Probing Nucleic Acid Structure with Shape‐Selective Rhodium and Ruthenium Complexes

Brian A. Jackson1, Jacqueline K. Barton1

1 California Institute of Technology, Pasadena, California
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 6.2
DOI:  10.1002/0471142700.nc0602s00
Online Posting Date:  May, 2001
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Abstract

In this unit, transition metal complexes are used as photochemical probes for the structure of RNA and DNA. The transition metal ion provides a rigid substitutionally inert framework and an octahedral geometry for ligand coordination. The complexes can be constructed to define shapes, symmetries, and functionalities that complement those of the nucleic acid target. Complex formation is easily detected by light‐induced nucleic acid cleavage. The modular construction of the complexes makes it possible to generate probes to examine a wide variety of structural characteristics of nucleic acids.

     
 
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Table of Contents

  • Basic Protocol 1: Mapping DNA Major and Minor Groove Characteristics
  • Support Protocol 1: Synthesis of Bis(1,10‐Phenanthroline) (9,10‐Phenanthrenequinone Diimine)Rhodium(III) Trichloride
  • Support Protocol 2: Separation of Enantiomers of Bis(1,10‐Phenanthroline) (9,10‐Phenanthrnenequinone Diimine)Rhodium(III) Trichloride
  • Support Protocol 3: Synthesis of Tris(Phenanthroline) CoplexesS of Ruthenium(II)
  • Basic Protocol 2: Shape‐Selective Cleavage of Unusual Structures in Nucleic Acids
  • Support Protocol 4: Synthesis of Tris(4,7‐Diphenyl‐1,10‐Phenanthroline) Rhodium(III) Trichloride
  • Basic Protocol 3: Recognition of Mismatches and Abasic Sites in DNA
  • Support Protocol 5: Synthesis of Bis(2,2′‐Bipyridine)(5,6‐Chrysenequinone Diimine)Rhodium(III) Trichloride
  • Basic Protocol 4: Photofootprinting of DNA‐Binding Molecules
  • Support Protocol 6: Synthesis of Bis(9,10‐Phenanthrenequinone Diimine)(2,2′‐Bipyridyl)Rhodium(III) Trichloride
  • Basic Protocol 5: Shape‐Selective Cleavage of RNA
  • Basic Protocol 6: Singlet Oxygen‐Mediated Cleavage at Guanine Residues in DNA and RNA
  • Support Protocol 7: Preparation and Labeling of DNA and RNA
  • Support Protocol 8: Photolysis of Metal Complexes
  • Support Protocol 9: Mapping Cleavage Sites on the Nucleic Acid
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Mapping DNA Major and Minor Groove Characteristics

  Materials
  • Labeled DNA solution (see protocol 13):
  •  for cleavage with [Rh(phen) 2(phi)]3+, typically 100 µM base pairs in 2× buffer
  •  for cleavage with [Ru(TMP) 3]2+ or [Ru(TMP) 3]2+, typically 200 µM base pairs in 2× Tris/NaCl/imidazole buffer (see recipe)
  • Metal complex solution (see recipe for preparation of stock solutions):
  •  rac‐ or Δ‐[Rh(phen) 2(phi)]3+ (typically 10 µM; see protocol 2 for  synthesis; see protocol 3 for separation of enantiomers)
  •  rac‐[Ru(TMP) 3]2+ (typically 60 µM; see protocol 4)
  •  rac‐[Ru(phen) 3]2+ (typically 60 µM; see protocol 4)
  • 1 M (10% v/v) piperidine solution in deionized water
  • Dry ice
  • Heating block or bath set to 90°C
  • Speedvac (Savant) or lyophilizer
  • Additional reagents and equipment for photolysing metal complexes (see protocol 14) and mapping cleavage sites (see protocol 15)

Support Protocol 1: Synthesis of Bis(1,10‐Phenanthroline) (9,10‐Phenanthrenequinone Diimine)Rhodium(III) Trichloride

