Use of Capillary Electrophoresis to Study the Binding Interaction of Aptamers with Wild-Type, K103N, and Double Mutant (K103N/Y181C) HIV-1 RT Studying the Binding Interaction of Wild-Type, K103N, and Double Mutant (K103N/Y181C) HIV-1 RT with Aptamers by Performing the Capillary Electrophoresis

作者：Aeksiri Niran; Warakulwit Chompunuch; Hannongbua Supa; Unajak Sasimanas; Choowongkomon Kiattawee^*

来源：Applied Biochemistry and Biotechnology, 2017, 182(2): 546-558.

DOI：10.1007/s12010-016-2343-x

• HIV-1 reverse transcriptase
• Aptamer
• Capillary electrophoresis
• NECEEM
• Binding affinity

A number of nucleic acid aptamers with high affinities to human immunodeficiency virus reverse transcriptase (HIV-1 RT) are currently known. They can potentially be developed as broad-spectrum antiviral drugs, but there is little known about their binding interaction with mutant HIV-1 RT. Therefore, we utilized non-equilibrium capillary electrophoresis of equilibrium mixture (NECEEM) to study the interaction of three HIV-1 RTs (wild type, K103N, and double mutant (K103N/Y181C)) with RT1t49 and RT12 aptamers. This approach was used to study and evaluate the K (d) values of these molecules. The results showed that the K-d values of the tested aptamers were lower than that of the DNA substrate. The results also pointed out that RT1t49 could bind with all HIV-1 RTs and compete with the DNA substrate at the active site. Moreover, we studied the binding stoichiometry of HIV-1 RT using aptamers as probes. The findings showed evidence of two binding stoichiometries with HIV-1 RT and the RT12 aptamer but only one binding stoichiometry for RT1t49. In addition, RT1t49 could bind specifically with the wild-type, K103N, and double mutants in Escherichia coli lysate. This result also indicated that the aptamer could detect HIV-1 RT in the presence of E. coli lysate.