Towards the selection of phosphorothioate aptamers optimizing in vitro selection steps with phosphorothioate nucleotides.
The high affinity of a given nucleic acid for a protein ligand can be used to isolate specific inhibitors of enzymes involved in pathological situations. The latter property is the basis of the SELEX (systematic evolution of ligands by exponential enrichment) technique. Recently, several potent nucleic acids inhibitors of HIV-1 replication have been isolated using the SELEX approach. However, phosphodiester oligodeoxynucleotides (PO-ODNs) were not used as antiviral agents because of their sensitivity to nucleases. Our goal in this work was to explore the possibility of selecting, from a fully substituted phosphorothioate library, oligonucleotides having both a strong affinity for HIV-1 reverse transcriptase (RT) and nuclease resistance. HIV-1 RT initiates in vivo reverse transcription from the 3' end of a host tRNALys. Although phosphorothioate ODNs (PS-ODNs) have been claimed to bind unspecifically to proteins, we have shown previously that an ODN corresponding to the acceptor stem of tRNALys was able to inhibit specifically HIV-1 replication in HIV-1 infected cells, without showing cytotoxicity up to 10 microM. As the SELEX strategy requires 'in vitro' transcription and reverse transcription of the selected DNA, we have assayed the available PS precursors as a model system by using PS-dNTPs and rNTPs. We have also developed an experimental procedure to optimize the incorporation of four PS-dNTPs during the PCR step of the SELEX approach. In the course of this work, we have showed that the PS-dGTP is a strong inhibitor of thermostable DNA polymerases as well as of HIV-1 RT.
UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, Bordeaux, France; Institut Fédératif de Recherches 'Pathologies Infectieuses' (IFR 66), Bordeaux, France. marie-line.andreola@reger.u-bordeaux2.fr
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ML
Andreola
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C
Calmels
J
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Michel
J J
JJ
Toulmé
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S
Litvak
D001483:Base Sequence; D017931:DNA Primers; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015497:HIV-1 / Q000187:drug effects* / Q000201:enzymology / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D020319:Oligodeoxyribonucleotides, Antisense / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D016133:Polymerase Chain Reaction / Q000379:methods; D012357:RNA, Transfer, Lys / Q000378:metabolism; D018894:Reverse Transcriptase Inhibitors / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D013698:Templates, Genetic; D013873:Thionucleotides; D014158:Transcription, Genetic; D014764:Viral Proteins; D014779:Virus Replication / Q000187:drug effects*
D017931:DNA Primers; D009838:Oligodeoxyribonucleotides; D020319:Oligodeoxyribonucleotides, Antisense; D012357:RNA, Transfer, Lys; D018894:Reverse Transcriptase Inhibitors; D013873:Thionucleotides; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D054303:HIV Reverse Transcriptase; D012321:DNA-Directed RNA Polymerases