Expanding DNA Origami Design Freedom with De Novo Synthesized Scaffolds.

The construction of DNA origami nanostructures is heavily dependent on the folding of the scaffold strand, which is typically a single-stranded DNA genome extracted from a bacteriophage (M13). Custom scaffolds can be prepared in a number of methods, but they are not widely accessible to a broad user base in the DNA nanotechnology community. Here, we explored new design and construction possibilities with custom scaffolds prepared in our cost- and time-efficient production pipeline. According to the pipeline, we de novo produced a variety of scaffolds of specified local and global sequence characteristics and consequent origami constructs of modular arrangement in morphologies and functionalities. Taking advantage of this strategy of template-free scaffold production, we also designed and produced three-letter-coded scaffolds that can fold into designated morphologies rapidly at room temperature. The expanded design and construction freedom immediately brings in many new research opportunities and invites many more on the horizon.

DNA Self-Assembly Optimization by Betaine and Its Analogs.

The self-assembly yield of DNA nanostructures can be exponentially lower with increasing structural complexity. Few optimizing strategies are available in the DNA nanotechnology field for the assembly yield improvement. Here, betaine and its analogs are applied as supplementary ingredients in DNA self-assembly. Such a simple implementation results in effective yield improvement. Through a comprehensive investigation, a reliable yield improvement of two- to threefold is achieved for a number of DNA nanostructures with considerable complexity.

Controllable assembly of synthetic constructs with programmable ternary DNA interaction.

Compared with the dual binding components in a binary interaction, the third component of a ternary interaction often serves as modulator or regulator in biochemical processes. Here, we presented a programmable ternary interaction strategy based on the natural DNA triplex structure. With the DNA triplex-based ternary interaction, we have successfully demonstrated controllable hierarchical assemblies from nanometer scale synthetic DNA nanostructure units to micrometer scale live bacteria. A selective signaling system responsive to orthogonal nucleic acid signals via ternary interaction was also demonstrated. This assembly method could further enrich the diversified design schemes of DNA nanotechnology.

Reconfiguration of DNA nanostructures induced by enzymatic ligation treatment.

Enzymatic ligation is a popular method in DNA nanotechnology for structural enforcement. When employed as stability switch for chosen components, ligation can be applied to induce DNA nanostructure reconfiguration. In this study, we investigate the reinforcement effect of ligation on addressable DNA nanostructures assembled entirely from short synthetic strands as the basis of structural reconfiguration. A careful calibration of ligation efficiency is performed on structures with programmable nicks. Systematic investigation using comparative agarose gel electrophoresis enables quantitative assessment of enhanced survivability with ligation treatment on a number of unique structures. The solid ligation performance sets up the foundation for the ligation-based structural reconfiguration. With the capability of switching base pairing status between permanent and transient (ON and OFF) by a simple round of enzymatic treatment, ligation induced reconfiguration can be engineered for DNA nanostructures accordingly.

Enzymatic Assembly of DNA Nanostructures and Fragments with Sequence Overlaps.

Homologous recombination, an evolutionarily conserved DNA double-strand break repair pathway to protect genome stability, has long been exploited for the in vivo and in vitro assembly of multiple DNA duplex fragments in molecular cloning. Whether such methods can also be applied in the self-assembly of DNA nanostructures remains underexplored. Here, we report an enzymatic approach for the self-assembly of high-order DNA constructs with overlapping segments. In our system, a DNA polymerase with exonuclease activity was introduced to produce ssDNA overhangs for specific sticky end cohesion, and as many as 25 DNA structural units were designed to be hierarchically assembled. Using this approach, we successfully constructed a variety of high-order DNA nanostructures, including tubes and extended oligomers, from homogeneous assembly and custom multimers from heterogeneous assembly. Our strategy expands the construction toolbox of complex DNA nanostructures and highlights the potential to enhance the assembly of duplex fragments in molecular cloning.

Mesojunction-Based Design Paradigm of Structural DNA Nanotechnology.

Mesojunctions were introduced as a basic type of crossover configuration in the early development of structural DNA nanotechnology. However, the investigations of self-assembly from multiple mesojunction complexes have been overlooked in comparison to their counterparts based on regular junctions. In this work, we designed standardized component strands for the construction of complex mesojunction lattices. Three typical mesojunction configurations with three and four arms were showcased in the self-assembly of 1-, 2-, and 3-dimensional lattices constructed from both a scaffold-free tiling approach and a scaffolded origami approach.

What does the anesthesiologist need to know about monkeypox?

