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Structure and Functional Role of G-Quadruplex Structures in Genome
Following sequencing of the human genome, many guanine-rich sequences that had the potential to form quadraplexes were discovered. Depending on cell type and cell cycle, mediating factors such as DNA-binding proteins like chromatin, composed of DNA tightly wound around histone proteins, and other environmental conditions and stresses affect the dynamic formation of quadraplexes. For instance, quantitative assessments of the thermodynamics of molecular crowding indicate that the antiparallel g-quadruplex is stabilized by molecular crowding. This effect seems to be mediated by alteration of the hydration of the DNA and its effect on Hoogsteen base pair bonding. These quadruplexes seemed to readily occur at the ends of chromosome. In addition, the propensity of g-quadruplex formation during transcription in RNA sequences with the potential to form mutually exclusive hairpin or G-quadruplex structures depends heavily on the position of the hairpin-forming sequence.

Because repair enzymes would naturally recognize ends of linear chromosomes as damaged DNA and would process them as such to harmful effect for the cell, clear signaling and tight regulation is needed at the ends of linear chromosomes. Telomeres function to provide this signaling. Telomeres, rich in guanine and with a propensity to form g-quadruplexes, are located at the terminal ends of chromosomes and help maintain genome integrity by protecting these vulnerable terminal ends from instability.

These telomeric regions are characterized by long regions of double-stranded CCCTAA:TTAGGG repeats. The repeats end with a 3’ protrusion of between 10 and 50 single-stranded TTAGGG repeats. The heterodimeric complex ribonucleoprotein enzyme telomerase adds TTAGGG repeats at the 3’ end of DNA strands. At these 3’ end protrusions, the G-rich overhang can form secondary structures such as G-quadraplexes if the overhang is longer than four TTAGGG repeats. The presence of these structures prevent telomere elongation by the telomerase complex.

G4-quadruplex structures appear experimentally to serve a critical role in regulating telomere maintenance. In fact, a helicase and nuclease responsible for cleavage of G4s, DNA2, has been found to be involved in maintenance of telomere integrity,. These kinds of processes provide potential mechanisms for cell proliferation control. Understanding these mechanisms could help elucidate mechanisms of malignant transformation of cells and provide new avenues for therapeutic intervention. Small G4-ligands such as telomestatin and G4-binding proteins such as TRF2 show proising anti-proliferative and potential anti-tumor activities that are mediated by telomere interference,. These are thought to function through their interactions with structures formed by G-rich overhangs. There is some evidence that complicates this mechanism by suggesting that telomestatin derivatives prevent telomerase independent activity through targesting G4s involved in tumor genesis elsewhere in the genome.

Outside of telomeric regions, G-quadruplexes are found in several other locations in the genome, such as in gene promoter regions and untranslated regions. For example, the experimental stabilization of G-quadruplex structures in the promoter region of oncogenes MYC, KIT, or KRAS gave rise to the down-regulation of the corresponding gene. This contributes to an emerging literature on the role of G-quadruplexes as key biological regulatory agents, making the structures attractive targets for anti-cancer therapies. Currently, much effort is underway to develop novel ligands with enhanced properties of g-quadruplex recognition. For instance, the fluoroquinolone derivative, Quarfloxin, designed to disrupt protein-DNA interaction through targeting of a g-quadruplex found on ribosomal DNA, has entered clinical trials for multiple malignancies.

Role in Neurological Disorders
4.4: FMRP is a G-quadruplex binding protein implicated in neurological disease -FMRP is a widely expressed protein that binds to G-quadruplex secondary structures in neurons 77, 110,

4.5: FMRP binding to RNA G-quadruplexes directly affects translation -FMRP binding may stabilize G-quadruplex structure and inhibit ribosome elongation of mRNA 123, 124

4.6: FMRP binding to RNA G-quadruplexes is required for neuronal trafficking of specific mRNAs -FMRP binding to RNA G-quadruplexes is used to signal for neurite mRNA signaling which, when formed, can alter the mRNAs in which they reside; this possibly links G-quadruplex formation to repeat expansion in neurological diseases. 129

4.7: CGG repeats in FMR1 can form DNA G-quadruplexes -Due to increasing evidence that FMR1 CGG DNA repeats have a propensity to form a parallel bimolecular G-quadruplex structure that allows for transcription, researchers believe these repeats and G-quadruplex structures may play a role in pathogenesis of Fragile X Tremor/Ataxia Syndrome by lowering FMRP production. 18, 60, 61-63

Working Paragraph: Role in Fragile X Syndrome FMRP is a widely expressed protein coded by the FMR1 gene that binds to G-quadruplex secondary structures in neurons. FMRP binding stabilizes G-quadruplex structures and inhibits ribosome elongation of mRNA. Fragile X Syndrome, an intellectual disability disorder, is caused by an increase of CGG repeats in the FMR1 gene, possibly altering the amount of G-quadruplex structures formed, linking G-quadruplex formation in neurological disease. Due to increasing evidence that FMR1 CCGG DNA repeats have a propensity to form a parallel bimolecular G-quadruplex structure, which decreases FMRP production, it is believed that these repeats and G-quadruplex structures may play a role in the pathogenesis of Fragile X Syndrome.

