Dimerization-based homogeneous fluorosensor proteins for the detection of specific dsDNA

While there are many hybridization-based DNA sensors, none of them can detect native double-stranded DNA (dsDNA), which is most commonly found in physiological conditions. Here we made novel fluorosensor proteins comprised of a pair of two zinc fingers with an N-terminal dimerization motif and a C-terminal GFP variant to detect specific dsDNA sequence in a homogeneous solution. When a pair of purified zinc finger-GFP color variant proteins (Zif12-eCFP, Zif12-eYFP) were mixed and added with specific dsDNA with 12 bp inverted repeat (IR), fluorescence spectra of the solution showed significant concentration-dependent enhancement of fluorescence resonance energy transfer (FRET), with the detection limit of ∼10 nM. No significant change in FRET was observed if nonspecific DNA was added, indicating dsDNA-dependent dimerization of the two proteins. This dimerization-based dsDNA sensors will have a range of applications where conventional hybridization-based assay is difficult.