Efficient DNA fluorescence labeling via base excision trapping.

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Current DNA fluorescence labeling methods are often expensive and time-consuming.
• There is a need for more accessible and efficient labeling techniques.

Purpose of the Study:

• To introduce a general, cost-efficient, and readily applicable method for DNA fluorescence labeling.
• To demonstrate the versatility of this new method for various biological applications.

Main Methods:

• Developed base excision trapping (BETr) using deaminated DNA bases and base excision repair enzymes to create AP sites.
• Utilized specially designed aminooxy-substituted rotor dyes to trap AP sites for high fluorescence emission.
• Applied BETr orthogonally to DNA synthesis and in situ labeling without washing.
• Demonstrated labeling of double-stranded DNA (dsDNA) using nick translation for high labeling density.
• Enabled two-color site-specific labeling using two different deaminated bases.
• Showcased conjugation of multi-labeled DNA constructs to antibodies for protein imaging.
• Harnessed double-strand selective repair enzymes for sensitive DNA/RNA detection.

Main Results:

• BETr provides a convenient and cost-efficient alternative to existing DNA labeling methods.
• The method allows for in situ labeling and is compatible with polymerase-based DNA synthesis.
• High labeling density on dsDNA, including genomic DNA, is achievable in a single tube.
• Site-specific two-color labeling is possible.
• Multi-labeled DNA constructs can serve as bright fluorescence tags for biomolecule imaging.
• Sensitive and specific DNA/RNA detection assays can be developed using BETr.

Conclusions:

• Base excision trapping (BETr) is a versatile and convenient method for general DNA fluorescence labeling.
• This technique offers significant advantages in terms of cost, efficiency, and applicability.
• BETr has broad potential in molecular biology, diagnostics, and bioimaging.