Abstract
A nucleotide repeat expansion (NRE), (G4C2)n, located in a classically noncoding region of C9orf72 (C9), is the most common genetic mutation associated with ALS/FTD. There is increasing evidence that nucleic acid structures formed by the C9-NRE may both contribute to ALS/FTD, and serve as therapeutic targets, but there is limited characterization of these nucleic acid structures under physiologically and disease relevant conditions. Here we show in vitro that the C9-NRE DNA can form both parallel and antiparallel DNA G-quadruplex (GQ) topological structures and that the structural preference of these DNA GQs can be dependent on the molecular crowding conditions. Additionally, 5-methylcytosine DNA hypermethylation, which is observed in the C9-NRE locus in some patients, has minimal effects on GQ topological preferences. Finally, molecular dynamic simulations of methylated and nonmethylated GQ structures support in vitro data showing that DNA GQ structures formed by the C9-NRE DNA are stable, with structural fluctuations limited to the cytosine-containing loop regions. These findings provide new insight into the structural polymorphic preferences and stability of DNA GQs formed by the C9-NRE in both the methylated and nonmethylated states, as well as reveal important features to guide the development of upstream therapeutic approaches to potentially attenuate C9-NRE-linked diseases.
Highlights
A nucleotide repeat expansion (NRE), (G4C2)n, located in a classically noncoding region of C9orf[72] (C9), is the most common genetic mutation associated with amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD)
We show that C9-NRE DNA GQ structures can transition from a GQ-AP topological preference to a GQ-P topology under certain molecular crowding conditions that attempt to recapitulate molecular crowding conditions encountered within the nuclear environment in cells or potentially membraneless o rganelles[36,37,38]
The disease associated C9-NRE DNA mutation has been previously shown to be structurally polymorphic; varying lengths of DNA oligos derived from the NRE region can form canonical Watson:Crick double-helix bDNA as well as non-canonical hairpins and GQs with the diversity of these structures possibly increasing with repeat length[7,27,28,29,30]
Summary
A nucleotide repeat expansion (NRE), (G4C2)n, located in a classically noncoding region of C9orf[72] (C9), is the most common genetic mutation associated with ALS/FTD. Molecular dynamic simulations of methylated and nonmethylated GQ structures support in vitro data showing that DNA GQ structures formed by the C9-NRE DNA are stable, with structural fluctuations limited to the cytosine-containing loop regions These findings provide new insight into the structural polymorphic preferences and stability of DNA GQs formed by the C9-NRE in both the methylated and nonmethylated states, as well as reveal important features to guide the development of upstream therapeutic approaches to potentially attenuate C9-NRE-linked diseases. NMR spectroscopy and X-ray crystallography studies have generated detailed atomic resolution structures for some of these possible C9-NRE GQ-AP and GQ-P structural c onformations[29,30] These atomic structures could be utilized to screen and identify therapeutic candidates that might modify the stability of these structures and alter the C9-NRE-linked ALS/FTD disease cascade in patients. It is currently unknown if more physiologically relevant cellular conditions or disease-relevant DNA modifications, such as Scientific Reports | (2021) 11:23213 |
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