RNA G-quadruplexes form scaffolds that promote neuropathological α-synuclein aggregation.

Synucleinopathies, including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy, are triggered by α-synuclein aggregation, triggering progressive neurodegeneration. However, the intracellular α-synuclein aggregation mechanism remains unclear. Herein, we demonstrate that RNA G-quadruplex assembly forms scaffolds for α-synuclein aggregation, contributing to neurodegeneration. Purified α-synuclein binds RNA G-quadruplexes directly through the N terminus. RNA G-quadruplexes undergo Ca2+-induced phase separation and assembly, accelerating α-synuclein sol-gel phase transition. In α-synuclein preformed fibril-treated neurons, RNA G-quadruplex assembly comprising synaptic mRNAs co-aggregates with α-synuclein upon excess cytoplasmic Ca2+ influx, eliciting synaptic dysfunction. Forced RNA G-quadruplex assembly using an optogenetic approach evokes α-synuclein aggregation, causing neuronal dysfunction and neurodegeneration. The administration of 5-aminolevulinic acid, a protoporphyrin IX prodrug, prevents RNA G-quadruplex phase separation, thereby attenuating α-synuclein aggregation, neurodegeneration, and progressive motor deficits in α-synuclein preformed fibril-injected synucleinopathic mice. Therefore, Ca2+ influx-induced RNA G-quadruplex assembly accelerates α-synuclein phase transition and aggregation, potentially contributing to synucleinopathies.

Modulation of dynamic DNA G-quadruplex structures in the hTERT promoter region by ligands.

Small molecules can inhibit cellular processes such as replication and transcription by binding to the promoter regions that are prone to form G-quadruplexes. However, since G-quadruplexes exist throughout the human genome, the G-quadruplex binders suffer from specificity issues. To tackle this problem, a G-quadruplex binder (Pyridostatin, or PDS) is conjugated with a ligand (Polyamide, or PA) that can specifically recognize DNA sequences flanking the G-quadruplex forming region. The binding mechanism of this hybrid ligand to the hTERT promoter region (hTERT 5-12) is then elucidated using optical tweezers. During mechanical unfolding processes, different intermediate structures of hTERT 5-12 in presence of PDS, PA, or PA-PDS conjugate are observed. These intermediate structures are consistent with two folding patterns of G-quadruplexes in the hTERT 5-12 fragment. While the duplex DNA binder PA facilitates the folding of a hairpin-G-quadruplex structure, the PDS assists the formation of two tandem G-quadruplexes. Both replication stop assay in vitro and dual luciferase assay in vivo established the effectiveness of the PA-PDS conjugate for hTERT 5-12 targeting. We expect such a ligand dependent folding dynamics will provide guidelines to the development of drugs that not only target hTERT expressions, but also other oncogenes via interactions with specific G-quadruplex structures formed in their promotor regions.

Structural polymorphism of the nucleic acids in pentanucleotide repeats associated with the neurological disorder CANVAS.

Short tandem repeats are inherently unstable during DNA replication depending on repeat length, and the expansion of the repeat length in the human genome is responsible for repeat expansion disorders. Pentanucleotide AAGGG and ACAGG repeat expansions in intron 2 of the gene encoding replication factor C subunit 1 (RFC1) cause cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS) and other phenotypes of late-onset cerebellar ataxia. Herein, we reveal the structural polymorphism of the RFC1 repeats associated with CANVAS in vitro. Single-stranded AAGGG repeat DNA formed a hybrid-type G-quadruplex, whereas its RNA formed a parallel-type G-quadruplex with three layers. The RNA of the ACAGG repeat formed hairpin structure comprising C-G and G-C base pairs with A:A and GA:AG mismatched repeats. Furthermore, both pathogenic repeat RNAs formed more rigid structures than those of the nonpathogenic repeat RNAs. These findings provide novel insights into the structural polymorphism of the RFC1 repeats, which may be closely related to the disease mechanism of CANVAS.

Small molecule-based detection of non-canonical RNA G-quadruplex structures that modulate protein translation.

Tandem repeats of guanine-rich sequences in RNA often form thermodynamically stable four-stranded RNA structures. Such RNA G-quadruplexes have long been considered to be linked to essential biological processes, yet their physiological significance in cells remains unclear. Here, we report a approach that permits the detection of RNA G-quadruplex structures that modulate protein translation in mammalian cells. The approach combines antibody arrays and RGB-1, a small molecule that selectively stabilizes RNA G-quadruplex structures. Analysis of the protein and mRNA products of 84 cancer-related human genes identified Nectin-4 and CapG as G-quadruplex-controlled genes whose mRNAs harbor non-canonical G-quadruplex structures on their 5'UTR region. Further investigations revealed that the RNA G-quadruplex of CapG exhibits a structural polymorphism, suggesting a possible mechanism that ensures the translation repression in a KCl concentration range of 25-100 mM. The approach described in the present study sets the stage for further discoveries of RNA G-quadruplexes.

