Structure of Tetrahelical DNA Homopolymers Supports Quadruplex World Hypothesis

[Image: see text] We previously reported a tetrahelical monomolecular architecture of DNA, tmDNA, which employs G-quartets and an all-parallel GGGTGGGTGGGTGGG (G3T) quadruplex as the repeating unit. Based on thermodynamic and kinetic studies, we proposed that covalently joined (G3T)(n) units formed an uninterrupted programmable homopolymer; however, structural evidence for the tmDNA architecture was lacking. Here, we used NMR spectroscopy of wild-type and single-inosine-substituted constructs to characterize both monomolecular (G3T)(2) and bimolecular quadruplex-Mg-coupled versions of tmDNA. The NMR results support an architecture consisting of uninterrupted stacked G-tetrads in both the monomolecular constructs and bimolecular assemblies. Taken together, these data support the formation of a stable programmable homopolymeric tmDNA architecture, which may have been a precursor to the modern-day Watson–Crick DNA duplex.