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Liposome-mediated transfection of circular TAR RNA decoys into human cells.




 

HIV Pathog Treat Conf. 1998 Mar 13-19;:42 (abstract no. 1014). Unique

The HIV-1 TAR RNA element is a 59 nucleotide stem-bulge-loop structure that forms the 5' end of all HIV-1 transcripts and is an important regulator of HIV-1 transcription. A small circular TAR RNA decoy was prepared using the self-splicing activity of a group I permuted intron-exon. Unspliced linear precursor RNA was synthesized in vitro using T7 RNA polymerase. This precursor RNA self-spliced to form a circular product and two linear products. The nucleotide sequence of the precursor RNA was modified such that the circular product contained stem, bulge, and loop sequences from the HIV-1 TAR RNA element. The autocatalytic splicing reaction was very efficient, converting greater than 95 percent of the precursor RNA into products. Gel filtration chromatography was used to purify milligram quantities of circular TAR decoys for biochemical and cellular transfection experiments. In comparison to linear TAR decoys, circular TAR RNA decoys were found to be superior biochemical reagents because they were very stable in cellular extracts whereas linear TAR decoys were rapidly degraded. Radiolabelled circular TAR decoys were also found to be very stable in 293T cells after transfection using a liposome carrier. Circular TAR decoys appear to mimic the endogenous TAR structure because they were able to specifically inhibit Tat-activated HIV-1 transcription in a cell-free transcription system. Current experiments involve testing if transfected circular TAR decoys inhibit HIV-1 transcription in vivo.

MEETING ABSTRACTS Chromatography, Gel Exons Human Introns Liposomes RNA, Messenger/*GENETICS/ISOLATION & PURIF RNA, Viral/*GENETICS/ISOLATION & PURIF Transfection/*METHODS Viral Proteins/GENETICS



 




Information in this article was accurate in December 30, 1998. The state of the art may have changed since the publication date. This material is designed to support, not replace, the relationship that exists between you and your doctor. Always discuss treatment options with a doctor who specializes in treating HIV.