Patent Application
20070111228
The present invention relates to RNA interference. The present invention also relates to methods of selecting interfering RNAs. The present invention also relates to methods of using interfering RNAs. The present invention also relates to RNA interference based assays to identify target genes.
RNA interference is a process by which specific mRNAs are degraded into short RNAs. RNA interference has been observed in organisms as diverse as nematodes, insects, trypanosomes, planaria, hydra, zebrafish, and mice. To mediate RNA interference, a double-stranded RNA with substantial sequence identity to the target mRNA is introduced into a cell. The target mRNA is then degraded in the cell, resulting in decreased levels of that mRNA and the protein it encodes.
Many different mRNAs have been targeted in this manner. Various labs have demonstrated that RNA interference is functional in vitro, e.g., in Drosophila extracts, and in vivo, e.g., in C. elegans.
In certain embodiments, a method of decreasing the level of a target mRNA in a host cell is provided. In certain embodiments, a host cell is contacted with a double-stranded RNA molecule, wherein the double-stranded RNA molecule comprises a sequence complementary to at least a portion of the target mRNA. In certain embodiments, the double-stranded RNA molecule further comprises at least one chemical modification. In certain embodiments, the at least one chemical modification is selected from 2′-F, 2′-OMe, and 2′-deoxy. In certain embodiments, the host cell is incubated under conditions whereby RNA interference occurs, thereby decreasing the level of the target mRNA.
In certain embodiments, a method of decreasing the level of a target mRNA in a host cell is provided. In certain embodiments, a vector is delivered to the host cell. In certain embodiments, the vector comprises a first nucleic acid sequence and a second nucleic acid sequence. In certain embodiments, the first nucleic acid sequence encodes a first RNA molecule comprising a first RNA sequence that is complementary to at least a portion of the target mRNA. In certain embodiments, the second nucleic acid sequence encodes a second RNA molecule comprising a second RNA sequence that is substantially identical to at least a portion of the target mRNA. In certain embodiments, the host cell is incubated under conditions that allow transcription of the first nucleic acid sequence and the second nucleic acid sequence. In certain embodiments, the host cell is incubated under conditions that allow RNA interference to occur, thereby decreasing the level of the target mRNA.
In certain embodiments, the first RNA sequence and the second RNA sequence are each longer than about 70 nucleotides. In certain embodiments, the vector further comprises at least one promoter selected from a phage promoter, a viral promoter, a pol II promoter, and a pol III promoter.
In certain embodiments, a method of selecting a double-stranded RNA molecule is provided. In certain embodiments, a target mRNA sequence is inputted into Oligo 5.0™ Primer Analysis software. In certain embodiments, the primer length is selected as 19. In certain embodiments, a primer is identified in the stability window, wherein the primer has a bell-shaped internal energy profile. In certain embodiments, a primer is identified in the stability window, wherein the primer has a substantially flat internal energy profile. In certain embodiments, a primer is identified in the stability window, wherein the primer has a maximum internal energy of less than −10 kcal/mol. In certain embodiments, a primer is identified in the stability window, wherein the primer has an internal energy of between −6 and −9 kcal/mol. In certain embodiments, a primer is identified in the stability window, wherein the primer has a melting temperature below 65° C. In certain embodiments, a primer is identified in the stability window, wherein the primer has a melting temperature below 50° C.
In certain embodiments, a BLAST search is performed on the primer against an EST database. In certain embodiments, a double-stranded RNA is synthesized, wherein the double-stranded RNA comprises a first RNA strand comprising a first RNA sequence that is identical to the nucleotide sequence of the primer and a second RNA strand comprising a second RNA sequence that is complementary to the nucleotide sequence of the primer.
In certain embodiments, a method of decreasing the level of a target mRNA in a mammalian host cell is provided. In certain embodiments, a mammalian host cell is contacted with an RNA hairpin molecule. In certain embodiments, the RNA hairpin molecule comprises a first region, a second region, and a third region. In certain embodiments, the first region comprises a sequence that is substantially identical to at least a portion of the target mRNA. In certain embodiments, the third region comprises a sequence that is substantially complementary to the first region. In certain embodiments, the first region and the third region hybridize, thereby forming an RNA hairpin molecule. In certain embodiments, the mammalian host cell is incubated under conditions whereby RNA interference occurs, thereby decreasing the level of the target mRNA in the host cell.
In certain embodiments, a method of decreasing the level of a target mRNA in a host cell is provided. In certain embodiments, a vector is delivered to the host cell. In certain embodiments, the vector comprises a nucleic acid sequence, wherein the nucleic acid sequence encodes an RNA hairpin molecule. In certain embodiments, the RNA hairpin molecule comprises a first region, a second region, and a third region. In certain embodiments, the first region comprises a sequence that is substantially identical to at least a portion of the target mRNA. In certain embodiments, the third region comprises a sequence that is substantially complementary to the first region. In certain embodiments, the first region and the third region hybridize, thereby forming an RNA hairpin molecule. In certain embodiments, the host cell is incubated under conditions that allow transcription of the nucleic acid sequence. In certain embodiments, the host cell is incubated under conditions that allow RNA interference to occur, thereby decreasing the level of the target mRNA in the host cell.
In certain embodiments, a method of constructing a library of RNA hairpin molecules is provided. In certain embodiments, a plurality of single-stranded DNA hairpin templates is synthesized. In certain embodiments, each single-stranded DNA hairpin template comprises a first region, a second region, and a third region. In certain embodiments, the first region comprises an RNA polymerase promoter sequence. In certain embodiments, the second region comprises a random nucleotide sequence having between 5 and 500 nucleotides. In certain embodiments, the third region comprises a first nucleotide sequence, a second nucleotide sequence, and a third nucleotide sequence, wherein the first nucleotide sequence hybridizes to the third nucleotide sequence, thereby forming a single-stranded DNA hairpin template. In certain embodiments, the 3′ end of the third nucleotide sequence of each of the plurality of single-stranded DNA hairpin templates is extended to form a plurality of double-stranded DNA hairpin templates. In certain embodiments, the plurality of double-stranded DNA hairpin templates is amplified to form a plurality of double-stranded DNA templates. In certain embodiments, the plurality of double-stranded DNA templates is transcribed to form a library of RNA hairpin molecules.
In certain embodiments, a method of identifying a target gene is provided. In certain embodiments, an array comprising a plurality of positions is formed. In certain embodiments, each position comprises at least one mammalian cell. In certain embodiments, the at least one mammalian cell at each position is contacted with at least one RNA hairpin molecule. In certain embodiments, the at least one mammalian cell is incubated under conditions that allow RNA interference to occur. In certain embodiments, an at least one mammalian cell exhibiting at least one biological endpoint is selected. In certain embodiments, the at least one RNA hairpin molecule associated with the plurality of cells exhibiting at least one biological endpoint is identified. A BLAST search on the nucleic acid sequence of the at least one RNA hairpin molecule is performed, thereby identifying the target gene.
In certain embodiments, a library comprising a plurality of RNA hairpin molecules is provided. In certain embodiments, each RNA hairpin molecule comprises a first region, a second region, and a third region, wherein the first region comprises a random nucleotide sequence having between 5 and 500 nucleotides and the third region comprises a nucleotide sequence that is substantially complementary to the first region.
Various nucleic acid triggers were delivered into NIH 293 cells grown in 96-well plates by transfection with Lipofectamin-2000. Twenty-four hours after the transfection, levels of two specific messages, PKC-θ and Cyclophilin, were quantified by branched-DNA (bDNA) capture detection method. Cyclophilin, a housekeeping gene, was used to detect any nonspecific gene inhibition induced by various nucleic acid triggers. What is shown is the ratio of the mRNA levels of the two genes as a function of various triggers. Arrows indicate the reduction of the ratio triggered by synthetic and transcribed hairpin RNA molecules. The two triangles indicate the lack of gene knockdown by the ss-SiRNA hairpin containing a single nucleotide deletion, indicating that the RNA interference process is selective and uses hairpin triggers containing specific sequences for effective gene silencing.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise. In this application, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including but not limited to patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose.
Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection, etc.). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose. Unless specific definitions are provided, the nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.
The following examples, including the experiments conducted and results achieved are provided for illustrative purposes only and are not to be construed as limiting the present invention in any way.
I. Gene Inactivation Through RNA Interference (RNAi)
Introduction
Double-stranded RNA (dsRNA)-mediated specific gene inactivation or gene silencing has been observed in many different organisms. This phenomenon, referred to as RNA interference (or RNAi), is expected to play a significant role in understanding gene function, signal transduction pathways and identifying therapeutic agents in the future. To date, the mechanism by which a dsRNA molecule inactivates the expression of a gene is not completely clear. Certain experimental evidence indicates that the process of RNA interference may be post-transcriptional and involves degradation of specific mRNA. The mRNA that is degraded is dictated by the sequence of the dsRNA that is introduced into the cell. As a result, the RNA interference process has been shown to be extremely sequence-specific. It has been observed that the dsRNA that mediates the target mRNA degradation is processed (or cleaved) into a collection of fragments of 21-25 nucleotides. According to a proposed model (Zamore, P. D. et al., Cell, 101, 25-33, 2000; Bass, B. L., Cell, 101, 235-238, 2000), the sense strands of RNA fragments derived from the dsRNA are hybridized to the target mRNA. This process is believed to be facilitated by a helicase and a protein catalyzing an ATP-dependent strand exchange activity. The target mRNA is subsequently cleaved, resulting in the inactivation of the message before being translated (
In certain embodiments RNA interference may be used in the following areas.
1) To use as a tool for target validation in functional genomics.
2) To generate therapeutic molecules that interfere in specific gene expression.
3) To generate transgenic animals and plants by introducing gene constructs that express dsRNA targeted to a given gene.
4) To use as a technique for gene therapy applications in controlling the expression of targeted genes.
5) To use in oligonucleotide therapeutics using synthetic nucleic acid molecules.
