DNA Strand Cleavage near a CC Mismatch Directed by a Metalloinsertor

Abstract

Reagents for recognition and efficient cleavage of mismatched DNA without photoactivation were designed. They contain a combination of a mismatch-directing metalloinsertor, [Rh (bpy) 2 (chrysi)] 3+ (chrysi = 5,6-chrysenequinone diimine), and an oxidative cleavage functionality, [Cu(phen) 2 ] + (Cu). Both unconjugated (Rh+Cu) and conjugated (Rh−Cu) frameworks of the Rh insertor and Cu were prepared. Compared to Cu, both constructs Rh+Cu and Rh−Cu exhibit efficient site-specific DNA scission only with mismatched DNA, confirmed by experiments with 32 P-labeled oligonucleotides. Furthermore, these studies indicate that DNA cleavage occurs near the mismatch in the minor groove and on both strands. Interestingly, the order of reactivity of the three systems with a CC mismatch is Rh+Cu > Rh−Cu ≫ Cu. Rh binding appears to direct Cu reactivity with or without tethering. These results illustrate advantages and disadvantages in bifunctional conjugation.Base pair mismatches in the genome can arise from damage by environmental agents (i.e. UV light), a wide range of genotoxic chemicals as well as errors made by DNA polymerase during synthesis. 1,2 These base mispairs are generally corrected by the mismatch repair machinery; if they are not repaired, however, mutations arise, which subsequently lead to increased cancer susceptibility. [1][2][3][4] To recognize DNA mismatches, we have developed rhodium complexes containing an intercalating ligand that is too bulky to insert at stable matched sites and instead preferentially binds to thermodynamically destabilized mismatched sites. [5][6][7][8] The complex [Rh (bpy) 2 (chrysi)] 3+ (Figure 1), designed by our laboratory, targets more than 80% of mismatches with high selectivity and promotes single-stranded cleavage of the DNA backbone next to the mismatch site upon photoactivation. [5][6][7] More interestingly, this Rh complex can detect and photocleave a single base mismatch in a linearized 2725 base pair plasmid. 6 We have recently elucidated how the complex [Rh(bpy) 2 (chrysi)] 3+ interacts with the mismatched sites in DNA by crystal structural determination and NMR analysis. 9,10 The binding mode of this complex to the mismatch site is not classical intercalation but rather insertion. The expansive chrysi ligand is deeply inserted into the mismatch site in the minor groove resulting in the complete ejection of mismatched nucleotides from the base stack.Recently, this mismatch-specific metalloinsertor has shown promise in cell-selective strategies for chemotherapeutic design. 11 The Rh complex selectively inhibits cellular proliferation in mismatch repair-deficient cells as compared to mismatch repair-proficient cells. In another possible chemotherapeutic approach, bifunctional conjugates that combine a metalloinsertor targeting DNA mismatches with a reactive species such as an aniline mustard or a cisplatin *To whom correspondence should be addressed. Email: jkbarton@caltech.edu. Supporting Information Available: Preparation of the conjugate Rh-Cu and Figures S1-S3. ...

Crystal structure of Δ-[Ru(bpy)2dppz]2+ bound to mismatched DNA reveals side-by-side metalloinsertion and intercalation

“…To determine the groove preference of ruthenium complex binding, we employed the minor groove-specific quencher, Cu(phen) 2 2+ , 30,31 to quench the luminescence from the ruthenium complex (Figure 5). Quenching by paramagnetic Cu(phen) 2 2+ is expected rather than a direct competition for binding sites; Cu(phen) 2 2+ , a non-specific groove binder, binds several orders of magnitude more weakly to DNA than the intercalative ruthenium complex.…”

A Ruthenium(II) Complex as a Luminescent Probe for DNA Mismatches and Abasic Sites

“…To further elucidate the binding mode of [Ru(bpy) 2 (BNIQ)] 2+ at the mismatch site, we employed the quencher Cu(phen) 2 2+ . Cu(phen) 2 2+ binds in the DNA minor groove 22 − 24 and has been used to selectively quench the luminescence of ruthenium complexes bound to a mismatch in the minor groove. 5 , 25 We applied the Cu(phen) 2 2+ quencher to samples containing [Ru(bpy) 2 (BNIQ)] 2+ with the well-matched and mismatched duplexes ( Figure 4 ).…”

Targeting DNA mismatches with rhodium metalloinsertors

“…In an effort to increase the potency of metalloinsertors and obtain a covalent complex with DNA, our laboratory synthesized several new complexes that conjugate a selective rhodium metalloinsertor to a potent cytotoxic agent [68–70]. Three conjugates were designed that combine a reactive platinum moiety with a rhodium metalloinsertor (Figure 7) [69,71,72].…”