Patent Application
20110027794
The instant application contains a Sequence Listing which submitted via EFS-Web concurrently herewith and which hereby is incorporated by reference in entirety. Said ASCII copy, created on 15 Oct. 2010, is named 021315081.txt and is 10,349 bytes in size.
The present invention is related to a method for the synthesis of nucleic acids and its use in methods for the selection of target molecule binding nucleic acids.
Since the discovery of Cech and Altman who described for the first time that RNA is not only active in C. elegans as a messenger molecule between the genome, i.e. DNA, and the protein synthesis machinery, i.e. the ribosomes, but exhibits catalytic activities, numerous papers have been published in the field of ribozymes, i.e. catalytic RNA oligonucleotides and aptamers, i.e. target molecule binding (deoxy)oligonucleotides. Both approaches are suitable as such or in combination, for use as therapeutics, diagnostics, for target validation or as media for affinity chromatography or specific adsorbers.
A further approach to industrial application of oligonucleotides is antisense molecules as well as siRNAs which may result in post-transcriptional suppression of a specific gene expression.
A significant limitation is imposed on the use of oligonucleotides by their rapid degradation through ubiquitous RNases and DNases. Particularly in biological systems RNases and DNases can be found in significant amounts and result in lifetimes of RNA and DNA oligonucleotides of a few seconds to minutes (Griffin et al., 1993; Jellinek et al., 1995; Lin et al., 1994).
Methods to protect oligonucleotides from being attacked by exonucleases are in most cases based on the modification of their ends by the addition of protective groups such as, e.g., a terminal inverted nucleotide, i.e. 3′-3′ or 5′-5′ linkages or other large groups such as, for example, polyethylene glycol (Bell et al., 1999). The replacement of natural substituents protects similarly against exonucleases and endonucleases, particularly at the 2′ carbon of the ribose and at the phosphor. Non-natural, nuclease-resistant alternatives to ribose (2′-OH) or deoxyribose (2′-H) are: 2′-amino, 2′-fluoro, 2′-azido, 2′-O-methyl, 2′-alkyl, 2′-allyl and arabino nucleotides (Eaton and Pieken, 1995). The most frequent phosphor modification is the replacement of oxygen by sulfur which results in so-called phosphorothioates.
Measures of this kind provide for significantly longer lifetimes in biological liquids, which may be up to several hours (Eaton and Pieken, 1995; Green et al., 1995; Jellinek et al., 1995; Lin et al., 1994).
Any of the aforementioned modifications, however, goes along with some limitations. In particular the synthesis of modified oligonucleotides by DNA or RNA polymerases is in most cases not possible. This, however, is frequently a requirement for the identification of functional oligonucleotides. It is particularly by the immediate link between the phenotype (the structure and thus the function) and the genotype (the nucleic acid sequence) inherent to the oligonucleotides, and by their characteristic that they can be copied, that it is possible to enrich suitable nucleic acid sequences from natural libraries, as represented by the genome and the transcriptome, or from synthetic libraries, for example combinatorial libraries, by selection and amplification to such an extent that single sequences having the desired characteristics can be isolated.
Such modifications which cannot be reconciled with the enzymatic processes, can only be introduced by chemical synthesis of the identified sequences subsequent to the identification of the functional oligonucleotides. At first, it is necessary that the identified sequences can be chemically synthesized. Therefore, the candidates being RNA hybrids typically having a length of about 60 to 90 nucleotides, are first shortened to 50 nucleotides or less in order to allow for an efficient chemical synthesis. Subsequently, the unmodified purines are replaced by 2′-modified nucleotides. Frequently, the function of the oligonucleotide is thus affected (Kujau et al., 1997). Therefore, in only a minority of cases all nucleotides can be modified afterwards. Because of this, most of the stabilized oligonucleotides have some weak points in the molecule which may be subject to an attack by a nuclease, thereby reducing their lifetime.
Phosphorothioate may be enzymatically introduced by RNA polymerases ((Jhaveri et al., 1998) or by Taq-DNA polymerases, whereby, however, it is only possible to incorporate up to three phosphorothioate DNTPs (King et al., 2002). Phosphorothioates are apart from that disadvantageous insofar as they are cytotoxic (Henry et al., 2001).
2′-fluoro and 2′-amino modifications can be introduced into RNA by transcription. A review on modified aptamers has been prepared by Kusser (Kusser, 2000). Also the enzymatic incorporation of 2′-O-methyl and 2′-azido nucleoside triphosphates using T7-RNA polymerase has been described (Lin et al., 1994; Padilla and Sousa, 1999; Padilla and Sousa, 2002). The incorporation of such modified nucleotides is, however, limited to modified cytidines and uridines for the time being (Aurup et al., 1992). In particular, 2′-modified guanosines are not tolerated by known RNA polymerases. The reason therefor seems to reside in the instability of the complex of polymerase and DNA during the initial phase of the RNA polymerization. This stage, comprising the polymerization of the first 8 to 12 nucleotides has to be performed as quickly as possible as otherwise the RNA polymerase rapidly leaves the complex again (Lin et al., 1994; Milligan and Uhlenbeck, 1989).
A prerequisite for a successful initiation of the RNA synthesis, however, is at least one guanosine at the first two positions to be transcribed. An optimum initiation sequence even requires three consecutive guanosines at positions 1 to 3 of the RNA for T7- and T3-RNA polymerase (Milligan and Uhlenbeck, 1989) and GAAGNG for the SP6-RNA polymerase, such as, for example, presented in Meador et al., 1995.
Therefore, it has factually been impossible to date to incorporate 2′-modified guanosine nucleotides into RNA molecules and to synthesize completely 2′-modified nucleic acids by means of RNA polymerases, respectively.
Therefore, the objective underlying the present invention was to provide for an enzymatic method which allowed the incorporation of modified nucleotides and in particular of 2′-fluoro modified nucleotides into a nucleic acid. In connection therewith it was a particular objective to provide a method allowing to incorporate all of the five naturally occurring bases (A, C, G, T, U), base modifications thereof and possible universal bases such as, for example, inosine (I), as nucleoside phosphates in their sugar modified form into nucleic acids.
