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.

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.

A cyclic pyrrole-imidazole polyamide reduces pathogenic RNA in CAG/CTG triplet repeat neurological disease models.

Expansion of CAG and CTG (CWG) triplet repeats causes several inherited neurological diseases. The CWG repeat diseases are thought to involve complex pathogenic mechanisms through expanded CWG repeat-derived RNAs in a noncoding region and polypeptides in a coding region, respectively. However, an effective therapeutic approach has not been established for the CWG repeat diseases.

RNA G-quadruplex organizes stress granule assembly through DNAPTP6 in neurons.

Consecutive guanine RNA sequences can adopt quadruple-stranded structures, termed RNA G-quadruplexes (rG4s). Although rG4-forming sequences are abundant in transcriptomes, the physiological roles of rG4s in the central nervous system remain poorly understood. In the present study, proteomics analysis of the mouse forebrain identified DNAPTP6 as an RNA binding protein with high affinity and selectivity for rG4s.

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 (T ) and surface plasmon resonance assays revealed the high binding affinity of the cPIP.

Potential roles of G-quadruplex structures in RNA granules for physiological and pathological phase separation.

Cellular liquid-liquid phase separation is a physiologically inevitable phenomenon in molecularly crowded environments inside cells and serves to compartmentalize biomolecules to facilitate several functions, forming cytoplasmic and nuclear RNA granules. Abnormalities in the phase separation process in RNA granules are implicated in the onset of several neurodegenerative diseases; the initial liquid-like phase-separated droplets containing pathogenic proteins are prone to aberrantly mature into solid-like droplets. RNAs are involved in the maturation of physiological and pathological RNA granules and are essential for governing the fate of phase-transition processes.

CGG repeat RNA G-quadruplexes interact with FMRpolyG to cause neuronal dysfunction in fragile X-related tremor/ataxia syndrome.

Fragile X-related tremor/ataxia syndrome (FXTAS) is a neurodegenerative disease caused by CGG triplet repeat expansions in , which elicit repeat-associated non-AUG (RAN) translation and produce the toxic protein FMRpolyG. We show that FMRpolyG interacts with pathogenic CGG repeat-derived RNA G-quadruplexes (CGG-G4RNA), propagates cell to cell, and induces neuronal dysfunction. The FMRpolyG polyglycine domain has a prion-like property, preferentially binding to CGG-G4RNA.

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.

A synthetic transcription factor pair mimic for precise recruitment of an epigenetic modifier to the targeted DNA locus.

We developed an epigenetically active, cooperative DNA binding transcription factor platform assisted by cucurbit[7]uril (CB7) host-guest modules. This new type of molecule termed ePIP-HoGu not only mimics the operation of transcription factors as a pair but also recruits the epigenetic modifier to a particular DNA locus.

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).

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.

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.

Orientation preferences of hairpin pyrrole-imidazole polyamides toward CGG 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 CGG target sequence. Thermal denaturation assays revealed that the five hairpin Py-Im polyamides, which were anticipated to recognize CGG in a forward orientation, bind to nontarget DNA, GGC, in a reverse orientation.

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.

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.

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.

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.

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.

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.

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 β-alanine to match the curvature and twist of long DNA helices.