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

Herein, we demonstrate context-dependent molecular recognition of DNA by synthetic bPNA iron and copper complexes, using oxidative backbone cleavage as a chemical readout for binding. Oligoethylenimine bPNAs displaying iron. EDTA or coppe center dot phenanthroline sites were found to be efficient chemical nucleases for designed and native structured DNAs with T-rich single-stranded domains. Cleavage reactivity depends strongly on structural context, as strikingly demonstrated with DNA substrates of the form (GGGTTA)(n). This repeat sequence from the human telomere is known to switch between parallel and antiparallel G-quadruplex (G4) topologies with a change from potassium to sodium buffer: notably, bPNA-copper complexes efficiently cleave long repeat sequences into similar to 22-nucleotide portions in sodium, but not potassium, buffer. We hypothesize preferential cleavage of the antiparallel topology (Na+) over the parallel topology (K+) due to the greater accessibility of the TTA loop to bPNA in the antiparallel (Na+) form. Similar ion-sensitive telomere shortening upon treatment with bPNA nucleases can be observed in both isolated and intracellular DNA from PC3 cells by quantitative polymerase chain reaction. Live cell treatment was accompanied by accelerated cellular senescence, as expected for significant telomere shortening. Taken together, the loop-targeting approach of bPNA chemical nucleases complements prior intercalation strategies targeting duplex and quadruplex DNA. Structurally sensitive loop targeting enables discrimination between similar target sequences, thus expanding bPNA targeting beyond simple oligo-T sequences. In addition, bPNA nucleases are cell membrane permeable and therefore may be used to target native intracellular substrates. In addition, these data indicate that bPNA scaffolds can be a platform for new synthetic binders to particular nucleic acid structural motifs.