A new small molecule DoNA binding to CAG repeat RNA.

We here report a new molecule DoNA binding to a CAG repeat RNA. DoNA is a dimer of the NA molecule that we previously reported. NA binds with high affinity to a CAG repeat DNA but not significantly to a CAG repeat RNA. Binding analyses using SPR and CSI-TOF MS indicated a significant increase in the affinity of DoNA to a single stranded CAG repeat RNA compared to NA. Systematic investigation of the RNA motifs bound by DoNA using hairpin RNA models revealed that DoNA binds to the CAG units at overhang and terminal positions, and notably, it binds to the structurally flexible internal and hairpin loop region.

FUS regulates RAN translation through modulating the G-quadruplex structure of GGGGCC repeat RNA in C9orf72-linked ALS/FTD.

Abnormal expansions of GGGGCC repeat sequence in the noncoding region of the C9orf72 gene is the most common cause of familial amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD). The expanded repeat sequence is translated into dipeptide repeat proteins (DPRs) by noncanonical repeat-associated non-AUG (RAN) translation. Since DPRs play central roles in the pathogenesis of C9-ALS/FTD, we here investigate the regulatory mechanisms of RAN translation, focusing on the effects of RNA-binding proteins (RBPs) targeting GGGGCC repeat RNAs. Using C9-ALS/FTD model flies, we demonstrated that the ALS/FTD-linked RBP FUS suppresses RAN translation and neurodegeneration in an RNA-binding activity-dependent manner. Moreover, we found that FUS directly binds to and modulates the G-quadruplex structure of GGGGCC repeat RNA as an RNA chaperone, resulting in the suppression of RAN translation in vitro. These results reveal a previously unrecognized regulatory mechanism of RAN translation by G-quadruplex-targeting RBPs, providing therapeutic insights for C9-ALS/FTD and other repeat expansion diseases.

Inhibitory Effects of Mismatch Binding Molecules on the Repair Reaction of Uracil-Containing DNA.

The stable R-loop formed during transcription induces enzyme-mediated deamination of cytosine, and the uracil in the DNA produced activates the base excision repair (BER) pathway. DNA cleavage involved in the BER pathway is thought to be one of the possible causes of trinucleotide repeat instability. Here, we performed an in vitro assay to investigate the effect of a DNA-binding small molecule, naphthyridine carbamate dimer (NCD), on BER enzyme reactions. The gel electrophoretic mobility shift assay (EMSA) and thermal melting analysis revealed the binding of NCD to a 5'-XGG-3'/5'-XGG-3' triad (X = C or U or apurinic/apyrimidinic site), which is a mimic of a BER enzyme substrate. Polyacrylamide gel electrophoresis (PAGE) of the reaction products of these substrates with hSMUG1 and APE1 enzymes in the presence of NCD showed that NCD interfered with the repair reaction in the 5'-XGG-3'/5'-XGG-3' triad. These findings would broaden the potential of small molecules in modulating trinucleotide repeat instability.

Method for Identifying Sequence Motifs in Pre-miRNAs for Small-Molecule Binding.

Non-coding RNAs are emerging targets for drug development because they are involved in various cellular processes. However, there are a few reliable design strategies for small molecules that can target RNAs. This paper reports a simple and efficient method to comprehensively analyze RNA motifs that can be bound by a specific small molecule. The method involves Dicer-mediated pre-miRNA cleavage and subsequent analysis of the reaction products by high-throughput sequencing. A pre-miRNA mutant library containing a randomized region at the Dicer cleavage site was used as the substrate for the reaction. Sequencing analysis of the products of the reaction carried out in the presence or absence of a synthetic small molecule identified the pre-miRNA mutants whose Dicer-mediated cleavage was significantly altered by the addition of the small molecule. The binding of the small molecule to the identified pre-miRNA mutants was confirmed by surface plasmon resonance, demonstrating the feasibility of our method.

Premature translation termination mediated non-ER stress induced ATF6 activation by a ligand-dependent ribosomal frameshifting circuit.

