Studying sparsely populated conformational states in RNA combining chemical synthesis and solution NMR spectroscopy

Methods : a Companion to Methods in Enzymology
Elisabeth StrebitzerChristoph Kreutz

Abstract

Using chemical synthesis and solution NMR spectroscopy, RNA structural ensembles including a major ground state and minor populated excited states can be studied at atomic resolution. In this work, atom-specific 13C labeled RNA building blocks - a 5-13C-uridine and a 2,8-13C2-adenosine building block - are used to introduce isolated 13C-1H-spin topologies into a target RNA to probe such structural ensembles via NMR spectroscopy. First, the 5-13C-uridine 2'-O-TBDMS-phosphoramidite building block was introduced into a 21 nucleotide (nt) tP5c stem construct of the tP5abc subdomain of the Tetrahymena group I ribozyme. Then, the 2,8-13C2-adenosine 2'-O-TBDMS-phosphoramidite building block was incorporated into a 9 kDa and a 15 kD construct derived from the epsilon (ε) RNA element of the duck Hepatitis B virus. The 2,8-13C2-adenosine resonances of the 9 kDa 28 nt sequence could be mapped to the full-length 53 nt construct. The isolated NMR active nuclei pairs were used to probe for low populated excited states (<10%) via 13C-Carr-Purcell-Meiboom-Gill (CPMG)-relaxation dispersion NMR spectroscopy. The 13C-CPMG relaxation dispersion experiment recapitulated a secondary structure switching event in the P5c hairpin of the group I intron ...Continue Reading

Citations

Apr 19, 2019·Chembiochem : a European Journal of Chemical Biology·Maja MarušičKatja Petzold
Mar 21, 2019·Biological Chemistry·Paria Asadi-AtoiStefanie Kellner
Jun 20, 2020·Angewandte Chemie·Felix NußbaumerChristoph Kreutz
Nov 7, 2019·Chemical Communications : Chem Comm·Sha ZhaoShenlin Wang
May 5, 2020·Journal of Biomolecular NMR·Hyeyeon NamTheodore K Dayie
Sep 29, 2019·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Owen BecetteTheodore K Dayie
Dec 10, 2019·Journal of the American Chemical Society·Nicole I OrlovskyTerrence G Oas

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