1. For Functional Genomics and Therapeutics
An RNAi technique is envisioned to play a role in functional genomics to validate gene targets in both tissue culture and animal models. At the tissue culture level, inactivation of a target gene has been demonstrated by introducing into cells exogenous dsRNA that has been produced by in vitro transcription. These natural dsRNAs are relatively long molecules consisting of 100-800 base pairs. It may be beneficial to identify RNA species that are more nuclease resistant than natural RNA. In certain embodiments, one may screen a variety of chemically-modified RNAs to identify candidates that are both nuclease resistant and active in the RNAi mechanism (
In certain instances, the dsRNA molecules that mediate the degradation of a specific mRNA may be expressed in vivo (
In certain embodiments, a host cell may be contacted with two or more interfering RNAs that may target one or more target mRNAs.
Although an antisense approach may be used in target validation using tissue culture models, the technology sometimes may be challenging in validating the same targets in animal models. This may be due to poor delivery and targeting of short synthetic oligonucleotides to desired tissues in animals. Expression of dsRNA molecules in animals using gene delivery vectors (Examples: Retroviral, Adenovirus and Adeno Virus-Associated vectors) may obviate this challenge and is a subject of this disclosure. Hence, an RNA interference approach could represent a method of choice for validating target genes in animal models.
2. Generating Transgenic Animals and Plants
Transgenic species that do not express certain genes could be produced by specific introduction of DNA cassettes that transcribe dsRNA specific to messages of genes of interest. In certain embodiments, viral resistant transgenic species may be produced by introducing dsRNA specific to certain viruses known to be pathogenic in that species.
3. Gene Therapy Applications
The RNA interference approach could be useful in inhibiting a gene product in patients using a gene therapy approach. For example, genes that are responsible for a multi drug resistance phenotype may be a useful target for an RNA interference approach. This has a direct impact in cancer patients undergoing chemotherapy.
Advantages of an RNAi Approach:
1. Accumulating experimental evidence suggests that the process of RNA interference may be a catalytic process mediated by interplay of several proteins. As a result, only a few copies of dsRNA per cell may be required to degrade a large pool of target RNA.
2. Double-stranded RNA molecules that carry out gene inactivation could be either introduced exogenously or expressed in vivo, facilitating the mode of delivery for different applications.
3. Double-stranded RNA molecules are readily taken up by cells. Unlike certain single-stranded oligonucleotides currently used in antisense research.
Technologies that allow specific inhibition of gene expression are becoming important for drug discovery efforts to identify better human therapeutics. Such techniques may play an important role in the following specific areas.
Furthermore, in addition to their role in facilitating drug discovery, molecules that mediate specific gene inhibition could become therapeutics as well. One widely used technique in target validation in cultured mammalian cells has been the antisense approach (reviewed in (Murray, 1992)) in which the specific destruction of target mRNA is triggered by the delivery of a ssDNA molecule that is complimentary to a region within the target. Upon hybridization of the antisense (AS) oligonucleotides to its target mRNA, it is believed that the enzyme RNase H is recruited to the site to cleave the message.
To compare the characteristics of RNA interference with those of antisense-mediated gene knockdown, PKC-θ, an endogenous gene expressed in the human kidney cell line HEK 293 (293 cells), was used in the following experiment as the primary target. After the delivery of either SiRNA triggers or antisense oligonucleotides, the level of PKC-θ message was measured by a branched DNA (bDNA) assay, an assay designed to capture and detect a specific nucleic acid sequence such as a unique mRNA. In parallel, cellular toxicity and nonspecific gene inactivation was studied by monitoring the level of a housekeeping gene, cyclophilin. In addition to these studies, experiments were designed to gain further understanding of the nature of SiRNA triggers with respect to their specificity, homology, length requirements, and their tolerance to a range of chemical modifications.
Materials and Methods
SiRNA
SiRNAs were prepared using several different methods. Certain chemically synthesized SiRNAs were synthesized using RNA phosphoramidites containing a 2′-O-TriisopropylsilylOxyMethyl (TOM) protection group from Glen Research (Sterling, Va.). Other SiRNAs were obtained from Dharmacon (Longmont, Colo.) employing 5′-Silyl-2′-bis(2-acetoethoxy)methyl (ACE) Orthoester chemistry. Synthesized SiRNAs using TOM phosphoramidites were HPLC purified, whereas those obtained from Dharmacon were used without further purification, due to their high purity resulting from extremely high coupling efficiency (Scaringe, 2001). Ss-SiRNAs produced by in vitro transcription were obtained using corresponding synthetic DNA templates containing the promoter for the T7 RNA polymerase (Milligan et al., 1987). After transcription, full-length RNAs were purified by polyacrylamide gel electrophoresis run under denaturing conditions. SiRNAs were annealed in an annealing buffer consisting of 100 mM KCl, 30 mM HEPES (pH 7.5), and 2 mM MgCl2 by heating to 75° C. for 2 minutes followed by slow cooling to ambient temperature. HPLC-purified antisense oligonucleotides (ASO) were obtained by solid phase synthesis using cyanoethyl phosphoramidites. The ASOs contained two chemical modifications: the middle seven nucleotides carried a 2′-OMe modification in the sugar and the remaining flanking regions contained phosphorothioate linkages.
Plasmid Expressing Secreted Alkaline Phosphatase (SEAP)
The SEAP gene (from pAP-1 SEAP vector from Clontech) was cloned into an adeno-associated vector (AAV #6).
Cultured Mammalian Cells
This work used the following cell lines cultured in DMEM with 10% FBS at 5% CO2 at 37° C.
Delivery of Nucleic Acid Triggers
Cells seeded in 96-well plates at approximately 25,000/well in the previous day were transfected with different nucleic acid triggers using Lipofectamin 2000 and Opti-MEM I (from Invitrogen). Briefly, SiRNA was diluted in Opti-MEM-I in 50 μL of volume. This was mixed with an equal volume of Lipofectamin 2000 diluted 12.5-fold in OPTI-MEM I. After incubating the mixture at ambient temperature for 20 minutes, 270 μL of the regular cell medium was added and 95 μL of the solution was immediately transferred onto the cells in the plate with no media. Plates were transferred to a 37° C. incubator with 5% CO2 for either 24 or 48 hours. To monitor the fate of SEAP transiently expressed in 293 cells, prAAV6-seap plasmid was included in the transfection mixture with and without SiRNAs.
Quantification of mRNA Levels
Specific mRNA levels of cells transfected with different nucleic acid triggers were quantified using QuantiGene High Volume Kit (from Bayer) that employs a branched-DNA (b-DNA) method for nucleic acid detection according to the manufacturer's instructions. Specific detection of a given mRNA is based on its selective capture on to the microtiter plate, which is dictated by the capture probes. Probe sets that are unique to each target mRNA were designed using the ProbeDSesigner software (Bayer) according to the manufacturer's instructions. Before using the probe sets in experiments, the probes were tested using the cells expressing each message to make sure that they worked.
Cytotoxicity Assays
After 24 or 48 hour incubation, 25 μL of AlamaBlue reagent (Trek Diagnostic Systems, Inc.) was added to each well and incubated at 37° C. with 5% CO2 for 2 hours. The absorbance at 570 nm was read in a SpectraMax UV/VIS 96-well plate format spectrophotometer. The MTT assay was carried out according to the manufacturer's instructions.
Secreted Alkaline Phosphatase (SEAP) Assay
Twenty-four hours after the transfection, 15 μL of medium from each well was transferred to a white opaque 96-well flat bottom microtiter plate, and the amount of SEAP was detected using chemiluminescent SEAP assay (Great EscAPe SEAP assay kit form Clontech) according to the manufacturer's instructions.
Results and Discussion
Design of SiRNA Triggers
SiRNA Triggers Based on Antisense Target Sites
The initial set of SiRNA triggers were based on three antisense DNA sequences that target three independent sites in the PKC-θ message. A computer program designed to identify the best target sites for antisense oligonucleotides had previously picked these sites. When targeted by antisense DNA molecules, sites 8 and 15 were more effective than site 4 in reducing the level of PKC-θ message.
Silencing of specific genes has been observed upon the introduction of RNA with the capacity to form long hairpin molecules in which one half of the folded sequence is homologous to the mRNA of the targeted gene (Piccin, 2001; Tavernarakis, 2000; Wang et al., 2000). The experiment here sought to determine whether, short chemically synthesized single-stranded RNA (ssRNA) with the propensity to fold-back and form short hairpin molecules may be used as triggers to induce RNAi in vivo. To investigate whether ss-RNA that forms short hairpin structures could trigger RNAi in mammalian cells, two such molecules for each target site were designed (
Impact of Different Nucleic Acid Triggers Upon Transfection into 293 Cells
We carried out parallel analysis of several end points within the cells transfected by a series of nucleic acids triggers such as single-stranded DNA and RNA antisense molecules, ds-SiRNA, and ss-SiRNA-HP. After delivery of each nucleic acid trigger into 293 cells, the following were measured: (1) reduction of specific mRNA, (2) reduction of nonspecific mRNA and (3) cellular toxicity.
Fate of Target-Specific and Nonspecific mRNA Levels in 293 Cells
The reduction of the specific message PKC-θ was evaluated by measuring the level of PKC-θ mRNA by a bDNA capture-detection method (
A nonspecific effect of nucleic acid triggers on 293 cells was probed by measuring the message level of a housekeeping gene, cyclophilin (
No nonspecific gene knock down by ss-SiRNA-HP triggers that could fold back to generate short hairpin structures was observed. This result along with the reduction of the specific message by ss-SiRNA suggests that ss-SiRNA-HP molecules, like ds-SiRNA molecules, function through the RNA interference mechanism to specifically reduce target mRNA levels.
