Site-Specific Labeling of RNA at Internal Ribose Hydroxyl Groups: Terbium-Assisted Deoxyribozymes at Work

A general and efficient single-step method was established for site-specific post-transcriptional labeling of RNA. Using Tb3+ as accelerating cofactor for deoxyribozymes, various labeled guanosines were site-specifically attached to 2′-OH groups of internal adenosines in in vitro transcribed RNA. Th...

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Bibliographic Details
Published in:Journal of the American Chemical Society 2014-06, Vol.136 (22), p.8131-8137
Main Authors: Büttner, Lea, Javadi-Zarnaghi, Fatemeh, Höbartner, Claudia
Format: Article
Language:English
Subjects:
Adenosine - chemistry
Biotinylation
Catalysis
Cross-Linking Reagents
DNA - chemistry
DNA, Catalytic - chemistry
Guanosine - chemistry
Protein Processing, Post-Translational
Ribonucleoprotein, U4-U6 Small Nuclear - chemistry
Ribose - chemistry
Riboswitch
RNA - chemistry
Terbium - chemistry
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Summary:A general and efficient single-step method was established for site-specific post-transcriptional labeling of RNA. Using Tb3+ as accelerating cofactor for deoxyribozymes, various labeled guanosines were site-specifically attached to 2′-OH groups of internal adenosines in in vitro transcribed RNA. The DNA-catalyzed 2′,5′-phosphodiester bond formation proceeded efficiently with fluorescent, spin-labeled, biotinylated, or cross-linker-modified guanosine triphosphates. The sequence context of the labeling site was systematically analyzed by mutating the nucleotides flanking the targeted adenosine. Labeling of adenosines in a purine-rich environment showed the fastest reactions and highest yields. Overall, practically useful yields >70% were obtained for 13 out of 16 possible nucleotide (nt) combinations. Using this approach, we demonstrate preparative labeling under mild conditions for up to ∼160-nt-long RNAs, including spliceosomal U6 small nuclear RNA and a cyclic-di-AMP binding riboswitch RNA.
ISSN:0002-7863
1520-5126
DOI:10.1021/ja503864v