A further objective underlying the present invention was to provide for a method for enzymatic synthesis of nucleic acids, whereby the nucleic acids completely consist of modified nucleoside phosphates, in particular 2′-fluoro-modified nucleoside phosphates.
Finally, another objective underlying the present invention was to provide a method for the selection of nucleic acids binding to a target molecule, whereby the nucleic acids partially or completely consist of modified nucleoside phosphates and in particular 2′-fluoro-modified nucleoside phosphates.
The objective is solved in accordance with the present invention by the methods of the independent claims. Preferred embodiments may be taken from the subclaims.
The problem underlying the present invention is solved in a first aspect by a method for the synthesis of a nucleic acid, whereby the nucleic acid comprises modified nucleotides, comprising the steps of:
In an embodiment the reverse transcriptase is selected from the group comprising reverse transcriptases of murine moloney leukemia virus (MMLV), avian myeloblastosis virus (AMV), thermostable reverse transcriptases, DNA polymerase of Carboxydothermus hydrogenoformans, respective mutants thereof, and mixtures thereof.
In an embodiment the modified nucleoside triphosphates are selected from the group comprising 2′-fluoro-modified nucleoside triphosphates, 2′-amino-modified nucleoside triphosphates, 2′-azido-modified nucleoside triphosphates, 2′-O-methyl-modified nucleoside triphosphates, 2′-alkyl-modified nucleoside triphosphates, 2′-allyl-modified nucleoside triphosphates, arabino-nucleoside triphosphates and nucleotide phosphorothioates.
In an embodiment the modified nucleoside triphosphates are 2′-fluoro nucleoside triphosphates.
In an embodiment the nucleoside triphosphates provided are exclusively modified nucleoside triphosphates and that the synthesized nucleic acid preferably essentially consists solely of modified nucleotides.
In an embodiment the template strand consists of RNA.
In an alternative embodiment the template strand consists of DNA.
In a further alternative embodiment the template strand consists of a modified nucleic acid, preferably a 2′-fluoro nucleic acid.
In an alternative the sequence of the primer is part of the nucleic acid to be synthesized.
In an embodiment the sequence of the primer is different from the nucleic acid to be synthesized.
In an embodiment the primer consists of modified nucleoside phosphates, whereby the modification of the nucleoside phosphates of the primer is the same modification as the one of the nucleoside triphosphates provided.
In an embodiment the primer consists of RNA.
In an embodiment the primer consists of DNA, whereby at least the 3′ terminal nucleotide of the primer is a deoxyribonucleotide.
In an embodiment the polymerase activity synthesizes a strand which is essentially complementary to the template strand, whereby it is preferably base paired with the template strand.
In a preferred embodiment the synthesized nucleic acid is separated from the template strand.
In an embodiment the primer or a part thereof is removed from the nucleic acid synthesized by the polymerase activity.
In an embodiment the template strand and/or the primer is digested or cleaved, preferably after the synthesis of the nucleic acid which is essentially complementary to the template strand.
In a preferred embodiment the separation and/or the cleavage is performed by alkaline cleavage or enzymatic activity.
The problem underlying the present invention is solved in a second aspect by the use of a reveres transcriptase for the synthesis of a nucleic acid, whereby the nucleic acid comprises at least a modified nucleoside phosphate.
In an embodiment according to the second aspect of the present invention the reverse transcriptase is selected from the group comprising reverse transcriptases of murine moloney leukemia virus (MMLV), avian myeloblastosis virus (AMV), thermostable reverse transcriptases, DNA polymerase of Carboxydothermus hydrogenoformans, mutants thereof, and mixtures thereof.
In an embodiment the modified nucleoside triphosphate is selected from the group comprising 2′-fluoro-modified nucleoside triphosphates, 2′-amino-modified nucleoside triphosphates, 2′-azido-modified nucleoside triphosphates, 2′-O-methyl-modified nucleoside triphosphates, 2′-alkyl-modified nucleoside triphosphates, 2′-allyl-modified nucleoside triphosphates, arabino nucleoside triphosphates and nucleoside phosphorothioates.
In an embodiment the nucleic acid essentially consists completely of modified nucleoside phosphates.
The problem underlying the present invention is solved in a third aspect by a method for the selection of a target molecule binding nucleic acid, in particular of aptamers, comprising the steps of:
(k) optionally sequencing the nucleic acid(s) obtained from step (f) or (g),
The problem underlying the present invention is solved in a fourth aspect by a method for the selection of a target molecule binding nucleic acid, in particular of aptamers, comprising the steps of:
The problem underlying the present invention is solved in a fifth aspect by a method for the selection of a target molecule binding nucleic acid, in particular of aptamers, in particular a method according to the third and the fourth aspect of the present invention, comprising the steps of:
In an embodiment of the fifth aspect of the present invention the reverse primer and a forward primer are used in the second strand synthesis, whereby the forward primer is at least partially complementary to a part of the forward primer binding site of the reverse transcription product, whereby the sequence of the synthesized second strand is essentially identical to the sequence of the nucleic acid of step (d) and additionally comprises at the 3′ end a sequence which is essentially complementary to the further partial region of the reverse primer.
In an embodiment of the fifth aspect of the present invention the further partial region of the reverse primer is a promoter sequence, whereby preferably the promoter sequence is selected from the group comprising promoter sequences of the T7-RNA polymerase, the T3-RNA polymerase and the SP6 polymerase.
In an embodiment of the fifth aspect of the present invention the strand synthesized in the second strand synthesis is used as a template strand in a transcription reaction, whereby the transcription product comprises in 3′→5′ direction the forward primer binding site, the complementary randomized sequence and the reverse primer sequence.
