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

2.1. Post Selection

To screen the high binding DNA aptamers on HIV-1 RTs (WT and KY), the GNPs-based colorimetric assay was used. This method was used to characterize the DNA aptamers that can complex with HIV-1 RTs with high affinities by detecting the color changes. 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. Sixty-one KY DNA aptamers were tested against WT and KY HIV-1 RTs. DNA aptamers were heated and chilled on ice to form the secondary structure before being used. The reactions were measured by a spectrophotometer in the scanning mode and the spectra were collected (Figure 1A). The fold change of 630/520 nm ratios continued to be calculated and Figure 1B shows the ratio of binding affinity of KY DNA aptamers to KY HIV-1 RT. Twenty-eight aptamers changed the color of GNPs to purple with at least a 2.5-fold increase in absorption. This approach is suitable for use as colorimetric systems for the selection of DNA aptamers binding to HIV-1 RTs. Thirteen DNA aptamers (KY1, KY22, KY23, KY24, KY28, KY41, KY42, KY43, KY44, KY56, KY57, KY58, and KY62) that bound to HIV-1 RTs with a high affinity (ranging from 2–4 fold change) were sampled to test for anti-HIV-1 RT activity.

2.2. Study of the Function of Aptamers on HIV-1 RTs Activity

The selected KY DNA aptamers were used to determine their activity against HIV-1 RTs. The HIV-1 RTs inhibitory experiment used the EnzChek^® Reverse Transcriptase Kit [22]. This experiment was used to determine the activity of DNA aptamers on HIV-1 RTs polymerase function and used 0.5 μM Efavirenz (EFV) as the positive control. Thirteen selected KY DNA aptamers (KY1, KY22, KY23, KY24, KY28, KY41, KY42, KY43, KY44, KY56, KY57, KY58, and KY62) were amplified and heated before being used. Figure 2A represents the relative inhibition of KY DNA aptamers on KY-mutant HIV-1 RT activity. The results show that KY1, KY22, KY23, KY28, and KY44 had more activity than other aptamers by inhibiting the KY-mutant HIV-1 RT activity at 60–85% relative inhibition and more than EFV which showed around 52% relative inhibition. Five KY aptamers were chosen to synthesize. To prove the function of synthesized KY aptamers, five selected KY aptamers were prepared to test against both WT and KY HIV-1 RTs. EFV at a concentration of 0.5 μM was used as the positive control for HIV-1 RTs activity. The results demonstrate that all five synthesized aptamers could inhibit both WT and KY HIV-1 RTs function by providing a % relative inhibition of more than 80% whereas EFV showed 85% and 50% relative inhibition on WT and KY HIV-1 RTs activity, respectively. This experiment confirmed that the 5 selected KY DNA aptamers could inhibit the HIV-1 RTs DNA polymerization function of both WT and KY double mutants. These five synthesized KY aptamers were further used to determine the IC50 values on the DNA polymerase function of HIV-1 RTs.

In a further study, the synthesized KY DNA aptamers were used to determine the IC50 values on HIV-1 RTs DNA polymerase function. The aptamer samples were diluted in the range of 0.46–9000 nM in 3-fold dilution with reaction buffer. The samples were heated and chilled on ice to form the secondary structure. The RT reaction contained 5 mL of each DNA aptamer, 5 mL of 25 nM HIV-1 RTs (WT or KY), and 15 mL of template/primer. Figure 3 shows the IC50 values of the five synthesized DNA aptamers on WT and KY HIV-1 RTs. EFV was used as the positive control. The synthesized KY DNA aptamers showed IC50 in the nanomolar range both of WT and KY HIV-1 RTs (Table 1). KY44 aptamer showed the highest activity on WT and KY HIV-1 RTs DNA polymerase function with IC50 values equal to 45.14 ± 5.75 and 63.82 ± 8.72 nM, respectively. Interestingly, EFV showed IC50 values at 16.03 + 3.83 nM on WT HIV-1 RT and 399.30 + 4.86 nM on KY HIV-1 RT. Five synthesized aptamers (KY1, KY22, KY23, KY28, and KY44) were studied for the binding kinetics by SPR experiment comparing WT and KY HIV-1 RTs.