Cellular Toxicity
This work included two commonly used cytotoxicity assays to evaluate cellular toxicity upon delivery of various nucleic acid triggers into cells. Twenty-four hours after the transfection, an AlamarBlue assay was carried out and completed within an hour (
The AlamarBlue assay revealed the toxicity associated with cells transfected with single-stranded antisense molecules, both DNA and RNA (
Randomly Picked ds-SiRNA
The above ds-SiRNA triggers were based on target sites picked by a computer program designed to identify the best sites for antisense targeting. They all worked as expected in reducing the target mRNA level by triggering the RNAi mechanism. This work also tested ds-SiRNA targeted to sites picked by random choice, without applying any pre-selected criteria. Several different ds-SiRNA triggers. Three of these had overlapping target sites within the PKC-θ mRNA (
The two dT residues at the 3′-end of the ds-SiRNAs studied so far do not recognize the target mRNA. These residues were changed to two dC residues and this trigger, containing dC residues in place of dT residues, worked effectively (
By targeting endogenous genes present in several other cell lines (
RNAi in a Transiently Expressed Reporter Gene
The above results were obtained with ds-SiRNA triggers directed against endogenous genes in cultured mammalian cells. This study was extended to a gene expressed transiently upon transfection into mammalian cells. A plasmid was designed to express a reporter gene. SEcreted Alkaline Phosphatase (SEAP), under the CMV promoter (prAAV6-seap) was transfected into 293 cells in the presence and absence of a ds-SiRNA trigger designed to target the SEAP mRNA. In this experiment, three different ds-SiRNA triggers picked randomly to target non-overlapping sites within the mRNA of SEAP were used. Out of the three ds-SiRNA tested, one trigger effectively silenced the transient expression of SEAP (
Duration of RNA Interference
Next, the duration of RNA interference triggered by a ds-SiRNA in 293 cells was examined.
Next, the duration of RNAi in silencing an endogenous gene, PKC-θ (
Certain SiRNA Triggers
Certain ds-SiRNA triggers may have 21 nucleotides in each strand, out of which 19 nucleotides form base pairs in the duplex leaving two nucleotides in the 3′ extension (Caplen, 2001; Elbashir, 2001a). In certain designs, all 21 nucleotides in the antisense strand are homologous to the target mRNA. Tests were conducted to test the effectiveness of different lengths of certain ds-SiRNAs, as well as to test the effectiveness of ds-SiRNAs having different homologies. The homology tests involved testing the number of mutations in a ds-SiRNA molecule that may be tolerated certain RNAi processes. These issues were addressed by using a series of ds-SiRNA triggers (
Homology of the SiRNA Trigger
Certain mutations were introduced in both strands of the ds-SiRNA-8A trigger to obtain two triggers (
Next, the tolerance of the RNA interference mechanism to the mutations localized only in the sense strand of a SiRNA trigger was investigated. Six ds-SiRNA triggers in which the sense strand carried 1-6 point mutations were constructed (
Short ds-SiRNA Triggers
In certain work, ds-SiRNA triggers of 21-25 nucleotides have been used to elicit RNA interference in mammalian cells (Caplen, 2001; Elbashir, 2001a). A rationale for choosing these lengths was based on the observation that long double-stranded RNAs are cleaved into 21-22 nucleotide fragments that serve as intermediates for the interference process (Elbashir, 2001b). The effect of RNAi triggered by ds-SiRNA triggers consisting of 14, 17, and 19 nucleotides in each strand was investigated (
To identify which factor might be important for RNA interference, three more ds-SiRNA triggers were designed to differentiate between the homology and the length of the trigger (
Nuclease Resistant SiRNA Triggers
Both ss- and ds-SiRNA triggers (unimolecular and bimolecular) that are stable to nucleases, and hence may be effective in initiating and maintaining an RNAi response in cells, were studied. Nuclease stable SiRNA triggers may survive longer in certain biological fluids both during and after their delivery into the cells. Once inside the cell, mere resistance to degradation may make nuclease stable triggers last longer, thereby establishing long lasting RNA interference. Moreover, certain modifications may facilitate cell penetration. The following modifications in either antisense or sense strand were made to explore the possibilities for creating chemically modified SiRNA triggers (
The inverted abasic residue and the inverted dT residue may function like 3′ caps that further protect an oligonucleotide from 3′-5′ exonucleases, in addition to the protection provided by two dT residues. Replacement of the 2′-OH group in the sugar by various groups such as NH2, F and OMe may make RNA more nuclease resistant (Lin et al., 1994; Pieken et al, 1991). As shown in
Ds-SiRNA triggers with one strand with a 2′-OMe substitution inhibited the RNAi process. However, it may be possible to have SiRNA triggers modified with 2′-OMe either in pyrimidines, or in purines, or at specific sites. SiRNA triggers with a single DNA strand may be analogous to SiRNA triggers with a 2′-OMe modification. RNA-DNA hybrids were not efficient in eliciting the RNAi process in this work. The lower level of RNAi in mammalian cells by SiRNA with DNA strands, as demonstrated here, is in agreement with a previous observation made in C. elegans with long RNA-DNA hybrid molecules. Previously, using relatively long dsRNA molecules Parrish, et al. observed the tolerance of 2′-F uracil in RNA interference in C. elegans (Parrish, 2000).
Next, a unimolecular trigger based on the fold-back hairpin structure was generated with 2′-F pyrimidines throughout the molecule and with a single inverted dT at the 3′ end (
The successful use of SiRNA triggers including both pyrimidine nucleotides carrying a 2′-F modification either on both strands (in the case of bimolecular ds-SiRNAs) or throughout the molecule (in the case of unimolecular ss-SiRNAs) was observed. Taken together, these results suggest that the design of SiRNA triggers carrying specific chemical modifications may be a viable approach to initiate RNA interference in mammalian cells. Certain additional modifications to SiRNA triggers include, but are not limited to, the addition of polyethylene glycol, the addition of lipids, and the addition of dyes, such as flourescein. It may be that these triggers will pave the way for creating effective SiRNA triggers for clinical applications.
Conclusions
RNA Interference Compared to Antisense
1. Relatively low or almost no toxicity to the cells transfected with SiRNA triggers was observed, whether they were ds-SiRNA or ss-SiRNA that form short hairpins, compared to the single stranded antisense molecules. Transfection with SiRNA triggers in this work did not lead to the reduction of nonspecific mRNAs. In certain instances, this may make RNAi technology more versatile than antisense techniques in general, allowing researchers to transfect cells at low cell density. This also suggests that RNA interference may be a useful tool to study cellular pathways that are sensitive to external insults.
2. RNAi in general may be more potent than antisense; about 10-fold lower concentration of SiRNA compared to the concentration of antisense oligonucleotides gave approximately the same level of mRNA reduction in this work. Effective RNA interference with SiRNA triggers transfected at 5 nM was observed.
3. Similar to antisense oligonucleotides, in certain instances, the efficiency of SiRNA triggers may be target site dependent, suggesting that targeting certain sites in a target mRNA may not be effective in eliciting gene silencing through RNAi.
4. SiRNA triggers may be picked randomly without any help from a computer algorithm. Since targeting to certain sites on mRNA may not work, a handful of triggers (between 3 and 5) may be tested. This is in contrast to the antisense approach that often involves screening 30-40 oligonucleotides. Since the conversion of a ds-SiRNA trigger that was not effective into a unimolecular trigger that produced effective interference was observed, it may be that the number of unimolecular triggers that are screened may be even lower.
Certain Novel SiRNA Triggers
1. Effective unimolecular SiRNA triggers that are single-stranded with the propensity to form short hairpins were observed. In those ss-SiRNA HP triggers, the nature of the nucleotides in the loop may not affect the efficiency of RNA interference.
2. Certain ss-SiRNA HP triggers appear to be sensitive to nucleotide deletions.
3. In certain embodiments, unimolecular siRNA triggers may contain synthetic loops. In certain embodiments, unimolecular siRNA triggers may contain polyethylene glycol loops.
Nuclease Stable SiRNA Triggers
1. The incorporation of a nuclease resistant cap, such as inverted dT and inverted abasic residues at the 3′ end of both strands of a ds-SiRNA trigger, may be effective in RNA interference. This also may work in ss-SiRNA triggers. Furthermore, the same strategy may work at the 5′ end as well.
2. In certain instances, pyrimidines in both strands of ds-SiRNA trigger may be replaced by 2′-F modified pyrimidines, making such triggers resistant to nucleases.
3. In the case of certain unimolecular ss-SiRNA triggers, the 2′-F modification may be introduced throughout the sequence in all pyrimidines without substantially compromising the efficacy of RNA interference.
Features of Certain SiRNA Triggers
1. Ds-SiRNA triggers may be designed to carry 3′ extensions made up of two dT residues that do not recognize the target. In other words, target sites on an mRNA may be any sequence, not restricted to the nature of N19-AA. This opens up a broad range of target sites within a target gene.
2. In certain instances, the length of each strand within a ds-SiRNA may be as short as 19 nucleotides out of which 17 nucleotides form a contiguous stretch of target homology with two nonhomologous dT residues at the 3′ extensions.
3. In certain instances, the region of homology may be as short as 15 nucleotides when the length of the trigger is 21 nucleotides. A 19 nucleotide long ds-SiRNA harboring 15 nucleotides of target homology may be effective in RNA interference.
4. In certain instances, ds-SiRNA triggers may tolerate a single base pair mutation.
5. In certain instances, mutations in the sense strand of the ds-SiRNA trigger also affect the efficiency of RNA interference. In certain instances, ds-SiRNA triggers with more than two mutations in the sense strand may decrease the RNAi effect. In certain instances, the effectiveness of RNA interference may vary depending on the mutation site in the sense strand, such that an effect may be observed when the mutation is located in the middle, as opposed to near the end of the RNA.
II. Gene Identification and Functional Analysis Using Short Interfering Random Sequence (SiRS)RNA Hairpin Libraries
Identification and characterization of novel genes combined with the functional analysis of identified genes may enable the discovery of novel drug targets. With the completion of the human genome database, companies are racing to identify novel genes. Among these may be genes that could be linked to various diseases.
