Catalytic-assembly of programmable atom equivalents.

Escape from metastable states in self-assembly of colloids is an intractable problem. Unlike the commonly adopted approach of thermal annealing, the recently developed enthalpy-mediated strategy provided a different option to address this dilemma in a dynamically controllable manner at room temperature. However, it required a complex catalytic-assembly DNA strand-displacement circuitry to mediate interaction between multiple components. In this work, we present a simple but effective way to achieve catalytic-assembly of DNA-functionalized colloidal nanoparticles, i.e., programmable atom equivalents, in a far-from-equilibrium system. A removable molecule named "catassembler" that acts as a catalyst was employed to rectify imperfect linkages and help the system escape from metastability without affecting the assembled framework. Notably, catalytic efficiency of the catassembler can be effectively improved by changing the seesaw catassembler in toehold length design or numbers of the repeat units. Leveraging this tractable catalytic-assembly approach, different ordered architectures were easily produced by directly mixing all reactants, as in chemical reactions. By switching bonding identities, solid-solid phase transformations between different colloidal crystals were achieved. This work opens up an avenue for programming colloid assembly in a far-from-equilibrium system.

Efficient precise integration of large DNA sequences with 3'-overhang dsDNA donors using CRISPR/Cas9.

CRISPR/Cas9 genome-editing tools have tremendously boosted our capability of manipulating the eukaryotic genomes in biomedical research and innovative biotechnologies. However, the current approaches that allow precise integration of gene-sized large DNA fragments generally suffer from low efficiency and high cost. Herein, we developed a versatile and efficient approach, termed LOCK (Long dsDNA with 3'-Overhangs mediated CRISPR Knock-in), by utilizing specially designed 3'-overhang double-stranded DNA (odsDNA) donors harboring 50-nt homology arm. The length of the 3'-overhangs of odsDNA is specified by the five consecutive phosphorothioate modifications. Compared with existing methods, LOCK allows highly efficient targeted insertion of kilobase-sized DNA fragments into the mammalian genomes with low cost and low off-target effects, yielding >fivefold higher knock-in frequencies than conventional homologous recombination-based approaches. This newly designed LOCK approach based on homology-directed repair is a powerful tool suitable for gene-sized fragment integration that is urgently needed for genetic engineering, gene therapies, and synthetic biology.

pH-Controlled Resettable Modular DNA Strand-Displacement Circuits.

Sophisticated dynamic molecular systems with diverse functions have been fabricated by using the fundamental tool of toehold-mediated strand displacement (TMSD) in the field of dynamic DNA nanotechnology. However, simple approaches to reset these TMSD-based dynamic systems are lacking due to the difficulty in creating kinetically favored pathways to implement the backward resetting reactions. Here, we develop a facile proton-driven strategy to achieve complete resetting of a modular DNA circuit by integrating a pH-responsive intermolecular CG-C+ triplex DNA and an i-motif DNA into the conventional DNA substrate. The pH-programmed strategy allows modular DNA components to specifically associate/dissociate to promote the forward/backward TMSD reactions, thereby enabling the modular DNA circuit to be repeatedly operated at a constant temperature without generating any DNA waste products. Leveraging this tractable approach, we further constructed two resettable DNA logic gates used for logical computation and two resettable catalytic DNA systems with good performance in signal transduction and amplification.

A Prospective Investigation of Patient Satisfaction and Psychosocial Status Following Facial Bone Contouring Surgery using the Face-Q.

OBJECTIVE: The objective of this study was to assess satisfaction and psychosocial status before and after facial bone contouring surgery using the Face-Q. METHODS: The Face-Q, a multimodular patient-reported outcome (PRO) instrument, comprises independently functioning scales and checklists designed to assess outcomes in facial aesthetic patients. A prospective cohort study was conducted from November 2020 to May 2022. Participants undergoing facial bone contouring surgery (reduction mandibuloplasty and/or malarplasty) were asked to complete the Face-Q preoperatively and 12 months postoperatively. Comparative analyses were conducted using normative Face-Q data from 534 matched normal individuals. Face-Q scores were evaluated for each domain on a scale of 0 to 100, with higher scores indicating greater satisfaction with appearance or a superior quality of life. RESULTS: A total of 284 patients (274 female and 10 male) completed the Face-Q preoperatively and 12 months postoperatively. Of these, 146 underwent reduction mandibuloplasty, 18 underwent malarplasty, and 120 underwent both procedures. Post-surgery, patients experienced significant improvements in overall appearance, features altered by surgery, and quality of life, excluding the patient-perceived age. Preoperatively, patients demonstrated significantly lower scores compared to normative data, with scores significantly increasing postoperatively to levels representative of the general population. Satisfaction with outcome was significantly correlated with postoperative Face-Q measurements but not preoperatively. CONCLUSION: Facial bone contouring surgery significantly improves the satisfaction and quality of life in patients with square faces, reaching a level at least equivalent to the normative population. The use of Face-Q should be highlighted in the clinic practice. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors   www.springer.com/00266.

