A Fluorogenic Covalent Chromone-Based Intercalator with a Mega-Stokes Shift for Sensing DNA Hybridization

Forced intercalation (FIT) probes have proven to be a reliable, rapid, inexpensive, and accurate method for the detection and visualization of specific nucleic acid sequences. The accommodation of a rationally designed chromone-based fluorogen within a double-stranded DNA structure was investigated by UV-Vis spectrophotometry and steady-state fluorescence spectroscopy under physiological pH conditions. After selective excitation matching with a 350 nm laser, the intrinsically negligible fluorescence of the tethered electroneutral label in a single-stranded context was increased 10-fold upon duplex formation. This fluorescence enhancement was also accompanied by a mega-Stokes shift (similar to 100 nm) that placed the emission in the cyan color range; both features are appreciable for bio-imaging purposes. In sum, its fluorogenic behavior and its marginal impact on the double helix make this dye a prospective tool for selectively sensing sequences of interest with a remarkable ON/OFF contrast.

Automated summary

Forced intercalation (FIT) probes are a reliable and rapid method for detecting and imaging specific nucleic acid sequences. A chromone-based fluorogen was successfully incorporated into double-stranded DNA, resulting in a 10-fold increase in fluorescence upon duplex formation. This dye shows promising potential for selective sensing of sequences with a high ON/OFF contrast, making it suitable for bio-imaging applications.