In a preferred embodiment of the fifth aspect of the present invention the transcription product is reacted with a reverse transcriptase together with a forward synthesis primer and modified nucleoside triphosphates, preferably 2′-fluoro nucleoside phosphates, whereby the forward synthesis primer hybridizes to the forward primer binding site in order to obtain a synthesis product, whereby the synthesis product comprises modified nucleoside phosphates, preferably 2′-fluoro nucleoside phosphates.
In a preferred embodiment of the fifth aspect of the present invention the forward synthesis primer consists of modified nucleoside triphosphates.
In an embodiment of the fifth aspect of the present invention the template strand is subjected to an alkaline treatment in order to obtain a single-stranded nucleic acid, whereby the nucleic acid comprises in 5′→3′ direction the forward primer sequence, the randomized region and the reverse primer binding site.
In a preferred embodiment of the fifth aspect of the present invention the forward primer comprises at its 5′ end a further partial region and that the synthesized second strand comprises at its 5′ end a sequence corresponding to the further partial region.
In a still more preferred embodiment of the fifth aspect of the present invention the strand synthesized in the second strand synthesis is subjected to a transcription reaction as a template strand, whereby the transcription product comprises in 3′→5′ direction the forward primer binding site including the sequence complementary to the further partial region of the forward primer, the complementary randomized region and the reverse primer sequence at its 5′ end, whereby the reverse primer sequence preferably lacks a sequence corresponding to the further partial region of the reverse primer.
In a further preferred embodiment of the fifth aspect of the present invention the transcription product is reacted with a reverse transcriptase together with a forward synthesis primer and modified nucleoside triphosphate, preferably 2′-fluoro nucleoside phosphates, whereby the forward synthesis primer is hybridized to the forward primer binding site, in order to obtain a synthesis product, whereby the synthesis product comprises modified nucleoside phosphates, preferably 2′-fluoro nucleoside phosphates.
In a preferred embodiment of the fifth aspect of the present invention the forward synthesis primer consists of ribonucleotides or of deoxyribonucleotides having at least one ribonucleotide at its 3′ end.
In a further preferred embodiment of the fifth aspect of the present invention the template strand is subjected to an alkaline cleavage and the forward synthesis primer is cleaved off, in order to obtain a single-stranded nucleic acid, whereby the nucleic acid comprises in 5′→3′ direction the forward primer sequence, the randomized region and the reverse primer binding site.
In an embodiment of the fifth aspect of the present invention the forward primer and the reverse primer consist of DNA.
The problem underlying the present invention is solved in a sixth aspect by a method for the selection of a target molecule binding nucleic acid, in particular of aptamers, comprising the steps of:
In an embodiment of the sixth aspect of the present invention the reverse primer and a forward primer are used in the second strand synthesis, whereby the forward primer is essentially complementary to the forward primer binding site, whereby the sequence of the synthesized second strand is essentially identical to the nucleic acid to be amplified.
In an embodiment of the sixth aspect of the present invention the synthesis after step (g) the amplified reverse transcription product is reacted with a forward synthesis primer, modified nucleoside triphosphates, preferably 2′-fluoro nucleoside triphosphates, and a reverse transcriptase, whereby the forward synthesis primer hybridizes to the forward primer binding site, in order to obtain a synthesis product, whereby the synthesis product comprises modified nucleoside phosphates, preferably 2′-fluoro nucleoside phosphates.
In an embodiment of the sixth aspect of the present invention the forward synthesis primer consists of modified nucleoside triphosphates.
In an embodiment of the sixth aspect of the present invention the template strand is subjected to digestion, preferably an enzymatic digestion, in order to obtain a single-stranded nucleic acid, whereby the nucleic acid comprises in 5′→3′ direction the forward primer sequence, the randomized region and the reverse primer binding site.
In a preferred embodiment of the sixth aspect of the present invention the second strand synthesis, the reverser primer and a forward primer are used, whereby the forward primer is essentially complementary to the forward primer binding site and comprises at its 5′ end a further partial region, whereby the partial region preferably has a length of about 10 to 25 and more preferably a length of about 10 to 15 nucleotides, whereby the partial region preferably is a binding site or a part thereof, for a forward synthesis primer, and an extended reverse transcription product is obtained, whereby the extended reverse transcription product corresponds to the reverse transcription product, whereby the reverse transcription product is supplemented at its 3′ end by a sequence, whereby the sequence is complementary to the sequence of the further partial region of the forward primer.
In a further preferred embodiment of the sixth aspect of the present invention the synthesis after step (g) the amplified reverse transcription product is reacted with a forward synthesis primer, modified nucleoside triphosphates, preferably 2′-fluoro nucleoside triphosphates, and a reverse transcriptase, whereby the forward synthesis primer hybridizes to the binding site for the forward synthesis primer in order to obtain a synthesis product, whereby the synthesis product comprises modified nucleoside phosphates, preferably 2′-fluoro nucleoside phosphates.
In an even more preferred embodiment of the sixth aspect of the present invention the forward synthesis primer consists of ribonucleotides or of deoxyribonucleotides having at least one ribonucleotide at its 3′ end.
In a preferred embodiment of the sixth aspect of the present invention the template strand is subjected to a digestion, preferably an enzymatic digestion, in order to obtain a single-stranded nucleic acid, whereby the nucleic acid comprises in 5′→3′ direction the forward primer sequence, the randomized region and the reverse primer binding site.
In an embodiment of the sixth aspect of the present invention the forward primer and the reverse primer consist of DNA.
The problem underlying the present invention is solved in a seventh aspect by a method for the selection of a target molecule binding nucleic acid, particularly of aptamers, comprising the steps of:
In an embodiment of the seventh aspect of the present invention the complementary strand in step (g) is separated by interaction between the label and the interaction partner.
In a preferred embodiment of the seventh aspect of the present invention the interaction partner is immobilized to a surface.
In an even more preferred embodiment of the seventh aspect of the present invention the amplification product is immobilized at the surface by the interaction between the label and the interaction partners.
