Expansion of d(GGCCTG)n hexanucleotide repeats in the NOP56 gene is the genetic cause of spinocerebellar ataxia type 36 (SCA36) which is an inheritable neurodegenerative disease. Non-B DNA is known to be the structural intermediate causing repeat expansions. Yet, the structure and mechanism of genetic instability of d(GGCCTG)n repeats remain elusive. In this work, we investigated the solution structures of sequences containing two to eight GGCCTG repeats using nuclear magnetic resonance (NMR) spectroscopy. They were found to form diverse secondary structures, including hairpin, duplex and G-quadruplex (G4). Intriguingly, the hairpin structure was present in all the investigated sequences. The NMR solution structure of the hairpin formed by d(GGCCTG)2 was determined, disclosing an unprecedented CCTGGG hexanucleotide loop in which the first and sixth loop residues formed a Watson-Crick loop-closing base pair, the second and third loop residues stacked in the major groove, whereas the fourth and fifth loop residues formed a G·G mismatch. Apart from the hairpin, antiparallel G4 and palindromic duplex structures were found to form in d(GGCCTG)2 and d(GGCCTG)3-8, respectively. Results of this work provide new insights into the genetic instability of d(GGCCTG)n repeats and structure-based drug design for SCA36.
Keywords: G-quadruplex; Hairpin structure; Hexanucleotide d(GGCCTG)(n) repeats; NMR spectroscopy; Spinocerebellar ataxia type 36.
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