PURPOSE: Monkeypox (or "mpox" as preferred by the World Health Organization) is an emerging infectious disease with sustained global transmission occurring outside of West Africa and the Democratic Republic of Congo. The recent 2022 mpox outbreak has involved widespread atypical presentations. Infected patients requiring surgery can increase the exposure of health care professionals and other patients to the virus. As it is a relatively new infectious disease internationally, there is less familiarity in managing this risk, especially in the surgical and anesthesia setting. This paper aims to provide information about mpox and how to manage suspected or confirmed cases. SOURCE: Various authorities such as the World Health Organization, Infection Prevention and Control Canada, Public Health Agency of Canada, the Centers for Disease Control and Prevention (USA), and the National Centre for Infectious Diseases (Singapore) have recommended that public health and hospital systems prepare to recognize, isolate, and care for suspected and confirmed cases appropriately, as well as manage any possible exposure of staff and patients. PRINCIPAL FINDINGS: Local authorities and hospitals should set up protocols for health care providers (HCPs) to minimize nosocomial transmission and risk to HCPs. Antivirals used in patients with more severe disease may cause renal or hepatic impairment and thus anesthetic drug pharmacology. Anesthesiologists and surgeons should be able to recognize mpox, and work with local infection control and epidemiologic programs to familiarize themselves with relevant infection prevention guidelines. CONCLUSION: Essential measures include clear protocols for transferring and managing surgical patients who are suspected or confirmed to be infected with the virus. Care in use of personal protective equipment and handling contaminated material is necessary to prevent inadvertent exposure. Risk stratification after exposure should be done to determine need for post-exposure prophylaxis for staff.

RéSUMé: OBJECTIF: La variole du singe (ou « mpox ¼, le terme privilégié en anglais par l'Organisation mondiale de la santé) est une maladie infectieuse émergente dont la transmission mondiale est soutenue en dehors de l'Afrique de l'Ouest et de la République démocratique du Congo. La récente épidémie de variole du singe de 2022 a donné lieu à des présentations atypiques généralisées. Les patients infectés nécessitant une intervention chirurgicale peuvent accroître l'exposition des professionnels de la santé et des autres patients au virus. Comme il s'agit d'une maladie infectieuse relativement nouvelle à l'échelle internationale, la gestion de ce risque d'exposition est moins familière, en particulier dans le cadre chirurgical et anesthésique. Cet article vise à fournir des informations sur la variole du singe et sur la prise en charge des cas suspects ou confirmés. SOURCES: Diverses autorités telles que l'Organisation mondiale de la Santé, Prévention et contrôle des infections Canada, l'Agence de la santé publique du Canada, les Centers for Disease Control and Prevention (États-Unis) et le National Centre for Infectious Diseases (Singapour) ont recommandé que les systèmes de santé publique et hospitaliers se préparent à reconnaître, isoler et soigner les cas suspects et confirmés de manière appropriée, ainsi qu'à gérer toute exposition possible du personnel et des patients. CONSTATATIONS PRINCIPALES: Les autorités locales et les hôpitaux devraient établir des protocoles pour les fournisseurs de soins de santé afin de minimiser la transmission nosocomiale et les risques pour eux. Les antiviraux utilisés chez les patients atteints d'une forme plus grave de la maladie peuvent entraîner une insuffisance rénale ou hépatique et, par conséquent, une altération de la pharmacologie anesthésique. Les anesthésiologistes et les chirurgiens devraient être en mesure de reconnaître la variole du singe et de travailler avec les programmes locaux de contrôle des infections et d'épidémiologie pour se familiariser avec les lignes directrices pertinentes en matière de prévention des infections. CONCLUSION: Les mesures essentielles comprennent des protocoles clairs pour le transfert et la prise en charge des patients chirurgicaux soupçonnés ou confirmés d'être infectés par le virus. Il faut faire preuve de prudence dans l'utilisation des équipements de protection individuelle et la manipulation des matières contaminées afin de prévenir une exposition accidentelle. La stratification du risque après l'exposition devrait être réalisée afin de déterminer la nécessité d'une prophylaxie post-exposition pour le personnel.

Design of Orthogonal DNA Sticky-End Cohesion Based on Configuration-Specific Molecular Recognition.

In sticky-end cohesion, sequence complementarity is the key to design specific recognition among many DNA nanostructure units. The binding orthogonality usually arises from sticky-end pairs of different sequences. Instead of creating orthogonal species of sticky-end bonds based on sequence complementarity, we restricted the sticky-end sequence diversity down to the fixed C-G pair and explored orthogonal recognition of the synthetic DNA constructs based solely on the configurational match. From our comprehensive investigations of 2D tessellation and 3D crystallization, we demonstrated the new configuration-specific sticky-end cohesion to program specific and precise molecular recognition of synthetic structural units for high-order DNA self-assembly.

Allostery of DNA nanostructures controlled by enzymatic modifications.