2.3. Affinity Analysis of KY DNA Aptamers and HIV-1 RTs

To characterize the binding kinetics of the HIV-1 RTs-aptamer complexes, an SPR experiment was performed. Generally, Surface Plasmon Resonance or SPR was used to study the interaction between targets immobilized at a solid-liquid interface and partners in solution of the label-free system; and the analysis was performed in real time [23]. The five selected KY aptamers were prepared in SPR running buffer in a 2-fold serial dilution ranging from 125–1000 nM. The results show that all DNA aptamers interacted with both WT and KY HIV-1 RTs. These bindings revealed KD values between 0.55 and 12 μM as shown in Table 2. KY23 displayed the highest binding affinity to both WT and KY HIV-1 RTs. These bindings showed KD values at 5.52 ± 0.4 × 10⁻⁸ and 1.46 ± 0.005 × 10⁻⁷, respectively. Moreover, ITC experiments were performed to study the protein-ligand interaction by measuring the heat change of the reaction.

The binding of protein and ligand normally induces a heat change, the interaction particularly provided the reduction in heat; and it is identified as an endothermic property, whereas the increase in heat is termed an exothermic property. Based on the activity of DNA aptamers, KY44 was chosen to study the interaction between aptamer-HIV-1 RTs binding complexes by an NMR experiment.

2.4. Investigation of HIV-1 RTs Binding to DNA Aptamer by NMR

Nuclear magnetic resonance or NMR was used to study the interaction between protein and ligand. In a previous study, Thammaporn et al. [24] showed the HIV-1 RT-NNRTIs interactions by NMR. 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 ¹H-¹³C 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. Figure 4 shows the HSQC spectra of HIV-1 RTs-KY DNA aptamer complexes. WT HIV-1 RT-KY44 aptamer complexes (green spectra) caused a chemical shift in the methionine at the position 357 and had a small effect on M184 (Figure 4A). The complex of KY HIV-1 RT-aptamer displayed a small effect on M184, M230, and M357 as shown in Figure 4B. These positions are related to the NNRTI drug binding pocket, meaning that the KY44 aptamer affected the NNRTI drug binding pocket of HIV-1 RTs.

2.5. Toxicity Testing on HEK293 Cells

The KY44 DNA aptamer and EFV were prepared to analyze the cytotoxicity on HEK293 cells. The inhibitors were diluted to 0.076-500 μM in a 3-fold serial dilution. HEK293 cells were treated with HIV inhibitors in a CO₂ incubator. After 72 h, the culture medium was removed then replaced with Presto Blue (10-fold dilution in culture medium) at 37 °C for at least 1 h. The treated cells were measured at the wavelengths of 570/610 nm and calculated for the percentage of cell survival. The results show that EFV affected HEK293 cells with IC50 values equal to 39.95 ± 2.25 μM; whereas KY44 had an effect at the high concentration with nearly 20% cell death as shown in Figure 5. Therefore, the viral infection was studied by using 20 μM of inhibitors.

2.6. Inhibition of HIV Infection

To elucidate that the KY44 DNA aptamer could inhibit HIV infection, an HIV pseudotyped virus system was investigated. Uninfected and pseudo-HIV-1 particle-infected cells were used as the infection control. The viral infections were determined by qPCR [25]. The results revealed that KY44 aptamer also inhibited the infection of the pseudo-HIV-1 particles in HEK293 cells compared with NVP and EFV in a co-culture system (Figure 6). Data were analyzed by Dunnett’s multiple comparisons test (Graphpad Prism 6) and compared between EFV and KY44 (p < 0.05). The analyzed data of KY44 could decrease pseudo-HIV-1 particles infecting HEK293 cells when compared to EFV.

4.1. DNA Aptamer Preparation

The selected KY DNA aptamers were amplified by specific primers: forward primer 5′-CTTCTGCCCGCCTCCTTCC-3′ and reverse primer 5′AGTGTCCGCCTATCTCGTCTC C-3′ (synthesized by Integrated DNA Technology; IDT Singapore). For the amplification step, 20 μL of the PCR mixture was prepared consisting of 2 μL of 10X reaction buffer, 2 μL of 2.5 mM dNTP mix, 1 μL of 5 μM forward primer, 1 μL of 5 μM reverse primer, 1  μL of each KY DNA aptamer plasmid, and 0.2 μL of DreamTaq™ DNA Polymerase (Thermo Scientific™, Waltham, MA, USA), and the volume was adjusted to 20 μL with nuclease free water. PCR products were purified and dissolved in nuclease free water. The samples were stored at −20 °C prior to use. The synthetic ssDNA aptamers were synthesized by Macrogen (Seoul, Korea) and dissolved in nuclease free water to a stock concentration of 200 μM and stored at −20 °C prior to use. The sequences of the synthetic ssDNA aptamers were as follows:

