Characterization of New DNA Aptamers for Anti‐HIV‐1 Reverse Transcriptase

Abstract

HIV‐1 RT is a necessary enzyme for retroviral replication, which is the main target for antiviral therapy against AIDS. Effective anti‐HIV‐1 RT drugs are divided into two groups; nucleoside inhibitors (NRTI) and non‐nucleoside inhibitors (NNRTI), which inhibit DNA polymerase. In this study, new DNA aptamers were isolated as anti‐HIV‐1 RT inhibitors. The selected DNA aptamer (WT62) presented with high affinity and inhibition against wild‐type (WT) HIV‐1 RT and gave a KD value of 75.10±0.29 nM and an IC50 value of 84.81±8.54 nM. Moreover, WT62 decreased the DNA polymerase function of K103 N/Y181 C double mutant (KY) HIV‐1 RT by around 80 %. Furthermore, the ITC results showed that this aptamer has small binding enthalpies with both WT and KY HIV‐1 RTs through which the complex might form a hydrophobic interaction or noncovalent bonding. The NMR result also suggested that the WT62 aptamer could bind with both WT and KY mutant HIV‐1 RTs at the connection domain.

Biophysical Characterization of Novel DNA Aptamers against K103N/Y181C Double Mutant HIV-1 Reverse Transcriptase

“…Generally, GNPs have an optical absorption spectrum around 520 nm and show a red color. Aggregation of GNPs shifts their optical absorption spectrum to a higher wavelength at around 630 nm and making them a purple color [ 20 , 21 ]. The complete reaction contained KY DNA aptamer, HIV-1 RT, GNPs, and NaCl and was performed to compare the binding affinity ratio between WT and mutant.…”

“…They reported that four methionines (M16, M184, M230, and M357) of HIV-1 RT represented the NNRTI drug binding pocket caused by the chemical shift upon binding to NNRTIs. Our previous report investigated the binding complex of HIV-1 RTs and a DNA aptamer by 1 H- 13 C heteronuclear single-quantum coherence (HSQC) and showed that the aptamer affected the NNRTI drug binding pocket of HIV-1 RT [ 20 ]. In this research, we proposed to study the interaction between KY DNA aptamers isolated from KY double mutant HIV-1 RT by NMR.…”

“…To select high-affinity KY-mutant HIV-1 RT DNA aptamers, a gold nanoparticles (GNPs)-based colorimetric assay was used. The GNPs-based colorimetric assay followed the procedures of our previous work [ 20 , 34 ]. The complete GNPs (Sigma-Aldrich, Saint Louis, MO, USA) reaction was composed of 5 μL of 100 ng/μL PCR products (DNA aptamers), 37.5 μL of 15 nm GNPs, 5 μL of 50 ng/μL protein target (WT or KY HIV-1 RTs) and 2.5 μL of 2 M NaCl.…”

“…SPR (OpenSPR™; Nicoya Lifesciences, ON, Canada) was used to determine the affinities of the aptamer complexes WT and KY HIV-1 RTs. Measurement of KD values by using OpenSPR™ was performed according to Ratanabunyong et al [ 20 ]. Each DNA aptamer which was used as the analyte was diluted in a 2-fold serial dilution ranging from 125 to 1000 nM in SPR running buffer containing 10 mM HEPES pH 7.5, 150 mM NaCl, 80 mM KCl, and 5 mM MgCl 2 .…”

“…In the anti-HIV-1 integrase aptamer T30175 the individual thymidine forming loops were replaced by 5-hydroxymethyl-2-deoxyuridine residues (H) [ 19 ]. Additionally, our research found that aptamers inhibited WT HIV-1 RT activity at an IC50 value of 84.81 ± 8.54 nM and a KD value of 75.10 ± 0.29 nM [ 20 ]. This research aimed to isolate potential specific anti-HIV-1 RT DNA aptamers against K103N/Y181C double mutant (KY) HIV-1 RT.…”