Some technologies exist to potentially identify a function of a gene based on sequence information. Antisense oligonucleotides upon pairing-up with a known mRNA sequence mediate the destruction of a message in a cell, leading to the inactivation of gene expression. This technique may be aided by prior knowledge of the mRNA sequence of a gene and may be used to validate gene function. On the other hand, there are many genes in the human genome database whose sequences are not known to date. Furthermore, it may be that alternate splicing will lead to the generation of many splice variants to generate different protein products. This may further complicate the gene identification process.
A reverse genetic strategy to identify genes exclusively based on their function may be possible, allowing discovery of new genes from the human genome as well as the analysis of the functions of known genes. Furthermore, the proposed strategy may also help identify novel pathways of complex biological processes. This strategy uses epigenetic interference of gene expression using double-stranded RNA (dsRNA) molecules. In this work, certain double stranded RNA (dsRNA) corresponding to a sense and antisense sequence of an mRNA was introduced into a cell, the corresponding mRNA was degraded and the gene was silenced. This post-transcriptional gene silencing phenomenon, mediated by double stranded RNA sequences, is commonly referred to as RNA interference or RNAi (Fire, 1998). RNAi has been observed in a wide range of organisms, including nematodes, insects, trypanosomes, planaria, hydra, zebrafish, and the mouse (Reviewed in Bosher, 2000; Hammond, 2001). RNAi may be functionally related to posttranscriptional gene silencing observed in plants and quelling observed in Neurospora crassa. Since double stranded RNA mediates all three processes, they can be collectively called “RNA silencing” (Voinnet, 2001; Waterhouse, 2001).
Biochemical studies carried out with a Drosophila in vitro system led to the discovery of short RNA duplexes (21-22-nucleotides) that could effectively trigger gene silencing through RNAi (Elbashir, 2001b; Yang, 2000; Zamore, 2000). Recently, this epigenetic interference of gene silencing by short interfering RNAs (SiRNAs) has been demonstrated in cultured cells of mammals (Caplen, 2001; Elbashir, 2001a), opening the door for using RNAi technology in characterizing the human genome. Although complete details of the cellular mechanism that initiates and propagates gene silencing through RNA interference is unknown, it may be an extremely specific and very potent mechanism to inhibit gene expression by degrading specific mRNA. Only a few molecules of dsRNAi triggers per cell may be needed to inhibit mRNA present at high concentrations, suggesting an inherent amplification step present in the RNAi process. In certain instances, its specificity, efficacy and generality offer an attractive approach for studying gene function.
Short Interfering Random Sequence (SIRS)RNA Hairpin Libraries
In certain embodiments, proposed strategies may be based on the use of random sequence RNA libraries to elicit an RNAi response in mammalian cells. In certain embodiments, a library of random sequence short RNA hairpins may be used to elicit RNAi-mediated gene silencing in mammalian cells. Long RNA hairpins have been used to elicit an RNAi response in several species including C. elegans (Parrish, 2000; Tavernarakis, 2000) Trypanosome (Ngo, 1998), Drosophila (Piccin, 2001) and in plants (Chuang, 2000). The use of short hairpin molecules to trigger RNAi in cultured mammalian cells was investigated. This proposition was based on preliminary data that supports successful and specific gene inactivation by short hairpin molecules with defined sequences (
Certain methods may be proposed to screen SIRS hairpin libraries to identify ss-SiRNA hairpins that elicit specific phenotypic responses in cells. Certain methods use a split screening approach; as described above, to cull the original library, whereas certain other methods utilize a biological screening approach using a retrovirus that expresses random sequence short hairpins.
Method 1:
Method 1 may be based on the use of in vitro synthesized SiRS RNA hairpin libraries from which RNAi triggers may be identified through repetitive screening of sub-libraries. These sub-libraries may be subjected to splitting and amplification to identify the sequence of interest.
A. Library Construction
A. 1. General Design of Synthetic DNA Templates
Short interfering random sequence (SIRS)RNA hairpin libraries may be generated from synthetic DNA templates. The design of the synthetic DNA template is illustrated in
A. 2. Sequence Complexity
The number of nucleotides in the random region dictates the sequence complexity of a library. A library with 15 randomized nucleotides has 1.0×109 theoretically possible individual molecules, whereas a library with 20 such nucleotides will have 1.0×1012 individual molecules. One would expect a single 15-nucleotide-long sequence within a library of approximately 109 individual molecules to occur only one time in the human genome. Based on this calculation, such a library may be expected to provide RNAi triggers for all possible genes in the human genome. However, a SiRS RNA hairpin library having one million unique RNAi triggers may be used. This library may be large enough to capture most genes.
A. 3. Synthesis of RNA Libraries
A general scheme for the production of an SiRS RNA hairpin library from the corresponding synthetic DNA template library using in vitro transcription is outlined in
Since the concentration of each template may not be sufficient to generate enough RNA hairpins of one type, copies of immobilized templates may be amplified by polymerase chain reaction (PCR). PCR-amplified DNA templates may be used for in vitro transcription using T7 RNA polymerase (when T7 RNA polymerase is included in the DNA template). The resulting single stranded RNA molecules may fold into intra-molecular hairpin structures. They also may undergo intermolecular hybridization and generate double-stranded RNA molecules. Both RNA hairpins and dsRNAs may generate an RNAi response. Thus, the formation of intra- or inter-molecular structures should not pose a problem in this strategy. Once RNA molecules are made, they may be ready for use in cell-based screening assays.
B. Functional Screening
Certain screening exercises may start with a dsDNA template library having approximately 1×106 molecules that has been distributed into five 1536-well microtiter “Master Plates” resulting in approximately 130 unique templates in a single well. Once biotin-conjugated DNA templates are immobilized in the streptavidin (SA) coated “master plates”, excess SA in the well may be blocked with free biotin, and the wells may be thoroughly washed. Subsequently, each template molecule immobilized on the “master plate” may be amplified by PCR using specific primers one of which may be biotinylated at the 5′-end. Upon PCR amplification, amplified, biotinylated, template molecules may be transferred to four 384-well “lead plates” from a single “master plate” (
Certain high-throughput cell-based assays may be used to screen an SiRS hairpin library. Each functional screening assay may give rise to the identification of one or more wells with biological endpoints of interest. Corresponding wells in the lead plate contain “lead SiRS libraries”. Since functional screening for various biological end-points using different assay formats and cell types may be carried out in parallel, several “lead SiRS libraries” may be identified simultaneously from a single lead plate. These lead libraries may be split into sub-libraries and the resulting sub-libraries may be used for further screening as described below.
Once a lead library for a particular functional assay is identified, the specific RNAi trigger hairpin may be delineated by further fractionation of the members within the library. This may be done by subjecting the corresponding DNA library in a “lead plate” to low-level PCR amplification, followed by distributing the amplified products into several 96-well “daughter plates” (
The biological end-point in the particular functional screen may be confirmed by synthesizing the specific RNA hairpin molecule derived from the consensus sequence. Once confirmed, nucleotide sequences in both arms of the specific hairpin may be used to perform a BLAST search against the human genome database to identify the candidate gene (
The success of the screening process may depend on the efficiency of library transfection and the amount of RNAi trigger molecules that silence a gene within a single cell. Approaches that make SiRS RNA hairpin libraries more nuclease resistant may help improve the survival of individual molecules during transfection, which may be important during the primary screening step in which the libraries are more diverse. Previous studies in C. elegans using long dsRNA triggers suggested that the substitution of 2′-F uracils in place of 2′-OH uracil in either sense or antisense strand did not interfere with the RNAi process (Parrish, 2000). These authors also reported successful RNA interference with long dsRNA triggers synthesized with a single type of α-thio NTP. Substitution of α-thio UTP may produce a somewhat reduced level of RNA interference. A successful reduction of specific mRNA in mammalian cells was observed using SiRNA triggers of 21 nucleotides in which all pyrimidines in both strands are modified with 2′-F sugars. The incorporation of 2′-F-modified pyrimidines and α-thio purines into hairpins may make them nuclease stable. SiRS RNA hairpin libraries with chemically-modified pyrimidines on the sugar and phosphorothioate backbone modifications at all purines may be obtained by in vitro transcription employing the appropriately modified NTPs.
Another approach to enrich the population of each hairpin molecule within target cells employs the transfection of ds-DNA templates (preferably linear or circular in the form of plasmids) carrying the T7 promoter fused to the template for hairpin synthesis. In this case, the target cells may carry the T7 RNA polymerase gene integrated into their genome. Upon transfection, T7 RNA polymerase will transcribe many copies from each template within the cell.
Method 2:
Method 2 uses a biological screening method using a retroviral vector carrying SiRS RNA hairpin libraries. Retroviruses that mediate a specific biological function in infected cells may be identified, amplified and used for the next cycle of selection.
A retroviral screening system employing random sequence combinatorial libraries containing the target recognition site for a hairpin ribozyme has been used for target validation. (Kruger, 2000; Li, 2000)
A. Library Construction
DNA constructs that express SiRS hairpin RNA libraries in vivo may be designed as illustrated in
Plasmid libraries may be obtained by growing the transformant pool followed by carrying out minipreps. The plasmid DNA library may be used to generate the retroviral vector library by triple transfection methods known in the art. This may include the cotransfection of the plasmid DNA library along with two vectors, one expressing Gag-Pol (Landau, 1992) and the other expressing VSG-G (Burns, 1993) into a packaging cell line. The supernatant containing the retroviral library may be harvested, filtered, and used for biological screening.
Biological screening, as in Method 1, may be performed in parallel. In each case, cells may be transduced with SiRS RNA hairpins carrying retroviral vector at a very low MOI (multiplicity of infection), in certain instances, as low as 1. Cells may be maintained in the presence of the antibiotic to which the retrovirus is resistant and screened for a desired biological response. Individual wells exhibiting a desired biological outcome may be identified and viruses may be rescued from them.