The -1 programmed ribosomal frameshifting (-1 PRF) has been explored as a gene regulatory circuit for synthetic biology applications. The -1 PRF usually uses an RNA pseudoknot structure as the frameshifting stimulator. Finding a ligand-responsive pseudoknot with efficient -1 PRF activity is time consuming and is becoming a bottleneck for its development. Inserting a guanine to guanine (GG)-mismatch pair in the 5'-stem of a small frameshifting pseudoknot could attenuate -1 PRF activity by reducing stem stability. Thus, a ligand-responsive frameshifting pseudoknot can be built using GG-mismatch-targeting small molecules to restore stem stability. Here, a pseudoknot requiring stem-loop tertiary interactions for potent frameshifting activity was used as the engineering template. This considerably amplified the effect of mismatch destabilization, and led to creation of a mammalian -1 PRF riboswitch module capable of mediating premature translation termination as a synthetic regulatory mode. Application of the synthetic circuit allowed ligand-dependent ATF6N mimic formation for the activation of protein folding-related genes involved in the unfolded protein response without an ER-stress inducing agent. With the availability of mismatch-targeting molecules, the tailored module thus paves the way for various mismatch plug-ins to streamline highly efficient orthogonal ligand-dependent -1 PRF stimulator development in the synthetic biology toolbox.

Mismatch binding ligand upregulated back-splicing reaction producing circular RNA in a cellular model.

Circular RNA (circRNA) is a covalently closed single-stranded RNA produced from pre-mRNAs via back-splicing reaction, an alternative form of splicing. Here, we show naphthyridine carbamate dimer (NCD) upregulating the production of a circRNA from a pre-mRNA containing NCD-binding site UGGAA/UGGAA in cells, demonstrating the feasibility of small-molecule mediated circRNA production.

HT-SELEX-based identification of binding pre-miRNA hairpin-motif for small molecules.

Selective targeting of biologically relevant RNAs with small molecules is a long-standing challenge due to the lack of clear understanding of the binding RNA motifs for small molecules. The standard SELEX procedure allows the identification of specific RNA binders (aptamers) for the target of interest. However, more effort is needed to identify and characterize the sequence-structure motifs in the aptamers important for binding to the target. Herein, we described a strategy integrating high-throughput (HT) sequencing with conventional SELEX followed by bioinformatic analysis to identify aptamers with high binding affinity and target specificity to unravel the sequence-structure motifs of pre-miRNA, which is essential for binding to the recently developed new water-soluble small-molecule CMBL3aL. To confirm the fidelity of this approach, we investigated the binding of CMBL3aL to the identified motifs by surface plasmon resonance (SPR) spectroscopy and its potential regulatory activity on dicer-mediated cleavage of the obtained aptamers and endogenous pre-miRNAs comprising the identified motif in its hairpin loop. This new approach would significantly accelerate the identification process of binding sequence-structure motifs of pre-miRNA for the compound of interest and would contribute to increase the spectrum of biomedical application.

2-Amino-1,8-naphthyridine Dimer (ANP77), a High-Affinity Binder to the Internal Loops of C/CC and T/CC Sites in Double-Stranded DNA.

Small molecules targeting DNA regions with structural fluctuation are an important class of molecule as chemical probes for studying the role of these structures in biological systems and the development of neurological disorders. The molecule ANP77 we described here, where a three-atom linker connects two 2-amino-1,8-naphthyridines at the C7 position, was found to form stacked structure with protonation of naphthyridine at low pH, and bound to the internal loop consisting of C/CC and T/CC in double-stranded DNA with affinities of 4.8 and 34.4 nM, respectively. Mass spectrometry and isothermal titration calorimetry analyses determined the stoichiometry for the binding as 1:1, and chemical footprinting with permanganate and NMR structural analysis revealed that the T in the T/CC was forced to flip out toward an extrahelical position upon ANP77 binding. Protonated stacked ANP77 interacted with two adjacent cytosines through hydrogen bonding and occupied the position in the duplex by flipping out the C or T opposite CC. Finally, this study demonstrated the potential of ANP77 for binding to the sequences of biological significance with the TG(T/C)CC repeat of the PIG3 promoter and the telomere repeat CCCTAA.

A small-molecule fluorescence probe ANP77 for sensing RNA internal loop of C, U and A/CC motifs and their binding molecules.

