CAG RNAs induce DNA damage and apoptosis by silencing NUDT16 expression in polyglutamine degeneration.

DNA damage plays a central role in the cellular pathogenesis of polyglutamine (polyQ) diseases, including Huntington's disease (HD). In this study, we showed that the expression of untranslatable expanded CAG RNA per se induced the cellular DNA damage response pathway. By means of RNA sequencing (RNA-seq), we found that expression of the Nudix hydrolase 16 (NUDT16) gene was down-regulated in mutant CAG RNA-expressing cells. The loss of NUDT16 function results in a misincorporation of damaging nucleotides into DNAs and leads to DNA damage. We showed that small CAG (sCAG) RNAs, species generated from expanded CAG transcripts, hybridize with CUG-containing NUDT16 mRNA and form a CAG-CUG RNA heteroduplex, resulting in gene silencing of NUDT16 and leading to the DNA damage and cellular apoptosis. These results were further validated using expanded CAG RNA-expressing mouse primary neurons and in vivo R6/2 HD transgenic mice. Moreover, we identified a bisamidinium compound, DB213, that interacts specifically with the major groove of the CAG RNA homoduplex and disfavors the CAG-CUG heteroduplex formation. This action subsequently mitigated RNA-induced silencing complex (RISC)-dependent NUDT16 silencing in both in vitro cell and in vivo mouse disease models. After DB213 treatment, DNA damage, apoptosis, and locomotor defects were rescued in HD mice. This work establishes NUDT16 deficiency by CAG repeat RNAs as a pathogenic mechanism of polyQ diseases and as a potential therapeutic direction for HD and other polyQ diseases.

Water and Air Stable Copper(I) Complexes of Tetracationic Catenane Ligands for Oxidative C-C Cross-Coupling.

Aqueous soluble and stable Cu(I) molecular catalysts featuring a catenane ligand composed of two dicationic, mutually repelling but mechanically interlocked macrocycles are reported. The ligand interlocking not only fine-tunes the coordination sphere and kinetically stabilizes the Cu(I) against air oxidation and disproportionation, but also buries the hydrophobic portions of the ligands and prevents their dissociation which are necessary for their good water solubility and a sustained activity. These catenane Cu(I) complexes can catalyze the oxidative C-C coupling of indoles and tetrahydroisoquinolines in water, using H2O2 as a green oxidant with a good substrate scope. The successful use of catenane ligands in exploiting aqueous Cu(I) catalysis thus highlights the many unexplored potential of mechanical bond as a design element for exploring transition metal catalysis under challenging conditions.

Mechanical Interlocking Enhances the Electrocatalytic Oxygen Reduction Activity and Selectivity of Molecular Copper Complexes.

Efficient O2 reduction reaction (ORR) for selective H2O generation enables advanced fuel cell technology. Nonprecious metal catalysts are viable and attractive alternatives to state-of-the-art Pt-based materials that are expensive. Cu complexes inspired by Cu-containing O2 reduction enzymes in nature are yet to reach their desired ORR catalytic performance. Here, the concept of mechanical interlocking is introduced to the ligand architecture to enforce dynamic spatial restriction on the Cu coordination site. Interlocked catenane ligands could govern O2 binding mode, promote electron transfer, and facilitate product elimination. Our results show that ligand interlocking as a catenane steers the ORR selectivity to H2O as the major product via the 4e- pathway, rivaling the selectivity of Pt, and boosts the onset potential by 130 mV, the mass activity by 1.8 times, and the turnover frequency by 1.5 fold as compared to the noninterlocked counterpart. Our Cu catenane complex represents one of the first examples to take advantage of mechanical interlocking to afford electrocatalysts with enhanced activity and selectivity. The mechanistic insights gained through this integrated experimental and theoretical study are envisioned to be valuable not just to the area of ORR energy catalysis but also with broad implications on interlocked metal complexes that are of critical importance to the general fields in redox reactions involving proton-coupled electron transfer steps.

Macrocycle Dynamics in a Branched [8]Catenane Controlled by Three Different Stimuli in Three Different Regions.

