The Metastasis Associated Lung Adenocarcinoma Transcript 1(MALAT1) long non-coding RNA (lncRNA) is an oncogenic RNA that is upregulated in multiple cancer types. The 8.7 kb nuclear-localized lncRNA contains a triple-helical molecular linchpin on the 3’ end that contributes to its cellular stability. Two candidate small molecules have been discovered to bind the MALAT1 RNA triple helix and change its conformation, as well as decrease its cellular abundance. To avoid off-target effects during future cancer therapeutic studies, we set to explore the specificity of these small molecules to the MALAT1 triplex. We used a homologous triple helix to MALAT1 found in the multiple endocrine neoplasia-β (MENβ) lncRNA to assess the specificity of these small molecules. Interestingly, MENβ does not bind the small molecules. Thus, we designed three different gain-of-function mutants to the MENβ RNA: 1) addition of either one or two ‘U●A-U’ base triplets; 2) mutate the bulge region from U to GG to mimic the MALAT1 triple helix structure. We anticipate that by performing these mutations to the MENβ structure we will be able to individually track what properties allow the MALAT1 triple helix to gain selectivity to the small molecules over MENβ. Using a High-Fidelity DNA Polymerase along with our custom mutagenic primers, we have successfully obtained the MENβ +1,+2 and bulge mutant sequences from the wildtype MENβ sequence. Our current goals include using isothermal calorimetry and UV melt experiments to examine the structural effects of the mutagenesis to MENβ RNA and their putative role as gain-of-function mutants.

Investigating the triple helix structural properties of the lncRNA Metastasis Associated Lung Adenocarcinoma Transcript 1 by site-directed mutagenesis

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.