Small-molecules interacting with particular RNAs and modulating their functions are vital tools for RNA-targeting drug discovery. Considering the substantial distribution of the internal loops involving two contiguous cytosines opposite to a single-nucleotide base (Y/CC; Y = C, U or A) within the biologically significant functional RNAs, developing small-molecule probes targeting Y/CC sites should provide profound insight into their functions and roles in biochemical processes. Herein, we report ANP77 as the small-molecule probe for sensing RNA internal loop of Y/CC motifs and molecules binding to the motifs. The Y/CC motifs interact with ANP77 via the formation of a 1:1 complex and quench the fluorescence of ANP77. The flanking sequence-dependent binding to C/CC and U/CC sites was assessed by fluorometric screening, provided the binding heat maps. The quenching phenomena of ANP77 fluorescence was confirmed with intrinsic potential drug target pre-miR-1908. Finally, the binding-dependent fluorescence quenching of ANP77 was utilized in the fluorescence indicator displacement assay to demonstrate the potential of ANP77 as an indicator by using the RNA-binding drugs, risdiplam and branaplam.

Speeding drug discovery targeting RNAs: An iterative "RNA selection-compounds screening cycle" for exploring RNA-small molecule pairs.

RNA is an emerging target of next-generation drug development. Recently, new small molecules targeting RNAs were discovered by several pharmaceutical companies. Methods have been reported to identify small molecules targeting a specific RNA sequence and structural motif, however, because of diverse sequence and structural motifs potentially present in the druggable functional RNAs, large sets of structure-activity relationships (SARs) information of small molecule - RNA interactions will be required for the acceleration and efficient startup of the discovery programs toward unprecedented RNA targets. Here we describe our iterative RNA selection and compounds screening to accumulate rich information about small molecules - RNA interaction. The RNAs that selectively bind to the initial molecular target, compound 1 from our in-house chemical library (JT-library), was isolated using in vitro selection technique from a hairpin-structured RNA library mimicking precursor microRNA (pre-miRNA). Then, we engineered pre-let-7f-2 to create its mutant that can bind to compound 1 by embedding the in vitro selected RNA motif for compound 1 in the hairpin loop region. The obtained mutant pre-let-7f-2-loop-mt was used as a target for screening 316 analogs of compound 1. A surface plasmon resonance (SPR) -based screening was performed against pre-let-7f-2-loop-mt-immobilized sensor surface and we obtained four compounds that can bind to the RNA. Among these four compounds, three compounds showed higher affinity to pre-let-7f-2-loop-mt than the parental compound 1, which suggests the feasibility of our strategy for gathering the SAR information on small molecule - RNA interactions. We demonstrated only one cycle of RNA selection and compounds screening in the present study, but can continue this cycle with the selected molecule to gain new RNAs and even new RNA motifs and gather much SAR information with improved accuracy.

Small Molecule-Induced Dimerization of Hairpin RNA Interfered with the Dicer Cleavage Reaction.

MicroRNAs are potential targets for drug development. Small molecules that can inhibit or promote a specific miRNA's biogenesis would be useful for regulating its target genes. Various types of small molecules have been investigated so far for their potential application in modulating miRNA biogenesis. They bind to the target primary or precursor miRNAs and inhibit the processing of these precursors by Drosha or Dicer. However, the binding site that effectively interferes with the Dicer cleavage reaction is still undetermined. Here we report that our designed small molecule restricted naphthyridine dimer (RND) binds to the hairpin loop of a hairpin RNA and induces its dimerization. This study shows that the binding of the RND to the hairpin loop was not effective in interfering with the Dicer cleavage reaction, but dimerization of the hairpin RNA by RND binding effectively interfered with the Dicer cleavage reaction.

The Dimeric Form of 1,3-Diaminoisoquinoline Derivative Rescued the Mis-splicing of Atp2a1 and Clcn1 Genes in Myotonic Dystrophy Type†1 Mouse Model.

Expanded CUG repeat RNA in the dystrophia myotonia protein kinase (DMPK) gene causes myotonic dystrophy type 1 (DM1) and sequesters RNA processing proteins, such as the splicing factor muscleblind-like 1 protein (MBNL1). Sequestration of splicing factors results in the mis-splicing of some pre-mRNAs. Small molecules that rescue the mis-splicing in the DM1 cells have drawn attention as potential drugs to treat DM1. Herein we report a new molecule JM642 consisted of two 1,3-diaminoisoquinoline chromophores having an auxiliary aromatic unit at the C5 position. JM642 alternates the splicing pattern of the pre-mRNA of the Ldb3 gene in the DM1 cell model and Clcn1 and Atp2a1 genes in the DM1 mouse model. In vitro binding analysis by surface plasmon resonance (SPR) assay to the r(CUG) repeat and disruption of ribonuclear foci in the DM1 cell model suggested the binding of JM642 to the expanded r(CUG) repeat in vivo, eventually rescue the mis-splicing.

