2′-fluoro-modified pyrimidines enhance affinity of RNA oligonucleotides to HIV-1 reverse transcriptase

  1. Donald H. Burke1,2,4,5
  1. 1Department of Biochemistry, University of Missouri, Columbia, Missouri 65211, USA
  2. 2Bond Life Sciences Center, University of Missouri, Columbia, Missouri 65211, USA
  3. 3Department of Veterinary Pathobiology, University of Missouri, Columbia, Missouri 65211, USA
  4. 4Department of Molecular Microbiology and Immunology, University of Missouri, Columbia, Missouri 65211, USA
  5. 5Department of Biological Engineering, University of Missouri, Columbia, Missouri 65211, USA
  1. Corresponding author: burkedh{at}missouri.edu
  • 6 Present address: Stemloop, Inc., Evanston, Illinois 60204, USA

Abstract

Nucleic acid aptamers can be chemically modified to enhance function, but modifying previously selected aptamers can have nontrivial structural and functional consequences. We present a reselection strategy to evaluate the impact of several modifications on preexisting aptamer pools. RNA aptamer libraries with affinity to HIV-1 reverse transcriptase (RT) were retranscribed 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 preenriched libraries. Inhibition was the strongest with the 2′-F-pyrimidine (2′-FY) RNAs, as compared to inhibition by the 2′-OMeY and 2′-NH2Y RNAs. Unexpectedly, a diverse panel of retroviral RTs were strongly inhibited by all 2′-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 2′-FY in the transcript. RT binding affinity by 2′-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 2′-FY RNA than for unmodified 2′-OH RNA. These surprising features of 2′-FY-modified RNA may have general implications for applied aptamer technologies.

Keywords

Footnotes

  • Received June 25, 2020.
  • Accepted July 24, 2020.

This article, published in RNA, is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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