Allostery is comprehensively studied for natural macromolecules, such as proteins and nucleic acids. Here, we present controllable allostery of synthetic DNA nanostructure-enzyme systems. Rational designs of the synthetic allosteric systems are based on an in-depth understanding of allosteric sites with several types of strand placements, whose varying stacking strengths determine the local conformation and ultimately lead to a gradient level of allosteric transition. When enzymes in a molecular cloning toolbox such as DNA polymerase, exonuclease and ligase are applied to treat the allosteric sites, the resulting local conformational changes propagate through the entire structure for a global allosteric transition.

Rational Design of Allosteric Nanodevices Based on DNA Triple Helix.

Inspired by allosteric regulation of natural molecules, we present a rational design scheme to build synthetic nucleic acid allosteric nanodevices. The clearly specified conformational states of switches obtained from systematic screening and analyses make the ON-OFF transition clear-cut and quantification ready. Under the rational design scheme, we have developed a series of DNA switches with triplex-forming oligos as allosteric modulators and implemented designated allosteric transitions, allosteric coregulation, and reaction pathway control. In conjunction with toehold-mediated strand displacement, our design scheme has also been applied to synthetic nucleic acid computing including a set of logic operations and complex algorithm.

Hybrid Wireframe DNA Nanostructures with Scaffolded and Scaffold-Free Modules.

The scaffolded DNA origami approach and the scaffold-free LEGO approach both demonstrate an extraordinary self-assembly capability for constructing all kinds of complex DNA nanostructures. Combining the construction elements of the two approaches, we introduce a hybrid framework to build wireframe structures in this study. A collection of two-dimensional (2D) and three-dimensional (3D) wireframe structures are presented to showcase the simple and versatile design. Our development reveals more of the ever-expanding design space of structural DNA nanotechnology.

Reconfigurable Two-Dimensional DNA Lattices: Static and Dynamic Angle Control.

Branched DNA motifs serve as the basic construction elements for all synthetic DNA nanostructures. However, precise control of branching orientation remains a key challenge to further heighten the overall structural order. In this study, we use two strategies to control the branching orientation. The first one is based on immobile Holliday junctions which employ specific nucleotide sequences at the branch points which dictate their orientation. The second strategy is to use angle-enforcing struts to fix the branching orientation with flexible spacers at the branch points. We have also demonstrated that the branching orientation control can be achieved dynamically, either by canonical Watson-Crick base pairing or non-canonical nucleobase interactions (e.g., i-motif and G-quadruplex). With precise angle control and feedback from the chemical environment, these results will enable novel DNA nanomechanical sensing devices, and precisely-ordered three-dimensional architectures.

DNA nanostructures constructed with multi-stranded motifs.

Earlier studies in DNA self-assembly have foretold the feasibility of building addressable nanostructures with multi-stranded motifs, which is fully validated in this study. In realizing this feasibility in DNA nanotechnology, a diversified set of motifs of modified domain lengths is extended from a classic type. The length of sticky ends can be adjusted to form different dihedral angles between the matching motifs, which corresponds to different connecting patterns. Moreover, the length of rigidity core can also be tuned to result in different dihedral angles between the component helices of a certain motif therefore different numbers of component helices. The extended set of motifs is used for self-assembly of complex one dimensional, two dimensional and three dimensional structures.

Hierarchical Assembly of DNA Nanostructures Based on Four-Way Toehold-Mediated Strand Displacement.

Because of its attractive cost and yield, hierarchical assembly, in which constituent structures of lower hierarchy share a majority of components, is an appealing approach to scale up DNA self-assembly. A few strategies have already been investigated to combine preformed DNA nanostructures. In this study, we present a new hierarchical assembly method based on four-way toehold-mediated strand displacement to facilitate the combination of preformed DNA structural units. Employing such a method, we have constructed a series of higher-order structures composed of 5, 7, 9, 11, 13, and 15 preformed units respectively.

Self-Assembly of Wireframe DNA Nanostructures from Junction Motifs.

Wireframe frameworks have been investigated for the construction of complex nanostructures from a scaffolded DNA origami approach; however, a similar framework is yet to be fully explored in a scaffold-free "LEGO" approach. Herein, we describe a general design scheme to construct wireframe DNA nanostructures entirely from short synthetic strands. A typical edge of the resulting structures in this study is composed of two parallel duplexes with crossovers on both ends, and three, four, or five edges radiate out from a certain vertex. By using such a self-assembly scheme, we produced planar lattices and polyhedral objects.

Complex shapes self-assembled from single-stranded DNA tiles.

