Photoswitchable molecular glue for RNA: reversible photocontrol of structure and function of the ribozyme.

Single-stranded RNA folds into a variety of secondary and higher-order structures. Distributions and dynamics of multiple RNA conformations are responsible for the biological function of RNA. We here developed a photoswitchable molecular glue for RNA, which could reversibly control the association of two unpaired RNA regions in response to light stimuli. The photoswitchable molecular glue, NCTA, is an RNA-binding ligand possessing a photoisomerizable azobenzene moiety. Z-NCTA is an active ligand for the target RNA containing 5'-WGG-3'/5'-WGG-3' (W = U or A) site and stabilizes its hybridized state, while its isomer E-NCTA is not. Photoreversible isomerization of NCTA enabled control of the secondary and tertiary structure of the target RNA. The RNA-cleaving activity of hammerhead ribozyme, where appropriate RNA folding is necessary, could be reversibly regulated by photoirradiation in cells treated with NCTA, demonstrating precise photocontrol of RNA structure and function by the photoswitchable molecular glue.

Unique Crosslinking Properties of Psoralen-Conjugated Oligonucleotides Developed by Novel Psoralen N-Hydroxysuccinimide Esters.

Psoralens and their derivatives, such as trioxsalen, have unique crosslinking features to DNA. However, psoralen monomers do not have sequence-specific crosslinking ability with the target DNA. With the development of psoralen-conjugated oligonucleotides (Ps-Oligos), sequence-specific crosslinking with target DNA has become achievable, thereby expanding the application of psoralen-conjugated molecules in gene transcription inhibition, gene knockout, and targeted recombination by genome editing. In this study, we developed two novel psoralen N-hydroxysuccinimide (NHS) esters that allow the introduction of psoralens into any amino-modified oligonucleotides. Quantitative evaluation of the photo-crosslinking efficiencies of the Ps-Oligos to target single-stranded DNAs revealed that the crosslinking selectivity to 5-mC is the unique feature of trioxsalen. We found that the introduction of an oligonucleotide via a linker at the C-5 position of psoralen can promote favorable crosslinking to target double-stranded DNA. We believe our findings are essential information for the development of Ps-Oligos as novel gene regulation tools.

Synthesis and Evaluation of Oligonucleotide-Containing 2'-O-{[(4,5',8-Trimethylpsoralen)-4'-ylmethoxy]ethylaminocarb-onyl}adenosine as Photo-crosslinkable Gene Targeting Tools.

Several psoralen-conjugated oligonucleotides (Ps-Oligos) have been developed as photo-crosslinkable oligonucleotides targeting DNA or RNA. To avoid potential off-target effects, it is important to investigate the selective photo-crosslinking reactivity of Ps-Oligos to DNA or RNA. However, the selectivity of these Ps-Oligos has not been reported in detail thus far. In this study, we evaluated the photo-crosslinking properties of two Ps-Oligos, 5'-Ps-Oligo and a novel Ps-Oligo containing 2'-O-{[(4,5',8-trimethylpsoralen)-4'-ylmethoxy]ethylaminocarbonyl}adenosine (APs2-Oligo). Notably, 5'-Ps-Oligo preferentially crosslinked with DNA, whereas APs2-Oligo preferentially crosslinked with RNA. These results demonstrate the interesting crosslinking properties of Ps-Oligos, which will provide useful information for the molecular design of novel Ps-Oligos in future studies.

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.

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.

