2ʹ-Fluoro-Modified Pyrimidines Enhance Affinity of RNA Oligonucleotides to HIV-1 Reverse Transcriptase

Abstract

Nucleic acid aptamers can be chemically modified to enhance function, but modifying previously selected aptamers can have non-trivial structural and functional consequences. We present a reselection strategy to evaluate the impact of several modifications on pre-existing aptamer pools. RNA aptamer libraries with affinity to HIV-1 reverse transcriptase (RT) were re-transcribed with 2′-F, 2′-OMe, or 2′-NH2 pyrimidines and subjected to three additional selection cycles. RT inhibition was observed for representative aptamers from several structural families identified by high-throughput sequencing when transcribed with their corresponding modifications. Thus, reselection identified specialized subsets of aptamers that tolerated chemical modifications from unmodified pre-enriched libraries. Inhibition was the strongest with the 2ʹ-F-pyrimidine (2ʹ-FY) RNAs, as compared to inhibition by the 2ʹ-OMeY and 2ʹ-NH2Y RNAs. Unexpectedly, a diverse panel of retroviral RTs were strongly inhibited by all 2ʹ-FY-modified transcripts, including sequences that do not inhibit those RTs as unmodified RNA. The magnitude of promiscuous RT inhibition was proportional to mole fraction 2ʹ-FY in the transcript. RT binding by 2ʹ-FY transcripts was more sensitive to salt concentration than binding by unmodified transcripts, indicating that interaction with retroviral RTs is more ionic in character for 2ʹ-FY-RNA than for unmodified 2ʹ-OH-RNA. These surprising features of 2ʹ-FY-modified RNA may have general implications for applied aptamer technologies.