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. 2007;2(2):357-71.
doi: 10.1038/nprot.2007.22.

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

Brian M Zeglis  1 , Jacqueline K Barton
Affiliations

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

Brian M Zeglis et al. Nat Protoc. 2007.

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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Conflict of interest statement

COMPETING FINANCIAL INTERESTS: The authors have no competing financial interests regarding this chemistry.

Figures

Figure 1
Figure 1
Schematic of the use of mismatch-selective metallointercalators in detection of both single nucleotide polymorphisms (SNPs) (top) and single base mismatches (bottom).
Figure 2
Figure 2
Δ-[Rh(bpy)2(chrysi)]3+ (left) and Δ-[Rh(bpy)2(phzi)]3+ (right). In some cases (ref. , for example), these complexes are referred to as Rh(chrysi) and Rh(phzi).
Figure 3
Figure 3
Structural model of Rh(bpy)2(chrysi)3+ bound to a CA mismatch (adapted from Pierre, Kaiser, Barton, submitted). The bulky metal complex (red) inserts into the DNA base stack (gray) and the mismatched bases (blue) are extruded.
Figure 4
Figure 4
Synthesis of 2,3-benzo[a]phenazine quinone.
Figure 5
Figure 5
Synthesis of 5,6-chrysene quinone.
Figure 6
Figure 6
Synthetic route to rac-Rh(bpy)2(chrysi)3+ and rac-Rh(bpy)2(phzi)3+.
Figure 7
Figure 7
UV-Visible spectra of Rh(bpy)2(chrysi)3+ (red) and Rh(bpy)2(phzi)3+ (blue) in water. Extinction coefficients for Rh(bpy)2(chrysi)3+: 303 nm (ε=57,000 M−1), 315 nm (ε=52,200 M−1), 391 nm (ε=10,600 M−1). Extinction coefficients for Rh(bpy)2(phzi)3+: 304 nm (ε=65,800 M−1), 314 nm (ε=67,300 M−1), 343 nm (ε=39,300 M−1).
Figure 8
Figure 8
Column set-up for enantiomeric separation of Rh(bpy)2(chrysi)3+.
Figure 9
Figure 9
Circular dichroism spectra of Δ- and Λ-[Rh(bpy)2(chrysi)](Cl)3 in water (blue and red, respectively). Δε values for Δ-[Rh(bpy)2(chrysi)](Cl)3: 233 (34), 264 (26), 286 (−12), 308 (−42), 318 (−100), 341 (6). Δε values for Λ- [Rh(bpy)2(chrysi)](Cl)3: 233 (−34), 264 (−26), 286 (12), 308 (42), 318 (100), 341 (−6).
Figure 10
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. Conditions employed: 1 μM duplex DNA, 50 mM NaCl/10mM NaPi pH 7.1 buffer, 1 μM Rh complex when applicable, irradiations performed on an Oriel Instruments solar simulator (320 nm–450 nm) for 15 min. Lanes 1 and 2: matched DNA, Maxam Gilbert sequencing reactions. Lane 3: matched DNA, sample irradiated without rhodium. Lane 4: matched DNA, sample with rhodium but no irradiation. Lane 5: matched DNA irradiated with Δ-Rh(bpy)2(chrysi)3+. Lane 6: mismatched DNA, sample irradiated without rhodium. Lane 7: mismatched DNA, sample with rhodium but no irradiation. Lane 8: mismatched DNA irradiated with Δ-Rh(bpy)2(chrysi)3+. Lanes 9 and 10: mismatched DNA, Maxam Gilbert Sequencing reactions.
Figure 11
Figure 11
Schematic of SNP detection with Rh(bpy)2(phzi)3+. Adapted from ref. .
Figure 12
Figure 12
Sample SNP Detection Gel Electrophoresis Traces. The trace above (a) illustrates the result obtained using two homozygous plasmids. Without an SNP, no mismatch is formed upon denaturing and re-annealing the two plasmids. Therefore, only full-length products (in this case 436 bases), the parent band, can be observed. In contrast, the trace below (b) shows the result when two different templates, one containing an SNP, are mixed to create a mismatched site. In this case, the denaturing and re-annealing process creates a photocleavable CC mismatch. Therefore, after irradiation at 442 nm for 30 min with 200 nM Rh(bpy)2(phzi)3+, a fragment corresponding to the photocleaved strand, 170 bases in length, marking the SNP site, can be seen in addition to the full-length parent band. Adapted from ref. .
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References

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