3′-Terminal labelling of RNA with T4 RNA ligase View Full Text


Ontology type: schema:ScholarlyArticle     


Article Info

DATE

1978-10

AUTHORS

THOMAS E. ENGLAND, OLKE C. UHLENBECK

ABSTRACT

T4 RNA LIGASE catalyses the formation of an internucleotide phosphodiester bond between an oligonucleotide donor molecule with a 5′-terminal phosphate and an oligonucleotide acceptor molecule with a 3′-terminal hydroxyl1–3. Although the minimal acceptor must be a trinucleoside diphosphate, dinucleoside pyrophosphates and mononucleoside 3′,5′-bisphosphates (pNps) are effective donors in the intermolecular reaction4–6. We demonstrate here that various high molecular weight RNA molecules are acceptors in the RNA ligase reaction even when present in very low concentrations in the reaction mixture. One immediate consequence of this observation is that a convenient method for labelling the 3′ end of RNA molecules in vitro becomes available. By using a [5′-32P]pNp as a donor and RNA as an acceptor, the product of the reaction is an RNA molecule one nucleotide longer, with a 3′-terminal phosphate and a 32P-phosphate in the last internucleotide linkage. This reaction is therefore analogous to the in vitro labelling of the 5′ termini of RNA chains with polynucleotide kinase and [γ-32P]ATP and can be used in situations where 5′ labelling is not possible. In addition, the ability to add various donors to an RNA molecule should allow the function of the 3′ terminus of the molecule to be investigated. More... »

PAGES

560-561

Identifiers

URI

http://scigraph.springernature.com/pub.10.1038/275560a0

DOI

http://dx.doi.org/10.1038/275560a0

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1004227700

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/692735


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