Efficient RNA 5′-adenylation by T4 DNA ligase to facilitate practical applications

Yangming Wang, Scott K. Silverman

Research output: Contribution to journalArticle

Abstract

We describe a simple procedure for RNA 5′-adenylation using T4 DNA ligase. The 5′-monophosphorylated terminus of an RNA substrate is annealed to a complementary DNA strand that has a 3′-overhang of 10 nucleotides. Then, T4 DNA ligase and ATP are used to synthesize 5′-adenylated RNA (5′-AppRNA), which should find use in a variety of practical applications. In the absence of an acceptor nucleic acid strand, the two-step T4 DNA ligase mechanism is successfully interrupted after the adenylation step, providing 40%-80% yield of 5′-AppRNA after PAGE purification with few side products (the yield varies with RNA sequence). Optimized reaction conditions are described for 5′-adenylating RNA substrates of essentially any length including long and structured RNAs, without need for sequestration of the RNA 3′-terminus to avoid circularization. The new procedure is applicable on the preparative nanomole scale. This 5′-adenylation strategy using T4 DNA ligase is a substantial improvement over our recently reported adenylation method that uses T4 RNA ligase, which often leads to substantial amounts of side products and requires careful optimization for each RNA substrate. Efficient synthetic access to 5′-adenylated RNA will facilitate a range of applications by providing substrates for in vitro selection; by establishing a new protocol for RNA 5′-capping; and by providing an alternative approach for labeling RNA with 32P or biophysical probes at the 5′-terminus.

Original languageEnglish (US)
Pages (from-to)1142-1146
Number of pages5
JournalRNA
Volume12
Issue number6
DOIs
StatePublished - Jun 2006

Keywords

  • In vitro selection
  • RNA 5′-adenylation
  • RNA capping
  • RNA labeling
  • T4 DNA ligase

ASJC Scopus subject areas

  • Molecular Biology

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