUCCUCCUCGCGGUdTsdT





 91
GAAAUGUAUAGUCUUCUUA
324
UAAGAAGACUAUACAUUUC
562
GaAAuGuAuAGucuucuuAdTsdT
713
puAAGAAGACuAuAcAUUUCdTsdT





 92
AUGUUCAAACACCUGGUAC
325
GUACCAGGUGUUUGAACAU
563
AuGuucAAAcAccuGGuAcdTsdT
862
pGUACcAGGUGUUUGAAcAUdTsdT





 92
AUGUUCAAACACCUGGUAC
325
GUACCAGGUGUUUGAACAU
563
AuGuucAAAcAccuGGuAcdTsdT
952
pGuACcAGGUGUUUGAAcAUdTsdT





 93
AGGGAAUUUCUCUUCAAUG
326
CAUUGAAGAGAAAUUCCCU
564
AgGGAAuuucucuucAAuGdTsdT
716
pcAUUGAAGAGAAAUUCCCUdTsdT





 94
CCCUGUUAAGUGGUGAAAU
327
AUUUCACCACUUAACAGGG
565
cccuGuuAAGuGGuGAAAudTsdT
789
pAUUUcACcACUuAAcAGGGdTsdT





 95
GAUGAGGGUUUACACUGUG
328
CACAGUGUAAACCCUCAUC
566
GauGAGGGuuuAcAcuGuGdTsdT
857
pcAcAGUGuAAACCCUcAUCdTsdT





 96
UGUGUGAGUUAAUUCAUUU
329
AAAUGAAUUAACUCACACA
567
uguGuGAGuuAAuucAuuudTsdT
922
pAAAUGAAUuAACUcAcAcAdTsdT





 97
UUGCCUGAUGUUCAAACAC
330
GUGUUUGAACAUCAGGCAA
568
uuGccuGAuGuucAAAcAcdTsdT
858
pGUGUUUGAAcAUcAGGcAAdTsdT





 98
AAACUUGUGUAGACUAAGC
331
GCUUAGUCUACACAAGUUU
569
AaAcuuGuGuAGAcuAAGcdTsdT
779
pGCUuAGUCuAcAcAAGUUUdTsdT





 99
UAUAUCCCAUGUUCUGGCU
332
AGCCAGAACAUGGGAUAUA
570
uauAucccAuGuucuGGcudTsdT
828
pAGCcAGAAcAUGGGAuAuAdTsdT





100
UUGUGUAGACUAAGCAUGU
333
ACAUGCUUAGUCUACACAA
571
uuGuGuAGAcuAAGcAuGudTsdT
781
pACAUGCUuAGUCuAcAcAAdTsdT





100
UUGUGUAGACUAAGCAUGU
333
ACAUGCUUAGUCUACACAA
571
uuGuGuAGAcuAAGcAuGudTsdT
945
pAcAUGCUuAGUCuAcAcAAdTsdT





101
AUGCUGUUCGGAUAGAACA
334
UGUUCUAUCCGAACAGCAU
572
AuGcuGuucGGAuAGAAcAdTsdT
730
pUGUUCuAUCCGAAcAGcAUdTsdT





102
AAUUAUCAAUGCUGUUCGG
335
CCGAACAGCAUUGAUAAUU
573
AauuAucAAuGcuGuucGGdTsdT
727
pCCGAAcAGcAUUGAuAAUUdTsdT





103
GCCUGAUGUUCAAACACCU
336
AGGUGUUUGAACAUCAGGC
574
GccuGAuGuucAAAcAccudTsdT
859
pAGGUGUUUGAAcAUcAGGCdTsdT





104
CAUAGCAGUGACAAUGGCA
337
UGCCAUUGUCACUGCUAUG
575
cauAGcAGuGAcAAuGGcAdTsdT
902
pUGCcAUUGUcACUGCuAUGdTsdT





105
UGUGAGUUAAUUCAUUUAU
338
AUAAAUGAAUUAACUCACA
576
uguGAGuuAAuucAuuuAudTsdT
923
pAUAAAUGAAUuAACUcAcAdTsdT





105
UGUGAGUUAAUUCAUUUAU
338
AUAAAUGAAUUAACUCACA
576
uguGAGuuAAuucAuuuAudTsdT
959
pAuAAAUGAAUuAACUcAcAdTsdT





106
AGUGCUGGUAGUAUCACCU
339
AGGUGAUACUACCAGCACU
577
AguGcuGGuAGuAucAccudTsdT
749
pAGGUGAuACuACcAGcACUdTsdT





107
UAUCAAUGCUGUUCGGAUA
340
UAUCCGAACAGCAUUGAUA
578
uaucAAuGcuGuucGGAuAdTsdT
865
puAUCCGAAcAGcAUUGAuAdTsdT





108
GACUAAAGUAAGUUAAACU
341
AGUUUAACUUACUUUAGUC
579
GacuAAAGuAAGuuAAAcudTsdT
772
pAGUUuAACUuACUUuAGUCdTsdT





109
AAUGCUGUUCGGAUAGAAC
342
GUUCUAUCCGAACAGCAUU
580
AauGcuGuucGGAuAGAAcdTsdT
729
pGUUCuAUCCGAAcAGcAUUdTsdT





110
AGAAUAUUUCACUGGAAGG
343
CCUUCCAGUGAAAUAUUCU
581
AgAAuAuuucAcuGGAAGGdTsdT
740
pCCUUCcAGUGAAAuAUUCUdTsdT





111
AUCUGGCAGAUGUAUAAGA
344
UCUUAUACAUCUGCCAGAU
582
AucuGGcAGAuGuAuAAGAdTsdT
825
