Single-stranded loops as end-label polarity markers for double-stranded linear DNA templates in atomic force...

Area of Science:

• Molecular Biology
• Biophysics
• Nanotechnology

Background:

• Atomic force microscopy (AFM) visualizes DNA-protein interactions, crucial for understanding transcription.
• Protein migration on DNA complicates AFM analysis of multi-protein systems.
• Determining DNA template polarity is key for detailed single-molecule AFM studies.

Purpose of the Study:

• To develop a nucleic acid-based method for end-labeling double-stranded DNA.
• To enable discrimination of linear DNA template polarity at the single-molecule level using AFM.
• To facilitate the study of DNA-protein interactions, particularly transcription, by improving AFM data interpretation.

Main Methods:

• Oligonucleotide loop-primed synthesis was employed for DNA end-labeling.
• Single-stranded oligonucleotide primers with 3'-single-strand extensions were designed for template annealing.
• DNA polymerase extension was used to create labeled DNA templates.

Main Results:

• The developed method successfully labeled double-stranded DNA templates.
• Labeled templates formed open promoter complexes with Escherichia coli RNA polymerases.
• AFM imaging confirmed that the added loops did not impede RNA polymerase recruitment.

Conclusions:

• A novel, generic method for end-labeling linear DNA using oligonucleotide loop-primed synthesis is presented.
• This technique allows for single-molecule polarity discrimination in DNA templates for AFM.
• The method is suitable for studying DNA-protein interactions, such as transcription, without compromising sample preparation.