  Materials
  • Rhodium trichloride monohydrate
  • 1,10‐Phenanthroline
  • Hydrazine monohydrochloride
  • 95% (v/v) ethanol, denatured
  • Concentrated (28% to 30%) ammonium hydroxide solution
  • Phenanthrene quinone
  • 3:1 (v/v) acetonitrile (reagent grade or better)/0.4 M NaOH solution in distilled water
  • 1 M HCl solution ( appendix 2A)
  • Sephadex SP‐C25 cation‐exchange resin
  • 1 M MgCl 2 solution ( appendix 2A) in distilled water
  • HPLC‐grade acetonitrile
  • 0.1% (v/v) trifluoroacetic acid (TFA) in distilled water (for Sep‐Pak elution) and in deionized, filtered water (for HPLC)
  • Reflux condenser
  • Temperature‐controlled magnetic stir plate
  • Oil bath or heating mantle
  • 50‐ and 100‐mL round‐bottom flasks
  • 60‐mL medium‐frit glass funnel
  • 250‐mL filter flasks
  • Filter adapter
  • Aspirator or vacuum pump
  • Rotary evaporator
  • Chromatography column: 1 to 2 feet (30 to 61 cm) long, 1 to 1.5 in. (2.5 to 3.8 cm) diameter
  • 500‐mL Erlenmeyer flasks
  • Waters Sep‐Pak 5‐g C18 cartridges
  • 15‐mL plastic centrifuge tube or 25‐ to 50‐mL round‐bottom flask
  • Lyophilizer (optional)
  • High‐pressure liquid chromatography (HPLC) system with C18 reversed‐phase column
  • Additional reagents and equipment for proton nuclear magnetic resonance (1H NMR), UV/visible spectrometry, and mass spectrometry

Support Protocol 2: Separation of Enantiomers of Bis(1,10‐Phenanthroline) (9,10‐Phenanthrnenequinone Diimine)Rhodium(III) Trichloride

  • Hexamminecobalt(III) chloride
  • Nitrogen or argon gas
  • L‐Cysteine
  • Potassium hydroxide
  • 30% (v/v) hydrogen peroxide
  • [Rh(phen) 2(phi)]Cl 3 (see protocol 2)
  • Sephadex CM‐C25 ion‐exchange resin
  • 0.1 M KCl solution in deionized water
  • Thermometer
  • 500‐mL, 2‐ or 3‐neck round‐bottom flasks with appropriate stoppers
  • Bubbler for nitrogen or argon gas, equipped with a long pipet
  • pH paper
  • 60‐mL coarse‐frit glass funnels
  • 500‐mL filter flasks
  • 1000‐mL Erlenmeyer flask
  • Chromatography columns:
  •  2 to 4 feet (61 to 122 cm) long, 1 to 1.5 in. (2.5 to 3.8 cm) diameter
  •  0.5 to 1 feet (15 to 30 cm) long, 1 to 1.5 in. (2.5 to 3.8 cm) diameter
  • Solvent bottles for reservoirs
  • Recirculating column pump (optional)
  • Column end caps and tygon tubing for connecting recirculating pump and solvent reservoirs
  • Additional reagents and equipment for circular dichroism (CD) spectroscopy

Support Protocol 3: Synthesis of Tris(Phenanthroline) CoplexesS of Ruthenium(II)

  • Ruthenium trichloride hydrate
  • 6 M HCl solution (see Table 97.80.4711A.2A.1)
  • 3,4,7,8‐Tetramethyl‐1,10‐phenanthroline (TMP) or 1,10‐phenanthroline (phen)
  • 30% (w/v) hypophosphorus acid solution
  • 2 M NaOH solution
  • 30‐mL medium‐frit glass funnels

Basic Protocol 2: Shape‐Selective Cleavage of Unusual Structures in Nucleic Acids

