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EPR Distance Measurements on Long Non-coding RNAs Empowered by Genetic Alphabet Expansion Transcription.

Angewandte Chemie (International ed. in English)

Authors: Christof Domnick, Frank Eggert, Christine Wuebben, Lisa Bornewasser, Gregor Hagelueken, Olav Schiemann, Stephanie Kath-Schorr

We present herein a novel nitroxide spin label-containing RNA triphosphate TPT3 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.

© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

PMID: 31981397

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