A branched [8]catenane from an efficient one-pot synthesis (72 % HPLC yield, 59 % isolated yield) featuring the simultaneous use of three kinds of templates and cucurbit[6]uril-mediated azide-alkyne cycloaddition (CBAAC) for ring-closing is reported. Design and assembly of the [8]catenane precursors are unexpectedly complex that can involve cooperating, competing and non-influencing interactions. Due to the branched structure, dynamics of the [8]catenane can be modulated in different extent by rigidifying/loosening the mechanical bonds at different regions by using solvent polarity, acid-base and metal ions as the stimuli. This work not only highlights the importance of understanding the delicate interplay of the weak and non-obvious supramolecular interactions in the synthesis of high-order [n]catenane, but also demonstrates a complex control of dynamics and flexibility for exploiting [n]catenanes applications.

Zinc excess increases cellular demand for iron and decreases tolerance to copper in Escherichia coli.

Transition metals serve as an important class of micronutrients that are indispensable for bacterial physiology but are cytotoxic when they are in excess. Bacteria have developed exquisite homeostatic systems to control the uptake, storage, and efflux of each of biological metals and maintain a thermodynamically balanced metal quota. However, whether the pathways that control the homeostasis of different biological metals cross-talk and render cross-resistance or sensitivity in the host-pathogen interface remains largely unknown. Here, we report that zinc (Zn) excess perturbs iron (Fe) and copper (Cu) homeostasis in Escherichia coli, resulting in increased Fe and decreased Cu levels in the cell. Gene expression analysis revealed that Zn excess transiently up-regulates Fe-uptake genes and down-regulates Fe-storage genes and thereby increases the cellular Fe quota. In vitro and in vivo protein-DNA binding assays revealed that the elevated intracellular Fe poisons the primary Cu detoxification transcription regulator CueR, resulting in dysregulation of its target genes copA and cueO and activation of the secondary Cu detoxification system CusSR-cusCFBA Supplementation with the Fe chelator 2,2'-dipyridyl (DIP) or with the reducing agent GSH abolished the induction of cusCFBA during Zn excess. Consistent with the importance of this metal homeostatic network in cell physiology, combined metal treatment, including simultaneously overloading cells with both Zn (0.25 mm) and Cu (0.25 mm) and sequestering Fe with DIP (50 µm), substantially inhibited E. coli growth. These results advance our understanding of bacterial metallobiology and may inform the development of metal-based antimicrobial regimens to manage infectious diseases.

AQAMAN, a bisamidine-based inhibitor of toxic protein inclusions in neurons, ameliorates cytotoxicity in polyglutamine disease models.

Polyglutamine (polyQ) diseases are a group of dominantly inherited neurodegenerative disorders caused by the expansion of an unstable CAG repeat in the coding region of the affected genes. Hallmarks of polyQ diseases include the accumulation of misfolded protein aggregates, leading to neuronal degeneration and cell death. PolyQ diseases are currently incurable, highlighting the urgent need for approaches that inhibit the formation of disaggregate cytotoxic polyQ protein inclusions. Here, we screened for bisamidine-based inhibitors that can inhibit neuronal polyQ protein inclusions. We demonstrated that one inhibitor, AQAMAN, prevents polyQ protein aggregation and promotes de-aggregation of self-assembled polyQ proteins in several models of polyQ diseases. Using immunocytochemistry, we found that AQAMAN significantly reduces polyQ protein aggregation and specifically suppresses polyQ protein-induced cell death. Using a recombinant and purified polyQ protein (thioredoxin-Huntingtin-Q46), we further demonstrated that AQAMAN interferes with polyQ self-assembly, preventing polyQ aggregation, and dissociates preformed polyQ aggregates in a cell-free system. Remarkably, AQAMAN feeding of Drosophila expressing expanded polyQ disease protein suppresses polyQ-induced neurodegeneration in vivo In addition, using inhibitors and activators of the autophagy pathway, we demonstrated that AQAMAN's cytoprotective effect against polyQ toxicity is autophagy-dependent. In summary, we have identified AQAMAN as a potential therapeutic for combating polyQ protein toxicity in polyQ diseases. Our findings further highlight the importance of the autophagy pathway in clearing harmful polyQ proteins.

Activity-Based Sensing of Ascorbate by Using Copper-Mediated Oxidative Bond Cleavage.

