Near Quantitative Ligation Results in Resistance of DNA Origami Against Nuclease and Cell Lysate.

There have been limited efforts to ligate the staple nicks in DNA origami which is crucial for their stability against thermal and mechanical treatments, and chemical and biological environments. Here, two near quantitative ligation methods are demonstrated for the native backbone linkage at the nicks in origami: i) a cosolvent dimethyl sulfoxide (DMSO)-assisted enzymatic ligation and ii) enzyme-free chemical ligation by CNBr. Both methods achieved over 90% ligation in 2D origami, only CNBr-method resulted in ≈80% ligation in 3D origami, while the enzyme-alone yielded 31-55% (2D) or 22-36% (3D) ligation. Only CNBr-method worked efficiently for 3D origami. The CNBr-mediated reaction is completed within 5 min, while DMSO-method took overnight. Ligation by these methods improved the structural stability up to 30 °C, stability during the electrophoresis and subsequent extraction, and against nuclease and cell lysate. These methods are straightforward, non-tedious, and superior in terms of cost, reaction time, and efficiency.

An Artificial Liposome Compartment with Size Exclusion Molecular Transport.

The cellular compartment plays an essential role in organizing the complex and diverse biochemical reactions within the cell. By mimicking the function of such cellular compartments, the challenge of constructing artificial compartments has been taken up to develop new biochemical tools for efficient material production and diagnostics. The important features required for the artificial compartment are that it isolates the interior from the external environment and is further functionalized to control the transport of target chemicals to regulate the interior concentration of both substrate and reaction products. In this study, an artificial compartment with size-selective molecular transport function was constructed by using a DNA origami-guided liposome prepared by modifying the method reported by Perrault et al. This completely isolates the liposome interior, including the DNA origami skeleton, from the external environment and allows the assembly of a defined number of molecules of interest inside and/or outside the compartment. By incorporating a bacterial membrane protein, OmpF, into the liposome, the resulting artificial compartment was shown to transport only the molecule of interest with a molecular weight below 600 Da from the external environment into the interior of the compartment.

Macropinoscope: Real-Time Simultaneous Tracking of pH and Cathepsin B Activity in Individual Macropinosomes.

A fluorescent sensor that allows simultaneous analysis of environmental factors in a limited cellular space is useful for understanding precise molecular interactions in live cells and their biological responses. Macropinocytosis is a ubiquitous endocytic pathway for massive uptake of extracellular fluids, resulting in the formation of macropinosomes. Although macropinocytosis may impact intracellular delivery and cancer proliferation, information on the intracellular behaviors of macropinosomes is limited. Here, we aimed to develop a macropinoscope, a sensor that simultaneously detects pH and cathepsin B activity in individual macropinosomes. A macropinosome-specific marker, dextran (70 kDa), was employed as a platform, onto which fluorescein, Oregon Green, and tetramethylrhodamine were loaded for ratiometric pH sensing and imaging. A cathepsin-B-cleavable peptide sequence bearing sulfo-Cy5 and the quencher BHQ-3 was also mounted; cleavage of the sequence was detected as an increase in sulfo-Cy5 fluorescence. A steep decrease in pH was observed 5-10 min after macropinosome formation, which was accompanied by an immediate increase in cathepsin B activity. Our design concept will lead to the development of other macropinoscopes for the simultaneous detection of other parameters in individual macropinosomes.

Enzyme Cascade Reactions on DNA Origami Scaffold.

The protocols for constructing, characterizing, and analyzing enzyme cascade reaction systems on the DNA scaffold are described. Two-step and three-step enzyme cascade reactions were adapted from the xylose metabolic pathway as the example of natural metabolic pathway and were assembled on the DNA scaffold by using the DNA binding adaptors.

Controlled Assembly of Fluorophores inside a Nanoliposome.

Cellular compartmentalization plays an essential role in organizing the complex and multiple biochemical reactions in the cell. An artificial compartment would provide powerful strategies to develop new biochemical tools for material production and diagnosis, but it is still a great challenge to synthesize the compartments that encapsulate materials of interest while controlling their accurate locations, numbers, and stoichiometry. In this study, we evaluated chemical characteristics of a liposome-encapsulated compartment, which has great potential to locate various materials of interest with precise control of their locations and numbers in the compartment. A nanoliposome was constructed inside a ring-shaped DNA origami skeleton according to the method of Yang et al., and further equipped with a double-stranded DNA platform to assemble molecules of interest in the nanoliposome. Upon formation of the nanoliposome, a pH-sensitive fluorophore on the bridged platform showed little or no response to the pH change of the outer buffer, ensuring that the molecules assembled on the platform are effectively shielded from the outer environment. The ring-shaped DNA skeleton equipped with a double-stranded DNA platform allows spatial assembly of several functional molecules inside the nanoliposome to isolate them from the outer environment.