In an embodiment of the seventh aspect of the present invention the two strands of the amplification product are separated from each other, whereby preferably the complementary strand remains immobilized.
In an embodiment of the seventh aspect of the present invention the label is selected from the group comprising biotin, digoxigenin and linker having reactive functional groups and whereby the reactive functional groups are preferably selected from the group comprising amino, carboxy, epoxy and thiol.
In an embodiment of the seventh aspect of the present invention the interaction partner is selected from the group comprising streptavidin, avidin, neutravidin and anti-digoxigenin antibodies and complementary functional groups, and whereby the reactive functional groups are preferably selected from the group comprising amino, carboxy, epoxy and thiol.
In an embodiment of the seventh aspect of the present invention the label is attached at the 5′ and of the reverse primer.
In an embodiment of the seventh aspect of the present invention the forward primer comprises modified nucleoside phosphates, in particular 3′-fluoro nucleoside phosphates.
In an embodiment of the seventh aspect of the present invention the reverse primer comprises deoxynucleoside phosphates.
The problem underlying the present invention is solved in an eight aspect by a method for the selection of a target molecule binding nucleic acid, in particular of aptamers, comprising
In an embodiment of the eighth aspect of the present invention the cleavage in step (k) is an alkaline cleavage and/or is performed by RNase digestion.
The problem underlying the present invention is solved in a ninth aspect by a method for the selection of a target molecule binding nucleic acid, in particular of aptamers, comprising
In an embodiment of the ninth aspect of the present invention the phosphorylating in step (fa) occurs by performing a kinase reaction.
In an embodiment of the ninth aspect of the present invention the cleavage site is provided by a restriction enzyme cleavage site and the cleavage occurs by a restriction enzyme.
In an embodiment of the ninth aspect of the present invention the cleavage site is provided by a ribonucleotide and the cleavage occurs via alkaline cleavage or via RNases.
In an embodiment of the ninth aspect of the present invention the cleavage in accordance with step (l) occurs in an enzymatic manner, preferably by DNase, and/or that the forward synthesis primer sequence is removed by an RNase.
In an embodiment of the ninth aspect of the present invention the nucleic acid of step (a) is a single-stranded nucleic acid consisting of modified nucleoside phosphates, in particular 2′-fluoro-modified nucleoside phosphates.
The problem underlying the present invention is solved in a tenth aspect by a method for the selection of a target molecule binding nucleic acid, in particular of aptamers, comprising the steps of
In an embodiment of the tenth aspect of the present invention the nucleic acid of step (a) is a deoxyribonucleic acid.
In an embodiment of the tenth aspect of the present invention the promoter sequence is selected from the group comprising the promoter sequences of T7-RNA polymerase, T3-RNA polymerase and SP6 polymerase.
In an embodiment of the third, the fourth, the fifth, the sixth, the seventh, the eighth, the ninth and/or the tenth aspect of the present invention the selected nucleic acid(s) is/are selected from the group comprising aptamers, ribozymes, aptazymes, antisense molecules and siRNA.
The present inventors have surprisingly found that by means of a reverse transcriptase activity, which is also referred to herein in the following as reverse transcriptase, completely modified oligonucleotides and in particular completely 2′ F-modified oligonucleotides can be synthesized. For such purpose, only a reverse-complementary counterstrand of the sequence to be synthesized consisting of RNA or DNA or 2′-amino-modified nucleic acid or FNA, i.e. a 2′-fluoro-modified nucleic acid or a mixture thereof, and a suitable primer for the FNA synthesis are required which can hybridize to the counterstrand and which is referred to herein also as forward synthesis primer or FS primer. Preferably, this primer also consists of FNA. If such a primer was not available, then a primer consisting of RNA could be used which is, subsequent to the FNA synthesis, destroyed by RNases or alkaline hydrolysis. In such case the primer has to hybridize upstream, i.e. at the 5′ end of the sequence to be synthesized, at the counterstrand, which, optionally, has to have additional nucleotides at the 3′ end. As an alternative to the FS primer consisting of RNA, also a primer having the identical sequence but consisting of DNA may be used the 3′ terminal nucleotide of which is a ribonucleotide which is destroyed after FNA synthesis by RNases or alkaline hydrolysis. Optionally, the primer may contain further ribonucleotide moieties.
Using the method according to the present invention for the synthesis of a nucleic acid, in principle, nucleic acids can be prepared independent of their sequence and function. Functional nucleic acids which can be produced by using the method in accordance with the present invention for the synthesis of nucleic acids, comprise in particular also aptamers, ribozymes, antisense molecules and RNAi molecules. The molecules synthesis by using the method of the present invention have in common that they are, due to the modification, nuclease-resistant nucleic acids, which is particularly advantageous for the use in biological systems or processes. Biological systems as used herein are in particular embodiments among others: reactions with biological material, preparation of cells, tissue and organs, cells, tissues, organs and organisms, including, but not limited to, single-cellular or multi-cellular organisms, humans, animals and plants and samples thereof.
The method of the present invention also allows its implementation in the so-called SELEX process as, for example, described in European patent EP 0 533 838. It is thus possible by applying the method of the present invention for the synthesis of nucleic acids, to generate with the SELEX process target molecule binding oligonucleotides, nuclease-resistant aptamers and ribozymes, which completely consist of 2′-modified nucleotides. This extends their lifetime in an environment which contains RNases and DNases such as, for example, biological liquids and allow for an enlargement of their potential uses, for example prolonged lifetime in blood and other biological liquids, in the gastrointestinal tract, in cells and tissues.
Resistance to nucleases is also advantageous for antisense molecules which contribute to gene silencing (Manoharan, 1999). Antisense molecules are frequently chemically synthesized. There are, however, also methods which identify potentially successful antisense molecules by the use of enzymes (Xu et al., 2003; Zhang et al., 2003). By modifying such a method, also 2′ F-modified antisense molecules can be identified now. Additionally, 2′-F sequences have a higher melting point which results in stronger RNA-FNA or FNA-DNA duplexes (Cummins et al., 1995).