Ascorbate is an important biological reductant and enzyme cofactor. Although direct detection through ascorbate-mediated reduction is possible, this approach suffers from poor selectivity due to the wide range of cellular reducing agents. To overcome this limitation, we leverage reduction potential of ascorbate to mediate a copper-mediated oxidative bond cleavage of ether-caged fluorophores. The copper(II) complexes supported by a {bis(2-pyridylmethyl)}benzylamine or a {bis(2-pyridylmethyl)}(2-methoxybenzyl)amine ligand were identified as an ascorbate responsive unit and their reaction with ascorbate yields a copper-based oxidant that enables rapid benzylic oxidation and the release of an ether-caged dye (coumarin or fluorescein). The copper-mediated bond cleavage is specific to ascorbate and the trigger can be readily derivatized for tuning photophysical properties of the probes. The probes were successfully applied for the fluorometric detection of ascorbate in commercial food samples, human plasma, and serum, and within live cells by using confocal microscopy and flow cytometry.

Surface optimization of gold nanoparticle mass tags for the sensitive detection of protein biomarkers via immuno-capture LI-MS.

HPV-induced cervical cancer is one of the most lethal cancers. Therefore, the development of a reliable and accurate method for the early diagnosis of HPV infections is highly important. Here, gold nanoparticles (AuNPs) were utilized as mass tags in an immuno-capture LI-MS assay for the detection of HPV marker proteins. Through the optimization of the amount of antibodies and surface charges on AuNPs, high antigen detection efficiency with minimal non-specific binding was achieved. With optimized antibody-conjugated AuNPs, low attomole amount of HPV proteins in HeLa cell lysate was quantified.

In vivo bioluminescence imaging reveals copper deficiency in a murine model of nonalcoholic fatty liver disease.

Copper is a required metal nutrient for life, but global or local alterations in its homeostasis are linked to diseases spanning genetic and metabolic disorders to cancer and neurodegeneration. Technologies that enable longitudinal in vivo monitoring of dynamic copper pools can help meet the need to study the complex interplay between copper status, health, and disease in the same living organism over time. Here, we present the synthesis, characterization, and in vivo imaging applications of Copper-Caged Luciferin-1 (CCL-1), a bioluminescent reporter for tissue-specific copper visualization in living animals. CCL-1 uses a selective copper(I)-dependent oxidative cleavage reaction to release d-luciferin for subsequent bioluminescent reaction with firefly luciferase. The probe can detect physiological changes in labile Cu+ levels in live cells and mice under situations of copper deficiency or overload. Application of CCL-1 to mice with liver-specific luciferase expression in a diet-induced model of nonalcoholic fatty liver disease reveals onset of hepatic copper deficiency and altered expression levels of central copper trafficking proteins that accompany symptoms of glucose intolerance and weight gain. The data connect copper dysregulation to metabolic liver disease and provide a starting point for expanding the toolbox of reactivity-based chemical reporters for cell- and tissue-specific in vivo imaging.

Radial Hetero[5]catenanes: Peripheral Isomer Sequences of the Interlocked Macrocycles.

A pair of radial [5]catenanes, with either an isomeric cyclic -AABB- or -ABAB- type sequence of the interlocked ß-cyclodextrin (ß-CD) and cucurbit[6]uril (CB[6]) units, has been efficiently synthesized. Because of a marked difference in the binding strength and interlocking sequence of the peripheral macrocycles, interesting sequence-dependent properties, characteristic of mechanically bonded macrocycles, were realized. Variable-temperature 1 H NMR studies showed that the -ABAB- isomer has a more independent ß-CD dynamic, whereas the ß-CD motions in the -AABB- isomer are coupled. Dynamics of the pH-insensitive ß-CD can also be further modulated upon base-triggered mobilization of the CB[6]. These unique properties of the mechanical bond expressed in a sequence-specific fashion and the transmission of the control on the macrocycle dynamics from one interlocked component to another, highlight the potential of similar complex hetero[n]catenanes in the design of advanced, multicomponent molecular machines.

Synthesis of a [6]rotaxane with singly threaded γ-cyclodextrins as a single stereoisomer.

