Poster abstracts

Poster number 20 submitted by Mirjana Dimovska

Site-Specific Cleavage of Modified 16S Ribosomal RNA

Mirjana Dimovska (Department of Chemistry, Wayne State University), Yogo Sakakibara (Department of Chemistry, Wayne State University), Santosh Mahto (Department of Chemistry, Wayne State University), Stephanie Bierlein (Department of Chemistry, Wayne State University), Christine Chow (Department of Chemistry, Wayne State University)

Abstract:
Ribosomes are molecular machines essential to the process of life in all organisms because they are the instruments of the cell that perform translation of genetic information into proteins. Ribosomal RNA (rRNA) is particularly important, because it serves as the functional unit of ribosomes. The part of the ribosome of focus in our experiments is the decoding region – the area where the ribosome binds to transfer RNA (tRNA) – within the small ribosomal subunit (30S). In the decoding region, there are several modified bases. Although it is already known that these modifications are associated with antibiotic resistance and sensitivity, the mechanism and the function of modified nucleotides remain unclear. To understand the effects of these modified nucleotides, a new methodology to incorporate desired nucleosides into a specific site without inhibiting ribosomal activity is required. To date, several ways to engineer RNAs have been reported, but those methods are not optimized for ribosome engineering, and they are also cost ineffective. To develop a cost effective and powerful way for RNA engineering, we are focusing on the DNA enzyme (DNAzyme) which can cleave RNA at a specific site. We used two major types of DNAzyme, the 8-17 DNAzyme and the 10-23 DNAzyme, to test their enzyme activity on rRNA. The 8-17 DNAzymes target GG dinucleotides at positions 1904 of 23S rRNA and 1421 of 16S rRNA, and the 10-23 DNAzymes target GU and AU dinucleotides at positions 1392, 1413 of 16S rRNA, respectively. These DNAzymes show cleavage activities only after incubation at room temperature in the presence of divalent metal ions. We tested the catalytic effect of several divalent metal ions on RNA cleavage activity and concluded that Mg2+ is the best divalent metal ion for efficient cleavage of rRNA by the DNAzyme. Interestingly, we also observed significant DNAzyme reactivity without annealing DNAzymes to rRNA in advance. In conclusion, the DNAzyme has thus far proven effective in site-specifically cleaving the 16S rRNA. The next steps would be to purify the products of the DNAzyme reactions and continue with ligation of rRNA segments through the use of T4 DNA ligase.

Keywords: DNAzyme, RNA, ribosome