  Materials
  • Supercoiled plasmid DNA (either commercial or containing a specific site of interest)
  • Appropriate restriction enzymes (see steps for details) and buffer systems (according to manufacturer's specifications)
  • 1 µg/mL ethidium bromide solution ( appendix 2A) in water or 1× TBE electrophoresis buffer
  • 7.5 M ammonium acetate solution in deionized water
  • Absolute ethanol (200 proof, dehydrated)
  • Dry ice
  • 10× Tris/acetate buffer, pH 7.0 (see recipe)
  • Metal complex solution (see recipe): 20 µM rac‐[Rh(DIP) 3]Cl 3 (for synthesis, see protocol 6)
  • S1 single‐strand‐specific nuclease and appropriate buffer systems (according to manufacturer's specifications)
  • 9 mM base pairs of calf thymus DNA solution in deionized water, buffered to pH 7 to 9
  • DNA molecular weight standards (e.g., commercially available 100‐base ladder)
  • 1× TBE electrophoresis buffer ( appendix 2A)
  • Loading buffer (e.g., formamide loading buffer or urea loading buffer; see recipe)
  • Maxam‐Gilbert sequencing reactions on labeled, unirradiated samples of 250‐ to 500‐bp DNA fragment of interest (see protocol 15)
  • UV transilluminator
  • Speedvac evaporator (Savant) or lyophilizer
  • UV/visible spectrometer
  • 90°C heating block
  • Phosphorimager (optional)
  • Additional reagents and equipment for restriction digests (e.g., CPMB UNIT ), agarose gel electrophoresis (e.g., CPMB UNIT ), photolysis (see protocol 14), radiolabeling DNA (see protocol 13), nondenaturing PAGE (e.g., CPMB UNIT ), denaturing PAGE (sequencing gels; appendix 3B), autoradiography (optional; e.g., CPMB APPENDIX 3A), and mapping cleavage sites (see protocol 15)
NOTE: Because of the low solubility of rac‐[Rh(DIP) 3]Cl 3 in water, a more concentrated solution of the material may need to be made in ethanol (or an ethanol/water mixture) and diluted to the desired stock concentration (20 µM) with water.

Support Protocol 4: Synthesis of Tris(4,7‐Diphenyl‐1,10‐Phenanthroline) Rhodium(III) Trichloride

  • 4,7‐Diphenyl‐1,10‐phenanthroline (DIP)
  • 2.5 mg/mL hydrazine monohydrochloride in water
  • Saturated NaCl solution
  • Acetone
  • 100‐mL Erlenmeyer flask
  • 30‐mL medium‐frit glass funnel
  • 100‐mL filter flask

Basic Protocol 3: Recognition of Mismatches and Abasic Sites in DNA

  Materials
  • Metal complex solution (see recipe): rac‐[Rh(bpy) 2(chrysi)]Cl 3 solution (typically 2 µM; for synthesis, see protocol 8)
  • Labeled DNA solution (see protocol 13): typically 20 µM polymers in 2× buffer
  • Additional reagents and equipment for photolysis (see protocol 14) and for mapping cleavage sites (see protocol 15)

Support Protocol 5: Synthesis of Bis(2,2′‐Bipyridine)(5,6‐Chrysenequinone Diimine)Rhodium(III) Trichloride

  • Chrysene
  • Sodium dichromate
  • Glacial acetic acid
  • 2,2′‐Bipyridine
  • Mortar and pestle
  • 250‐ and 1000‐ to 2000‐mL round‐bottom flasks
  • 250‐mL beaker
  • 60‐mL coarse‐frit glass funnels
  • 250‐ and 1000‐mL filter flasks
  • 2000‐mL Erlenmeyer flask

Basic Protocol 4: Photofootprinting of DNA‐Binding Molecules

  Materials
  • Metal complex solution (see recipe): [Rh(phi) 2(bpy)]Cl 3 solution (typically 10 µM; for synthesis, see protocol 10)
  • Labeled DNA solution (see protocol 13): typically 10 µM base pairs in a 2× buffer appropriate to the DNA binding molecule of interest
  • Labeled DNA/binding molecule solution: labeled DNA solution containing DNA‐binding molecule at twice the concentration desired in the photofootprinting experiment
  • Additional reagents and equipment for photolysis (see protocol 14) and mapping cleavage sites (see protocol 15)