A series of hetero [4]-, [5]- and [6]rotaxanes containing both cucurbit[6]uril (CB[6]) and γ-cyclodextrin (γ-CD) as the macrocyclic components have been synthesized via a threading-followed-by-stoppering approach. Due to the orthogonal binding of CB[6] to ammonium and γ-CD to biphenylene/tetra(ethylene glycol), the [n]rotaxanes display a specific sequence of the interlocked macrocycles. In addition, despite of the asymmetry of γ-CD with respect to the orthogonal plane of the axle, only one stereoisomer of the [6]rotaxane was obtained.

Manganese causes neurotoxic iron accumulation via translational repression of amyloid precursor protein and H-Ferritin.

For more than 150 years, it is known that occupational overexposure of manganese (Mn) causes movement disorders resembling Parkinson's disease (PD) and PD-like syndromes. However, the mechanisms of Mn toxicity are still poorly understood. Here, we demonstrate that Mn dose- and time-dependently blocks the protein translation of amyloid precursor protein (APP) and heavy-chain Ferritin (H-Ferritin), both iron homeostatic proteins with neuroprotective features. APP and H-Ferritin are post-transcriptionally regulated by iron responsive proteins, which bind to homologous iron responsive elements (IREs) located in the 5'-untranslated regions (5'-UTRs) within their mRNA transcripts. Using reporter assays, we demonstrate that Mn exposure repressed the 5'-UTR-activity of APP and H-Ferritin, presumably via increased iron responsive proteins-iron responsive elements binding, ultimately blocking their protein translation. Using two specific Fe2+ -specific probes (RhoNox-1 and IP-1) and ion chromatography inductively coupled plasma mass spectrometry (IC-ICP-MS), we show that loss of the protective axis of APP and H-Ferritin resulted in unchecked accumulation of redox-active ferrous iron (Fe2+ ) fueling neurotoxic oxidative stress. Enforced APP expression partially attenuated Mn-induced generation of cellular and lipid reactive oxygen species and neurotoxicity. Lastly, we could validate the Mn-mediated suppression of APP and H-Ferritin in two rodent in vivo models (C57BL6/N mice and RjHan:SD rats) mimicking acute and chronic Mn exposure. Together, these results suggest that Mn-induced neurotoxicity is partly attributable to the translational inhibition of APP and H-Ferritin resulting in impaired iron metabolism and exacerbated neurotoxic oxidative stress. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Strategies To Assemble Catenanes with Multiple Interlocked Macrocycles.

As a major class of mechanically interlocked molecules, not only are catenanes topologically intriguing targets that challenge the chemical synthesis to the efficient formation of mechanical bonds, but also the mechanical properties arising from the topology offer unique and attractive features for the development of novel functional molecular materials. Despite advancements in templated methods for different types of interlocked architectures, [ n]catenane possessing multiple numbers of interlocked macrocycles still remains a difficult synthetic target with very few reported examples. If the unique mechanical properties of catenanes are to be fully exploited, reliable, controllable, and efficient strategies for accessing [ n]catenanes will be necessary. In this Viewpoint, challenges, considerations, and strategies to [ n]catenanes are discussed.

Efficient catenane synthesis by cucurbit[6]uril-mediated azide-alkyne cycloaddition.

We report here the efficient synthesis of a series of [3]catenanes featuring the use of cucurbit[6]uril to simultaneously mediate the mechanical and covalent bond formations. By coupling the mechanical interlocking with covalent macrocyclization, formation of topological isomers is eliminated and the [3]catenanes are formed exclusively in good yields. The efficient access to these [3]catenanes and the presence of other recognition units render them promising building blocks for the construction of other high-order interlocked structures.

Dynamic mechanostereochemical switching of a co-conformationally flexible [2]catenane controlled by specific ionic guests.

Responsive synthetic receptors for adaptive recognition of different ionic guests in a competitive environment are valuable molecular tools for not only ion sensing and transport, but also the development of ion-responsive smart materials and related technologies. By virtue of the mechanical chelation and ability to undergo large-amplitude co-conformational changes, described herein is the discovery of a chameleon-like [2]catenane that selectively binds copper(I) or sulfate ions and its associated co-conformational mechanostereochemical switching. This work highlights not only the advantages and versatility of catenane as a molecular skeleton in receptor design, but also its potential in constructing complex responsive systems with multiple inputs and outputs.

