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

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2′-Fluoro-Modified Pyrimidines Enhance Affinity of RNA Oligonucleotides to HIV-1 Reverse Transcriptase Downloaded from rnajournal.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press 2′′′′′-fluoro-modified pyrimidines enhance affinity of RNA oligonucleotides to HIV-1 reverse transcriptase PAIGE R. GRUENKE,1,2 KHALID K. ALAM,1,2,6 KAMAL SINGH,2,3 and DONALD H. BURKE1,2,4,5 1Department of Biochemistry, University of Missouri, Columbia, Missouri 65211, USA 2Bond Life Sciences Center, University of Missouri, Columbia, Missouri 65211, USA 3Department of Veterinary Pathobiology, University of Missouri, Columbia, Missouri 65211, USA 4Department of Molecular Microbiology and Immunology, University of Missouri, Columbia, Missouri 65211, USA 5Department of Biological Engineering, University of Missouri, Columbia, Missouri 65211, 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-py- ′′′′′ ′′′′′ ′′′′′ rimidine (2 -FY) RNAs, as compared to inhibition by the 2 -OMeY and 2 -NH2Y RNAs. Unexpectedly, a diverse panel of ret- roviral 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: SELEX; in vitro selection; 2′′′′′ modifications; 2′′′′′-fluoro pyrimidines INTRODUCTION accessible to unmodified nucleic acids (Kuwahara and Sugimoto 2010; Stovall et al. 2014; Lapa et al. 2016). To Aptamers are structured nucleic acids that bind to molec- stabilize RNA aptamers against rapid degradation by se- ular targets, often with high affinity and specificity. They rum endonucleases for therapeutic and diagnostic appli- are generated through an iterative selection process cations, one strategy is to modify the 2′ position on the termed systematic evolution of ligands by exponential en- ribose ring of pyrimidines (Adler et al. 2008; Keefe et al. richment (SELEX) (Ellington and Szostak 1990; Tuerk and 2010; Dellafiore et al. 2016). Common 2′-modifications Gold 1990). In some cases, chemically modified nucleo- such as 2′-fluoro (-F), 2′-amino (-NH ), and 2′-O-methyl tide building blocks within an aptamer can improve the 2 (-OMe) significantly increase RNA serum half-life, but binding affinity of an aptamer to its target of interest. For they also can have structural and functional consequences example, very high affinity aptamers against the human for the aptamer by perturbing the binding interface, bind- immunodeficiency virus type 1 reverse transcriptase (HIV- ing affinity, three-dimensional structure, and overall phar- 1 RT) and HIV-1 integrase were recently selected using macokinetic and pharmacodynamic properties (Burnett the 2′-deoxy-2′-fluoroarabinonucleotide (FANA) modifica- and Rossi 2012). Among the above-mentioned modifica- tion (Alves Ferreira-Bravo et al. 2015; Rose et al. 2019), and tions, the 2′-F-pyrimidine (2′-FY) modification has been ex- various classes of modifications can improve the biostabil- tensively used in selecting aptamers for use in vivo or in ity of aptamers and enable reaction chemistries that are in- serum, such as cell-targeted or cell-internalizing aptamers (Zhou and Rossi 2016; Tawiah et al. 2017). Modifications at 6 Present address: Stemloop, Inc., Evanston, Illinois 60204, USA Corresponding author: [email protected] © 2020 Gruenke et al. This article, published in RNA, is available Article is online at http://www.rnajournal.org/cgi/doi/10.1261/rna. under a Creative Commons License (Attribution-NonCommercial 4.0 077008.120. Freely available online through the RNA Open Access International), as described at http://creativecommons.org/licenses/ option. by-nc/4.0/. RNA (2020) 26:1667–1679; Published by Cold Spring Harbor Laboratory Press for the RNA Society 1667 Downloaded from rnajournal.