Role in Neurological Disorders
G-quadruplexes have been implicated in neurological disorders through two main mechanisms. The first is through expansions of G-repeats within a gene that can form G-quadruplex structures that directly cause disease, as is the case with the C9orf72 gene and amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD). The second of which is through mutations that affect the expression of G-quadruplex binding proteins, as seen in the FMR1 gene and Fragile X Syndrome.

The C9orf72 gene codes for C9orf72, a protein that is found throughout the brain in neuronal cytoplasm and at presynaptic terminals. Mutations of the C9orf72 gene have been linked to the development of FTD and ALS. These two diseases have a causal relationship to GGGGCC (G4C2) repeats within the 1st intron of C9orf72 gene. In normal individuals there are around 2 G4C2 repeats, but individuals with FTD or ALS have from 500 to several thousand G4C2 repeats. The transcribed RNA of these repeats have been shown to form stable G-quadruplexes, with evidence showing that the G4C2 repeats in DNA have the ability to form mixed parallel-antiparallel G-quadruplex structures as well. These RNA transcripts containing G4C2 repeats were shown to bind and separate a wide variety of proteins, including nucleolin. Nucleolin is involved in the synthesis and maturation of ribosomes within the nucleus, and separation of nucleolin by the mutated RNA transcripts impairs nucleolar function and ribosomal RNA synthesis.

Fragile X mental retardation protein (FMRP) is a widely expressed protein coded by the fragile X mental retardation gene 1 (FMR1) that binds to G-quadruplex secondary structures in neurons. Mutations of this gene can cause the development of Fragile X Syndrome, autism, and other neurological disorders. FMRP binding stabilizes G-quadruplex structures in the mRNA transcript, which inhibits ribosome elongation of mRNA in the neuron's dendrite. Fragile X Syndrome, an intellectual disability disorder, is caused by an increase from 50 to over 200 CGG repeats within exon 13 of the FMR1 gene. This increase inhibits FMR1 transcription by increasing DNA methylation and other epigenetic heterochromatin modifications. Due to increasing evidence that FMR1 CGG DNA repeats have a propensity to form parallel bimolecular G-quadruplex structures and decrease FMRP production, it is believed that these repeats and G-quadruplex structures within the FMR1 gene may play a role in the pathogenesis of Fragile X Syndrome.

Therapeutic Approaches for Neurological Disorders via Targeting G-Quadruplexes
Antisense-mediated interventions and small-molecule ligands are common strategies used to target neurological diseases linked to G-quadruplex expansion repeats. Therefore, these techniques are especially advantageous for targeting neurological diseases that have a gain-of-function mechanism, which is when the altered gene product has a new function or new expression of a gene; this has been detected in the C9orf72 (chromosome 9 open reading frame 72).

Antisense therapy is the process by which synthesized strands of nucleic acids are used to bind directly and specifically to the mRNA produced by a certain gene, which will inactivate it. Antisense oligonucleotides (ASOs) are commonly used to target C9orf72 RNA of the G-quadruplex GGGGCC expansion repeat region, which has lowered the toxicity in cellular models of C9orf72. . ASOs have previously been used to restore normal phenotypes in other neurological diseases that have gain-of-function mechanisms, the only difference is that it was used in the absence of G-quadruplex expansion repeat regions.

Another commonly used technique is the utilization of small-molecule ligands. These can be used to target G-quadruplex regions that cause neurological disorders. Approximately 1,000 various G-quadruplex ligands exist in which they are able to interact via their aromatic rings; this allows the small-molecule ligands to stack on the planar terminal tetrads within the G-quadruplex regions. A disadvantage of using small-molecule ligands as a therapeutic technique is that specificity is difficult to manage due to the variability of G-quadruplexes in their primary sequences, orientation, thermodynamic stability, and nucleic acid strand stoichiometry. As of now, no single small-molecule ligand has been able to be 100% specific for a single G-quadruplex sequence. However, a cationic porphyrin known as TMPyP4 is able to bind to the C9orf72 GGGGCC repeat region, which causes the G-quadruplex repeat region to unfold and lose its interactions with proteins causing it to lose its functionality. Small-molecule ligands, composed primarily of lead, can target GGGGCC repeat regions as well and ultimately decreased both repeat-associated non-ATG translation and RNA foci in neuron cells derived from patients with Amyotrophic lateral sclerosis (ALS). This provides evidence that small-molecule ligands are an effective and efficient process to target GGGGCC regions, and that specificity for small-molecule ligand binding is a feasible goal for the scientific community.