pUCUuAuAcAUCUGCcAGAUdTsdT





112
UAUAGUGCUGGUAGUAUCA
345
UGAUACUACCAGCACUAUA
583
uauAGuGcuGGuAGuAucAdTsdT
747
pUGAuACuACcAGcACuAuAdTsdT





113
GGCCAGCAAGACCGCGAGG
346
CCUCGCGGUCUUGCUGGCC
584
GgccAGcAAGAccGcGAGGdTsdT
706
pCCUCGCGGUCUUGCUGGCCdTsdT





114
CCAUGAUAUCUGGCAGAUG
347
CAUCUGCCAGAUAUCAUGG
585
ccAuGAuAucuGGcAGAuGdTsdT
823
pcAUCUGCcAGAuAUcAUGGdTsdT





115
UUAAACUUGUGUAGACUAA
348
UUAGUCUACACAAGUUUAA
586
uuAAAcuuGuGuAGAcuAAdTsdT
777
pUUAGUCuAcAcAAGUUuAAdTsdT





115
UUAAACUUGUGUAGACUAA
348
UUAGUCUACACAAGUUUAA
586
uuAAAcuuGuGuAGAcuAAdTsdT
944
pUuAGUCuAcAcAAGUUuAAdTsdT





116
UUCAAUGCCAUUGAAACGA
349
UCGUUUCAAUGGCAUUGAA
587
uucAAuGccAuuGAAAcGAdTsdT
844
pUCGUUUcAAUGGcAUUGAAdTsdT





117
AGAAAGCUGAGACAUUGCA
350
UGCAAUGUCUCAGCUUUCU
588
AgAAAGcuGAGAcAuuGcAdTsdT
925
pUGcAAUGUCUcAGCUUUCUdTsdT





118
CUAUGGCUUCCAAAUUGCC
351
GGCAAUUUGGAAGCCAUAG
589
cuAuGGcuuccAAAuuGccdTsdT
837
pGGcAAUUUGGAAGCcAuAGdTsdT





119
AAGUGACUAAAGUAAGUUA
352
UAACUUACUUUAGUCACUU
590
AaGuGAcuAAAGuAAGuuAdTsdT
769
puAACUuACUUuAGUcACUUdTsdT





120
UGACUAAAGUAAGUUAAAC
353
GUUUAACUUACUUUAGUCA
591
ugAcuAAAGuAAGuuAAAcdTsdT
771
pGUUuAACUuACUUuAGUcAdTsdT





121
UGCUGUUCGGAUAGAACAG
354
CUGUUCUAUCCGAACAGCA
592
ugcuGuucGGAuAGAAcAGdTsdT
731
pCUGUUCuAUCCGAAcAGcAdTsdT





122
GCGAGUAUCAGAGGAUGGG
355
CCCAUCCUCUGAUACUCGC
593
GcGAGuAucAGAGGAuGGGdTsdT
895
pCCcAUCCUCUGAuACUCGCdTsdT





123
GGGCCUUGCGCUGGAUUGG
356
CCAAUCCAGCGCAAGGCCC
594
GgGccuuGcGcuGGAuuGGdTsdT
848
pCCAAUCcAGCGcAAGGCCCdTsdT





124
ACCUCACAACCAGUCCUGU
357
ACAGGACUGGUUGUGAGGU
595
AccucAcAAccAGuccuGudTsdT
899
pACAGGACUGGUUGUGAGGUdTsdT





124
ACCUCACAACCAGUCCUGU
357
ACAGGACUGGUUGUGAGGU
595
AccucAcAAccAGuccuGudTsdT
956
pAcAGGACUGGUUGUGAGGUdTsdT





125
ACUAAGUGACUAAAGUAAG
358
CUUACUUUAGUCACUUAGU
596
AcuAAGuGAcuAAAGuAAGdTsdT
768
pCUuACUUuAGUcACUuAGUdTsdT





126
AUUACAGAAGCCCGCUGUU
359
AACAGCGGGCUUCUGUAAU
597
AuuAcAGAAGcccGcuGuudTsdT
830
pAAcAGCGGGCUUCUGuAAUdTsdT





127
GAGUAGGCGAGUAUCAGAG
360
CUCUGAUACUCGCCUACUC
598
GaGuAGGcGAGuAucAGAGdTsdT
890
pCUCUGAuACUCGCCuACUCdTsdT





128
CAGUGACAAUGGCAGUCUU
361
AAGACUGCCAUUGUCACUG
599
caGuGAcAAuGGcAGucuudTsdT
904
pAAGACUGCcAUUGUcACUGdTsdT





129
GGCCUUGCGCUGGAUUGGG
362
CCCAAUCCAGCGCAAGGCC
600
GgccuuGcGcuGGAuuGGGdTsdT
849
pCCcAAUCcAGCGcAAGGCCdTsdT





130
UUCUUAUUGACACUUACAU
363
AUGUAAGUGUCAAUAAGAA
601
uucuuAuuGAcAcuuAcAudTsdT
843
pAUGuAAGUGUcAAuAAGAAdTsdT





131
UUCACUAAGUGACUAAAGU
364
ACUUUAGUCACUUAGUGAA
602
uucAcuAAGuGAcuAAAGudTsdT
765
pACUUuAGUcACUuAGUGAAdTsdT





132
GUGUGAGUUAAUUCAUUUA
365
UAAAUGAAUUAACUCACAC
603
GuGuGAGuuAAuucAuuuAdTsdT
791
puAAAUGAAUuAACUcAcACdTsdT





133
CCCGCUCGCGCCCAUCACG
366
CGUGAUGGGCGCGAGCGGG
604
cccGcucGcGcccAucAcGdTsdT