This is also true for siRNAs. Also in connection therewith nuclease-resistance is advantageous so that the siRNAs reach their target, i.e. the RISC complex in the cytoplasm of their target cells, without being degraded (Chiu and Rana, 2003). By using the methods described herein completely 2′-modified siRNAs can be prepared in such a way in an enzymatic manner upon selection of appropriate templates and matrices.
The thus synthesized, completely 2′-modified nucleic acids are also suitable for, e.g. mass spectrometric analysis of nucleic acids. In connection with mass spectrometric analysis it has been found that there may be a loss of individual bases, whereby this loss can be reduced by the use of FNA. Because of this also longer sequences can be measured at high resolution if they are present as 2′-modified nucleic acids.
In summary, the methods of the present invention allow for a synthesis of nucleic acids having and consisting, respectively, of modified nucleoside phosphates, in particular 2′-F-modified nucleoside phosphates, whereby the portion of termination products is very small and whereby the portion of full length products is very high. A further advantage of the methods of the present invention is that the mutation rate is very low and that they allow for the incorporation of all and not only individual modified nucleotides.
Although the various embodiments are represented herein by reference to the use of 2′-fluoro-modified nucleoside phosphates, the methods of the present invention are not limited thereto, but are, in principle, applicable to all of the modified nucleoside phosphate described herein.
The reverse transcriptases used in the practice of the present invention are thus in preferred embodiments RNA-dependent reverse transcriptases which may, in connection with the present invention, also be used in combination with DNA matrices and FNA matrices.
As used herein in preferred embodiments, the term “reverse transcriptase” refers to any enzymatic activity which is, starting from a (+) RNA strand which is used as a template, suitable to synthesize a complementary (−) DNA strand. It is an RNA-directed DNA polymerase (Stryer, 1995). For the practicing of the methods described herein, suitable reverse transcriptases are particularly also those summarized in the following table.
Thermus thermophilus
Carboxydothermus
hydrogenoformans
It is within the present invention that also DNA polymerases can be used in connection with the methods of the present invention, provided that they exhibit the enzymatic activity required in the methods, i.e. the activity of a reverse transcriptase. Such DNA polymerases are, for example, bacterial DNA polymerases having reverse transcriptase activity such as the enzymes of Thermus thermophilus and Carboxyhydrothermus hydrogenoformans mentioned in the above table. Further particularly preferred reverse transcriptases are those which do not have any RNaseH activity.
About the use of the various reverse transcriptases disclosed herein it is to be noted that mixtures of the individual reverse transcriptases may be used. It is within the present invention that the individual reverse transcriptase is used in its wildtype form or in its mutated form or as a mixture thereof, either alone or together with one or several other reverse transcriptases whether in wildtype form or in mutated form or as a mixture thereof.
As used herein, the term “essentially” in connection with the description of a nucleic acid which essentially consists of a distinct species of nucleoside phosphates, indicates in an embodiment that the respective nucleic acid may consist completely of the respective nucleoside phosphates, but that, to a certain extent, also other nucleoside phosphates may be contained in the nucleic acid, namely those which do have a different or no modification. Examples that a distinct nucleic acid consists only essentially of distinct modified nucleoside phosphates can also be established by the starting materials, i.e. the individual used nucleoside phosphates not containing in each and any case the respective modification due to impurities. Furthermore, it is within the skills of the one of the art to determine to what extent those nucleoside phosphates may be contained in a nucleic acid which is different from the majority of the modified nucleoside phosphate contained in the nucleic acid.
In connection with the present invention all of the nucleoside triphosphates may be modified or only one respective species or, also, a distinct portion of an individual species may be modified or several species may be modified proportionally. Preferred nucleoside triphosphates are 2′-F-ATP, 2′-F-CTP, 2′-F-GTP, 2′-F-TTP and 2′-F-UTP as well as the nucleoside triphosphate of the universal base inosine 2′-F-ITP. These may be exchanged against 2′-dNTPs as desired.
Furthermore, in a preferred embodiment the term “essentially complementary to” refers to nucleic acids which are capable of hybridizing to each other, whereby it is preferred that they hybridize with each other under conditions of medium stringency and in particular under conditions of higher stringency. Conditions for medium stringency and high stringency, respectively, are, for example, described in Current Protocols or Maniatis et al. The stringency can be adjusted by selecting the incubation temperature and the concentration of cations accordingly.
As used herein the term “alkyl” refers to a methyl, ethyl and propyl group. Additionally, the term alkyl and “allyl”, respectively, also comprises an ethenyl and a propenyl group as well as methoxy, ethoxy and propoxy group.
As used herein, the term “nucleoside phosphate” refers in an embodiment of the present invention also to derivatives, in particular nucleoside thiophosphates, however, is not limited thereto.
It is within the methods for the selection of a target molecule binding nucleic acid disclosed herein that, among others, aptamers may be selected. Furthermore it is possible that by using these methods, also ribozymes, antisense molecules and RNAi molecules are selected, whereby the target molecule is different in accordance therewith. In case of ribozymes the characteristic of the ribozyme is to bind to the target molecule and to modify it or itself subsequently to the binding. The modification can be the forming or cleaving of one or several chemical bonds or both. In case of applying the selection methods of the present invention for the selection of antisense molecules the target molecule is a nucleic acid, in particular an mRNA or precursors thereof.
In a further aspect the present invention is also related to a method for the enzymatic synthesis of single-stranded DNA and in particular the use thereof in a method for the selection of single-stranded DNA or DNA oligonucleotides.
Single-stranded DNA oligonucleotides can—analogous to single-stranded RNAs—recognize target structures and bind thereto (Bock et al., 1992; Green et al., 1996; Leva et al., 2002). They are also referred to as DNA-aptamers. DNA-aptamers have, in contrast to those aptamers which contain ribonucleotides or consist thereof, the advantage that they are stable to alkali and can be autoclaved. This is a tremendous advantage in case the aptamer is, for example, used in chromatography columns as affinity matrices which are to be regenerated by “cleaning in place” procedures. In connection therewith diluted lyes are commonly used. For applications in the medical field such as, for example, extracorporeal adsorber for blood dialysis sterility is an absolute requirement. Therefore, a sterilization is required, possibly also for re-use. The most common method is autoclaving. DNA aptamers survive this while staying intact, whereas RNA aptamers hydrolyse.