Programming of Supercrystals Using Replicable DNA-Functionalized Colloids.

The development of self-replicating systems is of great importance in research on the origin of life. As the most iconic molecules, nucleic acids have provided prominent examples of the fabrication of self-replicating artificial nanostructures. However, it is still challenging to construct sophisticated synthetic systems that can create large-scale or three-dimensionally ordered nanomaterials using self-replicating nanostructures. By integrating a template system containing DNA-functionalized colloidal seeds with a simplified DNA strand-displacement circuit programmed subsystem to produce DNA-functionalized colloidal copies, we developed a facile enthalpy-mediated strategy to control the replication and catalytic assembly of DNA-functionalized colloids in a time-dependent manner. The replication efficiency and crystal quality of the resulting superlattice structures can be effectively increased by regulating the molar ratio of the template to the copy colloids. By constructing binary systems from two types of gold nanoparticles (or proteins), superlattice structures with different crystal symmetries can be obtained through the replication and catalytic assembly processes. This programmable enthalpy-mediated approach was easily leveraged to achieve the phase transformation and catalytic amplification of colloidal crystals starting from different initial template crystals. This work offers a potential way to construct self-replicating artificial systems that exhibit complicated phase behaviors and can produce large-scale superlattice nanomaterials.

A Bone-Penetrating Precise Controllable Drug Release System Enables Localized Treatment of Osteoporotic Fracture Prevention via Modulating Osteoblast-Osteoclast Communication.

Improving local bone mineral density (BMD) at fracture-prone sites of bone is a clinical concern for osteoporotic fracture prevention. In this study, a featured radial extracorporeal shock wave (rESW) responsive nano-drug delivery system (NDDS) is developed for local treatment. Based on a mechanic simulation, a sequence of hollow zoledronic acid (ZOL)-contained nanoparticles (HZNs) with controllable shell thickness that predicts various mechanical responsive properties is constructed by controlling the deposition time of ZOL and Ca2+ on liposome templates. Attributed to the controllable shell thickness, the fragmentation of HZNs and the release of ZOL and Ca2+ can be precisely controlled with the intervention of rESW. Furthermore, the distinct effect of HZNs with different shell thicknesses on bone metabolism after fragmentation is verified. In vitro co-culture experiments demonstrate that although HZN2 does not have the strongest osteoclasts inhibitory effect, the best pro-osteoblasts mineralization results are achieved via maintaining osteoblast-osteoclast (OB-OC) communication. In vivo, the HZN2 group also shows the strongest local BMD enhancement after rESW intervention and significantly improves bone-related parameters and mechanical properties in the ovariectomy (OVX)-induced osteoporosis (OP) rats. These findings suggest that an adjustable and precise rESW-responsive NDDS can effectively improve local BMD in OP therapy.

Catalytic assembly of symmetric diblock copolymers in a thin film: a dissipative particle dynamics simulation study.

Obtaining a thin block copolymer film with a perfect structure by self-assembly is difficult because the system is, in general, trapped in a metastable state. We used dissipative particle dynamics (DPD) to investigate the self-assembly of AB symmetric diblock copolymers in a thin film. We discovered that addition of a small molecule (molecule C) as the third composition could help the system evade the metastable state. Therefore, imperfect structures could be corrected, and ordered structures formed. Analogous to the performance of a catalyst in catalytic chemistry, molecule C could promote assembly into an ordered structure, but was less involved within the polymer phase. Moreover, simulation results showed that the content of molecule C and its repulsive interactions with blocks A and B were quite important for promoting assembly into ordered structures effectively.

Shape-induced crystallization of binary DNA-functionalized nanocubes.

