Probing Nucleic Acid Structure with Nickel‐ and Cobalt‐Based Reagents

Steven E. Rokita1, Cynthia J. Burrows2

1 University of Maryland, College Park, Maryland, 2 University of Utah, Salt Lake City, Utah
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 6.4
DOI:  10.1002/0471142700.nc0604s00
Online Posting Date:  May, 2001
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The use of nickel and cobalt reagents is presented for characterizing the solvent exposure of guanine residues in DNA and RNA. These reagents promote guanine oxidation in the presence of a peracid such as monopersulfate, and the extent of reaction indicates the steric and electronic environment surrounding the N7 and aromatic face of this residue. Since oxidation does not itself perturb target structure or induce strand scission, it is coupled with fragmentation by treatment with piperidine (for smaller polynucleotides) or termination of primer extension (for larger polynucleotides).

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Table of Contents

  • Basic Protocol 1: Nickel‐ and Cobalt‐Dependent Oxidation of Nucleic Acids
  • Support Protocol 1: DNA Strand Scission by Piperidine Treatment
  • Reagents and Solutions
  • Commentary
  • Figures
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Basic Protocol 1: Nickel‐ and Cobalt‐Dependent Oxidation of Nucleic Acids

  • 1 µg/µL carrier DNA or RNA
  • 100 mM potassium phosphate buffer, pH 7 ( appendix 2A)
  • 1 M NaCl in water
  • RNA or DNA sample: 30,000 cpm end‐labeled (unit 6.1orunit 6.3) or 1 pmol unlabeled
  • 60 µM [NiCR](PF 6) 2 solution (see recipe and Fig. ) or 60 µM CoCl 2 in water
  • 0.6 mg/mL OXONE solution (see recipe)
  • 20 mM HEPES/100 mM EDTA, pH 7, in water
  • NaOAc/EDTA/Tris solution (see recipe)
  • 1 mM EDTA, pH 8 ( appendix 2A)
  • Microdialyzer
  • Lyophilizer
  • Additional reagents and equipment for phenol/chloroform extraction and ethanol precipitation ( appendix 2A and, e.g., CPMB UNIT ), piperidine treatment (see protocol 2), aniline acetate treatment (unit 6.1), PAGE (e.g., appendix 3B or CPMB UNIT ), partial alkaline hydrolysis and T1 nuclease digestion (unit 6.1), and primer extension (unit 6.1)

Support Protocol 1: DNA Strand Scission by Piperidine Treatment

  • Modified DNA sample pellet (see protocol 1)
  • 0.2 M piperidine, freshly prepared in sterile water
  • 1.5‐mL screw‐cap microcentrifuge tubes
  • 90°C water bath
  • Speedvac evaporator
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Literature Cited

