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

Abstract

The human immunodeficiency virus type-1 Reverse Transcriptase (HIV-1 RT) plays a pivotal role in essential viral replication and is the main target for antiviral therapy. The anti-HIV-1 RT drugs address resistance-associated mutations. This research focused on isolating the potential specific DNA aptamers against K103N/Y181C double mutant HIV-1 RT. Five DNA aptamers showed low IC50 values against both the KY-mutant HIV-1 RT and wildtype (WT) HIV-1 RT. The kinetic binding affinity forms surface plasmon resonance of both KY-mutant and WT HIV-1 RTs in the range of 0.06–2 μM and 0.15–2 μM, respectively. Among these aptamers, the KY44 aptamer was chosen to study the interaction of HIV-1 RTs-DNA aptamer complex by NMR experiments. The NMR results indicate that the aptamer could interact with both WT and KY-mutant HIV-1 RT at the NNRTI drug binding pocket by inducing a chemical shift at methionine residues. Furthermore, KY44 could inhibit pseudo-HIV particle infection in HEK293 cells with nearly 80% inhibition and showed low cytotoxicity on HEK293 cells. These together indicated that the KY44 aptamer could be a potential inhibitor of both WT and KY-mutant HIV-RT.

Application of the Nicoya OpenSPR to Studies of Biomolecular Binding: A Review of the Literature from 2016 to 2022

“…SPR is a powerful tool for real-time, label-free measurement of binding affinity and kinetics, and the vast majority of papers citing the use of the OpenSPR fit neatly underneath this umbrella [ 3 , 5 , 7 , 9 , 15 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 ,…”

“…There are a wide variety of reasons investigators use SPR, and some of the most common applications are assessing the binding of a potential therapeutic to its target [ 33 , 37 , 44 , 53 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 65 , 66 , 67 , 80 , 84 , 169 , 176 , 179 , 181 , 207 ] or to measure the affinity of a potential inhibitor [ 23 , 25 , 46 , 86 , 89 , 98 , 105 , 114 , 115 , 123 , 133 , 143 , 148 , 158 , 159 , 171 , 182 , 186 , 187 , 204 ]. The proposed therapeutics were sometimes novel compounds, such as the tasine derivate compounds synthesized by Yang and coworkers to suppress HeLa cells [ 33 ] or the bioactive iridium metal-complex developed by Ji et al to eliminate excess reactive oxygen species (ROS) induced by spinal cord injuries (SCI) [ 179 ].…”

“…Researchers also investigated repurposing existing drugs to address critical public health problems, such as the work by Ge and coworkers to assess whether the antihistamine azelastine could be used to treat COVID-19 [ 57 ]. Many proposed therapeutics are small molecules, but certainly not all of them, such as the work by Ratanabunyong et al to assess the efficacy of an aptamer (short, single-stranded nucleic acid) inhibitor of human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT) [ 37 ]. Antibodies and antibody derivatives are also used as drugs, such as the artificial antibody-like peptide that Zheng et al use to target the biomarker HER2 from the human epidermal growth factor receptor family [ 169 ].…”

“…LSPR can be accomplished using affordable bench-top instrumentation, one example of which is the Nicoya OpenSPR (Nicoya Life Sciences, Waterloo, ON, Canada). The instrument has been used with a broad range of analytes including small molecules [ 23 , 24 , 25 , 26 ], peptides [ 27 , 28 , 29 , 30 ], proteins [ 27 , 28 , 29 , 31 , 32 , 33 ], nucleic acids [ 34 , 35 , 36 , 37 ], carbohydrates [ 38 , 39 , 40 ], and lipids [ 41 , 42 ]. Nicoya offers a wide selection of surface chemistries to be used with the OpenSPR instrument, ranging from the most popular carboxyl immobilization to more specific applications, such as liposome sensors.…”

Targeted inhibition of methicillin-resistant Staphylococcus aureus biofilm formation by a graphene oxide-loaded aptamer/berberine bifunctional complex

“…selected aptamer 1, which is specific to the hemagglutinin (HA) protein of the H1N1 influenza virus and can inhibit virus-induced hemagglutination and proliferation in host cells (Li et al., 2016 ). Additionally, several other aptamers have been developed as therapeutics for the treatment of Mycobacterium tuberculosis , HIV, human papilloma virus, and SARS-CoV-2 via mechanisms impairing the function of target proteins (Nicol et al., 2013 ; Srivastava et al., 2021 ; Sun et al., 2021 ; Ratanabunyong et al., 2022 ). Hence, the use of aptamers as inhibitors is a promising development with potentially extensive therapeutic benefits.…”