Leveraging the anisotropic shape of DNA-functionalized nanoparticles holds potential for shape-directed crystallization of a wide collection of superlattice structures. Using coarse-grained molecular dynamics simulations, we study the self-assembly of a binary mixture of cubic gold nanoparticles, which are functionalized by complementary DNA strands. We observe the spontaneous self-assembly of simple cubic (SC), plastic body-centered tetragonal (pBCT), and compositionally disordered plastic body-centered tetragonal (d-pBCT) phases due to hybridization of the DNA strands. We systematically investigate the effect of length, grafting density, as well as rigidity of the DNA strands on the self-assembly behavior of cubic nanoparticles. We measure the potential of mean force between DNA-functionalized nanocubes for varying rigidity of the DNA strands and DNA lengths. Using free-energy calculations, we find that longer and flexible DNA strands can lead to a phase transformation from SC to the pBCT phase due to a gain in entropy arising from the orientational degrees of freedom of the nanocubes in the pBCT phase. Our results may serve as a guide for self-assembly experiments on DNA-functionalized cubic nanoparticles.

Programming colloidal bonding using DNA strand-displacement circuitry.

As a strategy for regulating entropy, thermal annealing is a commonly adopted approach for controlling dynamic pathways in colloid assembly. By coupling DNA strand-displacement circuits with DNA-functionalized colloid assembly, we developed an enthalpy-mediated strategy for achieving the same goal while working at a constant temperature. Using this tractable approach allows colloidal bonding to be programmed for synchronization with colloid assembly, thereby realizing the optimal programmability of DNA-functionalized colloids. We applied this strategy to conditionally activate colloid assembly and dynamically switch colloid identities by reconfiguring DNA molecular architectures, thereby achieving orderly structural transformations; leveraging the advantage of room-temperature assembly, we used this method to prepare a lattice of temperature-sensitive proteins and gold nanoparticles. This approach bridges two subfields: dynamic DNA nanotechnology and DNA-functionalized colloid programming.

Programmable Transcriptional Modulation with a Structured RNA-Mediated CRISPR-dCas9 Complex.

Multi-module dCas9 engineering systems have been developed for controllable transcriptional manipulation such as chemical- or light-induced systems. However, there is still a need for a separate module that can be used for internal control over the CRISPR-dCas9 system. Here, we describe a multi-module CRISPR-dCas9 system in which a separate structured RNA was applied as a programmable component that could control dCas9-based gene regulation and achieved a higher activation efficiency than dCas9-VPR that is traditionally used. By introducing a microRNA sensor, we generated a dCas9-based transcriptional regulation platform that responded to endogenous microRNAs and allowed controllable activation of endogenous genes. Moreover, we applied the platform to selectively identify HCT116 cells in a cell mixture. This work provides a flexible platform for efficient and controllable gene regulation based on CRISPR-dCas9.

Oral Administration of Omega-3 Fatty Acids Attenuates Lung Injury Caused by PM2.5 Respiratory Inhalation Simply and Feasibly In Vivo.

For developing an effective interventional approach and treatment modality for PM2.5, the effects of omega-3 fatty acids on alleviating inflammation and attenuating lung injury induced by inhalation exposure of PM2.5 were assessed in murine models. We found that daily oral administration of the active components of omega-3 fatty acids, docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) effectively alleviated lung parenchymal lesions, restored normal inflammatory cytokine levels and oxidative stress levels in treating mice exposed to PM2.5 (20 mg/kg) every 3 days for 5 times over a 14-day period. Especially, CT images and the pathological analysis suggested protective effects of DHA and EPA on lung injury. The key molecular mechanism is that DHA and EPA can inhibit the entry and deposition of PM2.5, and block the PM2.5-mediated cytotoxicity, oxidative stress, and inflammation.

Identifying and Differentiating Topological G-Quadruplex Structures with DNA-Encoded Plasmonic Gold Nanoparticles.

DNA G-quadruplexes (G4s) have been identified as critical elements in modulating genomic functions and many other biological processes. Their functions are highly dependent on the primary nucleotides and secondary folding structures. Therefore, to understand their functions, methods to identify and differentiate structures of G4 with speed and accuracy are required but limited. In this report, we have applied a synthetic G4 DNA-encoded nanoparticle approach to identify and differentiate G4 DNA molecules with different topologies and nucleotide residues. We found that the resulting plasmonic properties of the gold nanoparticles, monitored by UV/Vis spectroscopy, are quite sensitive to different G4 structures, including stacking layers, loop sequences, capping bases on G4s, and topological structures. Through these systematic investigations, we demonstrate that this G4-encoded gold nanoparticle approach can be used to profile the G4 structures and distinguish G4s from human telomeres. Such a method may have wide applications in G4 research.

Structural and Physicochemical Properties and Biocompatibility of Linear and Looped Polymer-Capped Gold Nanoparticles.

