Dynamic sampling of a surveillance state enables DNA proofreading by Cas9.

CRISPR-Cas9 has revolutionized genome engineering applications by programming its single-guide RNA, where high specificity is required. However, the precise molecular mechanism underscoring discrimination between on/off-target DNA sequences, relative to the guide RNA template, remains elusive. Here, using methyl-based NMR to study multiple holoenzymes assembled in vitro, we elucidate a discrete protein conformational state which enables recognition of DNA mismatches at the protospacer adjacent motif (PAM)-distal end. Our results delineate an allosteric pathway connecting a dynamic conformational switch at the REC3 domain, with the sampling of a catalytically competent state by the HNH domain. Our NMR data show that HiFi Cas9 (R691A) increases the fidelity of DNA recognition by stabilizing this "surveillance state" for mismatched substrates, shifting the Cas9 conformational equilibrium away from the active state. These results establish a paradigm of substrate recognition through an allosteric protein-based switch, providing unique insights into the molecular mechanism which governs Cas9 selectivity.

Light-Powered Propeller-like Transporter for Boosted Transmembrane Ion Transport.

Membrane-active molecular machines represent a recently emerging, yet important line of expansion in the field of artificial transmembrane transporters. Their hitherto demonstrated limited types (molecular swing, ion fishers, shuttlers, rotors, etc.) certainly call for new inspiring developments. Here, we report a very first motorized ion-transporting carrier-type transporter, i.e., a modularly tunable, light-powered propeller-like transporter derived from Feringa's molecular motor for consistently boosting transmembrane ion transport under continuous UV light irradiation. Based on the EC50 values, the molecular propeller-mediated ion transport activities under UV light irradiation for 300 s are 2.31, 1.74, 2.29, 2.80, and 2.92 times those values obtained without irradiation for Li+, Na+, K+, Rb+, and Cs+ ions, respectively, with EC50 value as low as 0.71 mol % for K+ ion under light irradiation.

Lighting Up Bispyrene-Functionalized Chiral Molecular Muscles with Switchable Circularly Polarized Excimer Emissions.

Aiming at the further extension of the application scope of traditional molecular muscles, a novel bispyrene-functionalized chiral molecular [c2]daisy chain was designed and synthesized. Taking advantage of the unique dimeric interlocked structure of molecular [c2]daisy chain, the resultant chiral molecular muscle emits strong circularly polarized luminescence (CPL) attributed to the pyrene excimer with a high dissymmetry factor (glum) value of 0.010. More importantly, along with the solvent- or anion- induced motions of the chiral molecular muscle, the precise regulation of the pyrene stacking within its skeleton results in the switching towards either "inversed" state with sign inversion and larger glum values or "down" state with maintained handedness and smaller glum values, making it a novel multistate CPL switch. As the first example of chiral molecular muscle-based CPL switch, this proof-of-concept study not only successfully widens the application scopes of molecular muscles, but also provides a promising platform for the construction of novel smart chiral luminescent materials for practical applications.

Beyond Single-Cycle Autonomous Molecular Machines: Light-Powered Shuttling in a Multi-Cycle Reaction Network.

Biomolecular machines autonomously convert energy into functions, driving systems away from thermodynamic equilibrium. This energy conversion is achieved by leveraging complex, kinetically asymmetric chemical reaction networks that are challenging to characterize precisely. In contrast, all known synthetic molecular systems in which kinetic asymmetry has been quantified are well described by simple single-cycle networks. Here, we report on a unique light-driven [2]rotaxane that enables the autonomous operation of a synthetic molecular machine with a multi-cycle chemical reaction network. Unlike all prior systems, the present one exploits a photoactive macrocycle, which features a different photoreactivity depending on the binding sites at which it resides. Furthermore, E to Z isomerization reverses the relative affinity of the macrocycle for two binding sites on the axle, resulting in a multi-cycle network. Building on the most recent theoretical advancements, this work quantifies kinetic asymmetry in a multi-cycle network for the first time. Our findings represent the simplest rotaxane capable of autonomous shuttling developed so far and offer a general strategy to generate and quantify kinetic asymmetry beyond single-cycle systems.

Aggregation-Induced Emissive Feringa-Type Motor: Toward the Dual-Functional Motor in a Single Molecular Aggregation System.

