Recognition of Nucleic Acid Mismatches by Luminescent Ruthenium Complexes

Abstract

Deficiencies in DNA mismatch repair (MMR) have been implicated in the development of several forms of cancers, and MMR-deficient cells tend to be resistant to commonly employed cancer therapeutics such as cisplatin. Mismatch-targeting metalloinsertors developed in our laboratory have shown great promise as therapeutic and diagnostic agents for MMR-deficient cancers. In this work, we examine fundamental aspects of binding interactions of octahedral rhodium and ruthenium complexes to DNA mismatches, and strive to develop a luminescent sensor for mismatches inside cells. We first demonstrate that the mismatch binding affinity of rhodium metalloinsertors directly correlates with their antiproliferative effect against MMR-deficient colorectal carcinoma cells. Smaller ancillary ligands on the rhodium center facilitate binding to mismatches via metalloinsertion from the narrow minor groove of DNA. Complexes with higher mismatch binding affinity in turn selectively inhibit the growth of MMR-deficient cells compared to MMR-proficient ones. This correlation suggests that DNA mismatches are indeed the biological target of rhodium metalloinsertors inside cells. Besides rhodium metalloinsertors, luminescent ruthenium complexes are found to bind DNA mismatches as well. Mismatch binding is accompanied by enhanced luminescence intensity. We determined two crystal structures of Δ-Ru(bpy)2dppz2+ bound to oligonucleotide duplexes. For an oligonucleotide containing AA mismatches, the atomic-resolution structure revealed that the ruthenium complex binds to DNA mismatches also through metalloinsertion: the complex inserts a planar ligand into the mismatched site from the minor groove, ejecting the mismatched bases out of the helix. Several binding geometries of the complex intercalated between well-matched DNA were also observed. To improve the mismatch selectivity of luminescent ruthenium complexes, we tethered the complexes to organic dye molecules in an effort to amplify mismatch-associated luminescence signal through resonance energy transfer. We also modified the structure of the inserting ligand in an attempt to improve the binding affinity to mismatches over well-matched DNA. Coupling mismatch binding to luminescence response has proved most challenging in these endeavors. Finally, we venture into the realm of RNA. Unlike their nonspecific binding to DNA, ruthenium complexes bind poorly to well-matched RNA but quite avidly to RNA mismatches. As a result, mismatched RNA produces a higher luminescence signal from bound ruthenium. We subsequently applied the ruthenium complex to image RNA mismatches inside live HeLa cells using fluorescence microscopy.