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In the neurodegenerative disorders amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), expansion of the G4C2 hexanucleotide repeat in the gene C9orf72 is a most common known cause of the disease. Here we use atomic force microscopy (AFM) and gel electrophoresis to visualize the formation of higher-order structures by RNA G4C2 repeats in physiologically relevant conditions. For the RNA sequence r[G4C2G4], we observed G-wires with left-handed undulating features of 4.4-nm periodicity and a uniform height which is consistently higher than that of a duplex B-DNA. These higher-order structures were not degraded fully when treated with a mixture of RNase A and RNase T1. Similarly, higher-order structures were observed for sequences containing three or four G4C2 repeats, pointing towards their potential formation in longer sequence contexts. Our observations suggest that RNA G-quadruplex blocks and G-wires can accumulate in cells containing G4C2 repeat transcripts. Ministry of Education (MOE) Nanyang Technological University This research was supported by Singapore Ministry of Education Academic Research Fund Tier 2 (MOE2015-T2-1-092) and grants from Nanyang Technological University to A.T.P.

G-rich DNA/RNA sequences are highly dynamic and diverse in nature as they can adopt a myriad of secondary structures in physiologically relevant conditions. Several biologically relevant G-rich repeat motif DNA sequences are known to form higher-order G-quadruplexes, but little is known about their atomic level structure. Atomic Force Microscopy (AFM) has been widely used to investigate higher-order G-quadruplexes for the past 3 decades. In the current work, AFM has been pushed to the limits to develop a new methodology for probing biomolecular structure with sub-nanometer resolution and thus bridge the gap between single-molecule and high-resolution ensemble techniques. The protocol for visualization of G-quadruplex DNA and RNA in aqueous solution was developed to resolve their intricate structural features. Higher-order structures formed by three different biologically relevant sequences containing G-rich repeat motif were investigated, viz., GGGGTT, GGGGCC and TTAGGG. It was found that short and long G-wires are formed by d[G4T2G4], d[G4C2G4] and r[G4C2G4]. All these G-wires revealed similar morphology (uniform height, variable length) and periodic surface pattern. We built several molecular models and found one that can explain the experimentally observed features of G-wires. Our work hint towards a unified architecture adopted by G-wires formed by different DNA and RNA oligonucleotides. These findings are useful for the design of G-wire based nanodevices and may find relevance in biology. Transcription of PCR-amplified DNA containing several repeats of TTAGGG was used to generate telomeric repeat containing RNA (TERRA), which was investigated using high-resolution AFM in aqueous solution. The higher-order structures formed by TERRA were grouped into two broad classes based on their heights and intricate structural periodicities. One class of observed structures were like the pearl necklace as proposed earlier for telomeric DNA and RNA. In summary, we conclude that G-rich DNA and RNA sequences are capable of self-assembling into higher-order intermolecular G-quadruplexes which are consistent with a model of stacked G-tetrads having molecular periodicities which are multiples of the distance between individual G-tetrad layers. However, individual strands of G-rich DNA and RNA could exist as a pearl necklace, where the beads are likely G-quadruplexes, which are connected by a single-stranded linker. The higher-order structures reported here could be broadly classified into just two groups: four-stranded parallel G-wire and single-stranded pearl necklace. ​Doctor of Philosophy (SPMS)