813
pCGUGAUGGGCGCGAGCGGGdTsdT





134
GUAAGUUAAACUUGUGUAG
367
CUACACAAGUUUAACUUAC
605
GuAAGuuAAAcuuGuGuAGdTsdT
775
pCUAcAcAAGUUuAACUuACdTsdT





134
GUAAGUUAAACUUGUGUAG
367
CUACACAAGUUUAACUUAC
605
GuAAGuuAAAcuuGuGuAGdTsdT
943
pCuAcAcAAGUUuAACUuACdTsdT





135
CGGAAGUUGGAAUCAGGUU
368
AACCUGAUUCCAACUUCCG
606
cgGAAGuuGGAAucAGGuudTsdT
931
pAACCUGAUUCcAACUUCCGdTsdT





136
AUGUGAGGAUUAACUUCUG
369
CAGAAGUUAAUCCUCACAU
607
AuGuGAGGAuuAAcuucuGdTsdT
810
pcAGAAGUuAAUCCUcAcAUdTsdT





137
UUAAGUGGUGAAAUCAACU
370
AGUUGAUUUCACCACUUAA
608
uuAAGuGGuGAAAucAAcudTsdT
919
pAGUUGAUUUcACcACUuAAdTsdT





138
UGUAGACUAAGCAUGUAAU
371
AUUACAUGCUUAGUCUACA
609
uguAGAcuAAGcAuGuAAudTsdT
783
pAUuAcAUGCUuAGUCuAcAdTsdT





139
AUAAUGUGAGGAUUAACUU
372
AAGUUAAUCCUCACAUUAU
610
AuAAuGuGAGGAuuAAcuudTsdT
934
pAAGUuAAUCCUcAcAUuAUdTsdT





140
GGCUGGCUGUGACUUACCA
373
UGGUAAGUCACAGCCAGCC
611
GgcuGGcuGuGAcuuAccAdTsdT
901
pUGGuAAGUcAcAGCcAGCCdTsdT





141
AAGAGGCUACCUAUGGUGA
374
UCACCAUAGGUAGCCUCUU
612
AaGAGGcuAccuAuGGuGAdTsdT
850
pUCACcAuAGGuAGCCUCUUdTsdT





141
AAGAGGCUACCUAUGGUGA
374
UCACCAUAGGUAGCCUCUU
612
AaGAGGcuAccuAuGGuGAdTsdT
951
pUcACcAuAGGuAGCCUCUUdTsdT





142
CAGAUUACAGAAGCCCGCU
375
AGCGGGCUUCUGUAAUCUG
613
caGAuuAcAGAAGcccGcudTsdT
829
pAGCGGGCUUCUGuAAUCUGdTsdT





143
UGAGGCCUUGCCUGUGAAG
376
CUUCACAGGCAAGGCCUCA
614
ugAGGccuuGccuGuGAAGdTsdT
869
pCUUcAcAGGcAAGGCCUcAdTsdT





144
AUAAUUAUCAAUGCUGUUC
377
GAACAGCAUUGAUAAUUAU
615
AuAAuuAucAAuGcuGuucdTsdT
726
pGAAcAGcAUUGAuAAUuAUdTsdT





145
GUGACUUACCAUAGCAGUG
466
CACUGCUAUGGUAAGUCAC
616
GuGAcuuAccAuAGcAGuGdTsdT
753
pcACUGCuAUGGuAAGUcACdTsdT





146
UAGGGCUACUUUGAAUUAA
378
UUAAUUCAAAGUAGCCCUA
617
uaGGGcuAcuuuGAAuuAAdTsdT
795
pUUAAUUcAAAGuAGCCCuAdTsdT





146
UAGGGCUACUUUGAAUUAA
378
UUAAUUCAAAGUAGCCCUA
617
uaGGGcuAcuuuGAAuuAAdTsdT
946
pUuAAUUcAAAGuAGCCCuAdTsdT





147
UGGCAGAUGUAUAAGAAGG
379
CCUUCUUAUACAUCUGCCA
618
ugGcAGAuGuAuAAGAAGGdTsdT
826
pCCUUCUuAuAcAUCUGCcAdTsdT





148
AUAGCUUGAUUUAUUUGGU
380
ACCAAAUAAAUCAAGCUAU
619
AuAGcuuGAuuuAuuuGGudTsdT
759
pACcAAAuAAAUcAAGCuAUdTsdT





149
CAGCAAGACCGCGAGGAGG
381
CCUCCUCGCGGUCUUGCUG
620
caGcAAGAccGcGAGGAGGdTsdT
707
pCCUCCUCGCGGUCUUGCUGdTsdT





150
GACUGAUGCCUGGCCUCAC
382
GUGAGGCCAGGCAUCAGUC
621
GacuGAuGccuGGccucAcdTsdT
854
pGUGAGGCcAGGcAUcAGUCdTsdT





151
UUACCUUGGAUGCUGACUU
383
AAGUCAGCAUCCAAGGUAA
622
uuAccuuGGAuGcuGAcuudTsdT
897
pAAGUcAGcAUCcAAGGuAAdTsdT





152
AUUCAGCACUGGGAAUCCC
384
GGGAUUCCCAGUGCUGAAU
623
AuucAGcAcuGGGAAucccdTsdT
709
pGGGAUUCCcAGUGCUGAAUdTsdT





153
AGCAAGACCGCGAGGAGGA
385
UCCUCCUCGCGGUCUUGCU
624
AgcAAGAccGcGAGGAGGAdTsdT
814
pUCCUCCUCGCGGUCUUGCUdTsdT





154
AGGGCUACUUUGAAUUAAU
386
AUUAAUUCAAAGUAGCCCU
625
AgGGcuAcuuuGAAuuAAudTsdT