Support Protocol 6: Synthesis of Bis(9,10‐Phenanthrenequinone Diimine)(2,2′‐Bipyridyl)Rhodium(III) Trichloride

  • 9,10‐Diaminophenanthrene
  • Dimethylformamide
  • Argon gas
  • Absolute ethanol (anhydrous)
  • Chloroform
  • Silver trifluoromethanesulfonate (silver triflate)
  • 2,2′‐Bipyridine
  • 50% (v/v) acetonitrile (reagent grade or better) in distilled water
  • Sephadex QAE‐25 anion‐exchange resin
  • 1 M HCl ( appendix 2A)
  • Sephadex CM‐C25 cation‐exchange resin
  • 1000‐mL three‐necked, round‐bottom flasks
  • Vacuum line with argon flush
  • 100‐, 250‐,and 500‐mL round‐bottom flasks
  • Septa to fit flasks
  • Heat gun
  • 60‐mL gas‐tight syringe with long needle
  • 25‐, 500‐, and 1000‐mL Erlenmeyer flasks
  • 60‐mL coarse‐ and fine‐frit glass funnels
  • Chromatography columns:
  •  0.5 to 1 feet (15 to 30 cm) long, 1 to 1.5 in. (2.5 to 3.8 cm) diameter
  •  1 to 2 feet (30 to 61 cm) long, 1 to 1.5 in. (2.5 to 3.8 cm) diameter

Basic Protocol 5: Shape‐Selective Cleavage of RNA

  Materials
  • Metal complex solution (prepared at twice the concentration desired in the photocleavage experiment; see recipe)
  • DNA or RNA stock solution containing radiolabeled and unlabeled carrier DNA or RNA (see protocol 13; prepared in buffered solution at twice the desired concentration of all components in the photocleavage experiment)
  • 9 mM base pairs calf thymus DNA solution in deionized water, buffered to pH 7 to 9 (optional; see protocol 13)
  • 7.5 M ammonium acetate solution in deionized water (optional)
  • Absolute ethanol (200 proof, dehydrated; optional)
  • Dry ice
  • Loading buffer (e.g., formamide loading buffer or urea loading buffer; see recipes)
  • 1.7‐ or 0.65‐mL silanized microcentrifuge tubes
  • Light source, such as:
  •  Hg‐Xe arc lamp (e.g., Oriel) equipped with an infrared (IR) filter, monochromator, and ultraviolet (UV) cut‐off filter (<300 nm)
  •  He‐Cd laser (e.g., Linconix model 4200 NB; 442 nm, 22 mW)
  •  Transilluminating light box (e.g., Spectroline model TR302 from Spectronics) with a broad band of irradiation centered at 302 nm
  • Speedvac evaporator (Savant) or lyophilizer
  • Liquid scintillation counter
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Figures