An advantage of the use of a DNA oligonucleotide in an amplification step in the context of the SELEX method as depicted in more detail in
As an alternative to the separation of the DNA strands obtained in the PCR by gel purification, the use of a biotinylated primer is described in the prior art. In such case the PCR product is immobilized on streptavidin beads and the strand which does not contain the biotinylated primer, will be eluted by means of sodium hydroxide solution, heating or similar measures. The capacity of the bead is frequently very limited as also biotinylated, but non-incorporated primers occupy the biotin binding sites. Also, the beads frequently suffer from the alkaline conditions and heat, respectively, so that streptavidin or other bead constituents are eluted together with the DNA strand. These have to be removed subsequently again by, e.g., phenol chloroform extraction which cannot be automatised easily.
A further advantage of the method of the invention is that the DNA for each following selection round is not only prepared by preparative PCR, but also by PCR and in vitro transcription. Therefore, less PCR cycles are necessary. As the PCR exerts a high selection pressure and as in connection with high cycle numbers PCR artifacts, so-called amplification parasites, occur frequently (Murphy et al., 2003), this is of great value.
The present invention shall be illustrated in the following referring to the following figures and examples, from which further features, embodiments and advantages may be taken. It is particularly mentioned that individual features as disclosed in the context of further features as described in connection with the subsequently described figures and examples, may be used as such individually also in other embodiments of the present invention.
In particular
The nucleic acid having at its 5′ end a phosphate group, is subsequently modified. This modification uses two adapter molecules. The first adapter molecule consists of a double-stranded nucleic acid of a first and a second nucleic acid strand, whereby the first strands of the first and of the second adapter molecules are DNA, RNA or FNA, preferably DNA, the second nucleic acid strand is a deoxyribonucleic acid and the 5′ end of the second nucleic acid strand provides for an overhang, whereby the overhang is at least partially complementary to the first constant partial sequence of the nucleic acid of step (a) and/or (d), i.e. the starting nucleic acid. The second adapter molecule also consists of a double-stranded nucleic acid and also comprises a first and a second nucleic acid strand, whereby the first nucleic acid strand carries a 5′ phosphate and the second nucleic acid strand is a deoxyribonucleic acid and the 3′ end of the second nucleic acid strand provides for an overhang which is at least partially complementary to the second constant partial sequence of the starting nucleic acid shown in
The reverse strand of the double-stranded amplification product is, as depicted in
In principle, it is to be acknowledged that the method is performed in a manner similar to the one described in
The polymerase chain reaction depicted in
Two primers are used in the following polymerase chain reaction (
The thus obtained double-stranded product is immobilized to a streptavidin comprising matrix by means of the label on the complementary strand as shown in
The ligation method shown in
As depicted in
For the herein described experiments the itemized materials were used whereby a detailed specification of the suppliers of itemized substances, solutions and enzyme is cited in the corresponding text passage. Unless otherwise indicated the reagents were purchased from Merck (Darmstadt, Germany). In all cases LiChromosolv water from Merck (Darmstadt, Germany) was used.
The protein (basic, pI of 9; molecular mass of 9 kDa, do not bind to nucleic acids per se) used for in vitro selection was labeled according to the manufacturer's instructions with a biotin linker at least one of the accessible amino groups of the amino acids using the Biotinylation-Kit “EZ-Link Sulfo-NHS-LC-LC-Biotin” (Pierce, Rockford, USA). Thus, the separation of unbound nucleic acids using the biotin-streptavidin- or biotin-neutravidin-linkage can be realized. For this purpose the selection matrices neutravidin agarose and “UltraLink Plus” immobilized streptavidin gel (both purchased from Pierce, Rockford, USA) was used. Excess linker was removed according to the manufacturer's instructions with a molecular sieve, the filter device “YM-3” (molecular cut-off limit of MW 3000, Amicon/Milliore, Bedford, USA).
The used oligonucleotides such as primers and die initial DNA library were synthesized by standard phosphoramidite chemistry at NOXXON Pharma AG. The sequences can be found in Example 6.
Synthesis of the Template for the FNA-Synthesis: Fill-in reaction+in Vitro Transcription
At first 3 nmol of a synthetic DNA library BSA-1C initial library was converted into double-stranded DNA by a Fill-in-reaction using the BSA 1C-Reverse Primer T7 and the Vent-(exo−)-DNA polymerase (NEB, Frankfurt a.M., Germany). By using this reaction with the BSA 1C-Reverse Primer T7 the T7 RNA polymerase promoter was incorporated.
At first the batch without enzyme was denatured at 95° C. for 10 min. and cooled for 5 minutes on ice. Then the enzyme was added and the batch was incubated for two hours at 63° C.
Afterwards the dsDNA was desalted by ethanol precipitation (20 μg GlycoBlue (Ambion) and 2.5 vol. absolute ethanol, 30 min at −80° C.; centrifugation at 13200 rpm (16100 g) for 30 minutes at 4° C.; the pellet was once washed with 70% ethanol).
The dsDNA library was used as template for in vitro transcription.
Incubation: 2 to 12 hours at 37° C.
Subsequent to the in vitro transcription the remaining dsDNA template was digested with 20 units DNAse I (Sigma) for 20 min at 37° C. After addition of loading buffer (7M urea, xylene cyanol, bromphenol blue) the batch was denatured and gel-purified (10% denaturing polyacrylamide gel). The band of the transcript was detected by UV-Shadowing, cut off from the gel and eluted by the Crush-and-Soak method. The resultant eluate was precipitated in ethanol, the pellet washed once with ice-cold 70% ethanol and resuspended in water.