796
pAUuAAUUcAAAGuAGCCCUdTsdT





155
UAAGUUAUUGUUACCUAAA
387
UUUAGGUAACAAUAACUUA
626
uaAGuuAuuGuuAccuAAAdTsdT
799
pUUuAGGuAAcAAuAACUuAdTsdT





156
UUUAUAGUGCUGGUAGUAU
388
AUACUACCAGCACUAUAAA
627
uuuAuAGuGcuGGuAGuAudTsdT
900
pAUACuACcAGcACuAuAAAdTsdT





156
UUUAUAGUGCUGGUAGUAU
388
AUACUACCAGCACUAUAAA
627
uuuAuAGuGcuGGuAGuAudTsdT
957
pAuACuACcAGcACuAuAAAdTsdT





157
GCAAGACCGCGAGGAGGAU
389
AUCCUCCUCGCGGUCUUGC
628
GcAAGAccGcGAGGAGGAudTsdT
815
pAUCCUCCUCGCGGUCUUGCdTsdT





158
UCUAUGGCUUCCAAAUUGC
390
GCAAUUUGGAAGCCAUAGA
629
ucuAuGGcuuccAAAuuGcdTsdT
836
pGCAAUUUGGAAGCcAuAGAdTsdT





159
AAAGACUAACUUCUUUGAG
391
CUCAAAGAAGUUAGUCUUU
630
AaAGAcuAAcuucuuuGAGdTsdT
886
pCUcAAAGAAGUuAGUCUUUdTsdT





160
ACCAUGAUAUCUGGCAGAU
392
AUCUGCCAGAUAUCAUGGU
631
AccAuGAuAucuGGcAGAudTsdT
822
pAUCUGCcAGAuAUcAUGGUdTsdT





161
GACCAUUUCCUAAUCAGUU
393
AACUGAUUAGGAAAUGGUC
632
GaccAuuuccuAAucAGuudTsdT
811
pAACUGAUuAGGAAAUGGUCdTsdT





162
UUACCAUAGCAGUGACAAU
394
AUUGUCACUGCUAUGGUAA
633
uuAccAuAGcAGuGAcAAudTsdT
754
pAUUGUcACUGCuAUGGuAAdTsdT





163
AAUGUGAGGAUUAACUUCU
395
AGAAGUUAAUCCUCACAUU
634
AauGuGAGGAuuAAcuucudTsdT
809
pAGAAGUuAAUCCUcAcAUUdTsdT





164
UAGUGUCCUGGGAUUCUCU
396
AGAGAAUCCCAGGACACUA
635
uaGuGuccuGGGAuucucudTsdT
916
pAGAGAAUCCcAGGAcACuAdTsdT





165
UGUUAAGUGGUGAAAUCAA
397
UUGAUUUCACCACUUAACA
636
uguuAAGuGGuGAAAucAAdTsdT
918
pUUGAUUUcACcACUuAAcAdTsdT





166
ACAAAUAUUCUUAAUAGGG
398
CCCUAUUAAGAAUAUUUGU
637
AcAAAuAuucuuAAuAGGGdTsdT
792
pCCCuAUuAAGAAuAUUUGUdTsdT





167
GCGGAAGUUGGAAUCAGGU
399
ACCUGAUUCCAACUUCCGC
638
GcGGAAGuuGGAAucAGGudTsdT
930
pACCUGAUUCcAACUUCCGCdTsdT





168
AACUUGUGUAGACUAAGCA
400
UGCUUAGUCUACACAAGUU
639
AacuuGuGuAGAcuAAGcAdTsdT
780
pUGCUuAGUCuAcAcAAGUUdTsdT





169
AUUCUUAAUAGGGCUACUU
401
AAGUAGCCCUAUUAAGAAU
640
AuucuuAAuAGGGcuAcuudTsdT
794
pAAGuAGCCCuAUuAAGAAUdTsdT





170
CCUAAAGUUAAUCCAGAUU
402
AAUCUGGAUUAACUUUAGG
641
ccuAAAGuuAAuccAGAuudTsdT
929
pAAUCUGGAUuAACUUuAGGdTsdT





171
UAUUGUUACCUAAAGUUAA
403
UUAACUUUAGGUAACAAUA
642
uauuGuuAccuAAAGuuAAdTsdT
803
pUUAACUUuAGGuAAcAAuAdTsdT





171
UAUUGUUACCUAAAGUUAA
403
UUAACUUUAGGUAACAAUA
642
uauuGuuAccuAAAGuuAAdTsdT
947
pUuAACUUuAGGuAAcAAuAdTsdT





172
GUGCUGGUAGUAUCACCUU
404
AAGGUGAUACUACCAGCAC
643
GuGcuGGuAGuAucAccuudTsdT
750
pAAGGUGAuACuACcAGcACdTsdT





173
CUGUGACUUACCAUAGCAG
405
CUGCUAUGGUAAGUCACAG
644
cuGuGAcuuAccAuAGcAGdTsdT
752
pCUGCuAUGGuAAGUcAcAGdTsdT





174
GAGCUUCUUAAGUUAAAUC
406
GAUUUAACUUAAGAAGCUC
645
GaGcuucuuAAGuuAAAucdTsdT
917
pGAUUuAACUuAAGAAGCUCdTsdT





175
CUGUUCGGAUAGAACAGGA
407
UCCUGUUCUAUCCGAACAG
646
cuGuucGGAuAGAAcAGGAdTsdT
732
pUCCUGUUCuAUCCGAAcAGdTsdT





176
GUUAUUGUUACCUAAAGUU
408
AACUUUAGGUAACAAUAAC
647
GuuAuuGuuAccuAAAGuudTsdT
