Preparation of Site-Specifically Spin-Labeled RNA by in Vitro Transcription Using an Expanded Genetic Alphabet

Lisa Bornewasser; Stephanie Kath-Schorr

doi:10.1007/978-1-0716-2047-2_15

Preparation of Site-Specifically Spin-Labeled RNA by in Vitro Transcription Using an Expanded Genetic Alphabet

Methods Mol Biol. 2022:2439:223-240. doi: 10.1007/978-1-0716-2047-2_15.

Authors

Lisa Bornewasser¹, Stephanie Kath-Schorr²

Affiliations

¹ Department of Chemistry, University of Cologne, Köln, Germany.
² Department of Chemistry, University of Cologne, Köln, Germany. skathsch@uni-koeln.de.

PMID: 35226325
DOI: 10.1007/978-1-0716-2047-2_15

Abstract

Recent advances in pulsed electron paramagnetic resonance (EPR) spectroscopy enable studying structure and folding of nucleic acids. An efficient introduction of spin labels at specific positions within the oligonucleotide sequence is a prerequisite. We here present a step-by-step guide to synthesize long RNA oligonucleotides bearing spin labels at specific positions within the sequence. RNA preparation is achieved enzymatically via in vitro transcription using an expanded genetic alphabet. Highly structured, several hundred nucleotides long RNAs with two nitroxide spin labels at specific positions can be prepared by this method.

Keywords: Electron paramagnetic resonance (EPR); Expanded genetic alphabet; In vitro transcription; Pulsed electron–electron double resonance (PELDOR); RNA; Spin labeling; Unnatural base pairs.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Electron Spin Resonance Spectroscopy / methods
Oligonucleotides* / chemistry
Oligonucleotides* / genetics
RNA* / chemistry
RNA* / genetics
Spin Labels

Substances

Oligonucleotides
Spin Labels
RNA