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Chemical Dual End-Labeling of Large Ribozymes


Ahunbay, Esra; Steffen, Fabio D; Zelger-Paulus, Susann; Sigel, Roland K O (2022). Chemical Dual End-Labeling of Large Ribozymes. In: Steger, Gerhard; Rosenbach, Hannah; Span, Ingrid. DNAzymes : Methods and Protocols. New York: Springer, 191-204.

Abstract

Fast and efficient site-specific labeling of long RNAs is one of the main bottlenecks limiting distance measurements by means of Förster resonance energy transfer (FRET) or electron paramagnetic resonance (EPR) spectroscopy. Here, we present an optimized protocol for dual end-labeling with different fluorophores at the same time meeting the restrictions of highly labile and degradation-sensitive RNAs. We describe in detail the dual-labeling of a catalytically active wild-type group II intron as a typical representative of long functional RNAs. The modular procedure chemically activates the 5′-phosphate and the 3′-ribose for bioconjugation with a pair of fluorophores, as shown herein, or with spin labels. The mild reaction conditions preserve the structural and functional integrity of the biomacromolecule and results in covalent, dual-labeled RNA in its pre-catalytic state in yields suitable for both ensemble and single-molecule FRET experiments.

Abstract

Fast and efficient site-specific labeling of long RNAs is one of the main bottlenecks limiting distance measurements by means of Förster resonance energy transfer (FRET) or electron paramagnetic resonance (EPR) spectroscopy. Here, we present an optimized protocol for dual end-labeling with different fluorophores at the same time meeting the restrictions of highly labile and degradation-sensitive RNAs. We describe in detail the dual-labeling of a catalytically active wild-type group II intron as a typical representative of long functional RNAs. The modular procedure chemically activates the 5′-phosphate and the 3′-ribose for bioconjugation with a pair of fluorophores, as shown herein, or with spin labels. The mild reaction conditions preserve the structural and functional integrity of the biomacromolecule and results in covalent, dual-labeled RNA in its pre-catalytic state in yields suitable for both ensemble and single-molecule FRET experiments.

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Additional indexing

Item Type:Book Section, refereed, original work
Communities & Collections:07 Faculty of Science > Department of Chemistry
Dewey Decimal Classification:540 Chemistry
Scopus Subject Areas:Life Sciences > Molecular Biology
Life Sciences > Genetics
Language:English
Date:1 January 2022
Deposited On:06 Jan 2023 14:18
Last Modified:27 Dec 2023 08:10
Publisher:Springer
Series Name:Methods in Molecular Biology
ISSN:1064-3745
ISBN:978-1-0716-2046-5
OA Status:Closed
Publisher DOI:https://doi.org/10.1007/978-1-0716-2047-2_13
Other Identification Number:eBook ISBN 978-1-0716-2047-2
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