Stimuli-Responsible SNARF Derivatives as a Latent Ratiometric Fluorescent Probe.

Fluorescence imaging is a powerful technique for continuous observation of dynamic intracellular processes of living cells. Fluorescent probes bearing a fluorescence switching property associated with a specific recognition or reaction of target biomolecule, that is, stimuli-responsibility, are important for fluorescence imaging. Thus, fluorescent probes continue to be developed to support approaches with different design strategies. When compared with simple intensity-changing fluorescent probes, ratiometric fluorescent probes typically offer the advantage of less sensitivity to errors associated with probe concentration, photobleaching, and environmental effects. For intracellular usage, ratiometric fluorescent probes based on small molecules must be loaded into the cells. Thus, probes having intrinsic fluorescence may obscure a change in intracellular signal if the background fluorescence of the remaining extracellular probes is high. To overcome such disadvantages, it is necessary to minimize the extracellular background fluorescence of fluorescent probes. Here, the design strategy of the latent ratiometric fluorescent probe for wash-free ratiometric imaging using a xanthene dye seminapthorhodafluor (SNARF) as the scaffold of fluorophore is discussed.

Mechanistic Aspects for the Modulation of Enzyme Reactions on the DNA Scaffold.

Cells have developed intelligent systems to implement the complex and efficient enzyme cascade reactions via the strategies of organelles, bacterial microcompartments and enzyme complexes. The scaffolds such as the membrane or protein in the cell are believed to assist the co-localization of enzymes and enhance the enzymatic reactions. Inspired by nature, enzymes have been located on a wide variety of carriers, among which DNA scaffolds attract great interest for their programmability and addressability. Integrating these properties with the versatile DNA-protein conjugation methods enables the spatial arrangement of enzymes on the DNA scaffold with precise control over the interenzyme distance and enzyme stoichiometry. In this review, we survey the reactions of a single type of enzyme on the DNA scaffold and discuss the proposed mechanisms for the catalytic enhancement of DNA-scaffolded enzymes. We also review the current progress of enzyme cascade reactions on the DNA scaffold and discuss the factors enhancing the enzyme cascade reaction efficiency. This review highlights the mechanistic aspects for the modulation of enzymatic reactions on the DNA scaffold.

FRET-Based Cathepsin Probes for Simultaneous Detection of Cathepsin B and D Activities.

Fluorescent cathepsin probes were prepared by modification of peptidic substrates for cathepsin B (CTSB) and cathepsin D (CTSD) with FRET pairs. Fluorophores with distinguishable emission characteristics were applied to CTSB and CTSD probes with their appropriate quenchers to simultaneously monitor the activity of CTSB and/or CTSD. Conjugation of both the CTSB and CTSD probes with short single-stranded DNA drastically increased their reactivity to cathepsins over the parent probes possibly by improving their solubility. The activity of CTSB and CTSD were simultaneously detected by using these orthogonal FRET-based cathepsin probes.

A two-step screening to optimize the signal response of an auto-fluorescent protein-based biosensor.

Auto-fluorescent protein (AFP)-based biosensors transduce the structural change in their embedded recognition modules induced by recognition/reaction events to fluorescence signal changes of AFP. The lack of detailed structural information on the recognition module often makes it difficult to optimize AFP-based biosensors. To enhance the signal response derived from detecting the putative structural change in the nitric oxide (NO)-sensing segment of transient receptor potential canonical 5 (TRPC5) fused to enhanced green fluorescent protein (EGFP), EGFP-TRPC5, a facile two-step screening strategy, in silico first and in vitro second, was applied to variants of EGFP-TRPC5 deletion-mutated within the recognition module. In in silico screening, the structural changes of the recognition modules were evaluated as root-mean-square-deviation (RMSD) values, and 10 candidates were efficiently selected from 47 derivatives. Through in vitro screening, four mutants were identified that showed a larger change in signal response than the parent EGFP-TRPC5. One mutant in particular, 551-575, showed four times larger change upon reaction with NO and H2O2. Furthermore, mutant 551-575 also showed a signal response upon reaction with H2O2 in mammalian HEK293 cells, indicating that the mutant has the potential to be applied as a biosensor for cell measurement. Therefore, this two-step screening method effectively allows the selection of AFP-based biosensors with sufficiently enhanced signal responses for application in mammalian cells.

