DNA aptamers to human immunodeficiency virus reverse transcriptase selected by a primer-free SELEX method: characterization and comparison with other aptamers.

Nucleic Acid Therapeutics
Yi-Tak Lai, Jeffrey J DeStefano

Abstract

A 30-nucleotide DNA aptamer (5'-AGGAAGGCTTTAGGTCTGAGATCTCGGAAT-3', denoted PF1) selected for high affinity to human immunodeficiency virus reverse transcriptase (HIV RT) using a primer-free SELEX (systematic evolution of ligands by exponential enrichment) method was characterized to determine features promoting tight binding. PF1's equilibrium dissociation constant for RT was ∼80 nM, over 10-fold lower than a random 30-mer. Changing the 2 terminal diguanosine repeats (underlined above) to diadenosine or dithymidine modestly decreased binding. Any changes to the 2 central diguanosines dramatically decreased binding. Binding was highly sensitive to length, with any truncations that deleted part of the 4 diguanosine motifs resulting in a 6-fold or more decrease in affinity. Even a construct with all the diguanosine motifs but lacking the 5' terminal A and 3 nucleotides at the 3' end showed ∼3-fold binding decrease. Changes to the nucleotides between the diguanosines, even those that did not alter PF1's low secondary structure (free energy of folding ΔG=-0.61 kcal/mol), dramatically decreased binding, suggesting sequence specificity. Despite the diguanosine motifs, circular dichroism (CD) spectra indicated that PF1 did not form a G...Continue Reading

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Citations

Jul 23, 2015·BioMed Research International·Ka Lok Hong, Letha J Sooter
Feb 7, 2018·International Journal of Molecular Sciences·Maria A VorobyevaAlya G Venyaminova
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Apr 17, 2020·Journal of the American Chemical Society·Matthew R DunnJohn C Chaput
Aug 28, 2021·International Journal of Molecular Sciences·Katja Schilling-LoefflerJacquelina Williams-Woods

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Methods Mentioned

BETA
electrophoresis
SELEX
circular dichroism
electrophoreses
Assay
competition binding
filter binding
filter
equilibrium binding
nuclear

Software Mentioned

mfold
SigmaPlot

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