Investigation of various fluorescent protein-DNA binding peptides for effectively visualizing large DNA molecules

Large DNA molecules were visualized with novel fluorescent protein-DNA binding peptides (FP-DBPs). We constructed FP-DBPs by linking fluorescent protein to the N- or C-terminal of one or two peptides. We designed these DNA binding peptides from various DNA binding motifs such as oligo-lysine (K) and Lys-Trp (KW) repeats, TPKRPRGRPKK from high mobility group (HMG) chromosomal protein, and Ser-Pro-Arg-Lys (SPRK) from histone protein. We demonstrated the use of FP-DBP to stain large DNA molecules, and then analysed the fluorescence brightness and their binding affinity to double stranded DNA. This investigation provided HMG-tagged FP-DBP as the best DNA staining reagent in terms of fluorescence intensity, signal-to-noise ratio, and DNA binding affinity (K_d = 586 nM). Furthermore, we measured elongation of FP-DBP-stained DNA molecules tethered on the surface in order to evaluate FP-DBP-induced structural deformation.