Topologically-Interlocked Minicircles as Probes of DNA Topology and DNA-Protein Interactions.

Invited for the cover of this issue are Prof. Takashi Morii and co-workers at Kyoto University and Ewha Womans University. The cover image depicts the graphical design and atomic force microscopic (AFM) images of the synthesized topologically-interlocked DNA catenane and rotaxanes inside a frame-shaped DNA origami. Read the full text of the article at 10.1002/chem.202200108.

Topologically-Interlocked Minicircles as Probes of DNA Topology and DNA-Protein Interactions.

DNA minicircles exist in biological contexts, such as kinetoplast DNA, and are promising components for creating functional nanodevices. They have been used to mimic the topological features of nucleosomal DNA and to probe DNA-protein interactions such as HIV-1 and PFV integrases, and DNA gyrase. Here, we synthesized the topologically-interlocked minicircle rotaxane and catenane inside a frame-shaped DNA origami. These minicircles are 183 bp in length, constitute six individual single-stranded DNAs that are ligated to realize duplex interlocking, and adopt temporary base pairing of single strands for interlocking. To probe the DNA-protein interactions, restriction reactions were carried out on DNAs with different topologies such as free linear duplex or duplex constrained inside origami and free or topologically-interlocked minicircles. Except the free linear duplex, all tested structures were resistant to restriction digestion, indicating that the topological features of DNA, such as flexibility, curvature, and groove orientation, play a major role in DNA-protein interactions.

Augmentation of an Engineered Bacterial Strain Potentially Improves the Cleanup of PCB Water Pollution.

Polychlorinated biphenyls (PCBs) are recalcitrant organohalide pollutants, consisting of 209 congeners. PCB cleanup in natural landscapes is expected to be achieved by the metabolic activity of microorganisms, but aerobic PCB-degrading bacteria that inhabit sites polluted by PCBs cannot degrade all PCB congeners due to the specificity of their enzymes. In this study, we investigated the degradability of PCBs when a genetically modified PCB-degrading bacterium was compounded with wild-type PCB-degrading bacteria. We used two bacterial strains, Comamonas testosteroni YAZ2 isolated from a PCB-uncontaminated natural landscape and Escherichia coli BL21(DE3) transformed with a biphenyl dioxygenase (BphA) gene from a well-known PCB degrader, Burkholderia xenovorans LB400. The enzymatic specificities of BphA were 2,3-dioxygenation in the YAZ2 and 2,3- and 3,4-dioxygenations in the recombinant E. coli. For the PCB-degrading experiment, a dedicated bioreactor capable of generating oxygen microbubbles was prototyped and used. The combined cells of the recombinant and the wild-type strains with an appropriate composite ratio degraded 40 mg/L of Kaneclor KC-300 to 0.3 ± 0.1 mg/L within 24 h. All of the health-toxic coplanar PCB congeners in KC-300 were degraded. This study suggested that the augmentation of an engineered bacterial strain could improve the cleanup of PCB water pollution. It also revealed the importance of the ratio of the strains with different PCB-degrading profiles to efficient degradation and that the application of oxygen microbubbles could rapidly accelerate the cleanup. IMPORTANCE PCB cleanup technique in a natural environment relies on the use of enzymes from microorganisms, primarily biphenyl dioxygenase and dehalogenase. Herein, we focused on biphenyl dioxygenase and created a recombinant PCB-degrading E. coli strain. Despite the development of environments for the field use of transgenic microbial strains around the world, verification of the applicability of transgenic microbial strains for PCB cleanup in the field has not yet been reported. We tentatively verified the extent to which degradability could be obtained by an augmentation model of a transgenic strain, the enzyme expression of which is easily regulated in rivers and lakes with PCB pollution. Our experiments used a dedicated bioreactor to model the natural landscape and produced results superior to those of bioremediation or biostimulation methods. The application of micro-nano bubbles, which has recently been discussed, to the cleanup of environmental pollution was also found to be useful in this study.

Tuning the Reactivity of a Substrate for SNAP-Tag Expands Its Application for Recognition-Driven DNA-Protein Conjugation.

