Rhodium-cyanine fluorescent probes for detection and signaling of mismatches in DNA

Mismatched (non-Watson-Crick) base pair damage in DNA occurs naturally from errors during the replication process. Deficiencies in the mismatch repair (MMR) machinery, a DNA repair pathway, strongly predispose cells to cancer development. Therefore, efficiently detecting DNA mismatches will greatly enable early detection of MMR-deficient precancerous cells. Herein, we report the synthesis and characterization of a bifunctional fluorescent probe that combines a rhodium metalloinsertor with indol trimethine cyanine, Cy(3), the luminescent reporter, via a PEG-type linker. The conjugate displays low luminescence when free in soln. or in the presence of well-matched DNA but exhibits a luminescence increase up to 9-fold in the presence of a 27-mer oligonucleotide contg. a central CC mismatch. DNA photocleavage expts. demonstrate that upon photoactivation, the conjugate can cleave the DNA backbone near the mismatch site on a 27-mer oligonucleotide, thus providing further evidence for mismatch targeting. Fluorescence titrns. of the Rh conjugate with genomic DNA (gDNA) extd. from MMR-deficient and MMR-proficient HCT116 cell lines show a luminescence differential between gDNA from MMR-deficient and - proficient cell lines, reflecting the sensitive detection of differences in mismatch frequency.