TO CREATE A NEW TARGET-SPECIFIC CYTOTOXIC RNA & EXAMINE ITS VIABILITY FOR USE AGAINST HIV

This research project aims to create a new target-specific cytotoxic RNA and assess its viability to combat HIV (Human Immunodeficiency Virus). If an RNA aptamer against the HIV glycoprotein-120 (gp120) is linked to a cytotoxic siRNA, this RNA chimera could be used as a new approach to destroy HIV infected cells. To test the effectiveness of the therapeutic aptamer-small interfering RNA (siRNA) chimera, human HeLa cells were transfected with a reporter system that consists of a plasmid that carries three genes.  This plasmid encodes two reporter genes for Firefly Luciferase and Green Fluorescence Protein, as well as HIV gp160 viral gene, enabling expression of HIV gp120 on the cell surface, thereby mimicking an HIV-infected cell. The aptamer-siRNA chimera was first designed using known DNA sequences of cytotoxic siRNAs and the gp120 aptamer. A T-7 promoter sequence was added to the aptamer’s DNA sequence and was linked to the cytotoxic Bcl-2 siRNA. A Durascribe T7 transcription kit was used to transcribe the construct DNA into RNA. To test the aptamer-siRNA chimera, we transfected cells with the reporter system, then added the aptamer-siRNA chimera to the transfected cells. Results indicate that the GFP-P2A-Ffluc-T2A-gp160 lentivirus was successful in the infection and subsequent gene expression in the cells. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) shows the siRNA is effective in killing the gp120-expressing cells, while the aptamer portion is functional in delivering the siRNA to the gp120-expressing cells. Therefore, the aptamer-siRNA chimera is proven to be effective in targeting and eliminating HIV cells.