Abstract
Threading intercalators bind DNA with high affinities. Here, we describe single‐molecule studies on a cell‐permeant luminescent dinuclear ruthenium(II) complex that has been previously shown to thread only into short, unstable duplex structures. Using optical tweezers and confocal microscopy, we show that this complex threads and locks into force‐extended duplex DNA in a two‐step mechanism. Detailed kinetic studies reveal that an individual stereoisomer of the complex exhibits the highest binding affinity reported for such a mono‐intercalator. This stereoisomer better preserves the biophysical properties of DNA than the widely used SYTOX Orange. Interestingly, threading into torsionally constrained DNA decreases dramatically, but is rescued on negatively supercoiled DNA. Given the “light‐switch” properties of this complex on binding DNA, it can be readily used as a long‐lived luminescent label for duplex or negatively supercoiled DNA through a unique “load‐and‐lock” protocol.
Highlights
Owing to its central role in the process of life, new methods to visualize DNA and its dynamics are constantly being sought
A number of dinuclear ruthenium complexes based on linked RuII(dppz) fragments have been developed that thread through DNA duplexes, see Figure 1 for examples.[18,19]
A DNA dumbbell was assembled using a microfluidic laminar flow cell (Figure 2 A, channels 1–3), and held at a constant force (20–50 pN) in the presence of the complex (2–512 nM) (Figure 2 A, channel 4). At these forces, the DNA remains double-stranded, the stability is reduced at higher forces (> 60 pN)
Summary
The properties of water-soluble salts of d6 metal centres, especially polypyridyl RuII cations, have proven to be promising; as these species typically exhibit photostable[3] metal-to-ligand charge transfer (MLCT) excited states, they frequently possess bright long-lived emission with large Stokes shifts.[4,5,6,7,8,9,10,11,12] Due to this attractive combination of properties, complexes with extended aromatic ligands capable of intercalating into DNA were investigated and key early studies revealed that [RuII (LL)2(dppz)]2+ (LL = 2,2’bipyridine or 1,10-phenanthroline, dppz = dipyrido[3,2a:2’,3’-cj phenazine), Figure 1, displays a “DNA light switch” effect.[13] Through hydrogen-bonding interactions with the dppz ligand, the emissive state of this complex is solventquenched until solvent shielding through DNA intercalation “switches on” luminescence.[14,15,16,17] a number of dinuclear ruthenium complexes based on linked RuII(dppz) fragments have been developed that thread through DNA duplexes, see Figure 1 for examples.[18,19] In this binding motif, one of the bulky ruthenium centres passes through the DNA duplex to produce a final structure where a metal centre resides in both DNA grooves Such systems exhibit increased affinity and enhanced binding specificity towards particular DNA structures.[20,21]. Through these experiments we found that L,L-14+ functions as a “lockable” luminescent stain for duplex DNA
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