Uncovering intergenic DNA i-motifs in the telomere-to-telomere human genome

Noncanonical DNA structures such as i-motifs (iMs) are emerging as versatile functional elements within the human genome, yet their distribution and properties outside regulatory regions remain poorly characterized. Leveraging the complete telomere-to-telomere (T2T-CHM13) genome assembly, we generated an updated genome-wide catalogue of putative iM-forming sequences by implementing a bioinformatics pipeline for the systematic detection and statistical assessment of cytosine-rich motifs consistent with canonical iM-forming patterns. The analysis identified 789,952 candidates, representing a 6.39% increase over our previous hg38-based catalogue. Sequence-level comparison showed that 707,333 candidates (89.54%) had an identical sequence on the same chromosome and strand in the hg38-derived candidate set, whereas 82,619 (10.46%) were T2T-unmatched under these criteria. From this dataset, we focused on two distal-intergenic candidates selected to represent different positions within the T2T-CHM13 intergenic PSSM-score distribution and contrasting sequence architectures. The two candidates were characterized through an integrated biophysical workflow combining circular dichroism spectroscopy, UV absorption analyses (including thermal and isothermal difference spectra), nuclear magnetic resonance spectroscopy, native polyacrylamide gel electrophoresis, and size-exclusion chromatography. Both sequences adopted canonical iM folds under mildly acidic conditions (pH ≤ 6.25), displaying reversible, pH-dependent folding and distinct thermodynamic transitions. Despite similar lengths, the two iMs exhibited distinct stability and pH responsiveness, which could be rationalized by differences in cytosine density, C-tract organization, and loop architecture. Overall, this study provides a comprehensive set of iMs candidates within the T2T-CHM13 assembly, and it defines key structural and thermodynamic features of intergenic iMs.