Cryo-EM structures and dynamics of substrate-engaged human 26S proteasome.

The proteasome is an ATP-dependent, 2.5-megadalton molecular machine that is responsible for selective protein degradation in eukaryotic cells. Here we present cryo-electron microscopy structures of the substrate-engaged human proteasome in seven conformational states at 2.8-3.6 Å resolution, captured during breakdown of a polyubiquitylated protein. These structures illuminate a spatiotemporal continuum of dynamic substrate-proteasome interactions from ubiquitin recognition to substrate translocation, during which ATP hydrolysis sequentially navigates through all six ATPases. There are three principal modes of coordinated hydrolysis, featuring hydrolytic events in two oppositely positioned ATPases, in two adjacent ATPases and in one ATPase at a time. These hydrolytic modes regulate deubiquitylation, initiation of translocation and processive unfolding of substrates, respectively. Hydrolysis of ATP powers a hinge-like motion in each ATPase that regulates its substrate interaction. Synchronization of ATP binding, ADP release and ATP hydrolysis in three adjacent ATPases drives rigid-body rotations of substrate-bound ATPases that are propagated unidirectionally in the ATPase ring and unfold the substrate.

Conformational Landscape of the p28-Bound Human Proteasome Regulatory Particle.

The proteasome holoenzyme is activated by its regulatory particle (RP) consisting of two subcomplexes, the lid and the base. A key event in base assembly is the formation of a heterohexameric ring of AAA-ATPases, which is guided by at least four RP assembly chaperones in mammals: PAAF1, p28/gankyrin, p27/PSMD9, and S5b. Using cryogenic electron microscopy, we analyzed the non-AAA structure of the p28-bound human RP at 4.5 Å resolution and determined seven distinct conformations of the Rpn1-p28-AAA subcomplex within the p28-bound RP at subnanometer resolutions. Remarkably, the p28-bound AAA ring does not form a channel in the free RP and spontaneously samples multiple "open" and "closed" topologies at the Rpt2-Rpt6 and Rpt3-Rpt4 interfaces. Our analysis suggests that p28 assists the proteolytic core particle to select a specific conformation of the ATPase ring for RP engagement and is released in a shoehorn-like fashion in the last step of the chaperone-mediated proteasome assembly.

Constructing Stiffness Tunable DNA Hydrogels Based on DNA Modules with Adjustable Rigidity.

DNA hydrogel represents a potent material for crafting biological scaffolds, but the toolbox to systematically regulate the mechanical property is still limited. Herein, we have provided a strategy to tune the stiffness of DNA hydrogel through manipulating the rigidity of DNA modules. By introducing building blocks with higher molecular rigidity and proper connecting fashion, DNA hydrogel stiffness could be systematically elevated. These hydrogels showed excellent dynamic properties and biocompatibility, thus exhibiting great potential in three-dimensional (3D) cell culture. This study has offered a systematic method to explore the structure-property relationship, which may contribute to the development of more intelligent and personalized biomedical platforms.

Advanced Smart Biomaterials for Regenerative Medicine and Drug Delivery Based on Phosphoramidite Chemistry: From Oligonucleotides to Precision Polymers.

Over decades of development, while phosphoramidite chemistry has been known as the leading method in commercial synthesis of oligonucleotides, it has also revolutionized the fabrication of sequence-defined polymers (SDPs), offering novel functional materials in polymer science and clinical medicine. This review has introduced the evolution of phosphoramidite chemistry, emphasizing its development from the synthesis of oligonucleotides to the creation of universal SDPs, which have unlocked the potential for designing programmable smart biomaterials with applications in diverse areas including data storage, regenerative medicine and drug delivery. The key methodologies, functions, biomedical applications, and future challenges in SDPs, have also been summarized in this review, underscoring the significance of breakthroughs in precisely synthesized materials.

Gradually Self-Strengthen DNA Supramolecular Hydrogels.

