Oligonucleotide-Recognizing Topoisomerase Inhibitors (OTIs): Precision Gene Editors for Neurodegenerative Diseases?

Topoisomerases are essential enzymes that recognize and modify the topology of DNA to allow DNA replication and transcription to take place. Topoisomerases are divided into type I topoisomerases, that cleave one DNA strand to modify DNA topology, and type II, that cleave both DNA strands. Topoisomerases normally rapidly religate cleaved-DNA once the topology has been modified. Topoisomerases do not recognize specific DNA sequences, but actively cleave positively supercoiled DNA ahead of transcription bubbles or replication forks, and negative supercoils (or precatenanes) behind, thus allowing the unwinding of the DNA-helix to proceed (during both transcription and replication). Drugs that stabilize DNA-cleavage complexes with topoisomerases produce cytotoxic DNA damage and kill fast-dividing cells; they are widely used in cancer chemotherapy. Oligonucleotide-recognizing topoisomerase inhibitors (OTIs) have given drugs that stabilize DNA-cleavage complexes specificity by linking them to either: (i) DNA duplex recognizing triplex forming oligonucleotide (TFO-OTIs) or DNA duplex recognizing pyrrole-imidazole-polyamides (PIP-OTIs) (ii) or by conventional Watson-Crick base pairing (WC-OTIs). This converts compounds from indiscriminate DNA-damaging drugs to highly specific targeted DNA-cleaving OTIs. Herein we propose simple strategies to enable DNA-duplex strand invasion of WC-OTIs giving strand-invading SI-OTIs. This will make SI-OTIs similar to the guide RNAs of CRISPR/Cas9 nuclease bacterial immune systems. However, an important difference between OTIs and CRISPR/Cas9, is that OTIs do not require the introduction of foreign proteins into cells. Recent successful oligonucleotide therapeutics for neurodegenerative diseases suggest that OTIs can be developed to be highly specific gene editing agents for DNA lesions that cause neurodegenerative diseases.

Structural and functional analysis of the nucleotide and DNA binding activities of the human PIF1 helicase.

Pif1 is a multifunctional helicase and DNA processing enzyme that has roles in genome stability. The enzyme is conserved in eukaryotes and also found in some prokaryotes. The functions of human PIF1 (hPIF1) are also critical for survival of certain tumour cell lines during replication stress, making it an important target for cancer therapy. Crystal structures of hPIF1 presented here explore structural events along the chemical reaction coordinate of ATP hydrolysis at an unprecedented level of detail. The structures for the apo as well as the ground and transition states reveal conformational adjustments in defined protein segments that can trigger larger domain movements required for helicase action. Comparisons with the structures of yeast and bacterial Pif1 reveal a conserved ssDNA binding channel in hPIF1 that we show is critical for single-stranded DNA binding during unwinding, but not the binding of G quadruplex DNA. Mutational analysis suggests that while the ssDNA-binding channel is important for helicase activity, it is not used in DNA annealing. Structural differences, in particular in the DNA strand separation wedge region, highlight significant evolutionary divergence of the human PIF1 protein from bacterial and yeast orthologues.

Coupling the core of the anticancer drug etoposide to an oligonucleotide induces topoisomerase II-mediated cleavage at specific DNA sequences.

Etoposide and other topoisomerase II-targeted drugs are important anticancer therapeutics. Unfortunately, the safe usage of these agents is limited by their indiscriminate induction of topoisomerase II-mediated DNA cleavage throughout the genome and by a lack of specificity toward cancer cells. Therefore, as a first step toward constraining the distribution of etoposide-induced DNA cleavage sites and developing sequence-specific topoisomerase II-targeted anticancer agents, we covalently coupled the core of etoposide to oligonucleotides centered on a topoisomerase II cleavage site in the PML gene. The initial sequence used for this 'oligonucleotide-linked topoisomerase inhibitor' (OTI) was identified as part of the translocation breakpoint of a patient with acute promyelocytic leukemia (APL). Subsequent OTI sequences were derived from the observed APL breakpoint between PML and RARA. Results indicate that OTIs can be used to direct the sites of etoposide-induced DNA cleavage mediated by topoisomerase IIα and topoisomerase IIß. OTIs increased levels of enzyme-mediated cleavage by inhibiting DNA ligation, and cleavage complexes induced by OTIs were as stable as those induced by free etoposide. Finally, OTIs directed against the PML-RARA breakpoint displayed cleavage specificity for oligonucleotides with the translocation sequence over those with sequences matching either parental gene. These studies demonstrate the feasibility of using oligonucleotides to direct topoisomerase II-mediated DNA cleavage to specific sites in the genome.