A direct and nondestructive approach to determine the folding structure of the I-motif DNA secondary structure by NMR.

I-motifs are four-stranded DNA secondary structures formed in C-rich DNA sequences and consist of parallel-stranded DNA duplexes zipped together in an antiparallel orientation by intercalated, hemiprotonated cytosine(+)-cytosine base pairs. I-motif structures have been indicated to form in various regions of the human genome as well as in nanotechnological applications. While NMR is a major tool for structural studies of I-motifs, the determination of the folding topologies of unimolecular I-motifs has been a challenging and arduous task using conventional NMR spectral assignment strategies, due to the inherent sequence redundancy of the C-rich strands in the formation of unimolecular I-motif structures. We report here a direct and nondestructive method that can be utilized to unambiguously determine the hemiprotonated C(+)-C base pairs and thus the folding topology of unimolecular I-motif structures formed from native C-rich DNA sequences. The reported approach uses affordable low-enrichment site-specific labeling. More significantly, the reported method can directly and unambiguously determine the equilibrating multiple conformations coexisting in a single DNA sequence, which would be a very difficult task using conventional assignment strategies. Additionally, this method can be applied to the direct detection of the base-paired thymines that are involved in the capping structures.