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 situatio ns. The latter property is the basis of the SELEX (systematic evolution of ligands by exponential enrichment) technique. Recently, several potent nucl eic acids inhibitors of HIV-1 replication have been isolated using the SELE X approach. However, phosphodiester oligodeoxynucleotides (PO-ODNs) were no t used as antiviral agents because of their sensitivity to nucleases. Our g oal 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 TCT initiates in vivo reverse transcription from the 3' end of a host tR NA(Lys). Although phosphorothioate ODNs (PS-ODNs) have been claimed to bind unspecifically to proteins, we have shown previously that an ODN correspon ding to the acceptor stem of tRNA(Lys) was able to inhibit specifically HIV -1 replication in HIV-1 infected cells, without showing cytotoxicity up to 10 mu M. As the SELEX strategy requires 'in vitro' transcription and revers e transcription of the selected DNA, we have assayed the available PS precu rsors 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 d uring the PCR step of the SELEX approach. In the course of this work, we ha ve showed that the PS-dGTP is a strong inhibitor of thermostable DNA polyme rases as well as of HIV-1 RT.