Strong and Specific Recognition of CAG/CTG Repeat DNA (5'-dWGCWGCW-3') by a Cyclic Pyrrole-Imidazole Polyamide.

Abnormally expanded CAG/CTG repeat DNA sequences lead to a variety of neurological diseases, such as Huntington's disease. Here, we synthesized a cyclic pyrrole-imidazole polyamide (cPIP), which can bind to the minor groove of the CAG/CTG DNA sequence. The double-stranded DNA melting temperature (Tm ) and surface plasmon resonance assays revealed the high binding affinity of the cPIP. In addition, next-generation sequencing showed that the cPIP had high specificity for its target DNA sequence.

Pharmacological prospects of G-quadruplexes for neurological diseases using porphyrins.

Genomic regions with guanine (G)-rich sequences make non-Watson-Crick base pairs, which result in the formation of unique nucleic acid structures called G-quadruplexes (G4s) in cells. Studies have suggested that abnormal G4s are involved in neurological diseases. For example, the formation of G4s caused by expansion of G-rich sequences is implicated in C9orf72-mediated amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD), and fragile X-related tremor/ataxia syndrome (FXTAS). In addition, the disruption and/or mutation of G4 binding proteins (G4BPs), such as heterogeneous nuclear ribonucleoproteins (hnRNPs) and DNA/RNA helicases, is related to neurological diseases. For instance, mutations in a G4BP called ATRX lead to a neurodevelopmental disorder, ATR-X syndrome, which is associated with intellectual disability. We found that porphyrins are potential candidate drugs for treating ATR-X syndrome through their G4 binding ability. Importantly, intracellular porphyrins are produced from 5-aminolevulinic acid (5-ALA) in vivo. Oral administration of 5-ALA improved cognitive dysfunction in an ATR-X syndrome model mouse, and language ability in an ATR-X syndrome patient. In this review, we suggest a novel therapeutic strategy targeting G4s using porphyrins in neurological diseases.

Identification and immunohistochemical characterization of G-quadruplexes in mouse brain.

Guanine-rich DNA and RNA can form a four-stranded structure, termed G-quadruplexes (G4s) in vitro as well as in cells. The formation of G4 is implicated in many physiological events, such as gene transcription, translation, and epigenetics. However, the presence of G4 has not been revealed in the brain. Here, we demonstrate the localization of G4 in the mouse brain by immunohistochemical analysis. In cultured mouse forebrain neurons, numerous punctate G4 foci were observed in nuclei as well as in cytoplasmic areas, including axons, dendrites, and postsynapses. Interestingly, the G4 foci in nuclei show more marked co-localizations with the bright spots of DAPI-positive heterochromatin clusters in mature neurons compared to immature ones. In slices from adult mouse brain, the G4 foci were distributed throughout the brain but were particularly prominent in the hippocampus, olfactory bulb, and cerebellum. In the hippocampus, G4 foci were strongly expressed in neurons and weakly in astrocytes. Consistent with the results in cultured neurons, the nuclear G4 foci were co-localized with heterochromatin in calbindin-positive mature granule cells but less in doublecortin-positive neuronal progenitor cells in the dentate gyrus. Electron microscopic immunolabeling revealed G4 foci on nucleolus-associated chromosomal domains (NADs) and cytoplasm in the adult mouse hippocampal CA1 region. These observations suggest potentially critical roles of G4 in neuronal developmental stages through regulating chromatin structures and cytoplasmic metabolism of RNA.

Control of Forward/Reverse Orientation Preference of Cyclic Pyrrole-Imidazole Polyamides.

