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Free keywords:
Azide–alkyne cycloaddition; Click chemistry; Electron paramagnetic resonance (EPR) spectroscopy; Electron spin resonance (ESR) spectroscopy; Enzymatic ligation of RNA; In vitro RNA labeling; Spin labeling; Spin-labeled RNA
Abstract:
Electron paramagnetic resonance (EPR) is a spectroscopic method for investigating structures, conformational changes, and dynamics of biomacromolecules, for example, oligonucleotides. In order to be applicable, the oligonucleotide has to be labeled site-specifically with paramagnetic tags, the so-called spin labels. Here, we provide a protocol for spin labeling of long oligonucleotides with nitroxides. In the first step, a short and commercially available RNA strand is labeled with a nitroxide via a copper-(I)-catalyzed azide-alkyne cycloaddition (CuAAC), also referred to as "click" reaction. In the second step, the labeled RNA strand is fused to another RNA sequence by means of enzymatic ligation to obtain the labeled full-length construct. The protocol is robust and has been shown experimentally to deliver high yields for RNA sequences up to 81 nucleotides, but longer strands are in principle also feasible. Moreover, it sets the path to label, for example, long riboswitches, ribozymes, and DNAzymes for coarse-grained structure determination and enables to investigate mechanistical features of these systems.
