DNA Binding Fluorescent Proteins for the Direct Visualization of Large DNA Molecules

Abstract

Fluorescent proteins that also bind DNA molecules are promising 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 partial intercalating between DNA bases (1). Importantly, this ubiquitous binding motif enables fluorescent proteins (Kd =14.7 µM, 7-8 bp/FP-DBP of base occupancies per dye) to confluently stain DNA molecules and such binding is reversible via pH shifts of buffer solution. These proteins offer robust advantages for lack of fluorophore mediated photocleavage, which makes them ideal staining reagents for imaging of DNA molecules over extended time periods even without anti-bleaching agent because the DNA binding moiety of the FP is separated from the fluorophore moiety. Further, the staining with FP-DBPs does not perturb polymer contour lengths, showing that the λ DNA monomer is about 48 502 bp × 0.34 nm/bp = 16.5 μm long, while most of the intercalating dyes, such as EtBr and YOYO-1, are known to distort the DNA structure and increase its full contour length. Moreover, the most significant advantage of FP-DBPs for DNA staining is its application to live cells, or entire living organisms. In a unique fashion, the FP-DBP fluorescence localized in three or four discrete regions within the bacterial cells: one, or two spots in the middle, and some at both ends of cells, as well as DAPI stained bacterial cells.1. Lee, S. et al., 2015, Nucleic Acids Research http://dx.doi.org/10.1093/nar/gkv834