Aggregation-induced emission (AIE)allows tunable photoluminescence via the simple regulation of molecular aggregation. The research spurt along this vein has also offered tremendous opportunities for light-responsive artificial molecular machines that are to be fully explored for performing versatile functions. Herein, the study reports a light-driven Feringa-type motor, when in the appropriate aggregation state, not only demonstrates the light-activated rotary motion but emits photons with good quantum yield. A semi-quantitative TD-DFT calculation is also conducted to aid the understanding of the competitive photoluminescence and photoisomerization processes of the motor. Cytotoxicity test shows this motor possesses good biocompatibility, laying a solid foundation for applying it in the bio-environment. The results demonstrated that the engagement of the aggregation-induced emission concept and light-driven Feringa-motor can lead to the discovery of the novel motorized AIEgen, which will further stimulate the rise of more advanced molecular motors capable of executing multi-functionalities.

Dual-driven AND molecular logic gates for label-free and sensitive ratiometric fluorescence sensing and inhibitors screening.

Due to the complexity of regulatory networks of disease-related biomarkers, developing simple, sensitive, and accurate methods has remained challenging for precise diagnosis. Herein, an "AND" logic gates DNA molecular machine (LGDM) was constructed, which was powered by the catalytic hairpin assembly (CHA). It was coupled with dual-emission CdTe quantum dots (QDs)-based cation exchange reaction (CER) for label-free, sensitive, and ratiometric fluorescence detection of APE1 and miRNA biomarkers. Benefiting from synergistic signal amplification strategies and a ratiometric fluorometric output mode, this LGDM enables accurate logic computing with robust and significant output signals from weak inputs. It offers improved sensitivity and selectivity even in cell extracts. Using dual-emission spectra CdTe QDs, with a ratiometric signal output mode, ensured good stability and effectively prevented false-positive signals from intrinsic biological interferences compared to the approach relying on a single signal output mode, which enabled the LGDM to achieve rapid, efficient, and accurate natural drug screening against APE1 inhibitors in vitro and cells. The developed method provides impetus to streamline research related to miRNA and APE1, offering significant promise for widespread application in drug development and clinical analysis.

Mechanical control of molecular machines at an air-water interface: manipulation of molecular pliers, paddles.

Many artificial molecular machines have been synthesized, and various functions have been expressed by changing their molecular conformations. However, their structures are still simple compared with those of biomolecular machines, and more energy is required to control them. To design artificial molecular machines with more complex structures and higher functionality, it is necessary to combine molecular machines with simple movements such as components. This means that the motion of individual molecular machines must be precisely controlled and observed in various environments. At the air - water interface, the molecular orientation and conformation can be controlled with little energy as thermal fluctuations. We designed various molecular machines and controlled them using mechanical stimuli at the air - water interface. We also controlled the transfer of forces to the molecular machines in various lipid matrices. In this review, we describe molecular pliers with amphiphilic binaphthyl, molecular paddles with binuclear platinum complexes, and molecular rotors with julolidine and BODIPY that exhibit twisted intramolecular charge transfer.

This review discusses the dependence of the behaviour of molecular machines around their environment through the mechanically control of simple molecular machines at the air ­ water interface.

Confined Space Nanoarchitectonics for Dynamic Functions and Molecular Machines.

Nanotechnology has advanced the techniques for elucidating phenomena at the atomic, molecular, and nano-level. As a post nanotechnology concept, nanoarchitectonics has emerged to create functional materials from unit structures. Consider the material function when nanoarchitectonics enables the design of materials whose internal structure is controlled at the nanometer level. Material function is determined by two elements. These are the functional unit that forms the core of the function and the environment (matrix) that surrounds it. This review paper discusses the nanoarchitectonics of confined space, which is a field for controlling functional materials and molecular machines. The first few sections introduce some of the various dynamic functions in confined spaces, considering molecular space, materials space, and biospace. In the latter two sections, examples of research on the behavior of molecular machines, such as molecular motors, in confined spaces are discussed. In particular, surface space and internal nanospace are taken up as typical examples of confined space. What these examples show is that not only the central functional unit, but also the surrounding spatial configuration is necessary for higher functional expression. Nanoarchitectonics will play important roles in the architecture of such a total system.

Visualizing chaperone-mediated multistep assembly of the human 20S proteasome.

