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

Area of Science:

• Molecular Biology
• Biophysics
• Biotechnology

Background:

• Traditional fluorescent DNA labeling methods often suffer from rapid fluorescence decay.
• Existing intercalating dyes can cause DNA damage and photobleaching.
• There is a need for stable and reliable fluorescent probes for single-molecule DNA analysis.

Purpose of the Study:

• To develop a novel double-stranded DNA (dsDNA) fluorescent labeling method using fluorescent protein (FP) fusion proteins.
• To evaluate the performance of this new method for single-molecule DNA imaging.
• To compare the stability and dynamic behavior of FP-fused DNA probes with conventional dyes.

Main Methods:

• Fusion proteins of fluorescent protein (FP) with 7 kDa DNA-binding proteins (Sso7d, Aho7c, ATSV7, Sto7) were constructed.
• The developed method was used for single-molecule imaging of bacteriophage λDNA.
• Fluorescence decay rates and dynamic behaviors in microflow channels were analyzed and compared to SYTOX Green (SxG) and YOYO-1.

Main Results:

• The FP fusion protein labeling method demonstrated significantly slower fluorescence decay (2.4- to 6.4-fold) compared to SYTOX Green.
• Dynamic behaviors of FP-fused Aho7c-λDNA were observed to be similar to those with conventional intercalating dyes.
• Successful single-molecule imaging of λDNA molecules was achieved using the novel fluorescent probes.

Conclusions:

• Fluorescent protein fusion proteins are effective and stable fluorescent DNA probes for single-molecule analysis.
• This method overcomes the limitation of rapid fluorescence decay associated with intercalating dyes.
• FP fusion proteins represent a viable alternative to compound-based fluorescent dyes for DNA research.