Viral rescue from selected wells may be achieved by co-transfecting DNAs of two helper vectors expressing Gag-Pol and VSV-G. After co-transfection, the vector supernatant may be selected, pooled, filtered, and used for the next round of selection with a fresh plate of cells. Alternatively, PCR rescue may be performed to rescue the sub-library of inserts that provided the desired biological outcome. A single PCR primer carrying the second restriction site in the original DNA construct may be used for PCR rescue. For this, high molecular weight DNA may be extracted from identified wells and PCR amplified with the primer. Resulting PCR products may be digested with the second restriction enzyme, gel purified, and cloned into the retroviral vector as described above. The resulting retroviral sub-library may be used for the next selection round.
After 2-3 cycles of selection-enrichment of retroviral libraries, inserts may be cloned and sequenced to identify a consensus sequence motif. Based on the consensus sequence motif that emerges, a hairpin RNAi trigger may be synthesized and used to confirm the biological effect of the trigger. Once confirmed, nucleotide sequences in both stems of the RNAi trigger may be used to perform a blast search against the human genome database to identify the candidate gene.
III. Certain Methods for Designing Functional SiRNA Triggers for Effective Gene Silencing by RNA Interference
RNA interference (RNAi) is an effective technique for gene silencing in many organisms, including mammals. Short RNA duplexes of 21-22 nucleotides with 2-3 nucleotide 3′ extensions, generally referred to as SiRNA molecules, have been effective in specific gene silencing in mammalian cells by triggering the RNAi process. However, not all SiRNA molecules bearing homology to a region within an mRNA sequence work effectively in silencing the cognate gene. A simple, effective, and universal approach is described here for designing productive SiRNA triggers using at least one of the following two criteria: (1) a relatively low calculated melting temperature in the range of 55°-70° C.; and (2) a calculated low energy (−6 to −9 kcal/mol) internal stability profile with either a flat profile or a bell-shaped distribution-suggesting a high internal stability concentrated in the middle of the duplex. Analysis of more than 35 SiRNA triggers targeted to several human genes revealed that triggers that satisfy these two criteria were generally effective in silencing the targeted gene. The proposed approach has been experimentally validated by designing functional SiRNA triggers according to the criteria outlined in the method. Hence, choosing SiRNA triggers guided by the proposed approach may be helpful in avoiding the synthesis of a large number of triggers for a given target. Therefore, this technique may represent a more economical and efficient way to analyze gene function.
A. Introduction
Specific gene silencing has a wide range of applications in biology. Some of these applications include the understanding of the function of a gene, elucidating the individual role of a gene in a complex biological pathway, and the identification of novel therapeutic targets. Validation of therapeutically relevant gene targets may be valuable for the pharmaceutical industry in general, and may also have a broader societal impact in improving the quality of human life. In this regard, techniques that allow for specific gene silencing may play a role in achieving these goals. Some have used the antisense approach at the tissue culture level for target validation. An approach called RNA interference (RNAi) has emerged as an effective way to achieve specific gene silencing. In certain instances, RNAi is a post-transcriptional phenomenon mediated by double-stranded RNA (ds-RNA) with one strand bearing homology to the mRNA of the gene to be silenced. RNAi has been described in the nematode Caenorhabditis elegans (C. elegans) using long dsRNA molecules (Fire, 1998; Guo, 1995). RNAi also has been demonstrated in a wide range of species (reviewed in Bosher, 2000; Hammond, 2001; Sharp, 2001; Zamore, 2001). Although the exact mechanism of RNAi is not completely understood, an in vitro system derived from Drosophila embryonic cells that recapitulates the silencing event has provided some mechanistic insights into the process (Hammond, 2000; Tuschl et al., 1999). These studies led to the demonstration that the long dsRNA molecules that activate the RNAi process are cleaved into fragments of approximately two helical turns (approximately 21-22 nucleotides) (Zamore, 2000) by a multidomain ribonuclease III protein called Dicer (Bernstein, 2001). These short dsRNA may then become a platform on to which an ensemble of proteins assembles to form an RNA induced silencing complex (RISC). While the antisense strand of RISC may guide substrate recognition, an endonuclease may perform the cleavage of the target RNA (Elbashir, 2001). The short dsRNA molecules, derived from a long ds-RNA sequence, that become a part of the RISC are called short interfering RNA molecules (SiRNAs) (Elbashir, 2001).
While certain long dsRNA molecules homologous to a target mRNA may work effectively in lower organisms, in certain instances, they pose a challenge in mammalian cells. In response to the exposure to long dsRNA, mammalian cells are known to activate cellular pathways that lead to the global shut down of gene expression. This nonspecific inhibition of gene expression may be a result of an antiviral response mediated by interferon gamma and RNA-dependent protein kinase pathways (Geiss, 2001; Stark, 1998). Consequently, in certain instances, the use of long dsRNA in mammalian cells for RNAi-mediated gene silencing has been unproductive. However, recent experiments in the field suggest the use of SiRNA molecules as an effective trigger for silencing genes in mammalian cells. In contrast to long dsRNA molecules, the short length of SiRNA may bypass the antiviral response. This discovery opened up the use of an RNAi approach for analyzing mammalian genes, including those of humans. SiRNAs have been designed to carry 3′ extensions with two nucleotides that mimic the products of Dicer cleavage. To confer protection from potential 3′-5′ exonucleases, the two nucleotides in the 3′ extensions may be substituted with dT, instead of natural RNA bases.
A procedure to design SiRNA molecules with dT 3′ extensions has been suggested by Tuschl and colleagues ((http://www.mpibpc.gwdg.de/abteilungen/100/105SiRNA.html) and (http://www.dharmacon.com/tech/tech003.html). This suggested approach for choosing SiRNAs involves the following criteria: (1) locate the first AA dimer 75 nucleotides downstream of the start codon within the mRNA of the gene; (2) record the next 19 nucleotides following the AA dimmer; (3) calculate the GC (guanosine and cytidine) content of the 21 nucleotide sequence to see if it is within 30-70%; (4) perform a BLAST search against the EST database with the 21 nucleotide sequence to make sure only the gene of interest is targeted.
In order to silence a gene of interest using RNAi, it is customary to synthesize a handful of SiRNA triggers that target 19-22 nucleotide regions in the target mRNA. Out of this collection of molecules, one SiRNA molecule may be effective. This has been the case for some genes, but not for every gene. Additionally, for some targets, there are instances where none of these molecules in the first round of screening will work in triggering the RNAi process. If this occurs, the researcher must screen another set of SiRNAs with the hope of identifying one that will work. This can become an expensive exercise, especially given that the cost of RNA synthesis can be ten times as high as that for DNA. This scenario still occurs whether one arbitrarily screens SiRNA molecules or designs them using the approach described by Tuschl and colleagues (http://www.mpibpc.gwdg.de/abteilungen/100/105/SiRNA.html). Hence, criteria to intelligently guide researchers through the process of designing effective SiRNA molecules may minimize the overall cost of target validation by effectively reducing the number of SiRNA molecules required for screening per target, as well as reducing the associated burden of running additional functional assays.
While SiRNA molecules may regulate specific gene expression through targeted mRNA degradation, small temporal RNAs (StRNA) may regulate developmental timing by causing sequence-specific repression of mRNA translation. Similar to SiRNA, StRNA appear to be excised from long RNA molecules by the Dicer ribonuclease, and hence may be of similar size, 21-23 nts. Certain StRNAs that have been identified by genetic analysis were lin-4 and let-7 in C. elegans (Hutvagner et al., 2001; Rasquinelli, 2000). During the search for SiRNAs in Drosophila embryonic cell extract, Tuschl and colleagues identified several other small RNAs of the same size (Largos-Quintana et al., 2001). This novel class of RNA, now referred to as microRNA (miRNA), is evolutionary conserved among invertebrates and vertebrates. Initial experiments suggest these miRNAs may serve as gene regulators during development mRNAs have been identified in C. elegans, Drosophila melanogaster embryonic extracts and cultured human cells (Largos-Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001).
These miRNAs served as a rich source of sequence information to look for cues to design effective SiRNA molecules. Certain criteria present in miRNA molecules were identified. A collection of arbitrarily designed and functionally validated SiRNA molecules were also analyzed. This analysis revealed that, in general, these criteria were met in certain SiRNA molecules that function in gene silencing, but were not found within certain nonfunctional SiRNA molecules. This result suggests that these characteristics may be important for functional SiRNAs. Certain functional SiRNA triggers were designed according to the guidelines that were identified. Analysis of these designed SiRNA triggers suggested that they are functional in RNA interference in mammalian cells. Therefore, rationally designed SiRNA triggers may not only be effective in eliciting RNAi response, but may also work at very low concentrations. These features may be helpful in therapeutic applications of SiRNA triggers by lowering the effective dose and improving the efficacy of the procedure.
B. Results and Discussion
The observed failure to silence a gene by certain SiRNA molecules bearing homology to an arbitrary chosen site within the cognate mRNA invokes certain non-exclusive possibilities. First, the possible inability of the SiRNA within the RISC to access the target site within the substrate mRNA may be responsible for this observation. Either the folding of mRNA to conceal the target site or the occupancy of the site by RNA binding proteins may dictate the target site accessibility. Unlike the RNAi process that is facilitated by a number of proteins, the antisense process relies on the passive hybridization of antisense oligonucleotide strands to the target mRNA. For several mRNAs, certain SiRNA molecules were designed to target the same sites that antisense molecules have successfully targeted, and no correlation between antisense effect and RNAi was observed (unpublished observation). These observations suggested that target site accessibility alone is not necessarily enough for SiRNA molecules to initiate RNAi. Furthermore, addressing the issue of target site accessibility may be complicated and rather complex, primarily due to the fact that the RNA folding pattern will vary from target to target. As a result, it may be optimal for interfering RNAs to be tailor made to target individual mRNAs.
Another possibility for the inefficiency of certain SiRNA molecules to trigger RNAi could lie in the innate characteristics of the targeting SiRNA. Consequently, identification and understanding of the characteristics of SiRNA molecules that favor the RNAi process may be useful.
