Modified nucleotides and nucleic acids for the discovery of antiretroviral agents targeting HIV-1 reverse transcriptase

The reverse transcriptase (RT) of the human immunodeficiency virus (HIV) has both polymerase and ribonuclease H (RNase H) activity, and is a key enzyme in the HIV life cycle as it converts the viral RNA genome into double-stranded DNA. A series of studies built around the theme of chemically modified nucleic acids are described in order to: (1) better understand the biochemical processes of reverse transcription in HIV-1, (2) synthesize antiretroviral agents directed to novel targets on HIV-1 (RT), and (3) develop screening methods incorporating fluorescent nucleobase analogues to uncover new drug candidates.;We synthesized 6-phenylpyrroloribocytidine (PhpC), a novel fluorescent cytidine analogue incorporated into RNA. The PhpC-containing RNA formed native-like duplex structures with complementary strands and fluorometrically reported upon binding to complementary RNA and DNA strands. PhpC was shown to rank among the brightest fluorescent cytidine analogues based on quantum yields. RNA containing PhpC was cleaved by HIV-1 RT RNase H with a 14-fold increase in fluorescence intensity. In contrast to the same fluorescein/DABCYL containing oligonucleotides used in older RNase H assays to screen for inhibitors, PhpC containing RNA did not disrupt catalytic activity of the enzyme. Furthermore, the PhpC RNase H assay was implemented on 96-well microplate format, the first example among fluorescent nucleobase analogues.;Small interfering RNAs (siRNAs) containing PhpC were synthesized and shown to possess enhanced thermal stability and good gene-silencing activity. Due to their emissive properties, the biodistribution of PhpC-containing siRNAs could be monitored by fluorescence microscopy and were shown to accumulate in the cytoplasm in HeLa cells.;We synthesized a series of oligonucleotide primers with 3'-terminated nucleosides of varying sugar conformations. This was accomplished on solid supports using a methodology that avoids the direct coupling of phosphoramidite building blocks. The synthesis of an analogous AZT 3'-terminated primer was accomplished using AZT 5'-H-phosphonate with greater ease, speed and yield compared to traditional enzymatic approaches. We tested the primers as substrates for RT-catalyzed nucleotide excision, the phenotypic mechanism of drug resistance in HIV strains carrying thymidine analogue mutations (TAMs). We determined that in general RNA-like sugar conformations are poorly excised by HIV-1 RT, while DNA-like sugar conformations are quickly excised. Fluorescence-based nucleotide excision assays incorporating the emissive nucleobase analogues PhpC and 2-AP were explored. The rate of nucleotide excision could be monitored in 96-well microplate format for the PhpC-containing assay only.;We demonstrated how chemically modified nucleic acid hairpins inhibited the RNase H activity of HIV-1 RT. For example, substituting natural RNA for 2'-deoxy-2'-fluoro-ribonucleotides, 2'-deoxy-2'-fluoro-arabinonucleotides, locked nucleic acids or conjugation of cholesterol at the 5'-terminus modulated the potency of hairpins for RNase H activity. Biochemical methods indicated that the substrate for HIV-1 RT, a short primer-long template, may be bound at the polymerase domain with its trajectory diverted away from the RNase H domain by the presence of the synthetic hairpins. Furthermore, the binding of hairpins to HIV-1 RT had no adverse affects on the potency of chain terminators such as 3'-azido-3'-deoxythymidine (AZT), a widely used antiviral agent. This discovery supports a model where the RNase H activity can be an antiretroviral target independent of the rest of RT.