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DNA base mismatch detection with bulky rhodium intercalators: synthesis and applications

Nature Protocols volume 2, pages 357–371 (2007)Cite this article

Abstract

This protocol describes the syntheses and applications of two metallointercalators, Rh(bpy)2(chrysi)3+ and Rh(bpy)2(phzi)3+, that target single base mismatches in DNA. The complexes bind mismatched DNA sites specifically and, upon photoactivation, promote strand scission neighboring the mismatch. Owing to their high specificity and sequence context independence, targeting mismatches with these complexes offers an attractive alternative to current mismatch- and SNP-detection methodologies. This protocol also describes the synthesis of these complexes and their use in marking mismatched sites. Irradiation of 32P-labeled duplex DNA with either intercalator followed by denaturing PAGE allows the detection of mismatches in oligonucleotides. The protocol also outlines a method for efficient detection of single nucleotide polymorphisms (SNPs) in larger genes or plasmids. Pooled genes are denatured and re-annealed to form heteroduplexes; they are then incubated with either complex, irradiated and analyzed using capillary electrophoresis to probe for mismatches (SNP sites). The synthesis of the metallointercalators requires approximately 5–7 d. The mismatch- and SNP-detection experiments each require approximately 3 d.

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Figure 1
Figure 2: Δ-[Rh(bpy)2(chrysi)]3+ (left) and Δ-[Rh(bpy)2(phzi)]3+ (right).
Figure 3: Structural model of Rh(bpy)2(chrysi)3+ bound to a CA mismatch (Adapted with permission from ref. 16).
Figure 4
Figure 5
Figure 6
Figure 7: UV-visible spectra of Rh(bpy)2(chrysi)3+ (blue) and Rh(bpy)2(phzi)3+ (red) in water.
Figure 8
Figure 9: Circular dichroism spectra of Δ- and Λ-[Rh(bpy)2(chrysi)](Cl)3 in water (blue and red, respectively).
Figure 10: Phosphorimagery of a 20% polyacrylamide denaturing gel showing mismatch-specific photocleavage by Δ-[Rh(bpy)2(chrysi)]3+ of a 5-32P-labeled 36-mer containing a CC-mismatch.
Figure 11: Schematic of SNP detection with Rh(bpy)2(phzi)3+.
Figure 12: Sample SNP detection gel electrophoresis traces.

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Acknowledgements

We are grateful to the National Institutes of Health (GM33309) for their financial support. We also thank ABI for their support. In addition, we are grateful to our co-workers, J. Hart, I. Lau, V. Pierre and R. Ernst, for their help in working out experimental details.

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  1. Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, 91125, California, USA

    Brian M Zeglis & Jacqueline K Barton

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  1. Brian M Zeglis
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  2. Jacqueline K Barton
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Correspondence to Jacqueline K Barton.

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Zeglis, B., Barton, J. DNA base mismatch detection with bulky rhodium intercalators: synthesis and applications. Nat Protoc 2, 357–371 (2007). https://doi.org/10.1038/nprot.2007.22

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