801
pAACUUuAGGuAAcAAuAACdTsdT





177
UAAUGUGAGGAUUAACUUC
409
GAAGUUAAUCCUCACAUUA
648
uaAuGuGAGGAuuAAcuucdTsdT
935
pGAAGUuAAUCCUcAcAUuAdTsdT





178
ACCACUAAUGGGAGCCAAU
410
AUUGGCUCCCAUUAGUGGU
649
AccAcuAAuGGGAGccAAudTsdT
764
pAUUGGCUCCcAUuAGUGGUdTsdT





179
UGUGUAGACUAAGCAUGUA
411
UACAUGCUUAGUCUACACA
650
uguGuAGAcuAAGcAuGuAdTsdT
782
puAcAUGCUuAGUCuAcAcAdTsdT





180
UGGGCCUUGCGCUGGAUUG
412
CAAUCCAGCGCAAGGCCCA
651
ugGGccuuGcGcuGGAuuGdTsdT
847
pcAAUCcAGCGcAAGGCCcAdTsdT





181
AGGAGCUUCUUAAGUUAAA
413
UUUAACUUAAGAAGCUCCU
652
AgGAGcuucuuAAGuuAAAdTsdT
788
pUUuAACUuAAGAAGCUCCUdTsdT





182
GGUGACCCUUUAGUGAGCU
414
AGCUCACUAAAGGGUCACC
653
GguGAcccuuuAGuGAGcudTsdT
757
pAGCUcACuAAAGGGUcACCdTsdT





183
AGAGUAGGCGAGUAUCAGA
415
UCUGAUACUCGCCUACUCU
654
AgAGuAGGcGAGuAucAGAdTsdT
889
pUCUGAuACUCGCCuACUCUdTsdT





184
GCAGUGACAAUGGCAGUCU
416
AGACUGCCAUUGUCACUGC
655
GcAGuGAcAAuGGcAGucudTsdT
903
pAGACUGCcAUUGUcACUGCdTsdT





185
AAACGAUGCCUUGUGUCAA
417
UUGACACAAGGCAUCGUUU
656
AaAcGAuGccuuGuGucAAdTsdT
719
pUUGAcAcAAGGcAUCGUUUdTsdT





186
GGACUGAUGCCUGGCCUCA
418
UGAGGCCAGGCAUCAGUCC
657
GgAcuGAuGccuGGccucAdTsdT
853
pUGAGGCcAGGcAUcAGUCCdTsdT





187
UGAGAGAUAAAUGUUGAUC
419
GAUCAACAUUUAUCUCUCA
658
ugAGAGAuAAAuGuuGAucdTsdT
797
pGAUcAAcAUUuAUCUCUcAdTsdT





188
UGGUUUCUACACCAAAUAC
420
GUAUUUGGUGUAGAAACCA
659
ugGuuucuAcAccAAAuAcdTsdT
760
pGUAUUUGGUGuAGAAACcAdTsdT





188
UGGUUUCUACACCAAAUAC
420
GUAUUUGGUGUAGAAACCA
659
ugGuuucuAcAccAAAuAcdTsdT
941
pGuAUUUGGUGuAGAAACcAdTsdT





189
UCUCUGUAAUAUGAUACAU
421
AUGUAUCAUAUUACAGAGA
660
ucucuGuAAuAuGAuAcAudTsdT
927
pAUGuAUcAuAUuAcAGAGAdTsdT





190
GAGAGAUAAAUGUUGAUCU
422
AGAUCAACAUUUAUCUCUC
661
GaGAGAuAAAuGuuGAucudTsdT
926
pAGAUcAAcAUUuAUCUCUCdTsdT





191
ACUCUAAUGAAGCAAUACA
423
UGUAUUGCUUCAUUAGAGU
662
AcucuAAuGAAGcAAuAcAdTsdT
736
pUGuAUUGCUUcAUuAGAGUdTsdT





192
UGAAGUGUUACCAACUAGC
424
GCUAGUUGGUAACACUUCA
663
ugAAGuGuuAccAAcuAGcdTsdT
743
pGCuAGUUGGuAAcACUUcAdTsdT





193
AAUGAAGCAAUACAUUGAG
425
CUCAAUGUAUUGCUUCAUU
664
AauGAAGcAAuAcAuuGAGdTsdT
873
pCUcAAUGuAUUGCUUcAUUdTsdT





194
ACGAUGCCUUGUGUCAAGA
426
UCUUGACACAAGGCAUCGU
665
AcGAuGccuuGuGucAAGAdTsdT
720
pUCUUGAcAcAAGGcAUCGUdTsdT





195
AGACCGCGAGGAGGAUCUU
427
AAGAUCCUCCUCGCGGUCU
666
AgAccGcGAGGAGGAucuudTsdT
817
pAAGAUCCUCCUCGCGGUCUdTsdT





196
UUGUUACCUAAAGUUAAUC
428
GAUUAACUUUAGGUAACAA
667
uuGuuAccuAAAGuuAAucdTsdT
804
pGAUuAACUUuAGGuAAcAAdTsdT





197
CAGAAGCCCGCUGUUUCUA
429
UAGAAACAGCGGGCUUCUG
668
caGAAGcccGcuGuuucuAdTsdT
833
puAGAAAcAGCGGGCUUCUGdTsdT





198
UUUGACUUUAUGGAGAAUA
430
UAUUCUCCAUAAAGUCAAA
669
uuuGAcuuuAuGGAGAAuAdTsdT
883
puAUUCUCcAuAAAGUcAAAdTsdT





199
UACCUAAAGUUAAUCCAGA
431
UCUGGAUUAACUUUAGGUA
670
