Context-Sensitive Cleavage of Folded DNAs by Loop-Targeting bPNAs

Area of Science:

• Chemical Biology
• Molecular Biology
• Synthetic Chemistry

Background:

• Synthetic peptide nucleic acids (bPNAs) can be functionalized with metal complexes to create DNA-binding agents.
• Oxidative cleavage of the DNA backbone is a known mechanism for detecting DNA binding.
• G-quadruplex (G4) structures, particularly telomeric repeats, are important targets in cancer research.

Purpose of the Study:

• To investigate the DNA cleavage activity of bPNA-copper and bPNA-iron complexes.
• To determine if this cleavage activity is sequence and structure-dependent.
• To evaluate the potential of bPNA nucleases for targeting telomeres and inducing cellular effects.

Main Methods:

• Synthesis of oligoethylenimine bPNAs with iron·EDTA or copper·phenanthroline sites.
• Design and use of DNA substrates with T-rich single-stranded domains and telomeric repeat sequences.
• Assessing DNA cleavage using oxidative backbone cleavage as a readout.
• Treatment of isolated and intracellular DNA from PC3 cells with bPNA nucleases.
• Quantitative polymerase chain reaction (qPCR) to measure telomere length.
• Observation of cellular senescence as an indicator of telomere shortening.

Main Results:

• bPNA iron and copper complexes function as efficient chemical nucleases for structured DNA with T-rich regions.
• Cleavage activity is highly dependent on the DNA's structural context and buffer conditions (Na+ vs. K+).
• bPNA-copper complexes selectively cleaved telomeric repeat sequences in sodium buffer, mimicking antiparallel G4 topology.
• Treatment of PC3 cells with bPNA nucleases resulted in telomere shortening and accelerated cellular senescence.
• bPNA nucleases demonstrated cell membrane permeability, allowing targeting of intracellular DNA.

Conclusions:

• bPNA chemical nucleases offer a loop-targeting strategy that complements existing methods for DNA targeting.
• The ability to discriminate between similar DNA sequences based on structure expands the utility of bPNAs.
• bPNA nucleases are cell-permeable and can target native intracellular DNA structures.
• bPNA scaffolds provide a versatile platform for developing novel synthetic binders for specific nucleic acid motifs.