Dedicated assembly factors orchestrate stepwise production of many molecular machines, including the 28-subunit proteasome core particle (CP) that mediates protein degradation. Here, we report cryo-EM reconstructions of seven recombinant human subcomplexes that visualize all five chaperones and the three active site propeptides across a wide swath of the assembly pathway. Comparison of these chaperone-bound intermediates and a matching mature CP reveals molecular mechanisms determining the order of successive subunit additions, and how proteasome subcomplexes and assembly factors structurally adapt upon progressive subunit incorporation to stabilize intermediates, facilitate the formation of subsequent intermediates, and ultimately rearrange to coordinate proteolytic activation with gated access to active sites. The structural findings reported here explain many previous biochemical and genetic observations. This work establishes a methodologic approach for structural analysis of multiprotein complex assembly intermediates, illuminates specific functions of assembly factors, and reveals conceptual principles underlying human proteasome biogenesis.

Kinetic Asymmetry and Directionality of Nonequilibrium Molecular Systems.

Scientists have long been fascinated by the biomolecular machines in living systems that process energy and information to sustain life. The first synthetic molecular rotor capable of performing repeated 360° rotations due to a combination of photo- and thermally activated processes was reported in 1999. The progress in designing different molecular machines in the intervening years has been remarkable, with several outstanding examples appearing in the last few years. Despite the synthetic accomplishments, there remains confusion regarding the fundamental design principles by which the motions of molecules can be controlled, with significant intellectual tension between mechanical and chemical ways of thinking about and describing molecular machines. A thermodynamically consistent analysis of the kinetics of several molecular rotors and pumps shows that while light driven rotors operate by a power-stroke mechanism, kinetic asymmetry-the relative heights of energy barriers-is the sole determinant of the directionality of catalysis driven machines. Power-strokes-the relative depths of energy wells-play no role whatsoever in determining the sign of the directionality. These results, elaborated using trajectory thermodynamics and the nonequilibrium pump equality, show that kinetic asymmetry governs the response of many non-equilibrium chemical phenomena.

Structural basis of membrane machines that traffick and attach heme to cytochromes.

We evaluate cryoEM and crystal structures of two molecular machines that traffick heme and attach it to cytochrome c (cyt c), the second activity performed by a cyt c synthase. These integral membrane proteins, CcsBA and CcmF/H, both covalently attach heme to cyt c, but carry it out via different mechanisms. A CcsB-CcsA complex transports heme through a channel to its external active site, where it forms two thioethers between reduced (Fe+2) heme and CysXxxXxxCysHis in cyt c. The active site is formed by a periplasmic WWD sequence and two histidines (P-His1 and P-His2). We evaluate each proposed functional domain in CcsBA cryoEM densities, exploring their presence in other CcsB-CcsA proteins from a wide distribution of organisms (e.g., from Gram positive to Gram negative bacteria to chloroplasts.) Two conserved pockets, for the first and second cysteines of CXXCH, explain stereochemical heme attachment. In addition to other universal features, a conserved periplasmic beta stranded structure, called the beta cap, protects the active site when external heme is not present. Analysis of CcmF/H, here called an oxidoreductase and cyt c synthase, addresses mechanisms of heme access and attachment. We provide evidence that CcmF/H receives Fe+3 heme from holoCcmE via a periplasmic entry point in CcmF, whereby heme is inserted directly into a conserved WWD/P-His domain from above. Evidence suggests that CcmF acts as a heme reductase, reducing holoCcmE (to Fe+2) through a transmembrane electron transfer conduit, which initiates a complicated series of events at the active site.

Engineered DNA molecular machine for ultrasensitive detection of environmental lead pollution.

Dynamic monitoring of environmental Pb2+ is of utmost importance for food safety and personal well-being. Herein, we report a novel, rapid, and practical fluorescence detection platform for Pb2+. The platform comprises two essential components: an engineered DNAzyme probe (EDP) and a responsive functionalized probe (RFP). The EDP demonstrates specific recognition of Pb2+ and the subsequent release of free DNA fragments. The released DNA fragments are then captured using the RFP to form DNA complexes, which undergo multiple cascade amplification reactions involving polymerases and nickases, resulting in the generation of a large number of fluorescence signals. These signals can detect Pb2+ at concentrations as low as 0.114 nmol/L, with a dynamic range spanning from 0.1 nmol/L to 50 nmol/L. Moreover, the platform exhibits excellent sensitivity and selectivity for Pb2+ detection. To further validate its effectiveness, we successfully quantitatively detected lead contamination in water from Chaohu Lake, and the results aligned closely with those obtained using inductively coupled plasma-mass spectrometry (ICP-MS). Moreover, this platform is suitable for detecting Pb2+ in seawater, soil, and fish samples. These findings confirm the suitability of the current detection platform for the dynamic assessment of Pb contamination in ecological environments, thereby contributing to environmental and food safety.

