Characterization of Hairpin Loops and Cruciforms Across 118,019 Genomes Spanning the Tree of Life

Inverted repeats (IRs) can form alternative DNA secondary structures, including hairpins and cruciforms, which have a multitude of functional roles and have been associated with genomic instability. However, their prevalence across diverse organismal genomes remains only partially understood. Here, we examine the prevalence of perfect IRs, which do not have mismatches in their arms, across 118,019 complete organismal genomes. Our comprehensive analysis across taxonomic subdivisions reveals significant differences in the distribution, frequency, and biophysical properties of perfect IRs among these genomes. We identify a total of 33,558,920 perfect IRs and show a highly variable density across different organisms, with strikingly distinct patterns observed in Viruses, Bacteria, Archaea, and Eukaryota. We report IRs with perfect arms of extreme lengths, which can extend to hundreds of thousands of base pairs. Our findings reveal that Bacteria possess the highest IR density. Additionally, this study reveals the enrichment of IRs at transcription start and end sites in prokaryotes and Viruses and underscores their potential roles in gene regulation and genome organization. Analysis of intraspecies variation shows elevated substitution burden in IR spacers and relative conservation of IR arms, particularly near transcriptional terminators. Through a comprehensive overview of the distribution and characteristics of IRs in a wide array of organisms, this largest-scale analysis to date sheds light on the functional significance of perfect IRs, their contribution to genomic instability, and their evolutionary impact across the tree of life.