Fluorescent proteins that also bind DNA molecules are useful reagents for a broad range of biological applications because they can be optically localized and tracked within cells, or provide versatile labels for in vitro experiments. We report a novel design for a fluorescent, DNA-binding protein (FP-DBP) that completely ‘paints’ entire DNA molecules, whereby sequence-independent DNA binding is accomplished by linking a fluorescent protein to two small peptides (KWKWKKA) using lysine for binding to the DNA phosphates, and tryptophan for intercalating between DNA bases. Importantly, this ubiquitous binding motif enables fluorescent proteins (Kd = 14.7 μM) to confluently stain DNA molecules and such binding is reversible via pH shifts. These proteins offer useful robust advantages for single DNA molecule studies: lack of fluorophore mediated photocleavage and staining that does not perturb polymer contour lengths. Accordingly, we demonstrate confluent staining of naked DNA molecules presented within microfluidic devices, or localized within live bacterial cells.

Here, we demonstrate the use of MALDI-TOF as a fast and simple analytical approach to evaluate the DNA-binding capability of various peptides. Specifically, by varying the amino acid sequence of the peptides consisting of lysine (K) and tryptophan (W), we identified peptides with strong DNA-binding capabilities using MALDI-TOF. Mass spectrometric analysis reveals an interesting novel finding that lysine residues show sequence selective preference, which used to be considered as mediator of electrostatic interactions with DNA phosphate backbones. Moreover, tryptophan residues show higher affinity to DNA than lysine residues. Since there are numerous possible combinations to make peptide oligomers, it is valuable to introduce a simple and reliable analytical approach in order to quickly identify DNA-binding peptides.