Exploration of molecular machines in supramolecular soft robotic systems.

Soft robotic system, a new era of material science, is rapidly developing with advanced processing technology in soft matters, featured with biomimetic nature. An important bottom-up approach is through the implementation of molecular machines into polymeric materials, however, the synchronized molecular motions, acumination of strain across multiple length-scales, and amplification into macroscopic actuations remained highly challenging. This review presents the significances, key design strategies, and outlook of the hierarchical supramolecular systems of molecular machines to develop novel types of supramolecular-based soft robotic systems.

Dithienylethene-Based Single Molecular Photothermal Linear Actuator.

By employing a mechanically controllable break junction technique, we have realized an ideal single molecular linear actuator based on dithienylethene (DTE) based molecular architecture, which undergoes reversible photothermal isomerization when subjected to UV irradiation under ambient conditions. As a result, open form (compressed, UV OFF) and closed form (elongated, UV ON) of dithienylethene-based molecular junctions are achieved. Interestingly, the mechanical actuation is achieved without changing the conductance of the molecular junction around the Fermi level over several cycles, which is an essential property required for an ideal single molecular actuator. Our study demonstrates a unique example of achieving a perfect balance between tunneling width and barrier height change upon photothermal isomerization, resulting in no change in conductance but a change in the molecular length, which results in mechanical actuation at the single molecular level.

Light-Driven Aqueous Dissipative Pseudorotaxanes with Tunable Fluorescence Enabling Deformable Nano-Assemblies.

Developing an artificial dynamic nanoscale molecular machine that dissipatively self-assembles far from equilibrium is fundamentally important but is significantly challenging. Herein, we report dissipatively self-assembling light-activated convertible pseudorotaxanes (PRs) that show tunable fluorescence and enable deformable nano-assemblies. A pyridinium-conjugated sulfonato-merocyanine derivative (EPMEH) and cucurbit[8]uril (CB[8]) form the 2EPMEH ⊂ CB[8] [3]PR in a 2:1 stoichiometry, which phototransforms into a transient spiropyran containing 1:1 EPSP ⊂ CB[8] [2]PR when exposed to light. The transient [2]PR thermally relaxes (reversibly) to the [3]PR in the dark accompanied by periodic fluorescence changes that include near-infrared emission. Moreover, octahedral and spherical nanoparticles are formed through the dissipative self-assembly of the two PRs, and the Golgi apparatus is dynamically imaged using fluorescent dissipative nano-assemblies.

Combination Cancer Treatment: Using Engineered DNAzyme Molecular Machines for Dynamic Inter- and Intracellular Regulation.

Despite the promise of combination cancer therapy, it remains challenging to develop targeted strategies that are nontoxic to normal cells. Here we report a combination therapeutic strategy based on engineered DNAzyme molecular machines that can promote cancer apoptosis via dynamic inter- and intracellular regulation. To achieve external regulation of T-cell/cancer cell interactions, we designed a DNAzyme-based molecular machine with an aptamer and an i-motif, as the MUC-1-selective aptamer allows the specific recognition of cancer cells. The i-motif is folded under the tumor acidic microenvironment, shortening the intercellular distance. As a result, T-cells are released by metal ion activated DNAzyme cleavage. To achieve internal regulation of mitochondria, we delivered another DNAzyme-based molecular machine with mitochondria-targeted peptides into cancer cells to induce mitochondria aggregation. Our strategy achieved an enhanced killing effect in zinc deficient cancer cells.

A new class of biological ion-driven rotary molecular motors with 5:2 symmetry.

