A new fluorescence labeling method for molecular analysis of double-stranded DNA

In this study, a double-stranded DNA (dsDNA) fluorescent labeling method was developed using the fusion proteins of fluorescent protein (FP), and 7 kDa DNA-binding family members including Sso7d from Sulfolobus solfataricus, Aho7c from Acidianus hospitalis, ATSV7 from Acidianus tailed spindle virus and Sto7 from Sulfo-lobus tokodaii. Using this fluorescent DNA labeling method, we succeeded in single-molecule imaging of bacteriophage lambda DNA molecules stretched on glass surfaces. The fluorescence of the lambda DNA with FP fusion proteins decayed 2.4-to 6.4-fold slower than that of the typical intercalating method with SYTOX Green (SxG). In addition, the dynamic behaviors of FP-fused Aho7c-lambda DNA were relaxed and stretched with and without buffer flow, respectively, in microflow channels and were similar to that with typical intercalating dye, such as YOYO-1 and SxG. this fluorescent DNA labeling method. This fluorescent DNA labeling method can solve the problem of rapid fluorescence decay due to the intercalating dyes and therefore can be expected as an alternative to compound-based fluorescent dye. Thus, this study establishes FP fusion proteins as useful fluorescent DNA probes at the single-molecule level.

Automated summary

In this study, a double-stranded DNA (dsDNA) fluorescent labeling method using fusion proteins of fluorescent protein (FP) and 7 kDa DNA-binding family members was developed. The method successfully enabled single-molecule imaging of stretched bacteriophage lambda DNA molecules on glass surfaces. The fluorescence decay of the labeled DNA was slower than that of the typical intercalating method, and the dynamic behaviors of the labeled DNA were similar to those with typical intercalating dyes. This study establishes FP fusion proteins as useful fluorescent DNA probes at the single-molecule level.