The RNA library was used as template for FNA-synthesis using a reverse transcriptase.
Subsequent to the FNA-synthesis the template-strand (RNA) was hydrolyzed under alkaline conditions.
Afterwards the FNA library was desalted by ethanol precipitation (20 μg GlycoBlue (Ambion) and 2.5 Vol. absolute ethanol, 30 min at −80° C.; centrifugation at 13200 rpm (16100 g) for 30 minutes at 4° C.; the pellet was once washed with 70% ethanol). After addition of loading buffer (7M urea, xylene cyanol, bromphenol blue) the batch was denatured and gel-purified (10% denaturing polyacrylamide gel). The band of the FNA was detected by UV-Shadowing, cut off from the gel and eluted by the Crush-and-Soak method. The resultant eluate was precipitated in ethanol, the pellet washed once with ice-cold 70% ethanol and resuspended in water.
In order to detect the binding of the FNA library during the in vitro selection, the 5′—OH— group of the FNA library was radioactively labeled with 32P by performing a kinase reaction.
”Forward“-buffer (Invitrogen)
The reaction was run at 37° C. for one hour and was stopped at 65° C. (10 min).
After addition of loading buffer (7M urea, xylene cyanol, bromphenol blue) the reaction was denatured and gel-purified (10% denaturing polyacrylamide gel). The FNA was detected by UV-Shadowing, cut off from the gel and eluted by the Crush-and-Soak method. The resultant eluate was precipitated in ethanol, the pellet washed once with ice-cold 70% ethanol and resuspended in water.
All non-enzymatic steps of the selection (except for the denaturation step) were carried out in selection buffer (20 mM Tris pH 7.4; 150 mM NaCl; 4 mM KCl; 1 mM MgCl2 (all from Ambion, Austin, USA); 1 mM CaCl2 (Merck, Darmstadt, Germany) and 0.1% Tween-20 (Roche Diagnostics, Mannheim, Germany). The denaturation step was carried out for five minutes at 95° C. in selection buffer. Subsequent to the denaturation step the FNA was cooled down to 37° C. for 15 minutes at 37° C.
Subsequent to the folding, the FNA was at first incubated with the selection matrix (either with neutravidin agarose or with “UltraLink Plus” immobilized streptavidin, both from Pierce, USA) without protein at 37° C. for 30 minutes. This so-called “pre-selection” was done in order to remove potentially matrix binding molecules. After this incubation step the selection matrix was sedimented and the non-bound FNA in the supernatant was separated. An aliquot of the FNA was incubated with the biotinylated protein for one hour at 37° C. The other aliquot of the FNA was incubated without protein for one hour at 37° C. Subsequently, the biotin-binding selection matrix was added to the binding reaction. After incubation for 30 min at 37° C. the selection matrix including the complexes of protein and FNA bound thereto, was separated from the solution by centrifugation and washed with selection buffer.
The FNA which remained on the selection matrix after the washing step was eluted twice from the matrix material with 200 μl 8 M urea/10 mM EDTA (both from Ambion, Austin, USA), respectively. The first elution step was carried out for 15 minutes at 65° C., the second elution was done at 95° C. To the eluted FNA 400 μl of a mixture of phenol/(chloroform/isoamylalcohol) (1:(1:1/24)) (Applichem, Darmstadt, Germany) was added, the mixture was centrifuged for 5 min at 13000 rpm at room temperature. The aqueous phase (supernatant) was recovered, the phenolic phase was once re-extracted with 100 μl water, the aqueous phases were combined and shaken with 500 μl of a mixture of chloroform and isoamylyalcohol (24:1) (Applichem, Darmstadt, Germany), centrifuged for 5 min at 13000 rpm room temperature and the upper aqueous phase was separated.
Thereupon the aqueous phase was ethanol-precipitated (2.5 fold volume of absolute ethanol (Merck, Darmstadt, Germany), 0.3 M sodium acetate, pH 5.5 (Ambion, Austin, USA) and 1 μl glycogen (Roche Diagnostics, Mannheim, Germany)) for 30 min at −80° C. and centrifuged for 30 min at 14000 rpm (4° C.). The pellet was washed once with ice-cold 70% ethanol (Merck, Darmstadt, Germany).
Synthesis of the cDNA
In order to amplify the FNA library via a PCR reaction the FNA was transcribed into DNA.
cDNA-Synthesis
In order to prepare the dsDNA for the following in vitro transcription, the DNA was ethanol-precipitated.
Subsequently, the RNA that had been synthesized by in vitro transcription, was transcribed into FNA. The RNA that was used as template and the BSA-1C FS-Primer 3rG were fragmented by alkaline hydrolysis and removed using a molecular sieve (YM-filter units, Amicon/Millipore, Bedford, USA). The FNA was radioactively labeled (as described). The thus enriched FNA library was used in the next selection round.
The products of the amplification steps to be performed during one selection round are depicted in
During selection radioactively labeled FNA was used and the binding was calculated as percentage based on the utilized amount of FNA. The radioactivity was determined with a scintillation counter (Beckman, Fullerton, USA). The course of selection is depicted in
At first the synthetic DNA library BSA-1A initial library was converted into double-stranded DNA by a Fill-in-reaction using the BSA 1A-Reverse Primer T7. Then the dsDNA was used in the in vitro transcription. BSA-1A FS-Primer 3rG+15 was used as a primer for the FNA synthesis of the FNA library. Subsequent to the FNA synthesis the RNA template strand and the primer were removed by alkaline hydrolysis which led to an FNA library consisting of 63 nucleotides including 40 randomized nucleotides at its 5′-end ((N)40-CACGAGTGAAGTCTGAGCTCC-3′) (SEQ ID NO:26). Finally the sample was precipitated with ethanol for de-salting the sample. The components and reaction conditions correspond to the previous described protocols for FNA-synthesis, alkaline hydrolysis and ethanol precipitation.