Videos

Literature Cited

Literature Cited
   Campisi, D., Morii, T., and Barton, J.K. 1994. Correlations of crystal structures of DNA oligonucleotides with enantioselective recognition by [Rh(phen)2(phi)]3+: Probes of DNA propeller twisting in solution. Biochemistry. 33:4130‐4139.
   Cartwright, P.S., Gillard, R.D., and Sillanpåå, E.R.J. 1987. Optically active coordination compounds—XLVI. Resolution of tris‐diimmine compounds of chromium(III) using fac‐(+)tris[L‐cysteinesulphinato(2‐)SN]cobaltate (III). Polyhedron. 6:105‐110.
   Chow, C.S. and Barton, J.K. 1992. Recognition of G‐U mismatches by tris(4,7‐diphenyl‐1,10‐phenanthroline)rhodium(III). Biochemistry. 31:5423‐5429.
   Chow, C.S., Behlen, L.S., Uhlenbeck, O.C., and Barton, J.K. 1992. Recognition of tertiary structure in tRNAs by [Rh(phen)2(phi)]3+, a new reagent for RNA structure‐function mapping. Biochemistry. 31:972‐982.
   Dollimore, L.S. and Gillard, R.D. 1973. Optically active co‐ordination compounds. Part XXXII. Potassium (+) tris‐[L‐cysteinesulphinato(2‐)‐SN]cobaltate(III): A versatile agent for resolution of 3+ species. J. Chem. Soc. Dalton Trans. (1973):934‐940.
   Gidney, P.M., Gillard, R.D., and Heaton, B.T. 1972. 1,10‐Phenanthroline and 2,2′‐bipyridyl complexes of rhodium(III). J. Chem. Soc. Dalton Trans. (1972):2621‐2628.
   Gillard, R.D., Osborn, J.A., and Wilkinson, G. 1965. Catalytic approaches to complex compounds of rhodium(III). J. Chem. Soc Dalton Trans. (1965):1951‐1965.
   Greabe, V.C. and Hönisberger, F. 1900. Ueber die Oxydationsproducte des Chrysens. Ann. Chem. 311:257‐265.
   Hall, D.B., Holmlin, R.E., and Barton, J.K. 1996. Oxidative DNA damage through long range electron transfer. Nature. 382:731‐735.
   Hartmann, B. and Lavery, R. 1996. DNA structural forms. Q. Rev. Biophys. 29:309‐368.
   Howells, R.D. and McCown, J.D. 1977. Trifluormethanesulfonic acid and derivatives. Chem. Rev. 77:69‐92.
   Huber, P.W., Morii, T., Mei, H.‐Y., and Barton, J.K. 1991. Structural polymorphism in the major groove of a 5S RNA gene complements the zinc finger domains of transcription factor IIIA. Proc. Natl. Acad. Sci. U.S.A. 88:10801‐10805.
   Jackson, B.A. and Barton, J.K. 1997. Recognition of mismatches by a rhodium intercalator. J. Am. Chem. Soc. 199:12986‐12987.
   Jackson, B.A., Alekseyev, V.A., and Barton, J.K. 1999. A versatile recognition agent: Specific cleavage of a plasmid DNA at a single base mispair. Biochemistry 38:4655‐4662.
   Kirshenbaum, M.R., Tribolet, R., and Barton, J.K. 1988. [Rh(DIP)3]3+: A shape‐selective metal complex which targets cruciforms. Nucl. Acids Res. 16:7943‐7960.
   Lee, I. and Barton, J.K. 1993. A distinct intron‐DNA structure in simian virus 40 T‐antigen and adenovirus 2 E1A genes. Biochemistry. 32:6121‐6127.
   Lim, A.‐C. and Barton, J.K. 1993. Chemical probing of tDNAPhe with transition metal complexes: A structural comparison of RNA and DNA. Biochemistry. 32:11029‐11034.
   Lim, A.‐C. and Barton, J.K. 1997. Targeting the Tat‐binding site of bovine immunodeficiency virus TAR RNA with a shape‐selective rhodium complex. Bioorg. Med. Chem. 5:1131‐1136.
   Lin, C‐T., Böttcher, W., Chou, M., Creutz, C., and Sutin, N. 1976. Mechanism of the quenching of the emission of substituted polypyridine‐ ruthenium(II) complexes by iron(III), chromium(III), and europium(III) ions. J. Am. Chem. Soc 98:6536‐6544.
   Maxam, A. and Gilbert, W. 1980. Sequencing end‐labeled DNA with base‐specific chemical cleavages. Methods Enzymol. 65:499‐560.
