Site-specific cleavage of tobacco mosaic virus RNA: A study of factors influencing the cleavage

Abstract

DNA oligomer directed ribonuclease H (RNase H) methodology is applied to specifically cleave tobacco mosaic virus (TMV) RNA. Using a synthetic DNA oligomer P(dT8)dCdC, complementary to a region from nucleotide 5545 to nucleotide 5554 at the 3′ end of TMV RNA, we have cleaved the RNA at the site of polynucleotides complementary to the DNA oligomer. Factors such as secondary structure of the RNA, concentrations of DNA oligomer, RNase H and magnesium ions in the reaction mixture, and time of incubation were optimized for the RNase H cleavage of TMV RNA – DNA oligomer complex. Denaturation of TMV RNA with 50% dimethyl sulphoxide at 50 °C is essential for the site-specific cleavage.