Poster abstracts
Poster number 62 submitted by Neha Deshpande
Enzymatic synthesis of ITP-incorporated RNA for NMR-based structural investigations
Neha Deshpande (Department of Chemistry, University of Michigan), Yaping Liu (Program in Biophysics, University of Michigan), Sarah C. Keane (Department of Chemistry and Program in Biophysics, University of Michigan)
Abstract:
The non-exchangeable protons (anomeric and aromatic protons) of sequential nucleotides in an RNA exhibit a structured connectivity pattern on the 2D NOESY NMR, characteristic of an A-helical structure. Among the four natural nucleobases, only adenosine possesses an aromatic C2 proton that points towards the opposite strand of the RNA. This makes adenosines valuable probes for gaining insights into cross-strand interactions and investigating stacking interactions with neighboring nucleobases along the same strand.
However, information on cross-strand interactions using adenosine C2 protons might be limited if the RNA does not have a sufficient number of adenosines. This limitation could be addressed by incorporating a non-natural nucleotide that also has a proton on the C2 carbon, such as inosine. Just like its structural analog, guanosine, inosine prefers to base pair with cytosine.
We hypothesize that the C2 proton of inosines would be capable of reporting on cross-strand interactions, thereby expanding the cross-strand information provided by adenosine C2 protons. With miR-20a frag2 as our model RNA, we aim to substitute the two guanosines closest to the 3′ end of the RNA with inosines. Traditional RNA synthesis techniques such as chemical synthesis or enzymatic synthesis using T7 RNA polymerase suffer from size constraints or inefficient inosine incorporation. Therefore, we intend to use TGK, an engineered DNA polymerase from T. gorgonarius (Tgo) that is capable of extending an RNA primer in an RNA:DNA hybrid duplex and tolerates non-natural or modified nucleotides as its substrates. Our goals are to assess if TGK can accept inosine as a substrate while extending an RNA, evaluate whether inosine C2 protons can serve as probes to understand cross-strand interactions within an RNA, and determine the changes in the stability of the RNA with each guanosine-to-inosine substitution.
References:
Cozens, C.; Pinheiro, V. B.; Vaisman, A.; Woodgate, R.; Holliger, P. A short adaptive path from DNA to RNA polymerases. Proc. Natl. Acad. Sci. U.S.A. 2012, 109 (21), 8067–8072.
Haslecker, R.; Pham, V. V.; Glänzer, D.; Kreutz, C.; Dayie, T. K.; D’Souza, V. M. Extending the toolbox for RNA biology with SegModTeX: a polymerase-driven method for site-specific and segmental labeling of RNA. Nat. Commun. 2023, 14 (1), 8422.
Mária Brunderová; Vojtěch Havlíček; Ján Matyašovský; Pohl, R.; Lenka Poštová Slavětínská; Matouš Krömer; Hocek, M. Expedient Production of Site Specifically Nucleobase-Labelled or Hypermodified RNA with Engineered Thermophilic DNA Polymerases. Nat. Commun. 2024, 15 (1).
Keywords: Inosine incorporation, Cross-strand interactions, RNA NMR