Solution structure of a DNA quadruplex containing ALS and FTD related GGGGCC repeat stabilized by 8-bromodeoxyguanosine substitution.

Jasna Brčić,Janez Plavec

doi:10.1093/nar/gkv815

Abstract

A prolonged expansion of GGGGCC repeat within non-coding region of C9orf72 gene has been identified as the most common cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), which are devastating neurodegenerative disorders. Formation of unusual secondary structures within expanded GGGGCC repeat, including DNA and RNA G-quadruplexes and R-loops was proposed to drive ALS and FTD pathogenesis. Initial NMR investigation on DNA oligonucleotides with four repeat units as the shortest model with the ability to form an unimolecular G-quadruplex indicated their folding into multiple G-quadruplex structures in the presence of K+ ions. Single dG to 8Br-dG substitution at position 21 in oligonucleotide d[(G4C2)3G4] and careful optimization of folding conditions enabled formation of mostly a single G-quadruplex species, which enabled determination of a high-resolution structure with NMR. G-quadruplex structure adopted by d[(G4C2)3GGBrGG] is composed of four G-quartets, which are connected by three edgewise C-C loops. All four strands adopt antiparallel orientation to one another and have alternating syn-anti progression of glycosidic conformation of guanine residues. One of the cytosines in every loop is stacked upon the G-quartet contributing to a very compact and stable structure.

Highlights

Guanine-rich nucleic acids can form in the presence of cations such as K+ or Na+ non-canonical four-stranded structures called G-quadruplexes, which are composed of stacked layers of G-quartets, formed by four guanine residues connected by Hoogsteen-type hydrogen bonds
A recent study indicated that d(GGGGCC)4 folds into a polymorphic mixture of G-quadruplex structures and recognized antiparallel topology as the major form in K+ ions containing solution
G-quadruplex formation in the DNA strand within the region containing C9orf72 hexanucleotide repeat has been suggested to cause impairment in RNA polymerase processivity that leads to an increase in abortive RNA transcripts, which are probably cytotoxic and cause downstream pathological effects [16]

Summary

Introduction

Guanine-rich nucleic acids can form in the presence of cations such as K+ or Na+ non-canonical four-stranded structures called G-quadruplexes, which are composed of stacked layers of G-quartets, formed by four guanine residues connected by Hoogsteen-type hydrogen bonds. The underlying mechanism of non-coding repeat expansion disorders most commonly involves an RNA dependent gain-of-function mechanism independently of the proteins encoded by their sequences [14]. RNA gain-of-function mechanism involves unusual secondary structure formation in RNA transcripts leading to conformation-dependent sequestration of functionally important cellular proteins, a mechanism that was most thoroughly studied in myotonic dystrophy [15]. A disease mechanism has been proposed in which structural polymorphism within expanded GGGGCC repeat, including G-quadruplex structures at both DNA and RNA level and transcriptionally induced RNA·DNA hybrids (Rloops) are implicated in the development of ALS and FTD [17]. RNA G-quadruplex formation in RNA transcripts in turn leads to conformation-dependent interaction

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