Repeat-length variation generates stable TTGGG-family G-quadruplex motifs in plant mitochondrial genomes

Repeat-length expansion generates stable, predominantly parallel G4 structures from TTGGG repeats in vitro, identifying expanded plant mitochondrial loci as candidate non-B-DNA-prone regions whose in vivo function remains unresolved. G-quadruplexes (G4s) are non-canonical DNA structures formed by stacked guanine tetrads stabilized by Hoogsteen base pairing and monovalent cations. Although G4s have been extensively studied in nuclear genomes and telomeric DNA, much less is known about G-rich tandem repeats with G4-forming potential in organellar genomes. Here, we investigated exact TTGGG tandem repeats in plant mitochondrial genomes and asked whether repeat expansion can generate local G4-compatible sequence architectures. We first used the exact (TTGGG)₄ motif as the shortest TTGGG-derived repeat module compatible with one intramolecular G4-like fold and re-annotated BLAST-positive Viridiplantae records to identify mitochondrion-annotated candidates. Across the curated mitochondrial subset, exact TTGGG tracts formed a repeat-length continuum. The strongest genome-level example was detected in the Zea perennis mitochondrial genome, where a continuous reverse-strand (TTGGG)₁₆ tract was recovered. A complementary G4Hunter analysis of four maize reference mitogenomes recovered the Z. perennis locus as an extended G4-prone region overlapping the (TTGGG)₁₆ tract, whereas no comparable TTGGG-overlapping extended G4Hunter-supported region was detected in the other Zea references examined. Biophysical characterization of synthetic TTGGG-derived oligonucleotides, (TTGGG)₄, (TTGGG)₈, and (TTGGG)₁₂, revealed robust folding into predominantly parallel G-quadruplex structures, supported by circular dichroism (CD) spectroscopy and native gel electrophoresis. Thermal denaturation experiments further showed high stability under potassium-rich conditions. Together, these results identify expanded TTGGG repeats as an empirical example of G-rich mitochondrial tandem repeats in which repeat expansion increases the number of overlapping minimal G4-compatible modules. More broadly, our data support the view that expanded G-rich repeats can mark local non-B DNA-prone regions in repeat-rich plant mitogenomes, while their actual structural and functional states in vivo remain to be tested experimentally.