Selection of 2′-deoxy-2′-fluoroarabinonucleotide (FANA) aptamers that bind HIV-1 reverse transcriptase with picomolar affinity

Abstract

Using a Systematic Evolution of Ligands by Exponential Enrichment (SELEX) protocol capable of selecting xeno-nucleic acid (XNA) aptamers, a 2′-deoxy-2′-fluoroarabinonucleotide (FANA) aptamer (referred to as FA1) to HIV-1 reverse transcriptase (HIV-1 RT) was selected. FA1 bound HIV-1 RT with KD,app values in the low pM range under different ionic conditions. Comparisons to published HIV-1 RT RNA and DNA aptamers indicated that FA1 bound at least as well as these aptamers. FA1 contained a 20 nucleotide 5′ DNA sequence followed by a 57 nucleotide region of FANA nucleotides. Removal of the fourteen 5′ DNA nucleotides did not affect binding. FA1's predicted structure was composed of four stems and four loops. All stem nucleotides could be modified to G-C base pairs (14 total changes) with a small effect on binding. Eliminating or altering most loop sequences reduced or abolished tight binding. Overall, results suggested that the structure and the sequence of FA1 were important for binding. FA1 showed strong inhibition of HIV-1 RT in extension assays while no specific binding to avian myeloblastosis or Moloney murine leukemia RTs was detected. A complete DNA version of FA1 showed low binding to HIV-1 RT, emphasizing the unique properties of FANA in HIV-1 RT binding.

In vitroevolution of chemically-modified nucleic acid aptamers: Pros and cons, and comprehensive selection strategies

“…reported a 2′-FANA aptamer to human immunodeficiency virus-1 (HIV-1) reverse transcriptase 51 . This 2′-FANA aptamer was isolated from a 2′-FANA modified DNA pool through SELEX using electrophoretic mobility shift assay 51 .…”

“…reported a 2′-FANA aptamer to human immunodeficiency virus-1 (HIV-1) reverse transcriptase 51 . This 2′-FANA aptamer was isolated from a 2′-FANA modified DNA pool through SELEX using electrophoretic mobility shift assay 51 . The developed 2′-FANA modified DNA aptamer, FA 1 had a K d value of 4 pM and showed greater resistance to nucleases 51 …”

Future strategies for the discovery of therapeutic aptamers

“…2'F, 2'OMe, and 2'NH 2 nucleotides have been incorporated into RNA aptamers with Y639F mutant T7 RNA polymerase [1]. Other chemically modified nucleotides such as 2'-deoxy-2'-fluoroarabinonucleotide (FANA) [11], 2'-O,4'-C-methylene bridged/locked nucleic acid (2',4'-BNA/LNA)[12], and C2'-O-methyl(C2'-OMe)/ C2'-Fluorine (C2'-F) [13] have been incorporated into aptamers with engineered polymerase. The 2'-O-carbamoyl uridine (U cm ) is successfully incorporated by a wild-type T7 RNA polymerase [14].…”

“…To increase serum stability, in-SELEX and post-SELEX have been applied to incorporate modified nucleotide into aptamers. So far, it is very encouraging that 2′F, 2'OMe, 2'NH 2 [1], and newly developed modified nucleotides [11–14] have been successfully incorporated with the engineered polymerases in the in-SELEX. Currently available modified nucleotides are modified at the 2′-position of ribose.…”

The expanding world of DNA and RNA

“…Kuwahara and co-workers used a mixture of mutant and/or wild type KOD DNA polymerases to “transcribe” and “reverse transcribe” mixed LNA/DNA oligonucleotides during selection of an anti-thrombin aptamer [15,16]. Holliger and co-workers have dramatically expanded the potential of these analogs by evolving a series of TgoT DNA polymerase mutants to better “transcribe” and “reverse transcribe” these modified nucleotides, and they have used them to select HNA aptamers targeting HIV trans-activating response RNA and hen egg lysozyme [17•], a FANA aptamer targeting HIV-1 RT [18], and catalytic nucleic acids containing ANA, FANA, HNA, and CeNA moieties [19]. In addition, we have evolved several mutants of the Stoffel fragment that better “transcribe” and “reverse transcribe” fully modified 2’-F and 2’-OMe oligonucleotides [11].…”