KY1: AGTAAGACTATAATTCAGTCCTCATTGATTTCCCACATCG

KY22: CACACTAAGGGAGTAGGAATCCGCGGTCACAATGTTCCTG

KY23: CTATAGGGGGTTGCATCTTAGACATTAATGTTCATTTACA

KY28: CCAGAAAGCTCGGGTTCGTTCCACAAATAACACTTGCCC

KY44: GTGCAAGCGTACGAAGGGATAATTCACTCTAGGAGCTTCT

4.2. In Vitro Selection of High-Affinity DNA Aptamers for KY-Mutant HIV-1 RT

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. Each DNA aptamer was heated at 95 °C for 5 min and chilled on ice for 5 min followed by the addition of GNPs, protein target, and NaCl. The samples were incubated at room temperature for 5 min before being transferred to a 1 cm path length quartz cuvette for measurement of the absorbance spectra. The color changes were also detected by spectrophotometer (Varian Cary^® 50 UV-Vis Spectrophotometer, Victoria, Australia) in the scanning mode. The absorption spectra changes of GNPs solutions in the presence or absence of targets were determined. The absorbance ratios were calculated from the absorbance of 630 nm divided by 520 nm and compared with or without target protein. A higher ratio of absorbance at 520 nm was obtained from GNPs and at 630 nm was obtained from DNA aptamers complexed with HIV-1 RTs. Fold change of the absorbance was calculated by using the following Equation (1).

C = Control: complete reaction without HIV-1 RT

T = Test: complete reaction contained HIV-1 RT

4.3. HIV-1 Reverse Transcriptase Inhibition Assay

The inhibition assay was conducted by using the EnzChek^® Reverse Transcriptase Assay Kit (Invitrogen™ E-22064, Life Technologies, Carlsbad, CA, USA) following Silprasit et al. [22]. In brief, to conduct the HIV-1 RT inhibitory assay, 5 µL of 2.5 µg/µL of each aptamer sample was pipetted into a 96-well black plate. The negative control consisted of 5 µL of sample buffer, and 5 µL of 2.5 µM Efavirenz as the positive control. After that, 15 µL of template/primer polymerization buffer was added into each well. In the background control the complete HIV-1 RT reaction containing each aptamer and 2 µL of 0.2 M EDTA (stop solution) were used. The reaction was started by adding 5 µL of 25 nM purified recombinant WT or KY HIV-1 RTs and incubated at 25 °C for 30 min. The reactions were stopped by adding 2 µL of 0.2 M EDTA and measured by using the PicoGreen fluorometric method with Tecan Infinite^® 200 PRO (DKSH (Thailand) Limited) with an excitation/emission wavelength at 485/535 nm. The inhibitory effects on HIV-1 RTs’ activity were compared by the percentage of relative inhibition, which was measured by using the following Equation (2).

4.4. Fifty Percent Inhibitory Concentration (IC50) Determination

For IC50 calculations, each DNA aptamer was prepared in a 3-fold serial dilution with increases through concentrations of 0.46–9000 nM. The reactions were performed in 96-well black plates by adding 5 µL of each KY DNA aptamer, 5 µL of 25 nM purified WT or KY HIV-1 RTs followed by 15 µL of the template/primer polymerization buffer into each well. The reactions were incubated at 25 °C for 30 min. Afterwards, 2 µL of 0.2 M EDTA, the stop solution, was added to stop the reaction. The background control consisted of the complete HIV-1 RT reaction components including each aptamer and 2 µL of 0.2 M EDTA added to stop the reaction. The reaction results were determined by using the PicoGreen fluorometric method. Nonlinear regression dose-response curves plotted percent inhibition and log inhibitor concentration. The 50% inhibition concentrations (IC50) were analyzed using the GraphPad Prism program (GraphPad Software Inc. version 6 access on 1 October 2021, San Diego, CA, USA).

4.5. Surface Plasmon Resonance (SPR)

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 MgCl₂. The WT or KY HIV-1 RTs (ligand) were prepared in SPR running buffer to make a concentration of 1 μM and was used for immobilization on NTA sensor chips (Nicoya Lifesciences; Kitchener, ON, Canada). NTA sensor chips were immobilized with 200 µL of 1 mM WT or KY HIV-1 RTs using a flow rate of 20 µL/min. The increasing concentrations of each DNA aptamer were injected over an NTA sensor chip. The binding was measured by using the association period of 90 s, followed by 180 s of running buffer for dissociation time. All of the SPR experiments were completed at 25 °C in running buffer using a flow rate of 50 µL/min. Binding parameters were calculated by using TraceDrawer Software version 1.6.1 access on 12 July 2021 (Ridgeview Instruments AB, Uppsala, Sweden) with the model of 1:1 binding, based on the theory that the immobilized target protein binds to the aptamers in a 1:1 ratio.

