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

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 32 P-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.

Binding of Ru(bpy)2(eilatin)2+ to Matched and Mismatched DNA

“…After synthesis and purification, the reverse strand (not shown) was radioactively labeled with 32 P-ATP at its 5′ terminus via standard protocols. 17 Then, samples (1 μM) of radiolabeled EL-M and EL-MM DNA in buffer (50 mM NaCl, 10 mM NaPi, pH 7.1) were incubated with 8 μM Rh(bpy) 2 (phi) 3+ and irradiated for 20 min using a solar simulator in the presence of variable amounts of Ru(bpy) 2 (eilatin) 2+ . A concentration of 8 μM Rh(bpy) 2 (phi) 3+ provides 1 Rh molecule/4 base pairs, enough to saturate the entire oligonucleotide with Rh complexes.…”

“…All PAGE experiments described employed denaturing 20% polyacrylamide gels (National Diagnostics) and were performed according to published procedures. 17 DNA strands were radioactively labeled with [γ-32 P]-ATP (MP Biomedicals) using literature protocols and purified by 20% denaturing PAGE. 17 All irradiations were performed using an Oriel Instruments solar simulator (300-440 nm).…”

“…17 DNA strands were radioactively labeled with [γ-32 P]-ATP (MP Biomedicals) using literature protocols and purified by 20% denaturing PAGE. 17 All irradiations were performed using an Oriel Instruments solar simulator (300-440 nm). Gels were developed using Molecular Dynamics phosphorimaging screens and a Molecular Dynamics Storm 820 phosphorimager and subsequently visualized and quantified with Molecular Dynamics ImageQuant software.…”

Capillary Electrophoresis in Bioanalysis

“…CE was also used to analyze DNA fragments that resulted from photoinduced cleavage in the presence of metallo-intercalators (102). Rh(bpy) (2) (chrysi) (3+) and Rh(bpy) (2) (phzi) (3+) bind to mismatched DNA sites specifically and promote scission next to the mismatch.…”

Integrated nanostructures for direct detection of DNA at attomolar concentrations

“…1 Nucleic acids are biomarkers of central importance, bringing a great deal of attention to the development of nucleic acids biosensors. 2 In previous reports, the surface of a sensor is typically modified with nucleic acids probes capable of generating an optical, 3-8 gravimetric, 9 piezoelectric, 10 electrical or electrochemical [11][12][13][14][15][16] signal when hybridization with a complementary sequence occurs.…”