Recognition-driven modification has been emerging as a novel approach to modifying biomolecular targets of interest site-specifically and efficiently. To this end, protein modular adaptors (MAs) are the ideal reaction model for recognition-driven modification of DNA as they consist of both a sequence-specific DNA-binding domain (DBD) and a self-ligating protein-tag. Coupling DNA recognition by DBD and the chemoselective reaction of the protein tag could provide a highly efficient sequence-specific reaction. However, combining an MA consisting of a reactive protein-tag and its substrate, for example, SNAP-tag and benzyl guanine (BG), revealed rather nonselective reaction with DNA. Therefore new substrates of SNAP-tag have been designed to realize sequence-selective rapid crosslinking reactions of MAs with SNAP-tag. The reactions of substrates with SNAP-tag were verified by kinetic analyses to enable the sequence-selective crosslinking reaction of MA. The new substrate enables the distinctive orthogonality of SNAP-tag against CLIP-tag to achieve orthogonal DNA-protein crosslinking by six unique MAs.

Conditional dependence of enzyme cascade reaction efficiency on the inter-enzyme distance.

A dual-enzyme cascade, xylitol dehydrogenase and xylulose kinase, derived from the xylose metabolic pathway, was constructed on a three-dimensional DNA scaffold which exhibited a dynamic shape transition from an open state to a closed hexagonal prism. Evaluation of the cascade reaction efficiencies in the open and closed states revealed little to no inter-enzyme distance dependence, presumably due to the far larger catalytic constant of the downstream enzyme. The inter-enzyme distance was not the dominant factor for cascade efficiency when the kinetic parameters of the cascade enzymes were imbalanced with the highly efficient downstream enzyme.

Dynamic Shape Transformation of a DNA Scaffold Applied for an Enzyme Nanocarrier.

Structural programmability and accurate addressability of DNA nanostructures are ideal characteristics for the platform of arranging enzymes with the nanoscale precision. In this study, a three-dimensional DNA scaffold was designed to enable a dynamic shape transition from an open plate-like structure to its closed state of a hexagonal prism structure. The two domains in the open state were folded together to transform into the closed state by hybridization of complementary short DNA closing keys at both of the facing edges in over 90% yield. The shape transformation of the DNA scaffold was extensively studied by means of the fluorescence energy transfer measurement, atomic force microscope images, and agarose gel electrophoretic analyses. A dimeric enzyme xylitol dehydrogenase was assembled on the DNA scaffold in its open state in a high-loading yield. The enzyme loaded on the scaffold was subsequently transformed to its closed state by the addition of short DNA closing keys. The enzyme encapsulated in the closed state displayed comparable activity to that in the open state, ensuring that the catalytic activity of the enzyme was well maintained in the DNA nanocarrier. The nanocarrier with efficient encapsulation ability is potentially applicable for drug delivery, biosensing, biocatalytic, and diagnostic tools.

Stabilization and structural changes of 2D DNA origami by enzymatic ligation.

The low thermal stability of DNA nanostructures is the major drawback in their practical applications. Most of the DNA nanotubes/tiles and the DNA origami structures melt below 60°C due to the presence of discontinuities in the phosphate backbone (i.e., nicks) of the staple strands. In molecular biology, enzymatic ligation is commonly used to seal the nicks in the duplex DNA. However, in DNA nanotechnology, the ligation procedures are neither optimized for the DNA origami nor routinely applied to link the nicks in it. Here, we report a detailed analysis and optimization of the conditions for the enzymatic ligation of the staple strands in four types of 2D square lattice DNA origami. Our results indicated that the ligation takes overnight, efficient at 37°C rather than the usual 16°C or room temperature, and typically requires much higher concentration of T4 DNA ligase. Under the optimized conditions, up to 10 staples ligation with a maximum ligation efficiency of 55% was achieved. Also, the ligation is found to increase the thermal stability of the origami as low as 5°C to as high as 20°C, depending on the structure. Further, our studies indicated that the ligation of the staple strands influences the globular structure/planarity of the DNA origami, and the origami is more compact when the staples are ligated. The globular structure of the native and ligated origami was also found to be altered dynamically and progressively upon ethidium bromide intercalation in a concentration-dependent manner.

A facile combinatorial approach to construct a ratiometric fluorescent sensor: application for the real-time sensing of cellular pH changes.

