Detection of Aberrant 2′‐5′ Linkages in RNA by Anion Exchange

J.R. Thayer1, Srinivasa Rao1, Nitin Puri2

1 Dionex Corporation, Sunnyvale, California, 2 Ambion, an Applied Biosystems Business, Austin, Texas
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 10.13
DOI:  10.1002/0471142700.nc1013s32
Online Posting Date:  March, 2008
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Formation of aberrant 2′‐5′ linkages can unintentionally occur in chemical synthesis of RNA. These linkages may arise by phosphoryl migration during deprotection, release, or subsequent steps during manufacture of therapeutic RNA. Their presence has been linked to a number of biochemical activities, so their potential for contribution to “off‐target” effects is significant. Assaying for their presence, to ensure safe and effective therapeutic activity, is not straightforward. Since these linkages do not alter the RNA mass or the ionic or hydrophobic character of the product, confirmation of their presence or absence is not readily addressable by conventional chromatographic, electrophoretic, or mass spectrometric techniques. Since 2′‐5′ linkages are known to alter RNA solution conformation, they may also alter stationary phase interactions. A method for identifying the presence of these aberrant linkages by pellicular anion‐exchange HPLC is presented in this unit. Curr. Protoc. Nucleic Acid Chem. 32:10.13.1‐10.13.11. © 2008 by John Wiley & Sons, Inc.

Keywords: aberrant linkage; 2′‐5′ linkage; RNA; anion exchange; HPLC; DNAPac

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Table of Contents

  • Introduction
  • Analytical Platform
  • RNA Standards
  • Aberrant Linkage Assays
  • Critical Parameters
  • Expected Results
  • Summary
  • Literature Cited
  • Figures
  • Tables
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Literature Cited

Literature Cited
   Anderson, C.W., Atkins, J.F., and Dunn, J.J. 1976. Bacteriophage T3 and T7 early RNAs are translated by eukaryotic 80s ribosomes: Active phage T3 coded S‐adenosylmethionine cleaving enzyme is synthesized. Proc. Natl. Acad. Sci. U.S.A. 73: 2752‐2756.
   Cheng, Z. and Menees, T.M. 2004. RNA branching and debranching in the yeast retrovirus‐like element Ty1. Science 303: 240‐243.
   Feldstein, P.A., Buzayan, J.M., van Tol, H., deBear, J., Gough, G.R., Gilham, P.T., and Bruening, G. 1990. Specific association between an endoribonucleolytic sequence from a satellite RNA and a substrate analogue containing a 2′‐5′ phosphodiester. Proc. Natl. Acad. Sci. U.S.A. 87: 2623‐2627.
   Giannaris, P.A. and Damha, M.J. 1993. Oligoribonucleotides containing 2′,5′‐phosphodiester linkages exhibit binding selectivity for 3′,5′‐RNA over 3′,5′‐ssDNA. Nucleic Acids Res. 21: 4742‐4749.
   Lamontagne, B., Hannoush, R.N., Damha, M.J., and Elela, S.A. 2004. Molecular requirements for duplex recognition and cleavage by eukaryotic RNase III: Discovery of an RNA‐dependent DNA cleavage activity of yeast Rnt1p. J. Mol. Biol. 338: 401‐418.
   Lesiak, K., Imai, J., Floyd‐Smith, G., and Torrence, P.F. 1983. Biological activities of phosphodiester linkage isomers of 2‐5A. J. Biol. Chem. 258: 13082‐13088.
   Lyttle, M.H., Wright, P.B., Sinha, N.D., Bain, J.D., and Chamberlin, A.R. 1991. New nucleoside phosphoramidites and coupling protocols for solid‐phase RNA synthesis. J. Org. Chem. 56: 4608‐4615.
   Morgan, M.A., Kazakoc, S., and Hecht, S.M. 1995. Phosphoryl migration during the chemical synthesis of RNA. Nucleic Acids Res. 23: 3949‐3953.
   Nielsen, H., Westhof, E., and Johansen, S. 2005. An mRNA is capped by a 2′‐5′ lariat catalyzed by a group I‐like ribozyme. Science 309: 1584‐1587.
   Plevnik, M., Gdaniec, Z., and Plavec, J. 2005. Solution structure of a modified 2′,5′‐linked RNA hairpin involved in an equilibrium with duplex. Nucleic Acids Res. 33: 1749‐1759.
   Rohatgi, R., Bartel, D.P., and Szostak, J.W. 1996. Kinetic and mechanistic analysis of nonenzymatic, template‐directed oligoribonucleotide ligation. J. Am. Chem. Soc. 118: 3332‐3339.
   Rose, J.K. 1975. Heterogeneous 5′‐terminal structures occur on vesicular stomatitis virus mRNAs. J. Biol. Chem. 250: 8098‐8104.
   Scaringe, S.A., Wincott, F.E., and Caruthers, M.H. 1998. Novel RNA synthesis method using 5′‐O‐silyl‐2′‐O‐orthoester protecting groups. J. Am. Chem. Soc. 120: 11820.
   Schlech, T., Cross, B.P., and Smith, I.C.P. 1976. A conformational study of adenylyl‐(3′,5′)‐adenosine and adenylyl‐(2′,5′)adenosine in aqueous solution by carbon‐13 magnetic resonance spectroscopy. Nucleic Acids Res. 3: 355‐370.
   Semenyuk, A., Ahnfelt, M., Estmer Nilsson, C., Yong‐Hao, X., Földesi, A., Kao, Y.‐S., Chen, H.‐H., Kao, W.‐C., Peck, K., and Kwiatkowski, M. 2006. Cartridge‐based high‐throughput purification of oligonucleotides for reliable oligonucleotide arrays. Anal. Biochem. 356: 132‐141.
   Shih, I‐H. and Been, M.D. 1999. Ribozyme cleavage of a 2′,5′‐phosphodiester linkage: Mechanism and a restricted divalent metal‐ion requirement. RNA 5: 1140‐1148.
   Thayer, J.R, McCormick, R.M., and Avdalovic, N. 1996. High‐resolution nucleic acid separations by high‐performance liquid chromatography. Methods Enzymol. 271: 147‐174.
   Thayer, J.R., Barreto, V., Rao, S., and Pohl, C. 2005. Control of oligonucleotide retention on a pH‐stabilized strong anion‐exchange column. Anal. Biochem. 338: 39‐47.
   Thayer, J.R., Rao, S., Puri, N., Burnett, C.A., and Young, M. 2007. Identification of aberrant 2′‐5′ RNA linkage isomers by pellicular anion‐exchange chromatography. Anal. Biochem. 361: 132‐139.
   Tsou, D., Hampel, A., Andrus, A., and Vinayak, R. 1995. Large scale synthesis of oligoribonucleotides on High‐Loaded Polystyrene (HLP) support. Nucleosides and Nucleotides 14: 1481‐1492.
   Wincott, F., DiRenzo, A., Shaffer, C., Grimm, S., Tracz, D., Workman, C., Sweedler, D., Gonzalez, C., Scaringe, S., and Usman, N. 1995. Synthesis, deprotection, analysis and purification of RNA and ribozymes. Nucleic Acids Res. 23: 2677‐2684.
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