A slipped-CAG DNA-binding small molecule induces trinucleotide-repeat contractions in vivo.

In many repeat diseases, such as Huntington's disease (HD), ongoing repeat expansions in affected tissues contribute to disease onset, progression and severity. Inducing contractions of expanded repeats by exogenous agents is not yet possible. Traditional approaches would target proteins driving repeat mutations. Here we report a compound, naphthyridine-azaquinolone (NA), that specifically binds slipped-CAG DNA intermediates of expansion mutations, a previously unsuspected target. NA efficiently induces repeat contractions in HD patient cells as well as en masse contractions in medium spiny neurons of HD mouse striatum. Contractions are specific for the expanded allele, independently of DNA replication, require transcription across the coding CTG strand and arise by blocking repair of CAG slip-outs. NA-induced contractions depend on active expansions driven by MutSß. NA injections in HD mouse striatum reduce mutant HTT protein aggregates, a biomarker of HD pathogenesis and severity. Repeat-structure-specific DNA ligands are a novel avenue to contract expanded repeats.

RT-Hpro-PCR: A MicroRNA Detection System Using a Primer with a DNA Tag.

MicroRNAs (miRNAs) are short RNAs that regulate the expression of complementary messenger RNAs and are involved in numerous human diseases. However, current detection techniques lack the sensitivity to detect miRNAs of low abundance. Moreover, at a length of 20-25 bases, miRNAs are too short for the reverse transcription (RT) polymerase chain reaction (PCR). Here we have developed a new, rapid, and simple miRNA detection system utilizing an RT primer containing a DNA tag at the 5'-end to increase the length of the cDNA. This strategy increases the length of the hybridized tagged primer and the complementary template DNA, as well as the melting temperature of the primer⋅template DNA duplex. PCR efficiency is thus increased, thereby enhancing miRNA detection sensitivity.

Structural insights into synthetic ligands targeting A-A pairs in disease-related CAG RNA repeats.

The trinucleotide repeat expansion disorders (TREDs) constitute of a group of >40 hereditary neurodegenerative human diseases associated with abnormal expansion of repeated sequences, such as CAG repeats. The pathogenic factor is a transcribed RNA or protein whose function in the cell is compromised. The disorders are progressive and incurable. Consequently, many ongoing studies are oriented at developing therapies. We have analyzed crystal structures of RNA containing CAG repeats in complex with synthetic cyclic mismatch-binding ligands (CMBLs). The models show well-defined interactions between the molecules in which the CMBLs mimic nucleobases as they form pseudo-canonical base pairs with adenosine residues and engage in extensive stacking interactions with neighboring nucleotides. The binding of ligands is associated with major structural changes of the CAG repeats, which is consistent with results of biochemical studies. The results constitute an early characterization of the first lead compounds in the search for therapy against TREDs. The crystallographic data indicate how the compounds could be further refined in future biomedical studies.

Modulating RNA secondary and tertiary structures by mismatch binding ligands.

Much recent attention has been focused on small organic molecules binding to non-canonical structures of nucleic acids, especially, RNA. The Human Genome Project and the ENCODE (encyclopedia of DNA elements) project revealed that more than 75% of the human genome is transcribed into RNA, while only ∼3% of the human genome encodes a protein. These non-protein-coding RNAs are thought to play significant roles in many cellular processes and are promising targets for drug discovery. Emerging roles of the non-coding RNAs in a variety of diseases provides enormous opportunities for pharmaceutical research on developing drugs targeting undruggable and rare diseases. During the last two decades, our laboratory has focused attention on small molecules binding to non-canonical DNA and RNA structures, especially to mismatched base pairs. Mismatch binding ligands (MBLs) we have developed are synthetic molecules designed in silico based on the hypothesis of hydrogen-bonding and semi-intercalation to DNA and RNA. Most of MBLs consists of two heterocycles having hydrogen bonding surfaces fully or partially complementary to that of nucleotide bases. In our design, each heterocycle binds to one of the mismatched bases by hydrogen bonding to form pseudo-base pairs, which would be stacked with the adjacent base pairs. The hypothesis allows us in principle to design small molecules binding to any mismatched base pairs, but it turned out not to be the case in reality. However, we have so far succeeded in developing several MBLs binding to DNA and RNA motifs of biological significance. In this review, we shall describe the hypothesis of molecular design of MBLs and its outcome regarding RNA targeting.