Cyclic mismatch binding ligands interact with disease-associated CGG trinucleotide repeats in RNA and suppress their translation.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder caused by a limited expansion of CGG repeats in the FMR1 gene. Degeneration of neurons in FXTAS cell models can be triggered by accumulation of polyglycine protein (FMRpolyG), a by-product of translation initiated upstream to the repeats. Specific aims of our work included testing if naphthyridine-based molecules could (i) block FMRpolyG synthesis by binding to CGG repeats in RNA, (ii) reverse pathological alterations in affected cells and (iii) preserve the content of FMRP, translated from the same FMR1 mRNA. We demonstrate that cyclic mismatch binding ligand CMBL4c binds to RNA structure formed by CGG repeats and attenuates translation of FMRpolyG and formation of nuclear inclusions in cells transfected with vectors expressing RNA with expanded CGG repeats. Moreover, our results indicate that CMBL4c delivery can reduce FMRpolyG-mediated cytotoxicity and apoptosis. Importantly, its therapeutic potential is also observed once the inclusions are already formed. We also show that CMBL4c-driven FMRpolyG loss is accompanied by partial FMRP reduction. As complete loss of FMRP induces FXS in children, future experiments should aim at evaluation of CMBL4c therapeutic intervention in differentiated tissues, in which FMRpolyG translation inhibition might outweigh adverse effects related to FMRP depletion.

Rational design of a photoswitchable DNA glue enabling high regulatory function and supramolecular chirality transfer.

Short, complementary DNA single strands with mismatched base pairs cannot undergo spontaneous formation of duplex DNA (dsDNA). Mismatch binding ligands (MBLs) can compensate this effect, inducing the formation of the double helix and thereby acting as a molecular glue. Here, we present the rational design of photoswitchable MBLs that allow for reversible dsDNA assembly by light. Careful choice of the azobenzene core structure results in excellent band separation of the E and Z isomers of the involved chromophores. This effect allows for efficient use of light as an external control element for duplex DNA formation and for an in-depth study of the DNA-ligand interaction by UV-Vis, SPR, and CD spectroscopy, revealing a tight mutual interaction and complementarity between the photoswitchable ligand and the mismatched DNA. We also show that the configuration of the switch reversibly dictates the conformation of the DNA strands, while the dsDNA serves as a chiral clamp and translates its chiral information onto the ligand inducing a preference in helical chirality of the Z isomer of the MBLs.

Small molecule-induced trinucleotide repeat contractions during in vitro DNA synthesis.

We demonstrated that a synthetic ligand NA, which selectively binds to a 5'-CAG-3'/5'-CAG-3' triad, induced repeat contractions during DNA polymerase-mediated primer extension through the CAG repeat template. A thorough capillary electrophoresis and sequencing analysis revealed that the d(CAG)20 template gave shortened nascent strands mainly containing 3-6 CTG units in the presence of NA.

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.

Recognition of expanded GGGGCC hexanucleotide repeat by synthetic ligand through interhelical binding.

An expanded GGGGCC hexanucleotide (G4C2) repeat within the non-coding region of C9ORF72 gene has been identified as the most common genetic cause of FTD/ALS kindred, and synthetic ligand targeting this pathological expansion sequence holds a promising approach for the disease interference. We here describe the naphthyridine carbamate tetramer, p-NCTB, as a binding ligand to hairpin G4C2 repeat. p-NCTB simultaneously recognizes two distal CGGG/CGGG sites in G4C2 repeat DNA and RNA leading to the formation of the interhelical (inter- and intrastrand) binding complexes. The intrastrand binding was predominant when p-NCTB bound to long repeat sequence that accommodates multiple binding sites by folding into hairpins, while the interstrand binding was exclusive for short repeat sequence. The binding of p-NCTB showed repeat-length selectivity: the longer repeat sequence is a better target for p-NCTB. p-NCTB demonstrated inhibition of transcription against G4C2 repeat template in vitro in a repeat length-dependent manner.

A novel naphthyridine tetramer that recognizes tandem G-G mismatches by the formation of an interhelical complex.

We have designed and synthesized a novel naphthyridine tetramer, p-NCTB, for the recognition of tandem guanine-guanine (G-G) mismatches in DNA. p-NCTB possesses a p-biphenyl linker connecting two naphthyridine carbamate dimer (NCD) moieties that recognize G-G mismatches. p-NCTB preferentially bound to tandem G-G mismatches in dCGGG/dCGGG over dCGG/dCGG. Two dCGGG/dCGGG sites were simultaneously recognized and were noncovalently cross-linked via the formation of inter- and intrastrand complexes with p-NCTB. The intrastrand binding was more favorable, which could allow p-NCTB to bind selectively to a sequence containing multiple dCGGG/dCGGG sites.

