Synthesis of fluorescence labelled aptamers for use as low‑cost reagents in HIV/AIDS research and diagnostics

Abstract

Aptamers are nucleic acids selected by systematic evolution of ligands by exponential enrichment. They have potential as alternatives to antibodies in medical research and diagnostics, with the advantages of being non-immunogenic and relatively inexpensive to produce. In the present study, gp120 aptamers conjugated with fluorescein isothiocyanate (FITC) were generated, which could interact with HIV-1 gp120. A previously isolated gp120 aptamer, CSIR 1.1, was conjugated with FITC by incubation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and imidazole. The conjugation and binding to the glycoprotein were confirmed by flow cytometry. FITC conjugated aptamers showed an increase in fluorescence emission 24-fold higher than baseline, and this difference was statistically significant (P=0.0016). Compared with a commercially available biotinylated anti-gp120 antibody, detected using FITC conjugated streptavidin, the emission of fluorescence obtained from the FITC-conjugated aptamer was 8-fold higher, suggesting a stronger interaction with gp120. In addition, the FITC conjugated aptamer neutralized HIV-1 pseudoviruses with an average IC 50 of 21.3 nM, similar to the parent aptamer that had an IC 50 of 19.2 nM. However, the difference in inhibition between the two aptamers was not statistically significant (P= 0.784). These results indicate that the FITC-conjugated aptamer generated in the present study could potentially be used as a low-cost reagent in HIV/AIDS research and diagnostics.