Structural polymorphism of the nucleic acids in pentanucleotide repeats associated with CANVAS

Short tandem repeats are highly unstable, depending on repeat length, and the expansion of the repeat length in the human genome is responsible for repeat expansion disorders. Pentanucleotide AAGGG and ACAGG repeat expansions in intron 2 of the gene encoding replication factor C subunit 1 (RFC1) cause cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS) and other phenotypes of late-onset cerebellar ataxia. Herein, we reveal the structural polymorphism of the RFC1 repeat sequences associated with CANVAS in vitro. Single-stranded AAGGG repeat DNA formed a hybrid-type G-quadruplex, whereas its RNA formed a parallel-type G-quadruplex with three layers. The RNA of the ACAGG repeat sequence formed double helical hairpin structures comprising C-G and G-C base pairs with A:A and GA:AG mismatched repeats. Furthermore, both pathogenic repeat RNAs formed more rigid structures than those of the non-pathogenic sequences. These findings provide novel insights into the structural polymorphism of the RFC1 repeat sequences, which may be closely related to the disease mechanism of CANVAS.