4.6. Nuclear Magnetic Resonance (NMR)

To determine the structure-activity relationship of HIV-1 RT complexed with DNA aptamers, an NMR experiment was performed following Thammaporn et al. [24]. NMR is an effective method for analyzing HIV-1 RT binding to drugs. The results show the ¹H-¹³C heteronuclear single-quantum coherence (HSQC) data of HIV-1 RT-NNRTIs. The ¹H-¹³C HSQC spectra of WT or KY HIV-1 RTs labeled at methyl-¹³C-methionine on the p66 subunit were measured in the presence and absence of DNA aptamers. The HIV-1 RT and DNA aptamers were prepared in 10 mM Tris-d11 buffer (pD 7.6) containing 200 mM KCl, 1.5 mM sodium azide, and 4 mM MgCl₂. Twenty-eight micromolar HIV-1 RT was complexed with 140 mM DNA aptamers (1:5 molar ratio). The reaction was measured by using an AVANCE800 (Bruker BioSpin; Karlsruhe, Germany) spectrometer supplied with a cryogenic probe. The spectra data were prepared and analyzed by the Topspin 3.2 (Bruker BioSpin; Karlsruhe, Germany) and SPARKY 3.115 programs (San Francisco, CA, USA).

4.7. Cytotoxicity on HEK293 Cells

The cytotoxicity assay was performed by following Engelberg et al. [35] (Engelberg, Netzer, Assaraf, and Livney, 2019). HEK293 cells (ATCC, CRL-1573) were cultured in DMEM containing 10% FBS and 1% antibiotics. Cells were seeded in a 96-well plate at the density of 1 × 10⁵ cells/mL and incubated at 37 °C in an atmosphere containing 5% CO₂ overnight. The KY44 aptamer and EFV were diluted in DMEM complete medium to make the concentration in the range of 0.076–500 μM (3-fold serial dilution). The culture medium was removed and fresh medium containing KY44 aptamer or EFV at the various concentrations were added into the desired wells. The treated cells were incubated at 37 °C in an atmosphere containing 5% CO₂ for 72 h. After that, culture medium was removed and replaced with Presto Blue^® Cell Viability Reagent (Carlsbad, CA, USA) diluted to 1:10 with the complete medium. Cells were incubated in a CO₂ incubator for at least 30 min and the absorbance of the reagent at 570 and 610 nm was measured. The percent cell survival was calculated by using the following Equation (3).

4.8. Pseudo-HIV-1 Particle Infection

The lentiviral system was used to propagate the pseudo-HIV-1 particles by co-transfected pfNL43-dE-EGFP (which was a gift from Mario Chin (Addgene plasmid # 36865) and was a gift from Didier Trono (Addgene plasmid # 12259) into HEK293 cells by using the lentiviral system following Tiscornia et al. [36]. HEK293 cells were seeded in a 100 mm dish at the density of 5 × 10⁶ cells and incubated at 37 °C in an atmosphere containing 5% CO₂ overnight. Cells were transfected with 10 μg pfNL43 and 5 µg pMD2.G with lipofectamine 3000 and incubated at 37 °C in an atmosphere containing 5% CO₂ for 72 h. The pseudo-HIV-1 particles were harvested and stored at −80 °C before use. For the anti-HIV infection determination, HEK293 cells were seeded in 6-well plates at the density of 5 × 10⁵ cells/well and incubated overnight at 37 °C in an atmosphere containing 5% CO₂ overnight. The culture medium was removed and cells were infected with 400 µL of pseudo-HIV-1 particles containing 20 µM of the KY44 aptamer, NVP, or EFV into desired wells. The infected cells were incubated for 1 h by rocking every 15 min. The infected cells were amended with DMEM complete medium and incubated at 37 °C in an atmosphere containing 5% CO₂ for 72 h. The infected cells were harvested and the viral DNA was extracted [37]. The percentage of pseudo-HIV-1 particle infection was determined by used qPCR with the HIV LTR specific primer (forward primer 5′-GCGCTTCAGCAAGCCGAGTCCT-3′ and reverse primer 5′-CACACCTCAGGTACCTTTAA GA-3′) [25].