Programmed self-assembly of strands of nucleic acid has proved highly effective for creating a wide range of structures with desired shapes. A particularly successful implementation is DNA origami, in which a long scaffold strand is folded by hundreds of short auxiliary strands into a complex shape. Modular strategies are in principle simpler and more versatile and have been used to assemble DNA or RNA tiles into periodic and algorithmic two-dimensional lattices, extended ribbons and tubes, three-dimensional crystals, polyhedra and simple finite two-dimensional shapes. But creating finite yet complex shapes from a large number of uniquely addressable tiles remains challenging. Here we solve this problem with the simplest tile form, a 'single-stranded tile' (SST) that consists of a 42-base strand of DNA composed entirely of concatenated sticky ends and that binds to four local neighbours during self-assembly. Although ribbons and tubes with controlled circumferences have been created using the SST approach, we extend it to assemble complex two-dimensional shapes and tubes from hundreds (in some cases more than one thousand) distinct tiles. Our main design feature is a self-assembled rectangle that serves as a molecular canvas, with each of its constituent SST strands--folded into a 3 nm-by-7 nm tile and attached to four neighbouring tiles--acting as a pixel. A desired shape, drawn on the canvas, is then produced by one-pot annealing of all those strands that correspond to pixels covered by the target shape; the remaining strands are excluded. We implement the strategy with a master strand collection that corresponds to a 310-pixel canvas, and then use appropriate strand subsets to construct 107 distinct and complex two-dimensional shapes, thereby establishing SST assembly as a simple, modular and robust framework for constructing nanostructures with prescribed shapes from short synthetic DNA strands.

Self-assembly of fully addressable DNA nanostructures from double crossover tiles.

DNA origami and single-stranded tile (SST) are two proven approaches to self-assemble finite-size complex DNA nanostructures. The construction elements appeared in structures from these two methods can also be found in multi-stranded DNA tiles such as double crossover tiles. Here we report the design and observation of four types of finite-size lattices with four different double crossover tiles, respectively, which, we believe, in terms of both complexity and robustness, will be rival to DNA origami and SST structures.

A predictive nomogram to identify factors influencing the success of a concomitant laparoscopic cholecystectomy with common bile duct exploration for choledocholithiasis.

BACKGROUND: Single-staged laparoscopic common bile duct exploration (LCBDE) offers clear benefits in terms of cost and shorter hospitalization stays. However, a failed LCBDE requiring conversion to open surgery is associated with increased morbidity. This study reviewed the factors determining success of LCBDE, and created a predictive nomogram to stratify patients for the procedure. METHODS: A retrospective analysis of 109 patients who underwent LCBDE was performed. A nomogram was developed from factors significantly associated with conversion to open surgery and validated. RESULTS: Sixty-two patients underwent a successful LCBDE, while 47 patients required a conversion to open CBDE. The presence of underlying cholangitis (crude OR 2.70, 95% CI: 1.12-6.56, p = 0.017), together with its subsequent interventions, seemed to adversely increase the rate of conversion to open surgery. The predictive factors included in the nomogram for a failed laparoscopic CBDE included prior antibiotic use (adjusted OR (AOR) 2.98, 95% CI: 1.17-7.57, p = 0.022), previous ERCP (AOR 4.99, 95% CI: 2.02-12.36, p = 0.001) and abnormal biliary anatomy (AOR 9.37, 95% CI: 2.18-40.20, p = 0.003). CONCLUSION: LCBDE is useful for the treatment of choledocholithiasis. However, patients who were predicted to have an elevated risk for open conversion might not be ideal candidates for the procedure.

Complex reconfiguration of DNA nanostructures.

Nucleic acids have been used to create diverse synthetic structural and dynamic systems. Toehold-mediated strand displacement has enabled the construction of sophisticated circuits, motors, and molecular computers. Yet it remains challenging to demonstrate complex structural reconfiguration in which a structure changes from a starting shape to another arbitrarily prescribed shape. To address this challenge, we have developed a general structural-reconfiguration method that utilizes the modularly interconnected architecture of single-stranded DNA tile and brick structures. The removal of one component strand reveals a newly exposed toehold on a neighboring strand, thus enabling us to remove regions of connected component strands without the need to modify the strands with predesigned external toeholds. By using this method, we reconfigured a two-dimensional rectangular DNA canvas into diverse prescribed shapes. We also used this method to reconfigure a three-dimensional DNA cuboid.

Synthetic molecular switches driven by DNA-modifying enzymes.

Taking inspiration from natural systems, in which molecular switches are ubiquitous in the biochemistry regulatory network, we aim to design and construct synthetic molecular switches driven by DNA-modifying enzymes, such as DNA polymerase and nicking endonuclease. The enzymatic treatments on our synthetic DNA constructs controllably switch ON or OFF the sticky end cohesion and in turn cascade to the structural association or disassociation. Here we showcase the concept in multiple DNA nanostructure systems with robust assembly/disassembly performance. The switch mechanisms are first illustrated in minimalist systems with a few DNA strands. Then the ON/OFF switches are realized in complex DNA lattice and origami systems with designated morphological changes responsive to the specific enzymatic treatments.