A Programmable Toolbox for Fragment-Specific DNA Labeling by Coupling a Chimeric γ‑Peptide Nucleic Acid-Peptide...

Area of Science:

• Molecular Biology
• Biochemistry
• Chemical Biology

Background:

• Site-specific internal labeling of double-stranded DNA (dsDNA) is challenging with existing methods.
• Conventional peptide nucleic acids (PNAs) struggle with binding efficiency in GC-rich dsDNA.

Purpose of the Study:

• To develop a programmable, enzyme-free method for site-specific internal labeling and cleavage of dsDNA.
• To overcome limitations of existing DNA labeling and PNA binding strategies.

Main Methods:

• Design of a chimeric γPNA-peptide invasion probe with modified backbone and SV40 NLS.
• Utilizing the probe with T7 endonuclease I (T7EI) for site-specific cleavage.
• Demonstrating probe programmability by altering the sequence for new targets.

Main Results:

• The chimeric probe enables programmable, site-specific invasion and internal fluorescence labeling of dsDNA under mild conditions.
• Enhanced helical preorganization and binding overcome GC-rich target challenges.
• The probe-T7EI system provides fragment-specific labeling and cleavage, surpassing end-labeling.

Conclusions:

• A versatile and programmable toolbox for fragment-specific dsDNA labeling and cleavage was established.
• The platform offers minimal sequence constraints and broad applicability.
• Potential applications include molecular diagnostics, gene regulation, and DNA nanotechnology.