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.

Molecular Glue for RNA: Regulating RNA Structure and Function through Synthetic RNA Binding Molecules.

We introduce the concept of molecular glues for RNA, in which specific RNA-binding small molecules induce designed structural changes in target functional RNAs, resulting in modulation of the functions. (Z)-NCTS is an RNA-mismatch-binding small molecule that recognizes 5'-r(XGG)-3'/5'-r(XGG)-3' sequences (X=U or A) and acts as a molecular glue for RNA. The binding of (Z)-NCTS brings two distinct 5'-r(XGG)-3' domains into contact with each other, and this can result in higher-order structural changes of target RNAs. We applied (Z)-NCTS to induce the formation of a proposed tertiary structure of a ribozyme together with activation of RNA-cleaving ability. The concept of a molecular glue could inspire new small-molecule-based strategies for regulating biological functions: a synthetic small molecule targeting functional RNAs could regulate the RNA structure and function.

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.

Restoration of Ribozyme Tertiary Contact and Function by Using a Molecular Glue for RNA.

Some RNA classes require folding into the proper higher-order structures to exert their functions. Hammerhead ribozyme (HHR) requires a folding conformation stabilized by tertiary interaction for full activity. A rationally engineered HHR was developed that was inactive, but could be activated by a synthetic RNA-binding ligand, naphthyridine carbamate tetramer with Z-stilbene linker (Z-NCTS). Binding of Z-NCTS could induce the formation of an active folding structure and thereby restore ribozyme activity, where Z-NCTS acts as a molecular glue to bring two isolated RNA loops into contact with each other. Next, we designed a Z-NCTS-responsive genetic switch using the HHR sequence inserted into the 3' untranslated region as a cis-acting element. We demonstrated that the rationally designed ribozyme switch enabled regulation of gene expression by Z-NCTS and was functional in mammalian cells.

Design and Synthesis of Cyclic Mismatch-Binding Ligands (CMBLs) with Variable Linkers by Ring-Closing Metathesis and their Photophysical and DNA Repeat Binding Properties.

Cyclophane-containing bis(2-amino-1,8-naphthyridine) moieties attached to variable linkers at the C2-position (linker B) were synthesized as cyclic mismatch-binding ligands (CMBLs). Ring-closing metathesis (RCM) is used as a key step for the introduction of double bonds at the linker B. Decreasing the size of the linker of the substrate, formation of the RCM products with an increasing trans/cis (E/Z) ratio was observed with moderate to high overall yields. Concentration-dependent fluorescence spectra were observed for CMBLs with longer linkers (n=3), whereas concentration-independent spectra were observed for CMBLs with shorter linkers (n=2 and/or 1) with a marked exception of the E-alkene 6 a. Concomitant changes in the absorption as well as in the fluorescence spectra were also observed for the CMBLs with an increasing hydrophobicity of the solvent. Absorption and fluorescence spectra of the CMBLs in solutions containing 99-100 % methanol resembled to that of the monomer. The binding behavior of these CMBLs with repeat DNA structures was investigated by using a surface plasmon resonance (SPR) assay and circular dichroism (CD) spectra. The cyclic E-alkenes 1 a (n=3) and 3 a (n=2) show an orthogonal binding relationship with d(CCTG)9 and d(CAG)9 . However, the selectivity for the cyclic Z-alkenes increased with decreasing the length of the linker from compound 2 b (n=3) to compound 7 b (n=1). These compounds display a large molecular diversity, which allowed the tuning of the binding affinity and selectivity of the CMBLs by varying the linkers towards various biologically significant repeat DNA structures.

Amphiphilic DNA tiles for controlled insertion and 2D assembly on fluid lipid membranes: the effect on mechanical properties.

