Fluorescent Markers For Computer Modeled Antisense Peptide Nucleic Acids Against Simian Virus 40 Large T-Antigen

Abstract

Peptide Nucleic Acids (PNA's) are DNA analogues which have had the traditional phosphodiester backbone replaced by an uncharged peptide backbone. PNA's have shown promise as possible antisense inhibitors of gene expression (Hanvey et al. 1992). The study of the antisense effects of a PNA is demonstrable using microinjection to deliver the PNA into a cell's nucleus along with a gene marker of microinjection efficiency, beta galaactocidase (Bgal), and target gene Simian Virus 40 large T antigen (5V40 TAg). Expression of SV40 TAg in comparison to marker Bgal expression is assayed by dual immunofluorescence. For this thesis, an antisense PNA was targeted against SV40 TAg, and a novel reporter system was devised. The first criterion for the selection of an antisense PNA against SV40 TAg was that the PNA mismatch any known human gene by at least two bases. The second criterion for antisense PNA selection was to find a target site as near to the SV40 TAg translation start site as possible. FastA searches of available genetic databases such as GenBank were used to locate a target site in SV40 TAg mRNA. Computer models of the SV40 TAg mRNA secondary structure predicted that this sequence, an 18mer, was part of a stem loop structure thirteen bases downstream of the start site with ten unpaired bases comprising a loop structure and the rest of the bases involved in intramolecular base pairing within the stem. The antisense PNA selected by these computer searches, AS7, was constructed and tested in several cultured cell types. The inhibitory effect achieved by AS7 was nearly 100% with no discernable antisense side effects. For the detection of antisense effect, a Green Fluorescent Protein - SV40 TAg plasmid (GFPT) was constructed which expressed SV40 TAg fused to the amino end of Green Fluorescent Protein(GFP). GFPT provided target gene expression with high efficiency. Three markers of microinjection efficiency were tested. Markers, in descending order of effectiveness, were BGal, red fluorescent beads, and blue fluorescent protein (BFP).