   Mei, H.Y. and Barton, J.K. 1986. Chiral probe for A‐form helices of DNA and RNA: Tris(tetramethylphenanthroline)ruthenium(II). J. Am. Chem. Soc. 108:7414‐7416.
   Mei, H.Y. and Barton, J.K. 1988. Tris(tetramethylphenanthroline)ruthenium(II): A chiral probe that cleaves A conformations. Proc. Natl. Acad. Sci. U.S.A. 85:1339‐1343.
   Müller, B.C., Raphael, A.L., and Barton, J.K. 1987. Evidence for altered DNA conformations in the simian virus genome: Site‐specific DNA cleavage by the chiral complex L‐tris(4,7‐diphenyl‐1,10‐phenanthroline)cobalt(III). Proc. Natl. Acad. Sci. U.S.A 84:1764‐1768.
   Mürner, H., Jackson, B.A., and Barton, J.K. 1998. A versatile synthetic approach to rhodium(III) diimine metallointercalators: Condensation of o‐quinones with coordinated cis‐ammines. Inorg. Chem. 37:3007‐3012.
   Neenhold, H.R. and Rana, T.M. 1995. Major groove opening at the HIV‐1 Tat‐binding site of TAR RNA evidenced by a rhodium probe. Biochemistry. 34:6303‐6309.
   Pyle, A.M., Long, E.C., and Barton, J.K. 1989. Shape‐selective targeting of DNA by (phenanthrenequinone)rhodium(III) photocleaving agents. J. Am. Chem. Soc 111:4520‐4522.
   Pyle, A.M., Chiang, M.Y., and Barton, J.K. 1990. Synthesis and characterization of physical, electronic, and photochemical aspects of 9,10‐phenanthrenequinone diimine complexes of ruthenium(II) and rhodium(III). Inorg. Chem. 29:4487‐4495.
   Sitlani, A., Barton, J.K. 1994. Sequence‐specific recognition of DNA by phenathrenequinone diimine complexes of rhodium(III): Importance of steric and van der Waals interactions. Biochemistry. 33:12100‐12108.
   Sitlani, A., Long, E.C., Pyle, A.M., and Barton, J.K. 1992. DNA photocleavage by phenanthrenequinone diimine complexes of rhodium(III): Shape selective recognition and reaction. J. Am. Chem. Soc 114:2303‐2312.
   Uchida, K., Pyle, A.M., Morii, T., and Barton, J.K. 1989. High resolution footprinting of EcoRI and distamycin with [Rh(phi)2(bpy)]3+, a new photofootprinting reagent. Nucl. Acids Res. 17:10259‐10279.
   Wilson, W.D., Tanious, F.A., Fernandez‐Saiz, M., and Rigl, C.T. 1997. Evaluation of drug‐nucleic acid interactions by thermal melting curves. In Methods in Molecular Biology, Vol. 90: Drug‐DNA Interaction Protocols (K.R. Fox, ed.) pp. 219‐240. Humana Press, Totowa, N.J.
   Yoshikawa, Y. and Yamasaki, K. 1979. Chromatographic resolution of metal complexes on Sephadex ion exchangers. Coord. Chem. Rev. 28:205‐229.
Key References
   Campisi et al., 1994. See above.
  Examples of mapping the major and minor grooves of DNA with [Rh(phen)2(phi)]3+ and [Ru(TMP)3]2+.
   Chow et al., 1992. See above.
  Examples of mapping RNA structure using rhodium probes.
   Hartmann and Lavery, 1996. See above.
  This is an excellent review of recent work in DNA structural determinations of A‐, B‐, and Z‐form DNA, mismatches, abasic sites, and bulges. As such, it provides an excellent overview of many of the targets these metal complex probes can be used to elucidate.
   Jackson and Barton, 1997. See above.
  Examples of using [Rh(bpy)2(chrysi)]3+ as a probe for DNA mismatches.
   Kirshenbaum et al., 1988. See above.
  Examples of site‐selective cleavage of unusual structures in nucleic acids using [Rh(DIP)3]3+.
   Lim and Barton, 1997. See above.
  Example of [Ru(phen)3]2+ used as a guanine‐specific sequencing reagent for nucleic acids.
   Uchida et al., 1989. See above.
  Examples of footprinting both major groove–and minor groove–binding molecules using rhodium probes.
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