Inhibition of pre-miRNA-136 processing by Dicer with small molecule BzDANP suggested the formation of ternary complex of pre-miR-136-BzDANP-Dicer.

Small-molecule modulators, along with antisense oligonucleotide, would be powerful tools and potential drug candidates for modulating miRNA-related gene expressions. The mechanism of the inhibitory effect of the C-bulge binding small molecule BzDANP for the Dicer processing reaction of pre-miR-136 was discussed on the data obtained by SPR, NMR, and kinetic analysis for Dicer processing. SPR and NMR analysis showed the preference of BzDANP binding to the C-bulge. Michaelis-Menten analysis suggested the formation of a ternary complex pre-miR-136-BzDANP-Dicer during the Dicer-cleavage reaction of pre-miR-136 in the presence of BzDANP. The inhibitory effect of BzDANP is likely attributed to the slower reaction from the ternary complex than that from the binary pre-miR-136-Dicer complex.

A Dimeric 2,9-Diamino-1,10-phenanthroline Derivative Improves Alternative Splicing in Myotonic Dystrophy Type†1 Cell and Mouse Models.

Expanded r(CUG) repeats are the cause of the neurological disorder myotonic dystrophy type 1 (DM1). The pathological features of DM1 include the formation of ribonuclear foci containing expanded r(CUG) repeats, which sequester the MBNL1 protein and lead to the misregulation of alternative pre-mRNA splicing. Small molecules that bind to the r(CUG) repeats and improve alternative splicing have therapeutic potential in the treatment of DM1. Herein, the synthesis of DDAP (a dimeric form of the CUG-binding molecule DAP reported previously), its binding properties to r(CUG) repeats, and its effect on the misregulation of splicing are reported. The surface plasmon resonance assay, circular dichroism spectra, and ESI-TOF mass spectrometry results confirmed the binding of DDAP to r(CUG)9 repeats. Studies on a DM1 cell model and a DM1 mouse model revealed that DDAP was partially effective in the recovery of the pre-mRNA splicing defects. The mechanism underlying this recovery was studied in vitro through a competitive binding assay, and suggested that DDAP could interfere with the binding of MBNL1 to r(CUG) repeats in a concentration-dependent manner.

Small synthetic molecule-stabilized RNA pseudoknot as an activator for -1 ribosomal frameshifting.

Programmed -1 ribosomal frameshifting (-1PRF) is a recoding mechanism to make alternative proteins from a single mRNA transcript. -1PRF is stimulated by cis-acting signals in mRNA, a seven-nucleotide slippery sequence and a downstream secondary structure element, which is often a pseudoknot. In this study we engineered the frameshifting pseudoknot from the mouse mammary tumor virus to respond to a rationally designed small molecule naphthyridine carbamate tetramer (NCTn). We demonstrate that NCTn can stabilize the pseudoknot structure in mRNA and activate -1PRF both in vitro and in human cells. The results illustrate how NCTn-inducible -1PRF may serve as an important component of the synthetic biology toolbox for the precise control of gene expression using small synthetic molecules.

Synthesis of Naphthyridine Dimers with Conformational Restriction and Binding to DNA and RNA.

One of the important determinants in the efficiency of a molecular interaction is the necessity for conformational changes in host and/or guest molecules upon binding. In small-molecule interactions with nucleic acids, conformational changes on both molecules are often involved, especially in intercalating binding. Mismatch binding ligands (MBLs) we described here consist of two heterocycles that predominantly exist in one conformation, so it is of interest to determine if such molecules can bind to any DNA and RNA structures. One molecule, 1-NHR, which predominantly exists as the unstacked conformation in aqueous solvent, has been successfully synthesized and characterized. Compound 1-NHR did not efficiently bind to GX/Y DNA and RNA sequences, but the binding pattern is different from that of authentic MBL naphthyridine carbamate dimer. In vitro selection of RNA that specifically binds to 1-NHR was performed from pre-miR-29a loop library RNA, and one RNA, to which 1-NHR bound with high affinity, has been successfully identified. Although it was anticipated that 1-NHR, with a predominantly unstacked conformation, would show entropy-driven binding, isothermal titration calorimetry analysis suggested that the binding of 1-NHR to RNA was enthalpy driven with an apparent Kd of about 100 nm.