Pyrrole-imidazole polyamides (PIPs) bind to predetermined double-stranded DNA sequences and selectively target a large variety of DNA sequences. Although the forward-binding (5'-3'/N-C) orientation, in which the N-terminus of PIPs faces the 5'-terminus of DNAs, is considered to be the main binding manner of PIPs, a reverse-binding (5'-3'/C-N) orientation, in which the C-terminus of PIPs faces the 3'-terminus of DNAs, sometimes causes unintended binding. Here, we synthesized optical or structural isomers of previously reported cyclic PIPs (cPIPs), which differ in the position of the amino groups in the γ-turn units, and we investigated their binding affinities both in the forward- and reverse-binding orientation. We show that cPIPs with (R)-α-amino-γ-turn units prefer the forward orientation as do hairpin PIPs. More importantly, we document for the first time the remarkable reverse-binding preference of cPIPs with (S)-α-amino-γ-turns. These results indicate that the orientation preference of cPIPs can be controlled by the position of the amino groups on the γ-turn units, which may markedly increase the number of DNA sequences that can be targeted by PIPs.

Ligand Design to Acquire Specificity to Intended G-Quadruplex Structures.

A G-quadruplex is a nucleic acid secondary structure that is adopted by guanine-rich sequences, and is considered to be relevant in various pharmacological and biological contexts. G-Quadruplexes have also attracted great attention in the field of DNA nanotechnology because of their extremely high thermal stability and the availability of many defined structures. To date, a large repertory of DNA/RNA G-quadruplex-interactive ligands has been developed by numerous laboratories. Several relevant reviews have also been published that have helped researchers to grasp the full scope of G-quadruplex research from its outset to the present. This review focuses on the G-quadruplex ligands that allow targeting of specific G-quadruplexes. Moreover, unique ligands, successful methodologies, and future perspectives in relation to specific G-quadruplex recognition are also addressed.

Orientation preferences of hairpin pyrrole-imidazole polyamides toward mCGG site.

Hairpin pyrrole-imidazole (Py-Im) polyamides are promising medium-sized molecules that bind sequence-specifically to the minor groove of B-form DNA. Here, we synthesized a series of hairpin Py-Im polyamides and explored their binding affinities and orientation preferences to methylated DNA with the mCGG target sequence. Thermal denaturation assays revealed that the five hairpin Py-Im polyamides, which were anticipated to recognize mCGG in a forward orientation, bind to nontarget DNA, GGmC, in a reverse orientation. Therefore, we designed five Py-Im polyamides that could recognize mCGG in a reverse orientation. We found that the two Py-Im polyamides containing Im/ß pairs preferentially bound to mCGG in a reverse orientation. The reverse binding Py-Im polyamide successfully inhibited TET1 binding on the methylated DNA. Taken together, this study illustrated the importance of designing reverse binding Py-Im polyamides for the target sequence, mCGG, which paved the way for Py-Im polyamides that can be used with otherwise difficult to access DNA with CG sequences.

Perspectives for Applying G-Quadruplex Structures in Neurobiology and Neuropharmacology.

The most common form of DNA is a right-handed helix or the B-form DNA. DNA can also adopt a variety of alternative conformations, non-B-form DNA secondary structures, including the DNA G-quadruplex (DNA-G4). Furthermore, besides stem-loops that yield A-form double-stranded RNA, non-canonical RNA G-quadruplex (RNA-G4) secondary structures are also observed. Recent bioinformatics analysis of the whole-genome and transcriptome obtained using G-quadruplex-specific antibodies and ligands, revealed genomic positions of G-quadruplexes. In addition, accumulating evidence pointed to the existence of these structures under physiologically- and pathologically-relevant conditions, with functional roles in vivo. In this review, we focused on DNA-G4 and RNA-G4, which may have important roles in neuronal function, and reveal mechanisms underlying neurological disorders related to synaptic dysfunction. In addition, we mention the potential of G-quadruplexes as therapeutic targets for neurological diseases.

Recent Progress of Targeted G-Quadruplex-Preferred Ligands Toward Cancer Therapy.

A G-quadruplex (G4) is a well-known nucleic acid secondary structure comprising guanine-rich sequences, and has profound implications for various pharmacological and biological events, including cancers. Therefore, ligands interacting with G4s have attracted great attention as potential anticancer therapies or in molecular probe applications. To date, a large variety of DNA/RNA G4 ligands have been developed by a number of laboratories. As protein-targeting drugs face similar situations, G-quadruplex-interacting drugs displayed low selectivity to the targeted G-quadruplex structure. This low selectivity could cause unexpected effects that are usually reasons to halt the drug development process. In this review, we address the recent research on synthetic G4 DNA-interacting ligands that allow targeting of selected G4s as an approach toward the discovery of highly effective anticancer drugs.

G-Quadruplex Induction by the Hairpin Pyrrole-Imidazole Polyamide Dimer.

