title: 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.
abstract: 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 Kdvalues of these molecules. The results showed that the Kdvalues 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.
authors.affiliation: Interdisciplinary Program in Genetic Engineering, Graduate School, Kasetsart University, Chatuchak, Bangkok, 10900, Thailand.|Department of Agricultural Science, Faculty of Agriculture, Natural Resources and Environment, Naresuan University, Phitsanulok, 65000, Thailand.
authors.forename: Niran
authors.initials: N
authors.lastname: Aeksiri
authors.affiliation: Department of Chemistry, Faculty of Science, Kasetsart University, Chatuchak, Bangkok, 10900, Thailand.
authors.forename: Chompunuch
authors.initials: C
authors.lastname: Warakulwit
authors.affiliation: Department of Chemistry, Faculty of Science, Kasetsart University, Chatuchak, Bangkok, 10900, Thailand.
authors.forename: Supa
authors.initials: S
authors.lastname: Hannongbua
authors.affiliation: Department of Biochemistry, Faculty of Science, Kasetsart University, Chatuchak, Bangkok, 10900, Thailand.
authors.forename: Sasimanas
authors.initials: S
authors.lastname: Unajak
authors.affiliation: Interdisciplinary Program in Genetic Engineering, Graduate School, Kasetsart University, Chatuchak, Bangkok, 10900, Thailand. fsciktc@ku.ac.th.|Department of Biochemistry, Faculty of Science, Kasetsart University, Chatuchak, Bangkok, 10900, Thailand. fsciktc@ku.ac.th.|Center for Advanced Studies in Tropical Natural Resources, NRU-KU, Kasetsart University, Chatuchak, Bangkok, 10900, Thailand. fsciktc@ku.ac.th.
authors.forename: Kiattawee
authors.initials: K
authors.lastname: Choowongkomon
mesh_terms: D019943:Amino Acid Substitution; D052157:Aptamers, Nucleotide / Q000737:chemistry*; D019075:Electrophoresis, Capillary; D054303:HIV Reverse Transcriptase / Q000737:chemistry / Q000235:genetics; D015497:HIV-1 / Q000201:enzymology / Q000235:genetics; D006801:Humans; D020125:Mutation, Missense; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D052157:Aptamers, Nucleotide; D011994:Recombinant Proteins; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase
keywords: Aptamer; Binding affinity; Capillary electrophoresis; HIV-1 reverse transcriptase; NECEEM