Various polymer brushes with linear and looped conformations have gained considerable attention in the application of biomaterials and nanotechnology. In this work, the linear and looped polymer brush shells based on PEG-SH and SH-PEG-SH chains binding to gold nanoparticles (AuNPs) are synthesized. The structure and topology of the PEGylated AuNPs are systematically investigated. The basic physicochemical parameters of these PEGylated AuNPs such as hydrodynamic size, grafting density, hydrophilicity, colloidal stability, and biocompatibility are determined intensively. The looped polymer topology can remarkably alter physicochemical properties of polymer brushes compared with the linear counterparts. When the molecular weight of PEG is low (1 and 5 kDa), the looped polymer shells have smaller hydrodynamic size and lower grafting density than their linear analogues; when the molecular weight of PEG is high (10 kDa), the looped shells are much thicker and denser than the linear ones. Interestingly, the looped PEGs on AuNPs are more stable in a high-salt environment and have better hydrophilicity, which endow excellent biocompatibility, including protein resistance and cell viability. These results provide a simple approach to design polymer brushes with different topologies on AuNPs, improve the biocompatibility of hybrid AuNPs, and acquire the potential application in the biomedical field.

A Scalable "Junction Substrate" to Engineer Robust DNA Circuits.

Versatile building blocks are essential for building complex and scaled-up DNA circuits. In this study, we propose a conceptually new scalable architecture called a "junction substrate" (J-substrate) that is linked by prepurified double-stranded DNA molecules. As a proof-of-concept, this novel type of substrate has been utilized to build multi-input DNA circuits, offering several advantages over the conventional substrate (referred to as a "linear substrate", L-substrate). First, the J-substrate does not require long DNA strands, thus avoiding significant synthetic errors and costs. Second, the traditional PAGE purification method is technically facilitated to obtain high-purity substrates, whereby the initial leakage is effectively eliminated. Third, the asymptotic leakage is eliminated by introducing the "junction". Finally, circuits with the optimized J-substrate architecture exhibit fast kinetics. We believe that the proposed architecture constitutes a sophisticated chassis for constructing complex circuits.

Optimizing the Toehold Strategy of On-Chip Nucleic Acid Hybridization Probe for the Discrimination of Single Nucleotide Polymorphism.

The synthetic DNA hybridization probe has proved its importance in biology and biotechnology. In this study, taking advantage of a novel analytical technique called dual polarization interferometry (DPI), the influence of the toehold strategy of on-chip DNA hybridization probe on the discrimination of single nucleotide polymorphism (SNP) was investigated. Through adjusting the toehold length, the toehold strategies of on-chip toehold exchange probe were thoroughly optimized. For the "6/5" probe, an optimal discrimination factor of 78% against the spurious target was achieved. Moreover, the ability of the on-chip probe in SNP discrimination was significantly enhanced compared to its pure solution counterpart. This simple and rapid detection method for SNP discrimination based on the on-chip toehold exchange probe will show great potential in disease diagnosis.

Theoretical Insights into the Interactions between Star-Shaped Antimicrobial Polypeptides and Bacterial Membranes.

A structurally nanoengineered antimicrobial polypeptide consisting of lysine and valine residues is a new class of antimicrobial agent with superior antibacterial activity against multidrug-resistant bacteria and low toxicity toward mammalian cells. Utilizing coarse-grained models, we studied the interactions of microbial cytoplasmic membranes with polypeptides of either (K2V1)5 (star-KV) or CM15 (star-CM15). Our computational results verify the low toxicity of polypeptides of (K2V1)5 toward the dipalmitoyl phosphatidylcholine bilayer. This low toxicity is demonstrated to originate from weakened hydrophobicity combined with its random coil conformation for (K2V1)5 because of the highly abundant valine residues, compared with the typical antimicrobial peptides, such as CM15. In the interactions with a palmitoyl-oleoyl-phosphatidylethanolamine/palmitoyl-oleoyl-phosphatidylglycerol bilayer, star-KV has greater ability in phase separation and generation of phase boundary defects not only in lipid redistribution but also in lateral dynamic movements, although both star-KV and star-CM15 can extract the phosphatidylglycerol lipids and purify the phosphatidylethanolamine lipids into continuum domains. We suggest that the polypeptide of (K2V1)5 can nondisruptively kill bacteria by hampering bacterial metabolism through reorganizing lipid domain distribution and simultaneously "freezing" lipid movement.

Reduction-Triggered Transformation of Disulfide-Containing Micelles at Chemically Tunable Rates.