uaccuAAAGuuAAuccAGAdTsdT
807
pUCUGGAUuAACUUuAGGuAdTsdT





200
UUCAAACACCUGGUACACA
432
UGUGUACCAGGUGUUUGAA
671
uucAAAcAccuGGuAcAcAdTsdT
864
pUGUGuACcAGGUGUUUGAAdTsdT





201
UUGCACUCUAAUGAAGCAA
433
UUGCUUCAUUAGAGUGCAA
672
uuGcAcucuAAuGAAGcAAdTsdT
870
pUUGCUUcAUuAGAGUGcAAdTsdT





202
UGUUACCUAAAGUUAAUCC
434
GGAUUAACUUUAGGUAACA
673
uguuAccuAAAGuuAAuccdTsdT
805
pGGAUuAACUUuAGGuAAcAdTsdT





203
CACUAAGUGACUAAAGUAA
435
UUACUUUAGUCACUUAGUG
674
cacuAAGuGAcuAAAGuAAdTsdT
767
pUUACUUuAGUcACUuAGUGdTsdT





203
CACUAAGUGACUAAAGUAA
435
UUACUUUAGUCACUUAGUG
674
cacuAAGuGAcuAAAGuAAdTsdT
942
pUuACUUuAGUcACUuAGUGdTsdT





204
UGCCAGAUAGAAGACAGGU
436
ACCUGUCUUCUAUCUGGCA
675
ugccAGAuAGAAGAcAGGudTsdT
786
pACCUGUCUUCuAUCUGGcAdTsdT





205
AAUGUAUAGUCUUCUUAUU
437
AAUAAGAAGACUAUACAUU
676
AauGuAuAGucuucuuAuudTsdT
840
pAAuAAGAAGACuAuAcAUUdTsdT





206
GACCACUAAUGGGAGCCAA
438
UUGGCUCCCAUUAGUGGUC
677
GaccAcuAAuGGGAGccAAdTsdT
914
pUUGGCUCCcAUuAGUGGUCdTsdT





207
GUUACCUAAAGUUAAUCCA
439
UGGAUUAACUUUAGGUAAC
678
GuuAccuAAAGuuAAuccAdTsdT
928
pUGGAUuAACUUuAGGuAACdTsdT





208
UGAUGCCUGGCCUCACAUU
440
AAUGUGAGGCCAGGCAUCA
679
ugAuGccuGGccucAcAuudTsdT
855
pAAUGUGAGGCcAGGcAUcAdTsdT





209
CCAACUUUAAAGUCAGUCC
441
GGACUGACUUUAAAGUUGG
680
ccAAcuuuAAAGucAGuccdTsdT
915
pGGACUGACUUuAAAGUUGGdTsdT





210
UAAACUUGUGUAGACUAAG
442
CUUAGUCUACACAAGUUUA
681
uaAAcuuGuGuAGAcuAAGdTsdT
778
pCUuAGUCuAcAcAAGUUuAdTsdT





211
AGUAGGUUGUGUGAGUUAA
443
UUAACUCACACAACCUACU
682
AguAGGuuGuGuGAGuuAAdTsdT
920
pUUAACUcAcAcAACCuACUdTsdT





211
AGUAGGUUGUGUGAGUUAA
443
UUAACUCACACAACCUACU
682
AguAGGuuGuGuGAGuuAAdTsdT
958
pUuAACUcAcAcAACCuACUdTsdT





212
GUUAAACUUGUGUAGACUA
444
UAGUCUACACAAGUUUAAC
683
GuuAAAcuuGuGuAGAcuAdTsdT
776
puAGUCuAcAcAAGUUuAACdTsdT





213
CUGACCACUAAUGGGAGCC
445
GGCUCCCAUUAGUGGUCAG
684
cuGAccAcuAAuGGGAGccdTsdT
762
pGGCUCCcAUuAGUGGUcAGdTsdT





214
UAUUCUUAAUAGGGCUACU
446
AGUAGCCCUAUUAAGAAUA
685
uauucuuAAuAGGGcuAcudTsdT
793
pAGuAGCCCuAUuAAGAAuAdTsdT





215
GUAGUGUCCUGGGAUUCUC
447
GAGAAUCCCAGGACACUAC
686
GuAGuGuccuGGGAuucucdTsdT
787
pGAGAAUCCcAGGAcACuACdTsdT





216
UAUCUGGCAGAUGUAUAAG
448
CUUAUACAUCUGCCAGAUA
687
uaucuGGcAGAuGuAuAAGdTsdT
824
pCUuAuAcAUCUGCcAGAuAdTsdT





217
AGGCUACCUAUGGUGAACG
449
CGUUCACCAUAGGUAGCCU
688
AgGcuAccuAuGGuGAAcGdTsdT
721
pCGUUcACcAuAGGuAGCCUdTsdT





218
UCAGACCAUUUCCUAAUCA
450
UGAUUAGGAAAUGGUCUGA
689
ucAGAccAuuuccuAAucAdTsdT
936
pUGAUuAGGAAAUGGUCUGAdTsdT





219
UUACCUAAAGUUAAUCCAG
451
CUGGAUUAACUUUAGGUAA
690
uuAccuAAAGuuAAuccAGdTsdT
806
pCUGGAUuAACUUuAGGuAAdTsdT





220
GGUUUCUACACCAAAUACA
452
UGUAUUUGGUGUAGAAACC
691
GguuucuAcAccAAAuAcAdTsdT
761
pUGuAUUUGGUGuAGAAACCdTsdT





221
GUUGGUGCCAGAUAGAAGA
453
UCUUCUAUCUGGCACCAAC
692