Realtime monitoring of the cellular environment, such as the intracellular pH, in a defined cellular space provides a comprehensive understanding of the dynamics processes in a living cell. Considering the limitation of spatial resolution in conventional microscopy measurements, multiple types of fluorophores assembled within that space would behave as a single fluorescent probe molecule. Such a character of microscopic measurements enables a much more flexible combinatorial design strategy in developing fluorescent probes for given targets. Nanomaterials with sizes smaller than the microscopy spatial resolution provide a scaffold to assemble several types of fluorophores with a variety of optical characteristics, therefore providing a convenient strategy for designing fluorescent pH sensors. In this study, fluorescein (CF) and tetramethylrhodamine (CR) were assembled on a DNA nanostructure with controlling the number of each type of fluorophore. By taking advantage of the different responses of CF and CR emissions to the pH environment, an appropriate assembly of both CF and CR on DNA origami enabled a controlled intensity of fluorescence emission and ratiometric pH monitoring within the space defined by DNA origami. The CF and CR-assembled DNA origami was successfully applied for monitoring the intracellular pH changes.

Evaluation of the role of the DNA surface for enhancing the activity of scaffolded enzymes.

The catalytic enhancements of enzymes loaded on DNA nanostructures have been attributed to the characteristics provided by highly negative charges on the surface of the DNA scaffold, such as the modulation of the local pH near enzymes. In this study, two types of enzymes with optimal activity at pH 6 and 8 equally displayed significant catalytic enhancements on the DNA scaffold surface. By using a ratiometric pH indicator, a lower local pH shift of 0.8 was observed near the DNA scaffold surface. The postulated local pH change near the DNA scaffold surface is unlikely to play a general role in enhancing the activity of the scaffolded enzymes.

Inositol 1,4,5-trisphosphate 3-kinase B promotes Ca2+ mobilization and the inflammatory activity of dendritic cells.

Innate immune responses to Gram-negative bacteria depend on the recognition of lipopolysaccharide (LPS) by a receptor complex that includes CD14 and TLR4. In dendritic cells (DCs), CD14 enhances the activation not only of TLR4 but also that of the NFAT family of transcription factors, which suppresses cell survival and promotes the production of inflammatory mediators. NFAT activation requires Ca2+ mobilization. In DCs, Ca2+ mobilization in response to LPS depends on phospholipase C γ2 (PLCγ2), which produces inositol 1,4,5-trisphosphate (IP3). Here, we showed that the IP3 receptor 3 (IP3R3) and ITPKB, a kinase that converts IP3 to inositol 1,3,4,5-tetrakisphosphate (IP4), were both necessary for Ca2+ mobilization and NFAT activation in mouse and human DCs. A pool of IP3R3 was located on the plasma membrane of DCs, where it colocalized with CD14 and ITPKB. Upon LPS binding to CD14, ITPKB was required for Ca2+ mobilization through plasma membrane-localized IP3R3 and for NFAT nuclear translocation. Pharmacological inhibition of ITPKB in mice reduced both LPS-induced tissue swelling and the severity of inflammatory arthritis to a similar extent as that induced by the inhibition of NFAT using nanoparticles that delivered an NFAT-inhibiting peptide specifically to phagocytic cells. Our results suggest that ITPKB may represent a promising target for anti-inflammatory therapies that aim to inhibit specific DC functions.

Cryogenic Far-Field Fluorescence Nanoscopy: Evaluation with DNA Origami.

Far-field fluorescence localization nanoscopy of individual fluorophores at a temperature of 1.8 K was demonstrated using DNA origami as a one-nanometer-accurate scaffold. Red and near-infrared fluorophores were modified to the scaffold, and the fluorophores were 11 or 77 nm apart. We performed the localization nanoscopy of these two fluorophores at 1.8 K with a far-field fluorescence microscope. Under the cryogenic conditions, the fluorophores were perfectly immobilized and their photobleaching was drastically suppressed; consequently, the lateral spatial precision (a measure of reproducibility) was increased to 1 nm. However, the lateral spatial accuracy (a measure of trueness) remained tens of nanometers. We observed that the fluorophore centroids were laterally shifted as a function of the axial position. Because the orientation of the transition dipole of the fluorophores was fixed under cryogenic conditions, the anisotropic emission from the single fixed dipole had led to the lateral shift. This systematic error due to the dipole-orientation effect could be corrected by the three-dimensional localization of the individual fluorophores with spatial precisions of (lateral) 1 nm and (axial) 17 nm. In addition, the xy-error arising from the three-dimensional (3D) orientation of the scaffold with the two fluorophores 11 nm apart was estimated to be 0.3 nm. As a result, the individual fluorophores on the DNA origami were localized at the designed position, and the lateral spatial accuracy was quantified to be 4 nm in the standard error.