A Minimal Load‐and‐Lock Ru ^II Luminescent DNA Probe

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, show this threads locks force-extended in two-step mechanism. Detailed kinetic reveal an individual stereoisomer of the exhibits highest binding affinity reported for such mono-intercalator. This better preserves biophysical properties than widely used SYTOX Orange. Interestingly, threading torsionally constrained decreases dramatically, but is rescued negatively supercoiled DNA. Given “light-switch” DNA, it can be readily as long-lived label or through unique “load-and-lock” protocol.