The dynamic mechanical strength of the extracellular matrix (ECM) has been demonstrated to play important role in determining the cell behavior. Growing evidences suggest that the gradual stiffening process of the matrix is particularly decisive during tissue development and wound healing. Herein, a novel strategy to prepare hydrogels with gradually enhanced mechanical strength is provided. Such hydrogels could maintain the dynamic properties at their initial states, such as self-healing and shear-thinning properties. With subsequent slow covalent crosslinking, the stability and mechanical properties would be gradually improved. This method is useful for sequence programmability and oxidation strategies, which has provided an alternated tool to study cell behavior during dynamic increase in mechanical strength of ECM.

Effects of Univariate Stiffness and Degradation of DNA Hydrogels on the Transcriptomics of Neural Progenitor Cells.

Mechanical interactions between cells and extracellular matrix (ECM) are critical for stem cell fate decision. Synthetic models of ECM, such as hydrogels, can be used to precisely manipulate the mechanical properties of the cell niche and investigate how mechanical signals regulate the cell behavior. However, it has long been a great challenge to tune solely the ECM-mimic hydrogels' mechanical signals since altering the mechanical properties of most materials is usually accompanied by chemical and topological changes. Here, we employ DNA and its enantiomers to prepare a series of hydrogels with univariate stiffness regulation, which enables a precise interpretation of the fate decision of neural progenitor cells (NPCs) in a three-dimensional environment. Using single-cell RNA sequencing techniques, Monocle pseudotime trajectory and CellphoneDB analysis, we demonstrate that the stiffness of the hydrogel alone does not influence the differentiation of NPCs, but the degradation of the hydrogel that enhances cell-cell interactions is possibly the main reason. We also find that ECM remodeling facilitates cells to sense mechanical stimuli.

Frame-Guided Assembly of Amphiphiles.

Amphiphiles tend to self-assemble into various structures and morphologies in aqueous environments (e.g., micelles, tubes, fibers, vesicles, and lamellae). These assemblies and their properties have made significant impact in traditional chemical industries, e.g., increasing solubility, decreasing surface tension, facilitating foaming, etc. It is well-known that the molecular structure and its environment play a critical role in the assembly process, and many theories, including critical packing factor, thermodynamic models, etc., have been proposed to explain and predict the assembly morphology. It has been recognized that the morphology of the amphiphilic assembly plays important roles in determining the functions, such as curvature-dependent biophysical (e.g., liposome fusion and fission) and biochemical (e.g., lipid metabolism and membrane protein trafficking) processes, size-related EPR (enhanced permeability and retention) effects, etc. Meanwhile, various nanomaterials have promised great potential in directing the arrangement of molecules, thus generating unique functions. Therefore, control over the amphiphilic morphology is of great interest to scientists, especially in nanoscale with the assistance of functional nanomaterials. However, how to precisely manipulate the sizes and shapes of the assemblies is challenged by the entropic nature of the hydrophobic interaction. Inspired by the "cytoskeleton-membrane protein-lipid bilayer" principle of the cell membrane, a strategy termed "frame-guided assembly (FGA)" has been proposed and developed to direct the arrangement of amphiphiles. The FGA strategy welcomes various nanomaterials with precisely controlled properties to serve as scaffolds. By introducing scattered hydrophobic molecules, which are defined as either leading hydrophobic groups (LHGs) or nucleation seeds onto a selected scaffold, a discontinuous hydrophobic trace along the scaffold can be outlined, which will further guide the amphiphiles in the system to grow and form customized two- or three-dimensional (2D/3D) membrane geometries.Topologically, the supporting frame can be classified as three types including inner-frame, outer-frame, and planar-frame. Each type of FGA assembly possesses particular advantages: (1) The inner-frame, similar to endoskeletons of many cellular structures, steadily supports the membrane from the inside and exposes the full surface area outside. (2) The outer-frame, on the other hand, molds and constrains the membrane-wrapped vesicles to regulate their size and shape. It also allows postengineering of the frame to precisely decorate and dynamically manipulate the membrane. (3) The planar-frame mediates the growth of the 2D membrane that profits from the scanning-probe microscopic characterization and benefits the investigation of membrane proteins.In this Account, we introduce the recent progress of frame-guided assembly strategy in the preparation of customized amphiphile assemblies, evaluate their achievements and limitations, and discuss prospective developments and applications. The basic principle of FGA is discussed, and the morphology controllability is summarized in the inner-, outer-, and planar-frame categories. As a versatile strategy, FGA is able to guide different types of amphiphiles by designing specific LHGs for given molecular structures. The mechanism of FGA is then discussed systematically, including the driving force of the assembly, density and distribution of the LHGs, amphiphile concentration, and the kinetic process. Furthermore, the applications of FGA have been developed for liposome engineering, membrane protein incorporation, and drug delivery, which suggest the huge potential of FGA in fabricating novel and functional complexes.

