Targeting DNA Mismatches with Luminescent Ruthenium Complexes

DNA base pair mismatches occur naturally in cells, typically as a result of errors during replication. Cells have evolved a DNA damage response pathway called mismatch repair (MMR) that identifies and corrects base pair mismatches in newly synthesized DNA. However, proteins involved in MMR can undergo mutations, rendering them incapable of correcting mismatches. Such deficiencies in MMR leads to an increase in genetic mutations and are associated with several forms of cancer. Because a higher mismatch frequency serves as an early indicator of cancer progression, DNA mismatches are a promising target in the design of small molecule therapeutics and diagnostics. In this context, transition metal complexes are prime candidates, owing to their valuable spectroscopic and photophysical properties and versatile coordination sphere geometries. Our laboratory focuses on generating octahedral rhodium and ruthenium complexes that selectively target DNA mismatches. A class of rhodium complexes bearing sterically expansive planar ligands bind DNA mismatches with high selectivity and exhibit preferential cytotoxicity towards MMR-deficient cancer cells. These compounds bind to DNA through metalloinsertion, in which the bulky ligand inserts into the duplex at the thermodynamically destabilized mismatch site, displacing the mismatched bases into the DNA groove. Herein we describe recent advances in the development of luminescent ruthenium complexes that selectively probe DNA mismatches. We demonstrate that [Ru(Me4phen)2(dppz)]2+ (Me4phen = 3,4,7,8-tetramethyl-1,10-phenanthroline; dppz = dipyrido[3,2-a:2’,3’-c]phenazine) is a DNA “light switch” that exhibits a significantly brighter steady-state emission in the presence of a DNA duplex containing a mismatch relative to completely well-matched DNA. Importantly, the bulky Me4phen ancillary ligands discourage deep intercalation of dppz between well-matched base pairs, and instead, [Ru(Me4phen)2(dppz)]2+ favors metalloinsertion at thermodynamically destabilized mismatches. [Ru(Me4phen)2(dppz)]2+ possesses a higher binding affinity towards a DNA mismatch relative to well-matched base pairs, and furthermore exhibits a longer excited-state emission lifetime when bound to a mismatch compared to that when intercalated at well-matched sites; both of these observations contribute to the dramatic steady-state emission enhancement detected with the mismatched DNA duplex. Additionally, we reveal that the right-handed delta (∆) isomer of [Ru(Me4phen)2(dppz)]2+ is the enantiomer which imparts all mismatch selectivity, consistent with the handedness of B-form DNA. Another mismatch-specific luminescent probe presented in this work is [Ru(bpy)2(BNIQ)]2+ (bpy = 2,2’-bipyridine; BNIQ = benzo[c][1,7]naphthyridine-1-isoquinoline). In contrast to [Ru(Me4phen)2(dppz)]2+, the BNIQ complex exploits a bulky inserting ligand that selectively undergoes metalloinsertion at a DNA mismatch. This compound too exhibits a brighter steady-state emission in the presence of a mismatched duplex compared to entirely well-matched DNA, which we attribute to the fact that [Ru(bpy)2(BNIQ)]2+ possesses nearly a 500-fold higher binding affinity for the mismatch site compared to well-matched base pairs. Taken together, [Ru(Me4phen)2(dppz)]2+ and [Ru(bpy)2(BNIQ)]2+ represent two different yet valid approaches in the rational design of mismatch-specific small molecules, one based on ancillary ligand functionalization and the other on incorporating a sterically expansive inserting ligand. A third approach towards the design of mismatch-specific luminescent ruthenium probes that is briefly explored here is the modification of the intercalating dppz ligand of [Ru(bpy)2(dppz)]2+. Bearing a dppz ligand substituted with four methyl groups, [Ru(bpy)2(tmdppz)]2+ (tmdppz = 3,4,7,8-tetramethyl dipyridophenazine) shows no luminescence discrimination between mismatched and well-matched duplexes. This observation ostensibly arises from the fact that the appended methyl groups shield the dppz phenazine nitrogen atoms from interactions with water when intercalated within the DNA. With mismatch-specific luminescent metalloinsertors such as [Ru(Me4phen)2(dppz)]2+ in hand, we have commenced biological investigations to see whether these compounds can serve as luminescent proxies for rhodium metalloinsertors in MMR-deficient cancer cells. Confocal microscopy of HCT116N and HCT116O cells reveals that [Ru(Me4phen)2(dppz)]2+ does preferentially localize to mitochondria, unlike potent cell-selective rhodium complexes such as [Rh(chrysi)(phen)(PPO)]2+ (PPO = 2-(pyridine-2-yl)propan-2-ol; chrysi = 5,6-chrysenequinone diimine); however, [Ru(Me4phen)2(dppz)]2+ shows some degree of nuclear entry. Here our goal is the application of the mismatch-specific luminescent probe in co-localization experiments to investigate what proteins are involved in the DNA damage response that is activated upon metalloinsertor binding in cellulo. The work presented here expands beyond the study of luminescent ruthenium complexes. Amino acid conjugates of the earlier-generation rhodium metalloinsertor [Rh(HDPA)2(chrysi)]3+ (HDPA = 2,2’-dipyridylamine) were synthesized. While these conjugates exhibit mismatch binding affinities comparable to other rhodium metalloinsertors, they lose cell-selective biological activity, which may arise from altered uptake and/or sub-cellular localization. Finally, preliminary investigations were conducted on [Re(CO)3(pyOEt)(dppn)]+ (pyOEt = ethyl 3-(pyridin-4-yl)propanoate; dppn = benzodipyridophenazine) and [Ru(CN)(tpy)(dppz)]+ (tpy = terpyridine; CN = cyano), which were designed as IR-active probes to study the kinetics of DNA-mediated charge transport (CT) by time-resolved infrared (TRIR) spectroscopy. While these complexes do not possess the desired spectral TRIR properties as originally intended, steady-state luminescence experiments do suggest that this donor-acceptor pair is capable of undergoing DNA-mediated electron transfer. Altogether, this work demonstrates the versatility of transition metal complexes as non-covalent probes for DNA. Importantly, through the rational modification of their three-dimensional ligand scaffold, one can achieve site-specific recognition of clinically relevant biomarkers such as DNA mismatches.