Several new structures of three types of protein complexes, obtained by cryo-electron microscopy (cryo-EM) and published between 2019 and 2021, identify a new family of natural molecular wheels, the "5:2 rotary motors." These span the cytoplasmic membranes of bacteria, and their rotation is driven by ion flow into the cell. They consist of a pentameric wheel encircling a dimeric axle within the cytoplasmic membrane of both Gram-positive and gram-negative bacteria. The axles extend into the periplasm, and the wheels extend into the cytoplasm. Rotation of these wheels has never been observed directly; it is inferred from the symmetry of the complexes and from the roles they play within the larger systems that they are known to power. In particular, the new structure of the stator complex of the Bacterial Flagellar Motor, MotA5B2, is consistent with a "wheels within wheels" model of the motor. Other 5:2 rotary motors are believed to share the core rotary function and mechanism, driven by ion-motive force at the cytoplasmic membrane. Their structures diverge in their periplasmic and cytoplasmic parts, reflecting the variety of roles that they perform. This review focuses on the structures of 5:2 rotary motors and their proposed mechanisms and functions. We also discuss molecular rotation in general and its relation to the rotational symmetry of molecular complexes.

Simultaneous detection of cancerous exosomal miRNA-21 and PD-L1 with a sensitive dual-cycling nanoprobe.

Simultaneous detection of specific exosomal surface proteins and inner microRNAs are hampered by their heterogeneity, low abundance and spatial segregation in nanovesicles. Here, we design a dual-cycling nanoprobe (DCNP) to enable single-step simultaneous quantitation of cancerous exosomal surface programmed death-ligand 1 (PD-L1) (ExoPD-L1) and miRNA-21 (ExomiR-21) directly in exosome lysates, without resorting to either RNA extraction or time-consuming transmembrane penetration. In this design, DNA molecular machine-based dual-recognition probes co-assemble onto gold nanoparticle surface for engineering 'silent' DCNPs, which enable signal-amplified synchronous response to dual-targets as activated by ExomiR-21 and ExoPD-L1 within 20 min. Benefiting from cycling amplification of the molecular machine, DCNPs sensor achieves detection limits of tumor exosomes, ExoPD-L1 and ExomiR-21 down to 10 particles/µL, 0.17 pg/mL and 66 fM, respectively. Such a sensitive dual-response strategy allows simultaneous tracking the dynamic changes of ExoPD-L1 and ExomiR-21 expression regulated by signaling molecules or therapeutics. This approach further detects circulating ExoPD-L1 and ExomiR-21 in human plasma to differentiate breast cancer patients from healthy individuals with high accuracy, showing great potential of DCNPs for simultaneous profiling exosomal surface and inside biomarkers, and for clinical precision diagnosis.

DNA Origami-Based Single-Molecule CRISPR Machines for Spatially Resolved Searching.

Repurposing the RNA-guided endonuclease Cas9 to develop artificial CRISPR molecular machines represents a new direction toward synthetic molecular information processing. The operation of CRISPR-Cas9-based machines, nevertheless, relies on the molecular recognition of freely diffused sgRNA/Cas9, making it practically challenging to perform spatially regulated localized searching or navigation. Here, we develop a DNA origami-based single-molecule CRISPR machine that can perform spatially resolved DNA cleavage via either free or localized searching modes. When triggered at a specific site on the DNA origami with nanoscale accuracy, the free searching mode leads to searching activity that gradually decays with the distance, whereas the localized mode generates spatially-confined searching activity. Our work expands the function of CRISPR molecular machines and lays foundations to develop integrated molecular circuits and high-throughput nucleic acid detection.

The Bacterial Flagellar Motor: Insights Into Torque Generation, Rotational Switching, and Mechanosensing.

The flagellar motor is a bidirectional rotary nanomachine used by many bacteria to sense and move through environments of varying complexity. The bidirectional rotation of the motor is governed by interactions between the inner membrane-associated stator units and the C-ring in the cytoplasm. In this review, we take a structural biology perspective to discuss the distinct conformations of the stator complex and the C-ring that regulate bacterial motility by switching rotational direction between the clockwise (CW) and counterclockwise (CCW) senses. We further contextualize recent in situ structural insights into the modulation of the stator units by accessory proteins, such as FliL, to generate full torque. The dynamic structural remodeling of the C-ring and stator complexes as well as their association with signaling and accessory molecules provide a mechanistic basis for how bacteria adjust motility to sense, move through, and survive in specific niches both outside and within host cells and tissues.