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

Area of Science:

• Biochemistry
• Molecular Biology
• Virology

Background:

• Human immunodeficiency virus type-1 Reverse Transcriptase (HIV-1 RT) is crucial for viral replication and a primary target for antiviral drugs.
• Drug resistance mutations in HIV-1 RT necessitate the development of new therapeutic strategies.

Purpose of the Study:

• To isolate and characterize DNA aptamers that specifically target the K103N/Y181C double mutant HIV-1 RT.
• To evaluate the inhibitory potential and binding characteristics of these aptamers against both mutant and wildtype HIV-1 RT.

Main Methods:

• Selection of DNA aptamers against the K103N/Y181C double mutant HIV-1 RT.
• Determination of IC50 values and kinetic binding affinity using surface plasmon resonance.
• NMR spectroscopy to elucidate the interaction between aptamer KY44 and HIV-1 RT.
• Assessment of KY44 aptamer's inhibitory activity against pseudo-HIV particle infection and cytotoxicity in HEK293 cells.

Main Results:

• Five DNA aptamers exhibited low IC50 values against both KY-mutant and wildtype (WT) HIV-1 RT.
• Surface plasmon resonance revealed binding affinities in the micromolar range for KY-mutant and WT HIV-1 RT.
• NMR studies confirmed that aptamer KY44 interacts with both WT and KY-mutant HIV-1 RT at the NNRTI drug binding pocket, specifically at methionine residues.
• Aptamer KY44 demonstrated nearly 80% inhibition of pseudo-HIV particle infection with low cytotoxicity in HEK293 cells.

Conclusions:

• The DNA aptamer KY44 effectively binds to and inhibits both wildtype and the K103N/Y181C double mutant HIV-1 RT.
• KY44 aptamer demonstrates potential as a novel therapeutic agent against HIV-1, including drug-resistant strains.
• The findings suggest aptamer-based therapies could overcome existing drug resistance mechanisms in HIV-1 treatment.