Future lipid membrane-associated DNA nanostructures are expected to find applications ranging from synthetic biology to nanomedicine. Here we have designed and synthesized DNA tiles and modified them with amphiphilic covalent moieties. dod-DEG groups, which consist of a hydrophilic diethylene glycol (DEG) and a hydrophobic dodecyl group, are introduced at the phosphate backbone to create amphiphilic DNA strands which are subsequently introduced into one face of the DNA tiles. In this way the tile becomes able to stably bind to lipid membranes by insertion of the hydrophobic groups inside the bilayer core. The functionalized tiles do not aggregate in solution. Our results show that these amphiphilic DNA tiles can bind and assemble into 2D lattices on both gel and fluid lipid bilayers. The binding of the DNA structures to membranes is dependent on the lipid phase of the membrane, the concentration of Mg2+ cations, the length of the amphiphilic modifications to the DNA as well as on the density of the modifications within the tile. Atomic force microscopy-based force spectroscopy is used to investigate the effect of the inserted DNA tiles on the mechanical properties of the lipid membranes. The results indicate that the insertion of DNA tiles produces an approx. 20% increase of the bilayer breakthrough force.

A Ligand That Targets CUG Trinucleotide Repeats.

The development of small molecules that can recognize specific RNA secondary and tertiary structures is currently an important research topic for developing tools to modulate gene expression and therapeutic drugs. Expanded CUG trinucleotide repeats, known as toxic RNA, capture the splicing factor MBNL1 and are causative of neurological disorder myotonic dystrophy type 1 (DM1). Herein, the rational molecular design, synthesis, and binding analysis of 2,9-diaminoalkyl-substituted 1,10-phenanthroline (DAP), which bound to CUG trinucleotide repeats, is described. The results of melting temperature (Tm ) analyses, surface plasmon resonance (SPR) assay, and electrospray spray ionization time-of-flight (ESI-TOF) mass spectrometry showed that DAP bound to r(CUG)9 but not to r(CAG)9 and r(CGG)9 . The dual luciferase assay clearly indicated DAP bound to the r(CUG)n repeat by affecting the translation in vitro.

Cyclic mismatch binding ligand CMBL4 binds to the 5'-T-3'/5'-GG-3' site by inducing the flipping out of thymine base.

A newly designed cyclic bis-naphthyridine carbamate dimer CMBL4: with a limited conformational flexibility was synthesized and characterized. Absorption spectra revealed that two naphthyridines in CMBL4: were stacked on each other in aqueous solutions. The most efficient binding of CMBL4: to DNA was observed for the sequence 5'-T-3'/5'-GG-3' (T/GG) with the formation of a 1:1 complex, which is one of possible structural elements involved in the higher order structures of (TGG)n repeat DNA triggering the genome microdeletion. Surface plasmon resonance assay also showed the binding of CMBL4: with TGG repeat DNA. Potassium permanganate oxidation studies of CMBL4: -bound duplex containing the T/GG site showed that the CMBL4: -binding accelerated the oxidation of thymine at that site, which suggests the flipping out of the thymine base from a π-stack. Preferential binding was observed for CMBL4: compared with its acyclic variants, which suggests the marked significance of the macrocyclic structure for the recognition of the T/GG site.

Synthesis of 1H-pyrrolo[3,2-h]quinoline-8-amine derivatives that target CTG trinucleotide repeats.

We describe a new molecular design, synthesis, and investigation of small molecules that bind to CTG trinucleotide repeats in DNA. 1H-Pyrrolo[3,2-h]quinoline-8-amine (PQA) has a tricyclic aromatic system with unique non-linear hydrogen-bonding surface complementary to thymine. We have synthesized a series of PQA derivatives with different alkylamino linkers. These PQAs showed binding to pyrimidine bulge DNAs and CNG (N=T and C) repeats depending on the linker structure, while quinoline derivatives lacking the pyrrole ring showed much lower binding affinity. PQA is a useful molecular unit for both CTG and CCG repeat binding.