GuuGGuGccAGAuAGAAGAdTsdT
785
pUCUUCuAUCUGGcACcAACdTsdT





222
GCUACCUAUGGUGAACGUG
454
CACGUUCACCAUAGGUAGC
693
GcuAccuAuGGuGAAcGuGdTsdT
723
pcACGUUcACcAuAGGuAGCdTsdT





223
UCACUAAGUGACUAAAGUA
455
UACUUUAGUCACUUAGUGA
694
ucAcuAAGuGAcuAAAGuAdTsdT
766
puACUUuAGUcACUuAGUGAdTsdT





224
UUAUUGUUACCUAAAGUUA
456
UAACUUUAGGUAACAAUAA
695
uuAuuGuuAccuAAAGuuAdTsdT
802
puAACUUuAGGuAAcAAuAAdTsdT





225
UAGCUGAAUAAUGUGAGGA
457
UCCUCACAUUAUUCAGCUA
696
uaGcuGAAuAAuGuGAGGAdTsdT
808
pUCCUcAcAUuAUUcAGCuAdTsdT





226
UGACCACUAAUGGGAGCCA
458
UGGCUCCCAUUAGUGGUCA
697
ugAccAcuAAuGGGAGccAdTsdT
763
pUGGCUCCcAUuAGUGGUcAdTsdT





227
GUAGCUACCUCACAACCAG
459
CUGGUUGUGAGGUAGCUAC
698
GuAGcuAccucAcAAccAGdTsdT
898
pCUGGUUGUGAGGuAGCuACdTsdT





228
UCCCGCUCGCGCCCAUCAC
460
GUGAUGGGCGCGAGCGGGA
699
ucccGcucGcGcccAucAcdTsdT
812
pGUGAUGGGCGCGAGCGGGAdTsdT





229
CUUGGCUUUAAAGUGAGGG
461
CCCUCACUUUAAAGCCAAG
700
cuuGGcuuuAAAGuGAGGGdTsdT
906
pCCCUcACUUuAAAGCcAAGdTsdT





230
AGAAGCCCGCUGUUUCUAU
462
AUAGAAACAGCGGGCUUCU
701
AgAAGcccGcuGuuucuAudTsdT
834
pAUAGAAAcAGCGGGCUUCUdTsdT





230
AGAAGCCCGCUGUUUCUAU
462
AUAGAAACAGCGGGCUUCU
701
AgAAGcccGcuGuuucuAudTsdT
948
pAuAGAAAcAGCGGGCUUCUdTsdT





231
ACUAAAGUAAGUUAAACUU
463
AAGUUUAACUUACUUUAGU
702
AcuAAAGuAAGuuAAAcuudTsdT
773
pAAGUUuAACUuACUUuAGUdTsdT





232
AGUAAGUUAAACUUGUGUA
464
UACACAAGUUUAACUUACU
703
AguAAGuuAAAcuuGuGuAdTsdT
774
puAcAcAAGUUuAACUuACUdTsdT





233
AAUAAUUAUCAAUGCUGUU
465
AACAGCAUUGAUAAUUAUU
704
AauAAuuAucAAuGcuGuudTsdT
725
pAAcAGcAUUGAuAAUuAUUdTsdT



















TABLE 6








SEQ ID


FPL Name
Function
Sequence
No.







hsRRM001
CE
cgggtttcagggattcccagTTTTTctcttggaaagaaagt
 997





hsRRM002
CE
gcttgctgcaaagaaagccaTTTTTctcttggaaagaaagt
 998





hsRRM003
CE
cttcttggctaaatcgctccaTTTTTctcttggaaagaaagt
 999





hsRRM004
CE
agcgggcttctgtaatctgaaTTTTTctcttggaaagaaagt
1000





hsRRM005
CE
gagaaattccctttctttgggaTTTTTctcttggaaagaaagt
1001





hsRRM006
CE
ggtagcctctttgtccccaatTTTTTctcttggaaagaaagt
1002





hsRRM007
LE
gaacatgggatataaaatatctctcctTTTTTaggcataggacccgtgtct
1003





hsRRM008
LE
ccaagttttcatttactatgccatcTTTTTaggcataggacccgtgtct
1004





hsRRM009
LE
catttcagaatgtatgttttccatgTTTTTaggcataggacccgtgtct
1005





hsRRM010
LE
catcgtttcaatggcattgaaTTTTTaggcataggacccgtgtct
1006





hsRRM011
LE
ccagcgcaaggcccagtTTTTTaggcataggacccgtgtct
1007





hsRRM012
LE
aaggctacaacacgttcaccataTTTTTaggcataggacccgtgtct
1008





hsRRM013
LE
aatgccttccactgcagcaTTTTTaggcataggacccgtgtct
1009





hsRRM014
BL
gcaatttggaagccatagaaac
1010





hsRRM015
BL
tcttttatgtaagtgtcaataagaagactata
1011





hsRRM016
BL
ctgccttcttcttgacacaagg
1012



















TABLE 7








SEQ ID


FPL Name
Function
Sequence
No.