Single-Molecular Poly(propylene oxide) (PPO) Nucleus-Guided Assembly for Hydrophobicity-Dependent Molecular Transport in the Nanopore.

By combining DNA nanotechnology and solid-phase nanopore technology, the aggregation behavior of polymer guided by a single-molecular poly(propylene) (PPO) nucleus in a 3D DNA network has been studied. At low temperature, the PPO chain is evenly dispersed in the rigid 3D DNA network; at higher temperature, the PPO chain self-collapses to a single-molecular nucleus; and upon addition of amphiphilic block copolymers below the critical micelle concentration (CMC), the chains tend to aggregate on the isolated hydrophobic nucleus through intermolecular hydrophobic interactions. The process has been characterized by a rheological test and an electrochemical test. This study not only provides a preliminary understanding of the nucleation and growth process of block copolymers but also offers a theoretical basis for the study of protein self-folding and aggregation in the future. On this basis, utilizing this nucleation and growth event, a novel smart nanopore has been developed for hydrophobicity-dependent molecular transport.

DNA Materials Assembled from One DNA Strand.

Due to the specific base-pairing recognition, clear nanostructure, programmable sequence and responsiveness of the DNA molecule, DNA materials have attracted extensive attention and been widely used in controlled release, drug delivery and tissue engineering. Generally, the strategies for preparing DNA materials are based on the assembly of multiple DNA strands. The construction of DNA materials using only one DNA strand can not only save time and cost, but also avoid defects in final assemblies generated by the inaccuracy of DNA ratios, which potentially promote the large-scale production and practical application of DNA materials. In order to use one DNA strand to form assemblies, the sequences have to be palindromes with lengths that need to be controlled carefully. In this review, we introduced the development of DNA assembly and mainly summarized current reported materials formed by one DNA strand. We also discussed the principle for the construction of DNA materials using one DNA strand.

Genetically Encoded Double-Stranded DNA-Based Nanostructure Folded by a Covalently Bivalent CRISPR/dCas System.

DNA nanotechnology has been widely employed in the construction of various functional nanostructures. However, most DNA nanostructures rely on hybridization between multiple single-stranded DNAs. Herein, we report a general strategy for the construction of a double-stranded DNA-ribonucleoprotein (RNP) hybrid nanostructure by folding double-stranded DNA with a covalently bivalent clustered regularly interspaced short palindromic repeats (CRISPR)/nuclease-dead CRISPR-associated protein (dCas) system. In our design, dCas9 and dCas12a can be efficiently fused together through a flexible and stimuli-responsive peptide linker. After activation by guide RNAs, the covalently bivalent dCas9-12a RNPs (staples) can precisely recognize their target sequences in the double-stranded DNA scaffold and pull them together to construct a series of double-stranded DNA-RNP hybrid nanostructures. The genetically encoded hybrid nanostructure can protect genetic information in the folded state, similar to the natural DNA-protein hybrids present in chromosomes, and elicit efficient stimuli-responsive gene transcription in the unfolded form. This rationally developed double-stranded DNA folding and unfolding strategy presents a new avenue for the development of DNA nanotechnology.

"Shutter" Effects Enhance Protein Diffusion in Dynamic and Rigid Molecular Networks.

