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.

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.

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.

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.

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.

Controllable dynamics of complex DNA nanostructures.

In the past four decades, a variety of self-assembly design frameworks have led to the construction of versatile DNA nanostructures with increasing complexity and controllability. The controllable dynamics of DNA nanostructures has garnered much interest and emerged as a powerful tool for conducting sophisticated tasks at the molecular level. In this minireview, we summarized the controllable reconfigurations of complex DNA nanostructures induced by nucleic acid strands, environmental stimuli and enzymatic treatments. We also envisioned that with the optimization of response time, sensitivity and specificity, dynamic DNA nanostructures have great promise in applications ranging from nanorobotics to life sciences.

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.

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.

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.

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.

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.

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.

Monochromatic Fluorescent Barcodes Hierarchically Assembled from Modular DNA Origami Nanorods.

With the rapid advancement of fluorescence microscopy, there is a growing interest in the multiplexed detection and identification of various bioanalytes (e.g., nucleic acids and proteins) for efficient sample processing and analysis. We introduce in this work a simple and robust method to provide combinations for micrometer-scale fluorescent DNA barcodes of hierarchically assembled DNA origami superstructures for multiplexed molecular probing. In addition to optically resolvable dots, we placed fluorescent loci on adjacent origami within the diffraction limit of each other, rendering them as unresolvable bars of measurable lengths. We created a basic set of barcodes and trained a machine learning algorithm to process and identify individual barcodes from raw images with high accuracy. Moreover, we demonstrated that the number of combinations can be increased exponentially by generating longer barcodes, by controlling the number of incorporated fluorophores to create multiple levels of fluorescence intensity, and by employing super-resolution imaging. To showcase the readiness of the barcodes for applications, we used our barcodes to capture and identify target nucleic acid sequences and for simultaneous multiplexed characterization of binding kinetics of several orthogonal complementary nucleic acids.

DNA dynamics and computation based on toehold-free strand displacement.

We present a simple and effective scheme of a dynamic switch for DNA nanostructures. Under such a framework of toehold-free strand displacement, blocking strands at an excess amount are applied to displace the complementation of specific segments of paired duplexes. The functional mechanism of the scheme is illustrated by modelling the base pairing kinetics of competing strands on a target strand. Simulation reveals the unique properties of toehold-free strand displacement in equilibrium control, which can be leveraged for information processing. Based on the controllable dynamics in the binding of preformed DNA nanostructures, a multi-input-multi-output (MIMO) Boolean function is controlled by the presence of the blockers. In conclusion, we implement two MIMO Boolean functions (one with 4-bit input and 2-bit output, and the other with 16-bit input and 8-bit output) to showcase the controllable dynamics.

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.

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.

Addressable DNA nanotubes with repetitive components.

Extended DNA nanostructures have already been constructed in a repetitive arrangement from millions of building blocks, many more than currently feasible with even the gold standard of addressable self-assembled structures. In order to construct addressable DNA nanostructures with more building blocks, it is desirable to arrange the addressable components repetitively. Accordingly, the overall size of the structure can be multiplied by the level of repetition in the addressable strands. In this study, we present a nanotube system that combines two seemingly conflicting features: addressability and repetitiveness. Based on an understanding of the tubulation resulting from the intrinsic curvature of the components, we produce DNA nanotubes with addressability available along the axial direction of the self-assembled tubes, which are also programmably repetitive along the lateral direction.

DNA nanostructures from double-C-shaped motifs with controllable twist and curvature.

We demonstrate twist and curvature engineering in DNA nanostructures from the scaffold-free approach. The DNA 'LEGO' bricks adopted in this study are double-C-shaped motifs, and extended nanostructures are constructed to visualize the structural details of twist or curvature. By systematically deleting and inserting base pairs at certain domains of the component motifs, we are able to study various levels of the twist and curvature of the resulting nanostructures comprehensively.

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.

Tumor infiltrating lymphocytes from acute myeloid leukemia marrow can be reverted to CD45RA+ central memory state by reactivation in SIP (Simulated Infective Protocol).

Simulated Infective Protocol (SIP) is an ex-vivo culture system modeled after the temporal changes of essential cytokines in an acute infection, and previously proven successful in converting T lymphocytes harvested and activated from peripheral blood of normal donors, to revertant CD45RA + Central Memory T lymphocytes (Tcmra) demonstrating properties akin to T Memory Stem Cells (Tscm). In this study, we applied similar SIP on tumor infiltrating lymphocytes (TIL) from bone marrow of patients diagnosed with acute myeloid leukemia (AML), and replicated the feasibility to convert activated TILs into Tcmra phenotype. These revertant Tcmra lymphocytes re-expressed CD45RA+, CCR7+, CD62L + and CD127+, shown improved survivability with longer telomere length, expressed memory properties including higher Eomes to Tbet ratio, and exhibited cytotoxicity against autologous AML blast cells.