For comparison DNA was synthesized in parallel. Instead of the FNA-synthesis a DNA-synthesis according to protocol of the FNA-synthesis was carried out, whereby the FNTPs were substituted for dNTPs (for protocols of all reactions using the herein specified oligonucleotides, see Example 1)
As additional controls libraries consisting of RNA and 2′-F-pyrimidine RNA were used. The libraries were synthesized by in vitro transcription. Starting from the BSA-1A initial library a PCR reaction using the BSA-1 FS-Primer 3′G and the BSA1 Reverse Primer T7 was carried out (protocol, see Example 1) and the PCR-product was subjected to an alkaline hydrolysis (in a manner analogous to the description in Example 1 for the alkaline hydrolysis subsequent to the FNA-synthesis) and an in vitro transcription was carried out for the synthesis of the RNA library (protocol, see Example 1) and for the synthesis of a 2′-F-pyrimidine library (protocol, see in the following) (length of the libraries: 63 nucleotides). To avoid any undesired inhibition of the RNases by the RNase-inhibitor “RNaseOut” (Invitrogen), the RNase inhibitor was removed by phenol chloroform extraction and subsequent ethanol precipitation.
Die 2′-F-pyrimidine synthesis was carried out as follows:
Incubation: 4 to 12 hours at 37° C.
Each 4 pmole of the FNA library and likewise synthesized DNA library (see Example 2) were incubated for 5 min at 37° C. in 10 μl of a buffer (100 mM NaOAc, 5 mM MgCl2) including different amounts of DNase I (Sigma) according to the manufacturer's instructions. The dilutions of DNase I were prepared in the same buffer immediately prior to their use.
The DNase digestion was stopped by addition of 6 M urea/15 mM EDTA and by a denaturation step (10 min at 95° C.).
The samples were separated on a 10% denaturing polyacrylamide gel and visualized by a UV-transilluminator upon staining with ethidium bromide (
For further characterization of the FNA the RNase stability of the enzymatically synthesized FNA library was determined and compared to RNA and to 2′-F-pyrimidine RNA (synthesis, see Example 2). Each 4 pmole of the FNA library, RNA or 2′-pyrimidine RNA library were incubated according to the manufacturer's instructions for 30 min at 37° C. in 10 μl of a buffer including different amounts of RNase T1 (1-1000 units) and RNase I (0.1-100 units). The RNase T1 originates from Aspergillus oryzae and cleaves after Gs. RNase I originates from E. coli and cleaves RNA in an unspecific manner. Both RNases were recombinantly produced in E. coli by Ambion (Austin, Tex., USA). RNase T1 buffer: 10 mM Tris, pH 8, 100 mM NaCl; RNase I buffer: 50 mM Tris, pH 7.5, 1 mM EDTA.
The reaction was stopped by addition of 20 μl STOP-solution (2% SDS, 50 mM EDTA, 6 M urea) and quick-freeze in a mixture of dry ice and ethanol. In the following the samples were stored on ice until denaturation (5 min at 95° C.), were quickly cooled on ice after denaturation and loaded onto an analytical denaturing 10% polyacrylamide gel.
After separation by gel electrophoresis the bands were stained with ethidium bromide and visualized under UV light on a UV-transilluminator (
The stability of FNA and RNA (for the synthesis, see Example 2) to serum nucleases was tested in human serum. In order to minimize pH-shifts in the course of the experiment (4 days) 50 mM sodium phosphate buffer was added to the serum. Each 4 pmole of nucleic acid were incubated in 20 μl batches with 14 μl human serum (70% serum) at 37° C. for a length of time as depicted in
For the different reactions as they were used in the examples the following oligonucleotides were used.
FNA-Selection 1 with FS Primer Consisting of FNA (A)
underlined: T7 RNA polymerase promoter or SP6 RNA polymerase promoter
TCT AAT ACG ACT CAC TAT AGG AGC TCA GAC TTC ACT CG
used for different tests:
FNA Selection 1 (B and C) with FS Primer Consisting of RNA or DNA
(using two overlapping primers and a Forward Synthesis Primer consisting of RNA or of DNA with a 3′-terminal ribo-nucleotide)
underlined: T7 RNA polymerase promoter or SP6 RNA polymerase promoter
NOH: ribo-nucleotide
TCT AAT ACG ACT CAC TAT AGG AGC TCA GAC TTC ACT CG
(using two overlapping primers and a Forward Synthesis Primer consisting of RNA or of DNA including a 3′-terminal ribo-nucleotide)
in italics: Nucleotides of the libraries and of the ligation matrices that hybridize with each other
underlined: T7 RNA-polymerase-promoter or SP6-RNA-polymerase-promoter
NOH: ribo-nucleotide
pN: 5′-phosphate of a nucleotide
3′dN: 2′-3′-dideoxynucleotide
FNA Selection 2 with FS Primer Consisting of FNA (A)
(using a Forward Synthesis Primer consisting of FNA)
FNA Selection 2 with FS-Primer Consisting of DNA or RNA (B)
(using one overlapping primer consisting of RNA or of DNA with a 3′-terminal ribo-nucleotide)
NOH: ribo-nucleotide
(using one overlapping primer consisting of RNA or of DNA with a 3′-terminal ribo-nucleotide)
in italics: Nucleotides of the libraries and of the ligation templates that hybridize with each other
pN: 5′-Phosphate of a nucleotide
3′ dN: 2′-3′-dideoxynucleotide
DNA Selection with FS Primer Consisting of DNA
underlined: T7 RNA polymerase promoter or SP6 RNA polymerase promoter
TCT AAT ACG ACT CAC TAT AGG AGC TCA GAC TTC ACT CG
The various references which are contained herein read completely as follows and their disclosure is incorporated herein by reference.
The features of the invention disclosed in the preceding description, the claims and the figures can be essential for the practice of the invention either alone or in any combination.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10342373.7 | Sep 2003 | DE | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10572291 | Feb 2007 | US |
| Child | 12905910 | US |
