Conformational features of intramolecular G4-DNA constrained by single-nucleotide loops

Conformation of the telomeric DNA fragment dG₃(TTAG₃)₃ depends on multiple factors including solution conditions, length, and the nucleotide sequence of the flanking regions. In potassium solution, this sequence tends to adopt hybrid (3 + 1) G-quadruplex (G4) Form 1 or Form 2 conformation contingent on the flanking nucleotides. Theoretically, other (3 + 1) G4 folds (beyond Forms 1 and 2) are not sterically forbidden, but are presumably energetically disfavored. We report here on the effect of substituting the TTA loop with a single T nucleotide for one, two, or three loops of telomeric DNA that allowed us to expand the conformational diversity of the G4 DNA. Circular dichroism, gel migration, and chemical probing with DMS and ZnP1 (a porphyrin derivative sensitive to G4 conformation) were applied to monitor conformations that occurred upon shortening each loop to a single nucleotide. We found that all oligonucleotide models formed an intramolecular quadruplex structure and that shortening the loops led to the prevalence of G4 with quartets of the same polarity. Despite similar CD signatures, each modified sequence had one of three specific patterns of light-induced oxidation with ZnP1. According to the predominant modification pattern, folding of each sequence could be assigned to one of three major G4 conformations: parallel and two different (3 + 1) G4 folds. We here provide novel experimental evidence of the propensity for modified telomeric sequences to form a (3 + 1) G4 conformer containing one lateral and two propeller loops.