RNA Ligases

Nicole M. Nichols1, Stanley Tabor2, Larry A. McReynolds1

1 New England Biolabs, Ipswich, Massachusetts, 2 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 3.15
DOI:  10.1002/0471142727.mb0315s84
Online Posting Date:  October, 2008
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Abstract

T4 RNA ligase 1 catalyzes the ATP‐dependent covalent joining of single‐stranded 5′‐phosphoryl termini of DNA or RNA to single‐stranded 3′‐hydroxyl termini of DNA or RNA. T4 RNA ligase 2 also catalyzes the joining of a 3′‐hydroxyl terminus of RNA to a 5′‐phosphorylated RNA or DNA; unlike T4 RNA ligase 1, this enzyme prefers double‐stranded substrates. A truncated form of T4 RNA ligase 2 requires a pre‐adenylated substrate for ligation. This unit describes specific reaction conditions, as well as applications such as radioactive labeling of the 3′ termini of RNA, circularizing oligodeoxyribonucleotides and oligoribonucleotides, ligating oligomers and nicks, creating hybrid and chimeric DNA/RNA molecules, and miRNA cloning. Curr. Protoc. Mol. Biol. 84:3.15.1‐3.15.4. © 2008 by John Wiley & Sons, Inc.

Keywords: RNA ligation; T4 RNA ligase; miRNA cloning

     
 
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Table of Contents

  • Enzyme: T4 RNA Ligase 1
  • Enzyme: T4 RNA Ligase 2
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

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Figures

Videos

Literature Cited

   Aravin, A. and Tuschl, T. 2005. Identification and characterization of small RNAs involved in RNA silencing. FEBS Lett. 579:5830‐5840.
   Brennan, C.A., Manthey, A.E., and Gumport, R.I. 1983. Using T4 RNA ligase with DNA substrates. Methods Enzymol. 100:38‐52.
   Bullard, D.R. and Bowater, R.P. 2006. Direct comparison of nick‐joining activity of the nucleic acid ligases from bacteriophage T4. Biochem. J. 398:135‐144.
   Edwards, J.B., Delort, J., and Mallet, J. 1991. Oligodeoxyribonucleotide ligation to single‐stranded cDNAs: A new tool for cloning 5′ ends of mRNAs and for constructing cDNA libraries by in vitro amplification. Nucleic Acid Res. 19:5227‐5232.
   Heckler, T.G., Chang, L.H., Zama, Y., Naka, T., Chorghade, M.S., and Hecht, S.M. 1984. T4 RNA ligase mediated preparation of novel “chemically misacylated” tRNAPheS. Biochemistry 23:1468‐1473.
   Ho, C.K., Wang, L.K., Lima, C.D., and Shuman, S. 2004. Structure and mechanism of RNA ligase. Structure 12:327‐339.
   Nandakumar, J. and Shuman, S. 2004. How an RNA ligase discriminates RNA versus DNA damage. Mol. Cell 16:211‐221.
   Romaniuk, P.J. and Uhlenbeck, O.C. 1983. Joining of RNA molecules with RNA ligase. Methods Enzymol. 100:52‐59.
   Uhlenbeck, O.C. and Gumport, R.I. 1982. T4 RNA ligase. In The Enzymes, Vol. 15B (P.D. Boyer, ed.) pp. 31‐60. Academic Press, New York.
Key Reference
   Uhlenbeck and Gumport, 1982. See above.
  A review of properties and applications of T4 RNA ligase 1.
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