QuadCleave: Enzymatic cleavage and analysis of DNA G-quadruplexes

Prediction of G-quadruplexes (G4), noncanonical DNA secondary structures containing guanine tetrads, can have profound relevance across cancer, neurodegenerative and other genetic diseases, but require experimental validation by instrument intensive methods including CD spectroscopy, fluorescence, NMR, or crystallography. We report a novel, inexpensive, efficient and scalable 2-enzyme system for confirmation of G4 formation and copy number repeat count. First, QuadCleave, in which Mismatch Endonuclease I was used to repeatably, site-specifically and selectively cleave oligodeoxynucleotides containing exclusively G4-forming sequences. Second, HaeIII enzyme identified G4-forming sequences that can also generate GG/CC base pairs, and a sequence’s cleavage by both established it in C9ORF72, the most mutated gene in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). These findings establish ubiquitously accessible experimental validation of G4 forming sequences and their copy number repeat assessment, with specificity to C9ORF72 oligodeoxynucleotides whose repeat expansion has critical clinical implications in a subpopulation of ALS and FTD patients.