Abstract
Reverse transcriptase (RT) inhibitors are core components of antiretroviral therapies; however, HIV’s high mutation rate prompts the emergence of drug resistance and necessitates new inhibitors with high genetic barriers to resistance. RNA aptamers that have been selected to bind RT exhibit potent RT inhibition and suppress viral replication when targeting the strain-specific RT that they were originally selected to bind, but some of these same, otherwise potent aptamers fail against both single-point mutant and phylogenetically-diverse RTs. We hypothesized that a subset of the total aptamer population in libraries pre-enriched against a single RT may exhibit broad-spectrum RT binding and inhibition, and we devised a multiplexed Poly-Target selection approach to elicit those phenotypes against a panel of diverse lentiviral RTs. High-throughput sequencing of starting, negative, and final libraries, followed by coenrichment analysis of parallel and duplicate selection trajectories, narrowed the list of candidate aptamers by orders of magnitude. Biochemical characterization of candidates identified a novel aptamer motif and several rare and unobserved variants of previously-known motifs that inhibited recombinant RTs from HIV-1, HIV-2 and SIV to varying degrees. These broad-spectrum aptamers also suppressed replication of viruses carrying phylogenetically-diverse RTs. The Poly-Target selection and coenrichment approach described herein is a generalizable strategy for identifying broad-spectrum behavior and cross-reactivity among related targets from combinatorial libraries.