hGAP001
CE
gaatttgccatgggtggaatTTTTTctcttggaaagaaagt
986





hGAP002
CE
ggagggatctcgctcctggaTTTTTctcttggaaagaaagt
987





hGAP003
CE
ccccagccttctccatggtTTTTTctcttggaaagaaagt
988





hGAP004
CE
gctcccccctgcaaatgagTTTTTctcttggaaagaaagt
989





hGAP005
LE
agccttgacggtgccatgTTTTTaggcataggacccgtgtct
990





hGAP006
LE
gatgacaagatcccgttctcTTTTTaggcataggacccgtgtct
991





hGAP007
LE
agatggtgatgggatttccattTTTTTaggcataggacccgtgtct
992





hGAP008
LE
gcatcgccccacttgattttTTTTTaggcataggacccgtgtct
993





hGAP009
LE
cacgacgtactcagcgccaTTTTTaggcataggacccgtgtct
994





hGAP010
LE
ggcagagatgatgacccttttgTTTTTaggcataggacccgtgtct
995





hGAP011
BL
ggtgaagacgccagtggactc
996





















TABLE 8











Cell-




p53
bDNA
Titer













status
expt. 1
expt. 2
Glo
xCELLigence











SEQ ID 497/711












HepG2
wt
10.1
33
540
210


HLF
mutant
4.7
37
180
80


A549
wt
5.2
165
520
140







SEQ ID 477/839












HepG2
wt
3.6
28
380
130


HLF
mutant
0.73
21
120
69


A549
wt
0.73
21
190
160




















TABLE 9







24 hr
48 hr
72 hr
















SEQ ID 497/711












HepG2
81
85
85



HLF
90
90
83



A549
84
59
ND







SEQ ID 477/839












HepG2
87
76
75



HLF
96
92
62



A549
91
54
ND









Claims
  • 1. A double-stranded ribonucleic acid molecule for inhibiting the expression of a ribonucleotide reductase M2 (RRM2) gene, wherein the double-stranded ribonucleic acid molecule comprises a sense strand and an antisense strand, wherein the antisense strand and the sense strand are each less than about 30 nucleotides in length, wherein the antisense strand comprises the nucleotide sequence of any of SEQ ID NO:234-SEQ ID NO:466, wherein the sense strand is at least substantially complementary to the antisense strand, and wherein the sense strand and/or the antisense strand comprises at least one modified nucleotide.
  • 2. The double-stranded ribonucleic acid molecule of claim 1, wherein the antisense strand and the sense strand are each 19-24 nucleotides in length.
  • 3. The double-stranded ribonucleic acid molecule of claim 2, wherein the modified nucleotide is selected from the group consisting of: 2′-O-methyl modified nucleotide, nucleotide comprising a 5′-phosphorothioate group, 2′-deoxy-2′-fluoro modified nucleotide, 2′-deoxy-modified nucleotide, locked nucleotide, abasic nucleotide, inverted deoxythymidine, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, phosphoramidate, and non-natural base comprising nucleotide.
  • 4. The double-stranded ribonucleic acid molecule of claim 2, wherein the sense strand and the antisense strand each comprise at least one modified nucleotide.
  • 5. The double-stranded ribonucleic acid molecule of claim 4, wherein the modified nucleotide in the sense strand and the modified nucleotide in the antisense strand is independently selected from the group consisting of: 2′-O-methyl modified nucleotide, nucleotide comprising a 5′-phosphorothioate group, 2′-deoxy-2′-fluoro modified nucleotide, 2′-deoxy-modified nucleotide, locked nucleotide, abasic nucleotide, inverted deoxythymidine, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, phosphoramidate, and non-natural base comprising nucleotide.
  • 6. The double-stranded ribonucleic acid molecule of claim 1, wherein the sense strand comprises the nucleotide sequence of any of SEQ ID NO: 1-SEQ ID NO:233.
  • 7. The double-stranded ribonucleic acid molecule of claim 6, wherein the sense strand and/or the antisense strand further comprises a 3′ overhang of 1-5 nucleotides in length.
  • 8. The double-stranded ribonucleic acid molecule of claim 7, wherein the 3′ overhang comprises nucleotides linked by one or more phosphorothioate groups.
  • 9. The double-stranded ribonucleic acid molecule of claim 8, wherein the 3′ overhang comprises uracil.
  • 10. The double-stranded ribonucleic acid molecule of claim 1, wherein the antisense strand of the double-stranded ribonucleic acid molecule comprises at least one 2′-O-methyl modified nucleotide, at least one 2′-deoxy-2′-fluoro modified nucleotide, and at least one nucleotide comprising a 5′-phosphorothioate group.
  • 11. The double-stranded ribonucleic acid molecule of claim 1, wherein the sense strand of the double-stranded ribonucleic acid molecule comprises at least one 2′-O-methyl modified nucleotide, at least one 2′-deoxy-2′-fluoro modified nucleotide, and at least one nucleotide comprising a 5′-phosphorothioate group.
  • 12. The double-stranded ribonucleic acid molecule of claim 6, wherein the sense strand and the antisense strand of the double-stranded ribonucleic acid molecule each comprises at least one 2′-O-methyl modified nucleotide, at least one 2′-deoxy-2′-fluoro modified nucleotide, and at least one nucleotide comprising a 5′-phosphorothioate group.
  • 13. The double-stranded ribonucleic acid molecule of claim 1, wherein the double-stranded ribonucleotic acid molecule is conjugated to a ligand.
  • 14. The double-stranded ribonucleic acid molecule of claim 6, wherein said double-stranded ribonucleotic acid molecule is conjugated to a ligand.
  • 15. The double stranded ribonucleic acid molecule of claim 1, wherein the antisense strand comprises any of SEQ ID NO:705-SEQ ID NO:985.
  • 16. The double stranded ribonucleic acid molecule of claim 6, wherein the sense strand comprises any of SEQ ID NO:467-SEQ ID NO:704.
  • 17. The double stranded ribonucleic acid molecule of claim 6, wherein the antisense strand comprises any of SEQ ID NO:705-SEQ ID NO:985, and wherein the sense strand comprises any of SEQ ID NO:467-SEQ ID NO:704.
  • 18. The double-stranded ribonucleic acid molecule of claim 15, wherein the double-stranded ribonucleotic acid molecule is conjugated to a ligand.
  • 19. The double-stranded ribonucleic acid molecule of claim 17, wherein the double-stranded ribonucleotic acid molecule is conjugated to a ligand.
  • 20. A pharmaceutical composition comprising: (i) the double-stranded ribonucleic acid molecule of claim 1, and (ii) a pharmaceutically acceptable carrier.
  • 21. A pharmaceutical composition comprising: (i) the double-stranded ribonucleic acid molecule of claim 6, and (ii) a pharmaceutically acceptable carrier.
Priority Claims (1)
Number Date Country Kind
10187851.0 Oct 2010 EP regional
PRIORITY TO RELATED APPLICATION(S)

This application is a continuation of U.S. patent application Ser. No. 15/906,054, filed Feb. 27, 2018, which is a continuation of U.S. patent application Ser. No. 15/415,415, filed 25 Jan. 2017, which is a continuation of U.S. patent application Ser. No. 14/578,716, filed 22 Dec. 2014, now abandoned, which is a continuation of U.S. patent application Ser. No. 13/275,377, filed 18 Oct. 2011, now U.S. Pat. No. 8,946,176, which claims the benefit of European Patent Application No. 10187851.0, filed Oct. 18, 2010, all of which are hereby incorporated by reference in their entirety.

Continuations (4)
Number Date Country
Parent 15906054 Feb 2018 US
Child 16846535 US
Parent 15415415 Jan 2017 US
Child 15906054 US
Parent 14578716 Dec 2014 US
Child 15415415 US
Parent 13275377 Oct 2011 US
Child 14578716 US