Analysis of a naturally occurring population of RNA molecules that may be processed by a mechanism similar to RNAi may yield some insight into the RNAi process. To that end, miRNA sequences were chosen that were identified recently in three organisms, C. elegans (Lau et al., 2001; Lee and Ambros, 2001), D. melanogaster and H. sapiens (Largos-Quintana et al., 2001). These miRNAs, embedded within long precursor RNA molecules as stem-loop structures, are processed by Dicer. The stems within which miRNAs exist before processing contain bulges and G/U wobble pairs. Hence, miRNAs depart structurally from certain SiRNAs that typically contain perfect helices. Furthermore, there may be differences between miRNAs and SiRNAs on the basis of Dicer processing. In the case of miRNAs, RNA from only one arm of the stem-loop precursor accumulates in the cell (Hutvagner et al., 2001), whereas both antisense and sense strands exist in SiRNA molecules. The nature of the two nucleotides in the 3′-extension (usually dTdT) may not affect the efficacy of RNAi (unpublished observation). So, SiRNA molecules may be treated as duplexes of 19 base pairs. To be consistent with this, each miRNA may also be calculated as 19 nucleotides in length. One may also use sense strands with perfect base pairing for miRNA duplexes, which may be different from their natural form.
The average internal stability of the miRNA duplexes of the three organisms was investigated (
The average internal stability of a nucleic acid duplex also reflects its propensity for melting. The calculated average melting temperatures (Tm values) of miRNA duplexes are relatively low, and hence agree with their low average internal stabilities. The average Tm values within the four groups of miRNA duplexes vary from 52°-58° C. (
MiRNA duplexes may be distinguished from the random RNA duplexes based on certain parameters; a bell-shaped calculated average internal stability profile and a low calculated Tm value. A collection of SiRNA molecules were analyzed. This collection included a series of SiRNA molecules that were experimentally validated against six different human genes, one mouse gene, and a single reporter gene SEAP (secreted alkaline phosphatase). In this collection, a total of 37 SiRNA molecules were tested, out of which 16 were effective in reducing the level of a targeted mRNA by greater than 70%. These molecules were mostly designed arbitrarily, although some were picked by using the suggestions made by Tuschl and colleagues (http://www.mpibpc.gwdg.de/abteilungen/f100/105/SiRNA.html). The average internal stability profiles of the two classes within the collection of experimentally validated SiRNA molecules were analyzed (
The calculated melting temperatures of the two classes of SiRNAs (
Interestingly, there may be SiRNAs in both classes, functional and nonfunctional, with calculated Tm values between 55° and 70° C. The subpopulation of SiRNAs with calculated Tm values between 55° and 70° C. from both classes were analyzed and their average internal stability profiles were calculated (
It may be that one will find SiRNA molecules that are either functional, but may not follow the criteria, or have the criteria fulfilled but fail to function. In fact, such outliers were searched for in the rather small collection of experimentally validated SiRNAs, and molecules in either category that failed both criteria were not found. However, two sets of examples in which SiRNAs fulfilled one criterion but not the other were found, (
An example in which three SiRNA molecules are targeted to a single gene is depicted in Panel C of
So far an analysis of the experimentally validated SiRNA collection with respect to certain criteria suggests a good correlation between the efficacy of SiRNA molecules and meeting the criteria. To put the proposed method of choosing functional SiRNA molecules using certain criteria to the test, six SiRNA molecules were designed to target mRNA of SEAP (
Next, the minimal effective concentration of three SiRNA triggers designed for SEAP was investigated. At a fixed 100 nM concentration, the degree of effectiveness of these three triggers in silencing SEAP expression was different (
It appears that in certain instances SiRNA triggers with low calculated Tm values may be functional triggers, provided the internal energy criteria are met. Target sites with low melting temperatures may be common within mRNAs that are low in G/C content. Consequently, the probability of success in identifying functional SiRNA molecules by random picking may be high. However, as indicated in
Certain criteria of SiRNA molecules, Tm in the range of 55° C., and a bell-shaped or flat internal stability profile, that promote RNAi emerged from the initial analysis of a set of naturally occurring miRNA molecules. Subsequently, the existence of these criteria in functional SiRNA molecules that were designed arbitrarily and validated experimentally was confirmed. The success of designing functional SiRNA molecules using the two criteria was demonstrated.
A possible, but nonlimiting, rationale for the benefits of the criteria follows. It is reasonable to assume that within the RISC the antisense strand of the SiRNA may come off from the duplex and subsequently anneal to the substrate mRNA. These events may be facilitated by either individual proteins or multi-domain proteins within the RISC. Under such a scenario, the two strands within an SiRNA would be expected to melt easily. In light of this view on the mechanism, the interplay of molecular forces between the two strands of an SiRNA to keep them in a dynamic environment may be important to an effective SiRNA trigger. Hence, SiRNA molecules with a relatively low Tm may be advantageous. SiRNA duplexes with a relatively high internal stability in the middle may keep the two strands together, while the two ends with low internal stability provide easy entry to a protein like helicase to facilitate strand separation when needed. In contrast, SiRNA duplexes that are highly stable, as characterized by a high Tm and high internal stability may resist the strand separation step in the RNAi mechanism and hence fail to trigger RNA interference. Recently, the participation of RNA-dependent RNA polymerase (RdRP) has been demonstrated in the RNA interference process in C. elegans (Sijen et al., 2001) and Drosophila embryo extracts (Lipardi et al., 2001). An antisense strand of SiRNA once annealed to the target mRNA may serve as a primer for an RdRP to convert mRNA into dsRNA that is degraded to generate more SiRNA molecules in situ. Even for this activity to take place, strand separation of an SiRNA molecule may be an important prerequisite.
C. Conclusion
As demonstrated experimentally, rational design of functional SiRNA triggers based on the criteria set forth herein may help facilitate target validation and other applications of RNA interference. However, there may be instances that SiRNA molecules may not necessarily adhere to these guidelines. This may be because the predictions are based on the pattern analysis of sequences alone and do not factor in the cellular environment in which the target mRNAs exist. In reality, RNA is not a linear target as treated in this analytical approach, but is folded and interacts with a host of RNA-binding proteins. Hence, these criteria are considered guidelines for a high probability for success, but may not always provide effective SiRNA molecules.
The following is a stepwise procedure for designing functional SiRNA molecules for a mRNA target sequence according to certain embodiments. Windows depicted in
D. Materials and Methods
SiRNA
SiRNAs were prepared using several different methods. Certain chemically synthesized SiRNAs were synthesized using RNA phosphoramidites containing a 2′-O-TriisopropylsilylOxyMethyl (TOM) protection group from Glen Research (Sterling, Va.). Other SiRNAs were obtained from Dharmacon (Longmont, Colo.) employing 5′-Silyl-2′-bis(2-acetoethoxy)methyl (ACE) Orthoester chemistry. Synthesized SiRNAs using TOM phosphoramidites were HPLC purified, whereas those obtained from Dharmacon were used without further purification due to their high purity resulting from extremely high coupling efficiency (Scaringe, 2001). SiRNAs were annealed in an annealing buffer including 100 mM KCl, 30 mM HEPES (pH 7.5), and 2 mM MgCl2 by heating to 75° C. for 2 minutes followed by slow cooling to ambient temperature.
Experimental Evaluation of SiRNAs in Tissue Culture
The efficiency of certain SiRNA molecules designed for each human gene target was evaluated upon transfection into cultured human cells expressing the target. The specific mRNA level of the target gene was measured following transfection. In parallel, the mRNA level of a housekeeping gene, cyclophilin, as a nonspecific target was also quantified. Changes in the cyclophilin mRNA levels with SiRNA triggers were not observed. The efficiency of SiRNA to specifically reduce the targeted mRNA was calculated as a ratio of the target mRNA to cyclophilin mRNA. SiRNA molecules that gave greater than 70% reduction of the target mRNA level were taken as functional SiRNAs.
Delivery of Nucleic Acid Triggers
Cells seeded in 96-well plates at approximately 25,000/well the previous day were transfected with different nucleic acid triggers using Lipofectamin 2000 and Opti-MEM I (from Invitrogen). Briefly, SiRNA was diluted in Opti-MEM-I in a 50 μL volume. This was mixed with an equal volume of Lipofectamin 2000 diluted 12.5-fold in OPTI-MEM I. After incubating the mixture at ambient temperature for 20 minutes, 270 μL of regular cell medium was added, and 95 μL of the solution was immediately transferred onto the cells in the plate with no media. Plates were transferred to a 37° C. incubator with 5% CO2 for either 24 or 48 hours. To monitor the fate of SEAP transiently expressed in 293 cells, prAAV6-seap plasmid was included in the transfection mixture with and without SiRNAs.
Quantification of mRNA Levels
Specific mRNA levels of cells transfected with different nucleic acid triggers were quantified using QuantiGene High Volume Kit (from Bayer) that employs a branched-DNA (b-DNA) method for nucleic acid detection according to the manufacturer's instructions. Specific detection of a given mRNA is based on its selective capture on to the microtiter plate, which is dictated by the capture probes. Probe sets that are unique to each target mRNA were designed using the ProbeDSesigner software (Bayer) according to the manufacturer's instructions. For each case, probe sets were validated using the cells expressing each message before being used for experiments.
Secreted Alkaline Phosphatase (SEAP) Assay
The SEAP gene (from pAP-1 SEAP vector from Clontech) was cloned into an adeno-associated vector (AAV #6) upstream of the EF1-α 3′ UTR. Twenty-four hours after the transfection, 15 μL of medium from each well was transferred to a white opaque 96-well flat bottom microtiter plate, and the amount of SEAP was detected using a chemiluminescent SEAP assay (Great EscAPe SEAP assay kit form Clontech) according to the manufacturer's instructions. RLU values obtained in the presence of an SiRNA trigger were normalized to that obtained in the absence of a trigger. SiRNA molecules that gave greater than 70% reduction of the RLU level compared to the control (no SiRNA added) were taken as functional SiRNAs.
