Selection of 2'-Deoxy-2'-Fluoroarabino Nucleic Acid (FANA) Aptamers That Bind HIV-1 Integrase with Picomolar Affinity.

Kevin M Rose,Mark A Ditzler,Philipp Holliger,Robert Craigie,Min Li,Irani Alves Ferreira-Bravo,Jeffrey J Destefano

doi:10.1021/acschembio.9b00237

Abstract

Systematic Evolution of Ligands by Exponential Enrichment (SELEX) is the iterative process by which nucleic acids that can bind with high affinity and specificity (termed aptamers) to specific protein targets are selected. Using a SELEX protocol adapted for Xeno-Nucleic Acid (XNA) as a suitable substrate for aptamer generation, 2′-fluoroarabinonucleic acid (FANA) was used to select several related aptamers to HIV-1 integrase (IN). IN bound FANA aptamers with equilibrium dissociation constants (KD,app) of ∼50–100 pM in a buffer with 200 mM NaCl and 6 mM MgCl2. Comparisons to published HIV-1 IN RNA and DNA aptamers as well as IN genomic binding partners indicated that FANA aptamers bound more than 2 orders of magnitude more tightly to IN. Using a combination of RNA folding algorithms and covariation analysis, all strong binding aptamers demonstrated a common four-way junction structure, despite significant sequence variation. IN aptamers were selected from the same starting library as FA1, a FANA aptamer that binds with pM affinity to HIV-1 Reverse Transcriptase (RT). It contains a 20-nucleotide 5′ DNA sequence followed by 59 FANA nucleotides. IN-1.1 (one of the selected aptamers) potently inhibited IN activity and intasome formation in vitro. Replacing the FANA nucleotides of IN-1.1 with 2′-fluororibonucleic acid (F-RNA), which has the same chemical formula but with a ribose rather than arabinose sugar conformation, dramatically reduced binding, suggesting that FANA adopts unique structural conformations that promote binding to HIV-1 IN.

Highlights

HIV-1 IN is a 32-kilodalton protein with three distinct domains: an N-terminus that coordinates zinc; a catalytic core domain that catalyzes the transfer of viral nucleic acid into the host cell chromosome via a conserved “D,D(35)E” motif commonly found in members of a nucleotidyltransferase superfamily, which includes HIV-1 IN, other retroviral integrases, and bacterial and eukaryotic transposases;[3,4] and a C-terminus.[5]
Despite chemical and conformational differences that are theorized to explain the nuclease resistance seen by these analogues, XenoNucleic Acid (XNA) retain the ability to adopt secondary structures and store information to their DNA and RNA counterparts.[45−47] Aptamers containing XNA can be made by modifying some of the nucleosides in aptamers produced with conventional nucleic acids (Macugen was produced using this approach48) or by direct selection using XNA nucleotides in a Systematic Evolution of Ligands by Exponential Enrichment (SELEX) protocol
Note that this approach produces “chimeric” aptamers with a 20-nucleotide fixed DNA sequence at the 5′ end followed by a ∼40-nucleotide region of random fluoroarabinonucleic acid (FANA) nucleotides, 19 nucleotides of the FANA fixed sequence

Summary

Introduction

Human immunodeficiency virus (HIV-1) integrase (IN), the enzyme responsible for incorporating virus-derived proviral double stranded DNA (dsDNA) into the host cell chromosome, has become an important drug target for HIV therapy.[1,2] HIV-1 IN is a 32-kilodalton (kDa) protein with three distinct domains: an N-terminus that coordinates zinc; a catalytic core domain that catalyzes the transfer of viral nucleic acid into the host cell chromosome via a conserved “D,D(35)E” motif commonly found in members of a nucleotidyltransferase superfamily, which includes HIV-1 IN, other retroviral integrases, and bacterial and eukaryotic transposases;[3,4] and a C-terminus.[5]. Aptamers can serve a myriad of purposes including, among others, replacement of antibodies in flow cytometry, cell-phenotyping, and ELISA assays; biosensors to detect and quantify proteins; and inhibitors for the neutralization of bacterial toxins and other targets.[10−24] Currently, only one aptamer has received FDA approval (Macugen) for the treatment of macular degeneration, but several other candidates are in or entering the drug pipeline.[16,25] Recently a “primer-template mimicking” aptamer was used to aid in the crystallization and characterization of HIV and hepatitis B virus (HBV) reverse transcriptases (RT), uncovering yet another potential use for aptamers.[21,26,27] In the case of HIV-1, aptamers have been successfully used in vitro and display potent antiviral activity with minor cytopathic effects, as well as demonstrating efficacy in animal models.[14,27−37] Current DNA aptamers to IN are potent inhibitors of enzyme activity and resemble G-quadruplex structures.[1,12,27] In some cases, these aptamers can be targeted to both IN and the RNase H domain of HIV RT, as these proteins are structurally related through their phosphotransferase domains.[27,38,39] RNA aptamers containing G-rich sequences have been raised to IN and exhibited low nanomolar binding KD values.[11,40]. FANA is structurally and chemically distinct from F-RNA or RNA due to its difference in sugar pucker as an arabinose-based analogue and the presence of an electronegative fluorine atom in the 2′ position in the β conformation in place of a hydroxyl group.[59,60] The sugar adopts a C2′/O4′-endo conformation much like DNA, in stark contrast to the C3′-endo conformation seen by both FRNA and RNA.[44,59,61−63]

Methods

Results

Conclusion