DNA binding fluorescent proteins for the direct visualization of large DNA molecules

Abstract

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.

TAMRA-polypyrrole for A/T sequence visualization on DNA molecules

“…Thus, simply increasing resolution may not be the solution to achieve high sensitivity. In addition, we tested a couple of extreme solutions such as pH 11 basic solution and salty buffer solution (1 × PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , pH 7.4), both of which detached fluorescent protein–DNA binding peptides from DNA in our previous report ( 34 ). However, we observed that TAMRA-polypyrrole DNA staining is successful in both the extreme solutions.…”

Nanochannel-Confined TAMRA-Polypyrrole Stained DNA Stretching by Varying the Ionic Strength from Micromolar to Millimolar Concentrations

“…Thus, light-induced DNA cleavage has been a notorious problem in using the intercalating dyes [20]. The chance of DNA cleavage increases for tethered or nanochannel-confined DNA, because there exist additional mechanical and thermodynamically pulling forces in such environments [21].…”

Shifting Retroviral Vector Integrations Away from Transcriptional Start Sites via DNA-Binding Protein Domain Insertion into Integrase

“… 24 , 25 In addition, the DNA-binding property of the inserted domains might support association of the integrase with host genomic DNA. 26 As DNA-binding domains, 10 zinc-finger domains (ZFDs) containing between two and five finger units were used (A1–A5 and B1–B5; Figure 1 B; Table S1 ). Because of the short length of the ZFD and the potential alteration of ZFD folding by the flanking viral protein domains, site-specific targeted integration could not be aimed for.…”