Abstract

Massive expansion of a DNA hexanucleotide sequence repeat (C2G4) within the human C9orf72 gene has been linked to a number of neurodegenerative diseases. In sodium or potassium salt solutions, single-stranded d(C2G4)n DNAs fold to form G-quadruplexes. We have found that in magnesium or lithium salt solutions, especially under slightly acidic conditions, d(C2G4)n oligonucleotides fold to form a distinctive higher order structure whose most striking feature is an “inverted” circular dichroism spectrum, which is distinguishable from the spectrum of the left handed DNA double-helix, Z-DNA. On the basis of CD spectroscopy, gel mobility as well as chemical protection analysis, we propose that this structure, which we call “iCD-DNA”, may be a left-handed Hoogsteen base-paired duplex, an unorthodox G-quadruplex/i-motif composite, or a non-canonical, “braided” DNA triplex. Given that iCD-DNA forms under slightly acidic solution conditions, we do not know at this point in time whether or not it forms within living cells.

Highlights

  • The repeat expansion of a hexanucleotide DNA sequence (CCGGGG) found in the 5’-untranslated region of the C9orf72 gene has been shown to be causally linked to Frontotemporal Lobar Dementia and familial Amyotrophic Lateral Sclerosis (FTD/ALS) [1, 2]

  • While preparing a negative control for a CD spectroscopic study of G-quadruplex formation by d(C2G4)7, we observed that this oligomer, dissolved at 700 μM concentration in TE-LiCl buffer (10 mM Tris, pH 7.4, 0.1 mM EDTA, 150 mM LiCl) and incubated at 37 ̊ C for up to 5 days, showed an unusual circular dichroism spectrum

  • Given that d(C2G4)7 contains only two of the four nucleobases, G and C, and the known important role of protonated cytosines in the formation of non-canonical secondary DNA structures like triplex and i-motif, we investigated whether pH values of < 7.0 impacted on the inverted CD spectrum

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Summary

Introduction

The repeat expansion of a hexanucleotide DNA sequence (CCGGGG) found in the 5’-untranslated region of the C9orf gene has been shown to be causally linked to Frontotemporal Lobar Dementia and familial Amyotrophic Lateral Sclerosis (FTD/ALS) [1, 2]. At the level of DNA, the d(GGGGCC) repeat expansion single strand and its complementary strand have been shown in vitro to form unusual secondary structures, namely hairpin folds, G-quadruplexes, i-motif and R loops [8,9,10]. These unusual structures, if present in repeatexpansion afflicted neurons, can potentially cause down-regulation in gene expression leading to reduced levels of the coded protein [11,12,13].

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