Algorithm for Searching and Testing the Activity of Antisense Oligonucleotides Exemplified by the mRNA of the rpoD Gene Encoding Staphylococcus aureus RNA Polymerase Sigma Factor

Abstract

One of the promising strategies in the development of efficient antibacterial drugs is the use of antisense oligonucleotides specifically interacting with mRNAs of genes controlling the key functions of the bacterial cell such as survival and reproduction. The present paper reports the development of an algorithm for searching and testing the antibacterial activity of antisense oligonucleotides using mRNA of the rpoD gene encoding the sigma factor of the Staphylococcus aureus RNA polymerase. Based on the calculated thermodynamic parameters and the rpoD mRNA 5'-fragment (1–377 nt region) secondary structure model, 11 antisense oligonucleotides 18–20 nt in length complementary to the rpoD mRNA regions with unstable secondary structure have been selected. Hybridization of the selected oligonucleotides with the total S. aureus RNA followed by selective in vitro digestion of antisense oligonucleotide/mRNA rpoD heteroduplexes with RNase H and assessment of rpoD mRNA digestion rate using PCR revealed six candidate sequences which reduced the rpoD mRNA level by 2.2–4 times. Phosphorothioate oligonucleotides targeted to the regions 1–20, 30–47, and 82–101 of the rpoD mRNA synthesized on the basis of these sequences efficiently suppressed the growth of S. aureus bacteria in vitro. It has been demonstrated that the efficiency of penetration of these oligonucleotides into the bacterial cell may be significantly increased by introducing arginine-rich peptide into the oligonucleotide structure. As a result of this work, we have developed a simple and efficient algorithm for selection of antisense oligonucleotides against bacterial RNAs encompassing all stages starting from the binding stage and ending with the analysis of biological activity.