Nicole M. Nichols1, Dongxian Yue1

1 New England Biolabs, Ipswich, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 3.13
DOI:  10.1002/0471142727.mb0313s84
Online Posting Date:  October, 2008
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Ribonucleases (RNases) with different sequence or structural specificities are used for a variety of analytical purposes, including RNA sequencing, mapping, and quantitation. The development of RNase protection assays, structural determination assays, and the production of small interfering RNAs (siRNA) employed in RNA interference (RNAi) experiments has depended on the unique substrate specificities of commercially available RNases, including RNases A, I, T1, V1, HI, III, and Dicer. One very common application for high purity RNase A is also presented in this unit and involves hydrolyzing RNA that contaminates DNA preparations. RNase HII and the placental RNase inhibitor are also discussed. Curr. Protoc. Mol. Biol. 84:3.13.1‐3.13.8. © 2008 by John Wiley & Sons, Inc.

Keywords: RNase; RNase protection assay; RNA footprinting; RNAi; siRNA

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Table of Contents

  • Introduction
  • Enzyme: Ribonuclease A
  • Enzyme: Ribonuclease I
  • Enzyme: Ribonuclease T1
  • Enzyme: Ribonuclease HI
  • Enzyme: Ribonuclease HII
  • Enzyme: Ribonuclease V1
  • Enzyme: Ribonuclease III
  • Enzyme: Dicer
  • Ribonuclease Inhibitor
  • Literature Cited
  • Figures
  • Tables
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Literature Cited

Literature Cited
   Berkower, I., Leis, J., and Hurwitz, J. 1973. Isolation and characterization of an endonuclease from Escherichia coli specific for ribonucleic acid in ribonucleic acid⋅deoxyribonucleic acid hybrid structures. J. Biol. Chem. 248:5914‐5921.
   Blackburn, P. and Moore, S. 1982. Pancreatic Ribonucleases. In The Enzymes, Vol. XV, Part B (P.D. Boyer, ed.) pp. 317‐433. Academic Press, New York.
   Blackburn, P., Wilson, G., and Moore, S. 1977. Ribonuclease inhibitor from human placenta. Purification and properties. J. Biol. Chem. 252:5904.
   Boehringer Mannheim Biochemicals. Biochemicals for Molecular Biology (catalog). Indianapolis.
   Donis‐Keller, H. 1979. Site specific enzymatic cleavage of RNA. Nucleic Acids Res. 7:179‐192.
   Libonati, M. and Sorrentino, S. 1992. Revisiting the action of bovine ribonuclease A and pancreatic‐type ribonucleases on double‐stranded RNA. Mol. Cell. Biochem. 117:139‐151.
   Myers, J.W., Jones, J.T., Meyer, T., and Ferrell, J.E. Jr. 2003. Recombinant Dicer efficiently converts large dsRNAs into siRNAs suitable for gene silencing. Nat. Biotechnol. 21:324‐328.
   Neu, H.C. and Heppel, L.A. 1964. The release of ribonuclease into the medium when Escherichia coli cells are converted to spheroplasts. J. Biol. Chem. 239:3893‐3900.
   Nicholson, A.W. 2003. The ribonuclease III superfamily: Forms and functions in RNA maturation, decay, and gene silencing. In RNAi: A guide to gene silencing (G.J. Hannon, ed.) pp. 149‐174. CSHL Press, New York.
   Richards, F.M. and Wyckoff, H.W. 1971. Bovine pancreatic ribonuclease. In The Enzymes, Vol. IV (P.D. Boyer, ed.) pp. 647‐806. Academic Press, New York.
   Robertson, H.D., Webster, R.E., and Zinder, N.D. 1968. Purification and Properties of Ribonuclease III from Escherichia coli. J. Biol. Chem. 243:82‐91.
   Sawadogo, M. and Roeder, R.G. 1985. Factors involved in specific transcription by human RNA polymerase II: Analysis by a rapid and quantitative in vitro assay. Proc. Natl. Acad. Sci. U.S.A. 82:4394‐4398.
   Spahr, P.F. and Hollingworth, B.R. 1961. Purification and mechanism of action of ribonuclease from Escherichia coli ribosomes. J. Biol. Chem. 236:823‐831.
   Tarentino, A., Plummer, J., and Maley, F. 1970. Studies on the oligosaccharide sequence of ribonuclease B. J. Biol. Chem. 245:4150‐4157.
   Uchida, T. and Egami, F. 1971. Microbial ribonucleases with special reference to RNases T1, T2, N1, and U2. In The Enzymes, Vol. IV (P.D. Boyer, ed.) pp. 205‐250. Academic Press, San Diego.
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