The G-quadruplex (G4) is one type of higher-order structure of nucleic acids and is thought to play important roles in various biological events such as regulation of transcription and inhibition of DNA replication. Pyrrole-imidazole polyamides (PIPs) are programmable small molecules that can sequence-specifically bind with high affinity to the minor groove of double-stranded DNA (dsDNA). Herein, we designed head-to-head hairpin PIP dimers and their target dsDNA in a model G4-forming sequence. Using an electrophoresis mobility shift assay and transcription arrest assay, we found that PIP dimers could induce the structural change to G4 DNA from dsDNA through the recognition by one PIP dimer molecule of two duplex-binding sites flanking both ends of the G4-forming sequence. This induction ability was dependent on linker length. This is the first study to induce G4 formation using PIPs, which are known to be dsDNA binders. The results reported here suggest that selective G4 induction in native sequences may be achieved with PIP dimers by applying the same design strategy.

Simultaneous Binding of Hybrid Molecules Constructed with Dual DNA-Binding Components to a G-Quadruplex and Its Proximal Duplex.

A G-quadruplex (quadruplex) is a nucleic acid secondary structure adopted by guanine-rich sequences and is considered to be relevant to various pharmacological and biological contexts. Although a number of researchers have endeavored to discover and develop quadruplex-interactive molecules, poor ligand designability originating from topological similarity of the skeleton of diverse quadruplexes has remained a bottleneck for gaining specificity for individual quadruplexes. This work reports on hybrid molecules that were constructed with dual DNA-binding components, a cyclic imidazole/lysine polyamide (cIKP), and a hairpin pyrrole/imidazole polyamide (hPIP), with the aim toward specific quadruplex targeting by reading out the local duplex DNA sequence adjacent to designated quadruplexes in the genome. By means of circular dichroism (CD), fluorescence resonance energy transfer (FRET), surface plasmon resonance (SPR), and NMR techniques, we showed the dual and simultaneous recognition of the respective segment via hybrid molecules, and the synergistic and mutual effect of each binding component that was appropriately linked on higher binding affinity and modest sequence specificity. Monitoring quadruplex and duplex imino protons of the quadruplex/duplex motif titrated with hybrid molecules clearly revealed distinct features of the binding of hybrid molecules to the respective segments upon their simultaneous recognition. A series of the systematic and detailed binding assays described here showed that the concept of simultaneous recognition of quadruplex and its proximal duplex by hybrid molecules constructed with the dual DNA-binding components may provide a new strategy for ligand design, enabling targeting of a large variety of designated quadruplexes at specific genome locations.

Orthogonal γPNA Dimerization Domains Empower DNA Binders with Cooperativity and Versatility Mimicking that of Transcription Factor Pairs.

Synthetic molecules capable of DNA binding and mimicking cooperation of transcription factor (TF) pairs have long been considered a promising tool for manipulating gene expression. Our previously reported Pip-HoGu system, a programmable DNA binder pyrrole-imidazole polyamides (PIPs) conjugated to host-guest moiety, defined a general framework for mimicking cooperative TF pair-DNA interactions. Here, we supplanted the cooperation modules with left-handed (LH) γPNA modules: i.e., PIPs conjugated with nucleic acid-based cooperation system (Pip-NaCo). LH γPNA was chosen because of its bioorthogonality, sequence-specific interaction, and high binding affinity toward the partner strand. From the results of the Pip-NaCo system, cooperativity is highly comparable to the natural TF pair-DNA system, with a minimum energetics of cooperation of -3.27 kcal mol-1 . Moreover, through changing the linker conjugation site, binding mode, and the length of γPNAs sequence, the cooperative energetics of Pip-NaCo can be tuned independently and rationally. The current Pip-NaCo platform might also have the potential for precise manipulation of biological processes through the construction of triple to multiple heterobinding systems.

DNA Interstrand Crosslinks by H-pin Polyamide (S)-seco-CBI Conjugates.

Although DNA interstrand crosslinking (ICL) agents are widely used as antitumor drugs, DNA sequence-specific ICL agents are quite rare. In this study, H-pin imidazole-pyrrole polyamide 1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-ol (seco-CBI) conjugates that produce sequence-specific DNA ICLs were designed and synthesized. Conjugates with H-pin polyamide and seco-CBI moieties were constructed to recognize a 7 bp DNA sequence, and their reactivity and selectivity in DNA alkylation were evaluated by using high-resolution denaturing gel electrophoresis and sequence-specific plasmid cleavage. One conjugate (6), which contained a chiral (S)-seco-CBI, exhibited greater sequence-specific ICL activity toward the target DNA sequence and was cytotoxic to a cancer cell line. Molecular modeling studies indicated that the greater activity of 6 resulted from the relative orientation of the cyclopropane group in the (S)-CBI unit.

