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EPR Distance Measurements on Long Non‐coding RNAs Empowered by Genetic Alphabet Expansion Transcription
Author(s) -
Domnick Christof,
Eggert Frank,
Wuebben Christine,
Bornewasser Lisa,
Hagelueken Gregor,
Schiemann Olav,
KathSchorr Stephanie
Publication year - 2020
Publication title -
angewandte chemie
Language(s) - English
Resource type - Journals
eISSN - 1521-3757
pISSN - 0044-8249
DOI - 10.1002/ange.201916447
Subject(s) - ribozyme , rna , nucleotide , transcription (linguistics) , non coding rna , electron paramagnetic resonance , pulsed epr , riboswitch , intron , chemistry , oligonucleotide , biophysics , computational biology , biology , genetics , dna , physics , nuclear magnetic resonance , gene , spin echo , medicine , radiology , magnetic resonance imaging , linguistics , philosophy
Abstract We present herein a novel nitroxide spin label‐containing RNA triphosphate TPT3 NO and its application for site‐specific spin‐labeling of RNA through in vitro transcription using an expanded genetic alphabet. Our strategy allows the facile preparation of spin‐labeled RNAs with sizes ranging from short RNA oligonucleotides to large, complex RNA molecules with over 370 nucleotides by standard in vitro transcription. As a proof of concept, inter‐spin distance distributions are measured by pulsed electron paramagnetic resonance (EPR) spectroscopy in short self‐complementary RNA sequences and in a well‐studied 185 nucleotide non‐coding RNA, the B. subtilis glmS ribozyme. The approach is then applied to probe for the first time the folding of the 377 nucleotide A‐region of the long non‐coding RNA Xist, by PELDOR.