Synthesis of a Fitc Conjugated Anti-gp120 Aptamer for the Detection of HIV Infected Cells By Flow Cytometry

Aptamers are nucleic acid molecules generated by systematic evolution of ligands by exponential Enrichment or SELEX. These molecules potentially have an important diagnostic and analytical application given their ability to bind different targets with high affinity and specificity. Currently, antibodies are the most commonly used molecules for these purposes in applications such as flow cytometry and ELISA. However, antibodies do also have various limitations. For example, they are often expensive to generate as they require the use of animals; and some target molecules may have limited immunogenicity. It is in this context that aptamers can be used as substitutes for antibodies since they are generated by a relatively inexpensive chemical synthesis. They can also be designed to bind any target.

The viral protein gp120 plays an important role in HIV-1 life cycle as it promotes the virus entry into target cells. Once a productive infection is established infected cells can be distinguished from uninfected ones by their expression of gp120 on their cellular membrane.

Thus, the aim of this study is to synthesize a FITC-conjugated anti-gp120 aptamer for the detection of HIV-1 infected cells by flow cytometry.    

We isolated a gp120 binding RNA aptamer and showed that it inhibit HIV-1 infectivity. We showed that this aptamer tightly binds a consensus HIV-1 subtype C gp120. We mapped its binding sites on gp120 and found that it interacts with the coreceptor binding site on the viral glycoprotein, at the base of the V3 loop, and in the bridging sheet within the conserved V1/V2 stem-loop. We now plan to conjugate this oligonucleotide to fluorescein (FITC) by standard solid phase chemistry using cyanoethyl phosphophoramidites. The ability of the FITC-conjugated aptamer to specifically bind gp120 will be tested by flow cytometry using the cell lines 293T expressing gp120 on their surfaces. Parent 293T cells, not expressing gp120, will be used as controls.

The final outcome of this project is a FITC-conjugated aptamer capable of detecting HIV-1 infected cells by flow cytometry.