Molecular imaging of labile iron(II) pools in living cells with a turn-on fluorescent probe.

Iron is an essential metal for living organisms, but misregulation of its homeostasis at the cellular level can trigger detrimental oxidative and/or nitrosative stress and damage events. Motivated to help study the physiological and pathological consequences of biological iron regulation, we now report a reaction-based strategy for monitoring labile Fe(2+) pools in aqueous solution and living cells. Iron Probe 1 (IP1) exploits a bioinspired, iron-mediated oxidative C-O bond cleavage reaction to achieve a selective turn-on response to Fe(2+) over a range of cellular metal ions in their bioavailable forms. We show that this first-generation chemical tool for fluorescence Fe(2+) detection can visualize changes in exchangeable iron stores in living cells upon iron supplementation or depletion, including labile iron pools at endogenous, basal levels. Moreover, IP1 can be used to identify reversible expansion of labile iron pools by stimulation with vitamin C or the iron regulatory hormone hepcidin, providing a starting point for further investigations of iron signaling and stress events in living systems as well as future probe development.

Catenane and Rotaxane Synthesis from Cucurbit[6]uril-Mediated Azide-Alkyne Cycloaddition.

The chemistry of mechanically interlocked molecules (MIMs) such as catenane and rotaxane is full of new opportunities for the presence of a mechanical bond, and the efficient synthesis of these molecules is therefore of fundamental importance in realizing their unique properties and functions. While many different types of preorganizing interactions and covalent bond formation strategies have been exploited in MIMs synthesis, the use of cucurbit[6]uril (CB[6]) in simultaneously templating macrocycle interlocking and catalyzing the covalent formation of the interlocked components is particularly advantageous in accessing high-order catenanes and rotaxanes. In this review, catenane and rotaxane obtained from CB[6]-catalyzed azide-alkyne cycloaddition will be discussed, with special emphasis on the synthetic strategies employed for obtaining complex [n]rotaxanes and [n]catenanes, as well as their properties and functions.

Molecular recognition and self-assembly special feature: Dynamic combinatorial synthesis of a catenane based on donor-acceptor interactions in water.

A new type of neutral donor-acceptor [2]-catenane, containing both complementary units in the same ring was synthesized from a dynamic combinatorial library in water. The yield of the water soluble [2]-catenane is enhanced by increasing either building-block concentrations or ionic strength, or by the addition of an electron-rich template. NMR spectroscopy demonstrates that the template is intercalated between the 2 electron-deficient naphthalenediimide units of the catenane.

Distinctive features and challenges in catenane chemistry.

From being an aesthetic molecular object to a building block for the construction of molecular machines, catenanes and related mechanically interlocked molecules (MIMs) continue to attract immense interest in many research areas. Catenane chemistry is closely tied to that of rotaxanes and knots, and involves concepts like mechanical bonds, chemical topology and co-conformation that are unique to these molecules. Yet, because of their different topological structures and mechanical bond properties, there are some fundamental differences between the chemistry of catenanes and that of rotaxanes and knots although the boundary is sometimes blurred. Clearly distinguishing these differences, in aspects of bonding, structure, synthesis and properties, between catenanes and other MIMs is therefore of fundamental importance to understand their chemistry and explore the new opportunities from mechanical bonds.

Development of Fluorescent Turn-On Probes for CAG-RNA Repeats.

Fluorescent sensing of nucleic acids is a highly sensitive and efficient bioanalytical method for their study in cellular processes, detection and diagnosis in related diseases. However, the design of small molecule fluorescent probes for the selective binding and detection of RNA of a specific sequence is very challenging because of their diverse, dynamic, and flexible structures. By modifying a bis(amidinium)-based small molecular binder that is known to selectively target RNA with CAG repeats using an environment-sensitive fluorophore, a turn-on fluorescent probe featuring aggregation-induced emission (AIE) is successfully developed in this proof-of-concept study. The probe (DB-TPE) exhibits a strong, 19-fold fluorescence enhancement upon binding to a short CAG RNA, and the binding and fluorescence response was found to be specific to the overall RNA secondary structure with A·A mismatches. These promising analytical performances suggest that the probe could be applied in pathological studies, disease progression monitoring, as well as diagnosis of related neurodegenerative diseases due to expanded CAG RNA repeats.