Calculation of Internal Stability Profiles and Melting Temperatures (Tm Values)
Internal stability profiles were calculated using the software program Oligo 5.0™ Primer Analysis Software (National Biosciences, Inc., Plymouth, Minn.), a program that is generally used for designing oligonucleotides for PCR and various nucleic acid hybridization applications. The ΔG value for each position reflects the average of all overlapping pentamer sequences of the 19 base-pair duplex. The program calculates the average ΔG value by adding ΔG values of the 4 nucleotide pairs within the pentamer. The terminal base pairs were excluded to avoid end effects and only the internal 17 nucleotide sequence was considered. Furthermore, the internal stability profiles of antisense strands were calculated using nearest neighbor calculations of DNA, not of RNA. Although the absolute values for RNA may vary, the general trends may still be valid.
Tm values were also calculated using the same software program according to the nearest neighbor thermodynamic values. Again, this calculation is also based on DNA and not on RNA, but the general trend for Tm also holds true.
IV. Certain Functional SiRNA Triggers in Mammalian Cells
It has been reported that SiRNA triggers with a 19-base pair helical region with two nucleotide 3′ extensions may be optimal triggers for gene silencing through RNA interference. These conclusions were born out based on two observations: (1) empirical design of RNA triggers to mimic cleavage products of Dicer, the enzyme involved in processing long double-stranded RNA into short interfering RNA or SiRNA; (2) Results obtained by using an in vitro system derived from Drosophila embryo extract. Certain 21 nucleotide triggers with a 19 nucleotide helicial region in fact work in silencing gene expression in mammalian cells.
The work described herein suggests that certain SiRNA triggers of variable functional anatomies work effectively in mammalian cells, a result that is different from certain results obtained using an in vitro system. Salient features of the current discovery are summarized below.
Certain SiRNA triggers with a 17-base pair RNA helical region with an antisense strand of 17 RNA nucleotides are nonfunctional in triggering RNA interference in mammalian cells. On the other hand, certain SiRNA triggers with a 17-base pair RNA helical region with an antisense strand of at least 19 RNA nucleotides are effective triggers.
Recently, it has been shown that instead of ssDNA antisense oligonucleotides, injection of dsRNA into the nematode C. elegans resulted in the loss of function of a gene to which the injected dsRNA had homology (Fire, 1998; Guo, 1995). RNAi is one manifestation of dsRNA-induced gene silencing. Other forms include post-transcriptional gene silencing (PTGS) and co-suppression observed in plants (Ketting, 2000), as well as quelling in the fungus Neurospora crassa (Reviewed in (Fire, 1999; Matzke, 2001; Waterhouse, 1999)). Nature may use the RNA silencing phenomenon to protect the cell from viral infections and from mobilization of transposons. A growing body of evidence suggests that it may also be used to regulate the expression of endogenous genes.
RNAi has been demonstrated in a host of species, including invertebrates such as hydra, planaria, trypanosomes, nematodes and insects as well as vertebrates (mouse and zebra fish) (reviewed in Bosher, 2000; Hammond, 2001; Sharp, 2001; Zamore, 2001). These studies revealed certain important aspects of RNA interference. In certain instances, it has been shown that only the gene to which the dsRNA shares homology becomes silenced (Fire, 1998; Kennerdell and Carthew, 1998). In certain instances, the dsRNA should be homologous to the exons of a gene to observe effective gene silencing, indicating that the silencing mechanism may not interfere with mRNA processing, but may occur post transcriptionally after splicing (Fire, 1998). In certain instances, the dsRNA molecule may be substantially complimentary to the coding or noncoding regions of the target mRNA. In certain instances, the dsRNA molecule may be substantially complimentary to the untranslated regions of the target mRNA, including but not limited to the 5′ and 3′ untranslated regions. In certain instances, only a few copies of the dsRNA trigger may be required to degrade mRNA present in large excess (Fire, 1998; Kennerdell and Carthew, 1998), suggesting that there may be a possible amplification step included within the molecular mechanism of RNA interference. In both worms and plants, RNA interference may spread across cell boundaries (Fire, 1998; Hamilton, 1999).
Mutants that are defective in RNA interference have been isolated in C. elegans (Grishok, 2000), Neurospora (Cogoni, 1999), Arabidopsis (Dalmay, 2000) and Chlamydomonas (Wu-Scharf, 2000). Biochemical analysis of the RNAi process was facilitated by the in vitro system derived from Drosophila embryonic cells (Hammond, 2000; Tuschl et al., 1999). Studies on the in vitro system that recapitulates the RNAi process provided some insight into the fate of the dsRNA. It was suggested that the dsRNA may be cleaved into discrete 21-23 nucleotide fragments by an ATP-dependent process which does not require the presence of target mRNA (Zamore, 2000). This suggests that the small 21-23 nucleotide dsRNA fragments may not be by products of the process, but may be intermediates in the RNAi process. These results obtained in vitro were in agreement with the previous observation of the existence of small dsRNA of 25 nucleotides in plants undergoing PTGS either by viruses or trans genes (Hamilton, 1999). These small dsRNAs found in plants undergoing PTGS included both sense and antisense strands corresponding to the silenced gene. The conversion of dsRNA into small dsRNAs was suggested in vivo in C. elegans and Drosophila as well (Parrish, 2000; Yang, 2000). The multidomain RNAse III protein Dicer may be the enzyme that processes dsRNA into 21-23 nucleotide short dsRNA (Bernstein, 2001) that are called SiRNAs (Short Interfering RNAs)(Elbashir, 2001b). According to one of the proposed models for RNAi (Hammond, 2001; Zamore, 2001), the SiRNA molecules generated upon the cleavage of the long dsRNA become a part of a ribonucleo-protein complex called RiSC(RNA-induced silencing complex). The RISC may then find and bind to the target mRNA through a homology searching mechanism facilitated by a protein(s) within the complex and the antisense strand of the SiRNA. Once the site of homology is identified, the target mRNA may be cleaved by an endonuclease, which may be a member of RISC as well. SiRNA guided cleavage of the target RNA has been suggested in vitro (Elbashir, 2001b). It has been suggested that the cleavage within the target RNA takes place near the center of the homology to the SiRNA.
Earlier attempts in applying relatively long (approximately 800 nts) dsRNA to initiate RNAi in mammalian cells failed (Calpan et al., 2000). However, RNAi has been observed in mouse oocytes and early embryos (Svoboda, 2000; Wianny, 2000), suggesting the possible existence of RNAi machinery in mammalian cells. The failed attempts in demonstrating RNAi in mammalian cells have been attributed to the nonspecific effects induced by long dsRNAs in mammalian cells. Certain dsRNAs are known to induce nonspecific effects in mammalian cells by activating several pathways through the rapid induction of IFNγ (Geiss, 2001; Stark, 1998). DsRNA is known to activate dsRNA-dependent protein kinase, PKR, which in turn phosphorylates and inactivates the translation factor elF2α. The overall result is the global shut down of protein synthesis in the cell and subsequent cell death. In addition, dsRNAs may also induce the production of 2′-5′-polyadenylic acid which in turn activates the nonspecific nuclease RNase L that nonspecifically degrades RNA. The induction of nonspecific effects by dsRNA in mammalian cells may be related to the length of the dsRNA; for example, the activation of PKR may require dsRNA that is longer than 30 base pairs. The efficiency of the activation of PKR increases with the length of RNA and 85 base pairs may provide optimal activation in certain instances (Manche, 1992).
The use of short dsRNA molecules, such as SiRNA, has the potential to keep the dsRNA-induced nonspecific pathways from activating. In other words, being intermediates of the RNA interference process, SiRNA may trigger RNAi in mammalian cells. Recent results with the delivery of SiRNA into mammalian cells suggest this theory may be accurate (Caplen, 2001; Elbashir, 2001a). These authors used SiRNA of 21-25 nucleotides to silence genes expressed either transiently or endogenously in cultured mammalian cells. The SiRNAs used in these studies were designed to have 2 nucleotide 3′-overhangs mimicking a digested RNA fragment resulting from cleavage of the ribonuclease III enzyme, Dicer. A phosphate group is present at the 5′ end of the Dicer cleavage products, yet it may not be required for a SiRNA molecule to trigger efficient RNA interference (Caplen, 2001; Elbashir, 2001a). Furthermore, the two RNA nucleotides in the 3′-overhang contain two dT residues to presumably protect the functional SiRNA triggers from possible 3′-5′ exonuclease activities (Elbashir, 2001a). Certain characteristics of certain SiRNA triggers have been delineated empirically. An attempt was made to understand the functional anatomy of SiRNA triggers using an in vitro system derived from Drosophila melanogaster embryo lysate (Elbashir et al., 2001). In this system, SiRNA triggers with 21 nucleotides in each strand and a 19-base pair helical region with 2 nucleotide 3′ extensions were the most efficient triggers for mediating RNA interference.
The results herein suggest that the functional anatomy of SiRNA molecules may be quite flexible in silencing genes in mammalian cells, since triggers with three possible end structures (3′-extension, 5′-extension and blunt) mediate effective gene silencing. Also, the single-stranded RNA molecules that fold into hairpin structures may be equally in triggering RNA interference in mammalian cells. These results may suggest either the mechanism by which RNA interference is mediated in mammalian cells has differences compared to the one found in lower organisms or there are some limiting factors in the in vitro system based on Drosophila embryo extract.
Materials and Methods
SiRNA
All SiRNA triggers and hairpin molecules used in this work were chemically synthesized in house using RNA phosphoramidites based on 5′-Silyl-2′-bis(2-acetoethoxy)methyl (ACE) Orthoester chemistry purchased from Dharmacon (Longmont, Colo.). After deprotection, short RNA molecules (all SiRNA triggers and short RNA hairpins) were used without further purification, due to their high purity resulting from extremely high coupling efficiency (Scaringe, 2001). The long RNA hairpin, SP-HP uucg AS-S, was purified by reverse phase high pressure liquid chromatography. Two strands of SiRNA molecules were annealed in an annealing buffer including 100 mM KCl, 30 mM HEPES (pH 7.5), and 2 mM MgCl2 by heating to 75° C. for 2 minutes followed by slow cooling to ambient temperature. Hairpin molecules were also heated and slowly cooled down to ambient temperature in the same annealing buffer.
