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Site-Specific Dual-Color Labeling of Long RNAs


Zhao, Meng; Börner, Richard; Sigel, Roland K O; Freisinger, Eva (2020). Site-Specific Dual-Color Labeling of Long RNAs. In: Heise, Tilman. RNA Chaperones. New York: Springer, 253-270.

Abstract

Labeling of large RNAs with reporting entities, e.g., fluorophores, has significant impact on RNA studies in vitro and in vivo. Here, we describe a minimally invasive RNA labeling method featuring nucleotide and position selectivity, which solves the long-standing challenge of how to achieve accurate site-specific labeling of large RNAs with a least possible influence on folding and/or function. We use a custom-designed reactive DNA strand to hybridize to the RNA and transfer the alkyne group onto the targeted adenine or cytosine. Simultaneously, the 3′-terminus of RNA is converted to a dialdehyde moiety under the experimental condition applied. The incorporated functionalities at the internal and the 3′-terminal sites can then be conjugated with reporting entities via bioorthogonal chemistry. This method is particularly valuable for, but not limited to, single-molecule fluorescence applications. We demonstrate the method on an RNA construct of 275 nucleotides, the btuB riboswitch of Escherichia coli.

Abstract

Labeling of large RNAs with reporting entities, e.g., fluorophores, has significant impact on RNA studies in vitro and in vivo. Here, we describe a minimally invasive RNA labeling method featuring nucleotide and position selectivity, which solves the long-standing challenge of how to achieve accurate site-specific labeling of large RNAs with a least possible influence on folding and/or function. We use a custom-designed reactive DNA strand to hybridize to the RNA and transfer the alkyne group onto the targeted adenine or cytosine. Simultaneously, the 3′-terminus of RNA is converted to a dialdehyde moiety under the experimental condition applied. The incorporated functionalities at the internal and the 3′-terminal sites can then be conjugated with reporting entities via bioorthogonal chemistry. This method is particularly valuable for, but not limited to, single-molecule fluorescence applications. We demonstrate the method on an RNA construct of 275 nucleotides, the btuB riboswitch of Escherichia coli.

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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 2020
Deposited On:16 Apr 2020 06:03
Last Modified:27 Jan 2022 01:50
Publisher:Springer
Series Name:Methods in Molecular Biology
Number:2106
ISSN:1064-3745
ISBN:978-1-0716-0230-0
OA Status:Green
Publisher DOI:https://doi.org/10.1007/978-1-0716-0231-7_16

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