A dilemma exists between the circulation stability and cargo release/mass diffusion at desired sites when designing delivery nanocarriers and in vivo nanoreactors. Reported herein are disulfide-crosslinked (DCL) micelles exhibiting reduction-triggered switching of crosslinking modules and synchronized hydrophobic-to-hydrophilic transition. Tumor cell targeted DCL micelles undergo cytoplasmic milieu triggered disulfide cleavage and self-immolative decaging reactions at chemically adjustable rates, generating primary amine moieties. Extensive amidation reactions with neighboring ester moieties then occur because of the high local concentration and suppression of the apparent amine pKa  value within the hydrophobic cores, thus leading to the transformation of crosslinking modules and formation of tracelessly crosslinked (TCL) micelles, with hydrophilic cores, inside live cells. We further integrate this design principle with theranostic nanocarriers for selective intracellular drug transport guided by enhanced magnetic resonance (MR) imaging performance.

Localized degradation of foreign DNA strands in cells: Only excising the first nucleotide of 5' region.

Intracellular delivery of foreign DNA probes sharply increases the efficiency of various biodetection protocols. Spherical nucleic acid (SNA) conjugate is a new type of probe that consists of a dense oligonucleotide shell attached typically to a gold nanoparticle core. They are widely used as novel labels for in vitro biodetection and intracellular assay. However, the degradation of foreign DNA still remains a challenge that can cause significant signal leakage (false positive signal). Hence, the site and behavior of intracellular degradation need to be investigated. Herein, we discover a localized degradation behavior that only excises the first nucleotide of 5' terminal from a DNA strand, whereas the residual portion of this strand is unbroken in MCF-7 cell. This novel degradation action totally differs from previous opinion that foreign DNA strand would be digested into tiny fragments or even individual nucleotides in cellular environment. On the basis of these findings, we propose a simple and effective way to avoid degradation-caused false positive that one can bypass the degradable site and choose a secure region to label fluorophore along the DNA stand, when using DNA probes for intracellular biodetection.

Doubly Caged Linker for AND-Type Fluorogenic Construction of Protein/Antibody Bioconjugates and In Situ Quantification.

In situ quantification of the conjugation efficiency of azide-terminated synthetic polymers/imaging probes and thiol-functionalized antibodies/proteins/peptides was enabled by a doubly caged profluorescent and heterodifunctional core molecule C1 as a self-sorting bridging unit. Orthogonal dual "click" coupling of C1 with azide- and thiol-functionalized precursors led to highly fluorescent bioconjugates, whereas single-click products remained essentially nonfluorescent. Integration with FRET processes was also possible. For the construction of antibody-probe conjugates from an anti-carcinoembryonic antigen and a quinone-caged profluorescent naphthalimide derivative, the dual "click" coupling process with C1 was monitored on the basis of the emission turn-on of C1, whereas prominent changes in FRET ratios occurred for antibody-imaging-probe conjugates when specifically triggered by quinone oxidoreductase (NQO1), which is overexpressed in various types of cancer cells.

pH Dependence of Adsorbed Fibrinogen Conformation and Its Effect on Platelet Adhesion.

Quartz crystal microbalance with dissipation (QCM-D) and dual polarization interferometry (DPI) were used to investigate fibrinogen (Fib) adsorption behavior on different surfaces by changing the pH value. Moreover, integrin adhesion to the adsorbed Fibs was studied using DPI. Qualitative and quantitative studies of platelet adhesion to the adsorbed Fibs were performed using scanning electron microscopy (SEM), confocal laser scanning microscope (CLSM), and released lactate dehydrogenase (LDH) assay. Experimental results indicated that the conformation and orientation of the absorbed Fibs depended on surface property and pH cycling. For the hydrophilic surface, Fibs adsorbed at pH 7.4 and presented a αC-hidden orientation. As a result, no integrin adhesion was observed, and a small number of platelets were adhered because the αC-domains were hidden under the Fib molecule. By changing the rinsing solution pH from 7.4 to 3.2 and then back to 7.4, the adsorbed Fib orientation became αC-exposed via the transformation of Fib conformation during pH cycling. Therefore, integrin adhesion was more likely to occur, and more platelets were adhered and activated. For the hydrophobic surface, the adsorbed Fibs became more spread and stretched due to the strong interaction between the Fibs and surface. αC-exposed orientation remained unchanged when the rinsing solution pH changed from 7.4 to 3.2 and then back to 7.4. Therefore, a large number of integrins and platelets were adhered to the adsorbed Fibs, and almost all of the adhered platelets were activated.