Delivery of Nucleic Acid Triggers
Cells seeded in 96-well plates at approximately 95% confluence were transfected with different nucleic acid triggers using Lipofectamin 2000 and Opti-MEM I (from Invitrogen). Briefly, SiRNA was diluted in Opti-MEM-I in 100 μL volume. This was mixed with an equal volume of Lipofectamin 2000 diluted 25-fold in OPTI-MEM I with SuperRNAsin at 1.4 U/μL (from Ambion) and prAAV6-seap plasmid (1 ng/μL). After incubating the mixture at ambient temperature for 5-20 minutes, 550 μL of regular cell medium was added and 100 μL of the solution was immediately transferred onto cells in the plate with no media. Plates were transferred to a 37° C. incubator with 5% CO2 for either 24 or 48 hours. In each case, the final concentration of SiRNA triggers was 100 nM.
Secreted Alkaline Phosphatase (SEAP) Assay
Twenty-four hours after the transfection, 15 μL of medium from each well was transferred to a white opaque 96-well flat bottom microtiter plate, and the amount of SEAP was detected using a chemiluminescent SEAP assay (Great EscAPe SEAP assay kit form Clontech) according to the manufacturer's instructions.
Results and Discussion
RNA interference may be used to study gene function in mammalian cells. Two to three nucleotide 3′-extensions within SiRNA triggers for effective silencing of cognate genes in lower organisms has been reported (Elbashir et al., 2001). Effective gene silencing by hairpin SiRNA triggers in mammalian cells has been observed. In order to investigate the effect of the end structure in effective gene silencing in mammals, a reporter gene, SEcreted Alkaline Phosphatase (SEAP) was expressed under a strong CMV promoter and was used as a target gene. A plasmid expressing SEAP mixed with SiRNA triggers was transfected into HEK 293 cells. Silencing of the SEAP gene was monitored 24 hours after transfection using a chemiluminescence assay directed to detect the activity of alkaline phosphatase.
A site within the SEAP mRNA that was previously characterized to provide effective reduction of gene expression by SiRNA was chosen as the target site (
Variation of SiRNA Trigger Length
It has been demonstrated that SiRNA triggers with a 19 base pair helical region and two nucleotides at the 3′ overhang may be optimal for effective gene silencing in Drosophila extract (Elbashir et al., 2001). These triggers may be effective in mammalian cells as well (Caplen, 2001; Elbashir, 2001a). The effect of the change in the helical length of certain SiRNA triggers was investigated using triggers with 17, 19, 21, 23, and 25 RNA base pairs. Although these triggers carry helical regions of different lengths they have the same end structure; two dT residues as 3′ overhangs. As shown in
SiRNA Triggers with Different End Structures
The effect of gene silencing mediated by SiRNA triggers possessing asymmetric lengths in the two strands was explored. Several series of SiRNA triggers were designed in which the length of the sense strand was kept constant and the length of the antisense strand, as well as the nature of its end structure, was changed. Sequences of SiRNA triggers belonging to eight series are listed in
A similar series of triggers, keeping the antisense strand constant and varying the length of the sense strand, was constructed. Sequences of SiRNA triggers of eight series are illustrated in
Certain end structures that effectively work in silencing the SEAP gene in mammalian cells are shown below.
In the current work, certain anatomical structures of SiRNA triggers that efficiently work well in certain mammalian cells were different than certain triggers that work in certain in vitro systems. Caplen et al. have shown gene silencing in primary mouse embryonic fibroblasts, 293, and HeLa cells with SiRNA triggers longer than a 19 base pair helical region with 2-3 nucleotides at the 3′ extensions (Caplen, 2001). Taken together these results suggest that it may be possible that certain factors that are involved in effective utilization of double-stranded RNA with a helical region longer than 19 base pair in mammalian cells may be missing in an in vitro system. These factors may include nucleases for trimming the long double stranded RNA into effective short triggers or other proteins that help activate and/or facilitate this process.
Substitution of G-U Base Pairs
During an attempt to rationally design functional SiRNA triggers, SiRNA triggers were observed with relatively low internal stability that tend to be functional in silencing target genes in mammalian cells. In certain instances, SiRNAs containing contiguous G-C base pairs may not be optimal in eliciting RNA interference. In certain instances, one may desire SiRNA triggers that do not have higher than 4 contiguous G-C base pairs in an SiRNA trigger. An approach to make G-C base pairs less stable by substituting uridines in place of cytosines to generate G-U base pairs with a higher propensity for melting. In fact, G-U base pairs are present in certain microRNA molecules that are also processed by Dicer, the same enzyme that processes SiRNA molecules. Hence, the inclusion of G-U base pairs in an SiRNA molecule may not affect the RNA interference process.
Two SiRNA triggers, one functional (SP-1795) and the other non functional (SP-1260), were used to explore the effect of substituting G-U base pairs for G-C base pairs (C-U substitution). In each SiRNA trigger, all cytosines in either antisense or sense strand were replaced with uridines. These strands containing uridines were combined with complimentary strands with cytosines to generate SiRNA triggers with uridines in either antisense or sense strands.
The effect of gene silencing by these SiRNA triggers is shown in
SiRNA triggers with a 17-base pair RNA helical region with an antisense strand of greater than or equal to 19 RNA nucleotides may be effective triggers.
SiRNA triggers carrying an RNA helical region(s) greater than or equal to 19 base-pairs with different end structures may be functional in eliciting RNA interference in mammalian cells.
SiRNA triggers having a 17-base pair RNA helical region with an antisense strand of greater than or equal to 19 RNA nucleotides with different end structures may be effective triggers.
The 3′-ends may be either ribo- or two deoxy-nucleotides in certain functional SiRNA triggers.
SiRNA triggers in which a sense strand having 17 nucleotides is annealed to an antisense strand that is greater than or equal to 19 nucleotides may be functional in mammalian cells.
Unimolecular RNA molecules with the propensity to fold into hairpin structures may serve as functional SiRNA triggers in silencing gene expression in mammalian cells.
C-U substitutions in the sense strand but not in the antisense strand may be tolerated in functional SiRNA triggers.
These results suggest that SiRNA triggers with a 19-base pair helical region with two nucleotide 3′ extensions may not be the only structure with the ability to mediate highly efficient RNA interference in mammalian cells.
V. RNA Interference Using DNA Delivery
Gene silencing using RNA interference may be an attractive approach as a tool for understanding gene function and as a therapeutic approach to inhibit undesirable gene expression implicated in disease. A double stranded RNA molecule having homology to the target mRNA mediates the silencing process.
It has been demonstrated that the introduction of certain synthetic SiRNA molecules into cultured cells elicits silencing of the target gene, suggesting that the initial cleavage by the Dicer enzyme may be bypassed. Not all SiRNA triggers identified against a target gene are equally efficacious in silencing that gene, and hence screening of several triggers may be carried out. On the other hand, a relatively long dsRNA molecule cleaved by the Dicer enzyme generates several different such triggers inside the cell. The latter approach may have a higher probability of getting an effective trigger for the silencing process. Transfection of dsRNA longer than 70 base pairs has been shown to elicit cytotoxicity, and, therefore, may not be used as a functional trigger in certain instances. However, the intracellular expression of longer dsRNA may not induce cellular toxicity. Furthermore, linear dsDNA fragments having a U6 promoter upstream of either antisense or sense strands of a targeted reporter gene were constructed using PCR. These PCR fragments were intended to produce either antisense or sense strands of RNA approximately of 22 nucleotide homology to the target gene. The transfection of both types of PCR fragments (antisense and sense), along with a plasmid expressing the target reporter gene, into HEK 293 cells was able to silence the reporter gene expression.
1. One may use DNA constructs with the capacity to express RNA having significant homology to a target gene of interest in RNAi. In certain embodiments, these constructs may encode dsRNAs of 70 to 150 nucleotides.
a. RNA may be single stranded with either antisense or sense polarity to the target mRNA. DNA constructs expressing both polarities may be used in certain embodiments. However, due to the possibility of aberrant RNA generation with an orientation opposite to the promoter, even one of the constructs expressing either sense or antisense RNA may be used.
b. RNA may have a double-stranded nature due to the presence of self-complimentary regions. An example of this type of RNA is a fold-back stem loop. When RNA molecules having a double-stranded nature are expressed, a single type of DNA construct may be used.
2. In DNA constructs described in 1, the promoters that drive RNA synthesis may be of phage derived, virus-derived, pol II, or pol III type.
3. DNA constructs described in 1, may or may not contain extra nucleotides that serve additional functions such as termination of transcription or a poly A signal.
4. DNA constructs described in 1, may be either linear or circular. In the case of circular DNA, it may be a plasmid with additional genes conferring different functions such as resistance to one or more antibiotics.
5. DNA constructs described in 1, may be synthetic, derived from PCR, or derived from growing inside a host. Alternatively, DNA constructs may be derived from one or more methods described above.
6. For application in tissue culture, DNA constructs described in 1, may be introduced into cells by transfection, electroporation, or microinjection.
7. For in vivo applications in animals and humans, DNA constructs described in 1, may be delivered by:
a. Simple injection into tissues or blood or any other body fluid;
b. Under pressure;
c. Electroporation;
d. Using micro pumps;
e. Using DNA guns;
f. Orally.
8. Adjuvants or formulations that may either stabilize DNA constructs or facilitate a delivery method may be used in the delivery methods outlined in 7.
This application claims priority benefit of U.S. Patent Application No. 60/436,849, filed Dec. 27, 2002. The entire contents of U.S. Patent Application No. 60/436,849 is specifically incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 60436849 | Dec 2002 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10745395 | Dec 2003 | US |
| Child | 11496966 | Jul 2006 | US |