Ligand-Mediated G-Quadruplex Induction in a Double-Stranded DNA Context by Cyclic Imidazole/Lysine Polyamide.

G-quadruplex (G4) DNA is often observed as a DNA secondary structure in guanine-rich sequences, and is thought to be relevant to pharmacological and biological events. Therefore, G4 ligands have attracted great attention as potential anticancer therapies or in molecular probe applications. Here, we designed cyclic imidazole/lysine polyamide (cIKP) as a new class of G4 ligand. It was readily synthesized without time-consuming column chromatography. cIKP selectively recognized particular G4 structures with low nanomolar affinity. Moreover, cIKP exhibited the ability to induce G4 formation of the promoter of G4-containing DNA in the context of stable double-stranded DNA (dsDNA) under molecular crowding conditions. This cIKP might be applicable as a molecular probe for the detection of potential G4-forming sequences in dsDNA.

Effect of ATRX and G-Quadruplex Formation by the VNTR Sequence on α-Globin Gene Expression.

ATR-X (α-thalassemia/mental retardation X-linked) syndrome is caused by mutations in chromatin remodeler ATRX. ATRX can bind the variable number of tandem repeats (VNTR) sequence in the promoter region of the α-globin gene cluster. The VNTR sequence, which contains the potential G-quadruplex-forming sequence CGC(GGGGCGGGG)n , is involved in the downregulation of α-globin expression. We investigated G-quadruplex and i-motif formation in single-stranded DNA and long double-stranded DNA. The promoter region without the VNTR sequence showed approximately twofold higher luciferase activity than the promoter region harboring the VNTR sequence. G-quadruplex stabilizers hemin and TMPyP4 reduced the luciferase activity, whereas expression of ATRX led to a recovery in reporter activity. Our results demonstrate that stable G-quadruplex formation by the VNTR sequence downregulates the expression of α-globin genes and that ATRX might bind to and resolve the G-quadruplex.

Comparative Analysis of DNA-Binding Selectivity of Hairpin and Cyclic Pyrrole-Imidazole Polyamides Based on Next-Generation Sequencing.

Many long pyrrole-imidazole polyamides (PIPs) have been synthesized in the search for higher specificity, with the aim of realizing the great potential of such compounds in biological and clinical areas. Among several types of PIPs, we designed and synthesized hairpin and cyclic PIPs targeting identical sequences. Bind-n-Seq analysis revealed that both bound to the intended sequences. However, adenines in the data analyzed by the previously reported Bind-n-Seq method appeared to be significantly higher in the motif ratio than thymines, even though the PIPs were not expected to distinguish A from T. We therefore examined the experimental protocol and analysis pipeline in detail and developed a new method based on Bind-n-Seq motif identification with a reference sequence (Bind-n-Seq-MR). High-throughput sequence analysis of the PIP-enriched DNA data by Bind-n-Seq-MR presented A and T comparably. Surface plasmon resonance assays were performed to validate the new method.

Sequence-specific DNA binding by long hairpin pyrrole-imidazole polyamides containing an 8-amino-3,6-dioxaoctanoic acid unit.

With the aim of improving aqueous solubility, we designed and synthesized five N-methylpyrrole (Py)-N-methylimidazole (Im) polyamides capable of recognizing 9-bp sequences. Their DNA-binding affinities and sequence specificities were evaluated by SPR and Bind-n-Seq analyses. The design of polyamide 1 was based on a conventional model, with three consecutive Py or Im rings separated by a ß-alanine to match the curvature and twist of long DNA helices. Polyamides 2 and 3 contained an 8-amino-3,6-dioxaoctanoic acid (AO) unit, which has previously only been used as a linker within linear Py-Im polyamides or between Py-Im hairpin motifs for tandem hairpin. It is demonstrated herein that AO also functions as a linker element that can extend to 2-bp in hairpin motifs. Notably, although the AO-containing unit can fail to bind the expected sequence, polyamide 4, which has two AO units facing each other in a hairpin form, successfully showed the expected motif and a KD value of 16nM was recorded. Polyamide 5, containing a ß-alanine-ß-alanine unit instead of the AO of polyamide 2, was synthesized for comparison. The aqueous solubilities and nuclear localization of three of the polyamides were also examined. The results suggest the possibility of applying the AO unit in the core of Py-Im polyamide compounds.