Hydrogels have been widely applied to understand the fundamental functions and mechanism of a natural extracellular matrix (ECM). However, revealing the high permeability of ECM through synthetic hydrogels is still challenged by constructing analogue networks with rigid and dynamic properties. Here, in this study, taking advantage of the rigidity and dynamic binding of DNA building blocks, we have designed a model hydrogel system with structural similarity to ECM, leading to enhanced diffusion for proteins compared with a synthetic polyacrylamide (PAAm) hydrogel. The molecular diffusion behaviors in such a rigid and dynamic network have been investigated both in experiments and simulations, and the dependence of diffusion coefficients with respect to molecular size exhibits a unique transition from a power law to an exponential function. A "shutter" model based on the rigid and dynamic molecular network has been proposed, which has successfully revealed how the rigidity and dynamic bond exchange determine the diffusion mechanism, potentially providing a novel perspective to understand the possible mechanism of enhanced diffusion behaviors in ECM.

Studies on the Synergistic Effect of Tandem Semi-Stable Complementary Domains on Sequence-Defined DNA Block Copolymers.

Tandem semi-stable complementary domains play an important role in life, while the role of these domains in the folding process of nucleic acid molecules has not been systematically studied. Here, we designed a clean model system by synthesizing sequence-defined DNA-OEG copolymers composed of ssDNA fragments with palindromic sequences and orthogonal oligo(tetraethylene glycol) (OEG) linkers. By altering the lengths of DNA units (6-12 nt) and OEG linkers (Xn = 0-4) separately, we systematically studied how stabilities of tandem complementary domains and connecting flexibilities affect the assembly topology. Combining experimental methods and coarse-grained molecular simulation analysis, distributions of multiple assembled conformations (mainly monomers, dimers, and clusters) were characterized. Both results indicated that tandem semi-stable complementary domains tend to form homogeneous closed circular dimers instead of larger clusters due to the synergistic enhancement effect, and the distributions of each conformation highly depend on flexibilities.

A Biostable l-DNA Hydrogel with Improved Stability for Biomedical Applications.

DNA hydrogels have attracted increasing attention owing to their excellent permeability and high mechanical strength, together with thixotropy, versatile programmability and good biocompatibility. However, the moderate biostability and immune stimulation of DNA have arisen as big concerns for future potential clinical applications. Herein, we report the self-assembly of a novel l-DNA hydrogel, which inherited the extraordinary physical properties of a d-DNA hydrogel. With the mirror-isomer deoxyribose, this hydrogel exhibited improved biostability, withstanding fetal bovine serum (FBS) for at least 1 month without evident decay of its mechanical properties. The low inflammatory response of the l-DNA hydrogel has been verified both in vitro and in vivo. Hence, this l-DNA hydrogel with outstanding biostability and biocompatibility can be anticipated to serve as an ideal 3D cell-culture matrix and implanted bio-scaffold for long-term biomedical applications.

Kinetically Interlocking Multiple-Units Polymerization of DNA Double Crossover and Its Application in Hydrogel Formation.

A novel kinetically interlocking multiple-units (KIMU) supramolecular polymerization system with DNA double crossover backbone is designed. The rigidity of DX endows the polymer with high molecular weight and stability. The observed concentration of the formed polymers is insensitive and stable under ultralow monomer concentration owing to the KIMU interactions, in which multiple noncovalent interactions are connected by the phosphodiester bonds. Furthermore, a pH-responsive DNA supramolecular hydrogel is constructed by introducing a half i-motif domain into the DNA monomer. The rigidity of DNA polymer endows the hydrogel with high mechanical strength and low gelation concentration. This study enriches the KIMU strategy and offers a simple but effective way to fabricate long and stable supramolecular polymers by balancing the reversibility and stability. It also shows great potentials to construct next generation of smart materials, such as DNA nanostructures, DNA motors, and DNA hydrogels.

Construction and Characterization of a Mirror-Image l-DNA i-Motif.

The first thermally stable and pH-responsive quadruplex intercalated motif (i-motif) structure formed by l-DNA is presented. Although this l-type i-motif exhibits the same physiochemical properties as its d isomer, its inverted chirality and good enzymatic resistance potentially open the way to the development of new DNA materials of pharmaceutical and biological interest.

