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

摘要

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 are described. The complexes selectively bind thermodynamically destabilized mismatched DNA sites and, upon photoactivation, promote strand scission neighboring the mismatch. Owing to their high specificity, independent of sequence context, targeting mismatches with these complexes offer an effective and convenient alternative to current mismatch- and SNP-detection methodologies. Syntheses of these complexes are described as well as protocols for the application of these complexes in marking mismatched sites. In the first, the irradiation of [³²P]-labeled duplex DNA with either intercalator followed by denaturing PAGE allows for the detection of mismatches in oligonucleotide sequences. The second protocol describes a method for the efficient detection of single nucleotide polymorphisms (SNPs) in larger genes or plasmids. Pooled genes are denatured and re- annealed to form heteroduplexes, incubated with Rh(bpy)2(chrysi)³⁺ or Rh(bpy)2(phzi)³⁺, irradiated, and analyzed by capillary electrophoresis for fragments indicative of mismatch and thus SNP sites. The synthesis of the metallointercalators requires about five to seven days. Both the mismatch and SNP detection experiments require approximately three days.