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

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 (K D,app ) of ∼50−100 pM in a buffer with 200 mM NaCl and 6 mM MgCl 2 . 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 20nucleotide 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.

Evolution of abiotic cubane chemistries in a nucleic acid aptamer allows selective recognition of a malaria biomarker

“…In contrast, preselection chemical modification-where modified nucleotides are directly included in the SELEX process-truly opens up directed evolution to extending the repertoire of aptamermediated molecular recognition (16)(17)(18)(19). Many chemical modifications have been directed at the level of the sugar-phosphate backbone in order to improve the nuclease resistance of aptamers (18,(20)(21)(22)(23). Other approaches include aptamers with artificially expanded genetic information systems going beyond the simple four building blocks of natural nucleic acids (24), even most recently to the eight-building-block Hachimoji system (25).…”

Improving Suppression of Hemipteran Vectors and Bacterial Pathogens of Citrus and Solanaceous Plants: Advances in Antisense Oligonucleotides (FANA)

“…Some of the credit in improved activity is the increased resistance to nuclease degradation, thus providing more "trigger" to the target within cells (Adeyinka et al, 2020;Crooke et al, 2020;Gruenke et al, 2020). One key trait is the gymnotic, "self-delivery" property of FANA oligos comes from the modifications of the phosphorothioate backbone, and modifications that produce a high affinity for proteins on the cell surface membrane promoting increase adsorptive endocytosis (Lebedeva et al, 2000;Souleimanian et al, 2012;Alves Ferreira-Bravo et al, 2015;Rose et al, 2019;Scoles et al, 2019;Ochoa and Milam, 2020). FANA are readily absorbed by the roots of citrus trees, potato, tomato, and okra, basil, and ornamental plants (sunflower, periwinkle, Orange jasmine, Murraya paniculata) and grapevines (see text footnote 1) (Hunter and Aishwarya, 2020;Pelz-Stelinski et al, 2020;Sandoval-Mojica et al, 2021).…”

Chemical Modification of Aptamers for Increased Binding Affinity in Diagnostic Applications: Current Status and Future Prospects

“…Recently, 2′F-ANA-modified aptamers have been identified via SELEX which possess affinity for the human integrase enzyme of the human immunodeficiency virus (HIV). This enzyme is responsible for the integration of viral DNA into the genome of the host and is crucial for the infection cycle of the virus [ 136 ]. The two 2′F-ANA-modified aptamers obtained after the selection process have dissociation constants in the 50–100 pM range, which is more than two orders of magnitude stronger compared to previously reported unmodified DNA and RNA aptamers.…”