Literature Cited
   Burrows, C.J. and Muller, J.G. 1998. Oxidative nucleobase modifications leading to strand scission. Chem. Rev. 98:1109‐1151.
   Burrows, C.J., Muller, J.G., Shih, H.‐C., and Rokita, S.E. 1995. Recognition of B vs. Z‐form DNA using nickel and cobalt complexes. In Supramolecular Stereochemistry (J.S. Siegel, ed.) pp. 57‐62. Kluwer Academic Publishers, Dordrecht, the Netherlands.
   Chen, X., Burrows, C.J., and Rokita, S.E. 1992. Conformation specific detection of guanosine in DNA: Ends, mismatches, bulges and loops. J. Am. Chem. Soc. 114:322‐325.
   Chen, X., Woodson, S.A., Burrows, C.J., and Rokita, S.E. 1993. A highly sensitive probe for guanine N7 in folded structures of RNA: Application to tRNAphe and Tetrahymena group I intron. Biochemistry 32:7610‐7616.
   Conway, L. and Wickens, M. 1989. Modification interference analysis of reactions using RNA substrates. Methods Enzymol. 180:369‐377.
   Ehresmann, C., Baudin, F., Mougel, M., Romby, P., Ebel, J.‐P., and Ehresmann, B. 1987. Probing the structure of RNAs in solution. Nucl. Acids Res. 15:9109‐9128.
   Heinemann, U. and Saenger, W. 1985. Mechanism of guanosine recognition and RNA hydrolysis by RNase T1. Pure Appl. Chem. 57:417‐422.
   Hickerson, R.P., Watkins‐Sims, C.D., Burrows, C.J., Atkins, J.F., Gesteland, R.F., and Felden, B. 1998. A nickel complex cleaves uridines in folded RNA structures: Application to E. coli tmRNA and related engineered molecules. J. Mol. Biol. 279:577‐587.
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   Knapp, G. 1989. Enzymatic approaches to probing of RNA secondary and tertiary structure. Methods Enzymol. 180:192‐212.
   Martin, L.Y., Sperati, C.R., and Busch, D.H. 1977. The spectrochemical properties of tetragonal complexes of high spin nickel(II) containing macrocyclic ligands. J. Am. Chem. Soc. 99:2968‐2981.
   Muller, J.G., Zheng, P., Rokita, S.E., and Burrows, C.J. 1996. DNA modification promoted by [Co(H2O)6]Cl2: Probing temperature‐dependent conformations. J. Am. Chem. Soc. 118:2320‐2325.
   Nielsen, P.E. 1990. Chemical and photochemical probing of DNA complexes. J. Mol. Recognit. 3:1‐25.
   Peattie, D.A. and Gilbert, W. 1980. Chemical probes for higher‐order structure in RNA. Proc. Natl. Acad. Sci. U.S.A. 77:4679‐4682.
   Ross, S.A., and Burrows, C.J. 1996. Cytosine‐specific chemical probing of DNA using bromide and monoperoxysulfate. Nucl. Acids Res. 24:5062‐5063.
   Schmidt, M., Zheng, P., and Delihas, N. 1995. Secondary structures of Escherichai coli antisense micF RNA, the 5′‐end of the target ompF mRNA, and the RNA/RNA duplex. Biochemistry 34:3621‐3631.
   Shih, H.‐C, Tang, N., Burrows, C.J., and Rokita, S.E. 1998. Nickel‐based probes of nucleic acid structure bind to guanine but do not perturb a dynamic equilibrium of extrahelical guanine residues J. Am. Chem. Soc. 120:3284‐3288.
   Sugiyama, H. and Saito, I. 1996. Theoretical studies of GG‐specific photocleavage of DNA via electron transfer: Significant lowering of ionization potential and 5′‐localization of HOMO of GG bases in B‐form DNA. J. Am. Chem. Soc. 118:7063‐7068.
   Woodson, S.A., Muller, J.G., Burrows, C.J., and Rokita, S.E. 1993. A primer extension assay for modification of guanine by Ni(II) complexes. Nucl. Acids Res. 21:5524‐5525.
   Zheng, P., Burrows, C.J., and Rokita, S.E. 1998 Nickel‐ and cobalt‐dependent reagents identify structural features of RNA that are not detected by dimethyl sulfate or RNase T1. Biochemistry 37:2207‐2214.
Key References
   Burrows, C.J. and Rokita, S.E. 1994. Probing guanine structure in nucleic acid folding using nickel complexes. Acc. Chem. Res. 27:295‐301.
  A comprehensive review on various nickel‐based reagents.
   Burrows, C.J. and Rokita, S.E. 1995. Nickel complexes as probes of guanine sites in nucleic acid folding. In Metal Ions in Biological Systems (H. Sigel, ed.) pp. 537‐560. Marcel Dekker, New York.
  The most recent review covering applications from various laboratories.
   Rokita, S.E., Zheng, P., Tang, N., Cheng, C.‐C., Yeh, R.‐H., Muller, J.G., and Burrows, C.J. 1995. Nickel complexes in modification of nucleic acids. In Genetic Response to Metals (B. Sarkar, ed.) pp. 201‐216. Marcel Dekker, New York.
  A summary of initial mechanistic studies.
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