Observing nucleotide flipping in DNA using indirect 2'-F nucleotide probes and 19F NMR

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Proteins interacting with DNA/RNA often use nucleotide flipping.
• This conformational change is typically monitored using spectroscopy like NMR.
• Previous methods using 19F NMR were limited to canonical nucleotides.

Purpose of the Study:

• To develop an indirect method for monitoring nucleotide flipping using 2'-F probes at proximal sites.
• To assess the utility of this indirect method for studying thymine DNA glycosylase (TDG) activity.
• To evaluate the impact of 2'-F probes on TDG enzyme kinetics.

Main Methods:

• Utilized 2'-fluoroarabino-substituted nucleotides as probes in DNA.
• Employed 19F NMR spectroscopy to monitor nucleotide flipping.
• Performed kinetic assays to measure TDG activity with the probes.

Main Results:

• Successfully monitored TDG-mediated flipping of deoxythymidine from G.T mispairs using proximal 2'-F probes.
• Demonstrated efficient monitoring of 5-carboxyl-2'-deoxycytidine (cadC) flipping by TDG using complementary strand 2'-F probes.
• Found that 2'-F probes had minimal to no effect on TDG enzymatic activity.

Conclusions:

• Developed a general and accessible approach for studying nucleotide flipping and other nucleic acid conformational changes.
• The indirect 19F NMR method expands the scope of nucleotide flipping studies.
• This technique provides valuable insights into DNA repair mechanisms and protein-DNA interactions.