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press Gruenke et al. the 2′-position of the pentose sugar in nucleic acids can af- post-SELEX chemical optimization of aptamers that were fect sugar puckering and the types of hydrogen bond pat- originally selected as native DNA, RNA, or other polymers terns (Shigdar et al. 2013) and alters electronic distribution by systematically introducing modified nucleotides into throughout the nucleotide (Guschlbauer and Jankowski preexisting aptamers in various combinations and deter- 1980). The high electronegativity of the fluorine makes it mining their impact on binding affinity, serum stability, a weak hydrogen bond acceptor relative to 2′-OH, and en- and function through systematic screens (Eaton et al. hances the C3′-endo character of the sugar pucker relative 1997; Aaldering et al. 2015; Darmostuk et al. 2015; to unmodified ribose to reinforce the canonical A-form he- Meek et al. 2016). This process is repeated until the mod- lix (Guschlbauer and Jankowski 1980; Zhou et al. 2009; ified aptamer has the desired properties, which typically ′ Patra et al. 2012). In contrast, the 2 -NH2 substitution requires screening a large number of variants (Lapa et al. more strongly prefers the C2′-endo sugar pucker found 2016), which can be laborious, expensive, and hit-or-miss. in DNA-like B-form helices (Guschlbauer and Jankowski We developed a reselection strategy that identifies 1980). 2′-OMe and unmodified 2′-OH nucleotides are in- aptamers that can either accommodate or adapt to nucle- ′ ′ termediate between the two extremes (2 -F and 2 -NH2), otide chemical modifications and that reduces many of the favoring the C3′-endo sugar pucker to approximately the obstacles commonly faced with the de novo modified same degree (Guschlbauer and Jankowski 1980). Many SELEX and post-SELEX optimization approaches. We other nucleotide analogs are available with modifications began with a preenriched library of RNA aptamers on the sugar, nucleobase, or internucleotide linkages, (2′-OH) that had previously undergone 14 rounds of affinity each associated with their own potential for modulating selection against HIV-1 RT (Burke et al. 1996). This library ′ ′ ′ nucleic acid function. was retranscribed with either 2 -F, 2 -OMe, or 2 -NH2 py- Two approaches are conventionally used to generate rimidines, and each new set was subjected to three addi- chemically modified aptamers. In the first approach de tional rounds of selection followed by high-throughput novo selection is performed with chemically modified ran- sequencing and bioinformatics analyses. From the result- dom libraries from the beginning. An advantage of this in- ing enrichment data, we chose candidate aptamers to fur- SELEX strategy is that it leverages the full sequence diver- ther characterize their ability to bind and inhibit HIV-1 RT sity of the random library to investigate a large number of enzymatic activity in primer extension assays. This ap- modified sequences simultaneously (Meek et al. 2016). proach identified 2′-modified RNA aptamers that main- Many types of modified nucleotides can be incorporated tained their originally selected function of binding to into selections to enhance aptamer functionality and HIV-1 RT despite the 2′ modification. Unexpectedly, all stability, such as modifications within the nucleobases, transcripts carrying 2′-FY modifications—including non- phosphate backbone, or other sugar modifications binding control sequences—were found to bind and inhib- (Dellafiore et al. 2016). However, the modified nucleotides it several retroviral RTs, but either did not inhibit or only must be recognized and/or incorporated by relevant poly- very weakly inhibited bacterial and human DNA polymer- merases (Lauridsen et al. 2012), and many modified nucle- ases. The enhanced inhibition by the 2′-FY-modified tran- otides are expensive or require specialized synthetic scripts is proportional to the mole fraction of 2′-FY and is expertise to produce them in-house (Darmostuk et al. due, in part, to increased ionic interactions of the 2′-FY 2015). Polymerase engineering has yielded many new transcripts with HIV-1 RT in comparison with 2′-OH tran- polymerases that incorporate diverse modified nucleo- scripts. These observed properties of 2′-FY-modified tides efficiently and greatly expanded the alphabet of ac- RNA may provide new insights on the binding interactions cessible nucleotide analogs for in vitro selection of 2′-FY aptamers to their respective targets. (Pinheiro et al. 2012; Larsen et al. 2016; Kimoto et al. 2017).
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