Cryo-EM structure of the DNA-PK holoenzyme.

DNA-dependent protein kinase (DNA-PK) is a large protein complex central to the nonhomologous end joining (NHEJ) DNA-repair pathway. It comprises the DNA-PK catalytic subunit (DNA-PKcs) and the heterodimer of DNA-binding proteins Ku70 and Ku80. Here, we report the cryo-electron microscopy (cryo-EM) structures of human DNA-PKcs at 4.4-Å resolution and the DNA-PK holoenzyme at 5.8-Å resolution. The DNA-PKcs structure contains three distinct segments: the N-terminal region with an arm and a bridge, the circular cradle, and the head that includes the kinase domain. Two perpendicular apertures exist in the structure, which are sufficiently large for the passage of dsDNA. The DNA-PK holoenzyme cryo-EM map reveals density for the C-terminal globular domain of Ku80 that interacts with the arm of DNA-PKcs. The Ku80-binding site is adjacent to the previously identified density for the DNA-binding region of the Ku70/Ku80 complex, suggesting concerted DNA interaction by DNA-PKcs and the Ku complex.

Construction of Liposomes Mimicking Cell Membrane Structure through Frame-Guided Assembly.

The shape of eukaryotic cells is determined by the cytoskeleton associated with membrane proteins; however, the detailed mechanism of how the integral morphologies with structural stability is generated and maintained is still not fully understood. Here, based on the Frame-Guided Assembly (FGA) strategy, we successfully prepared hetero-liposomes with structural composition similar to that of eukaryotic cells by screening a series of transmembrane peptides as the leading hydrophobic groups (LHGs). It was demonstrated that the conformation and transmembrane mode of the LHGs played dominant roles during the FGA process. The FGA liposomes were formed with excellent stability, which may further provide evidence for the cytoskeleton-membrane protein-lipid bilayer model. Taking advantage of the biocompatibility and stability, the FGA liposomes were also applied to prepare novel drug delivery vehicles, which is promising in diagnostic imaging and cancer therapy applications.

DNA Origami as Scaffolds for Self-Assembly of Lipids and Proteins.

Since first being reported in 2006, the DNA origami approach has attracted increasing attention due to programmable shapes, structural stability, biocompatibility, and fantastic addressability. Herein, we provide an account of recent developments of DNA origami as scaffolds for templating the selfassembly of distinct biocomponents, essentially proteins and lipids, into a diverse spectrum of integrated supramolecular architectures. First, the historical development of the DNA origami concept is briefly reviewed. Next, various applications of DNA origami constructs in controllable directed assembly of soluble proteins are discussed. The manipulation and self-assembly of lipid membranes and membrane proteins by using DNA origami as scaffolds are also addressed. Furthermore, recent progress in applying DNA origami in cryoelectron microscopy analysis is discussed. These advances collectively emphasize that the DNA origami approach is a highly versatile, fast evolving tool that may be integrated with lipids and proteins in a way that meets future challenges in molecular biology and nanomedicine.

Tuning the Mechanical Properties of a DNA Hydrogel in Three Phases Based on ATP Aptamer.

By integrating ATP aptamer into the linker DNA, a novel DNA hydrogel was designed, with mechanical properties that could be tuned into three phases. Based on the unique interaction between ATP and its aptamer, the mechanical strength of the hydrogel increased from 204 Pa to 380 Pa after adding ATP. Furthermore, with the addition of the complementary sequence to the ATP aptamer, the mechanical strength could be increased to 570 Pa.

Folding DNA into a Lipid-Conjugated Nanobarrel for Controlled Reconstitution of Membrane Proteins.

Building upon DNA origami technology, we introduce a method to reconstitute a single membrane protein into a self-assembled DNA nanobarrel that scaffolds a nanodisc-like lipid environment. Compared with the membrane-scaffolding-protein nanodisc technique, our approach gives rise to defined stoichiometry, controlled sizes, as well as enhanced stability and homogeneity in membrane protein reconstitution. We further demonstrate potential applications of the DNA nanobarrels in the structural analysis of membrane proteins.