Multi-enzyme complexes on DNA scaffolds capable of substrate channelling with an artificial swinging arm.

Molecular association is one of the most fundamental principles of chemistry and biology. Biomolecular complexes need to colocalize sequential catalytic and chemical processes such that the intermediates are precisely positioned to diffuse efficiently. The relative distance and orientation between the active sites within these complexes may affect the intermediate binding, which may also be different under different cell environments. In this work, we used Brownian dynamics simulations to model an intermediate substrate association in the tryptophan biosynthesis pathway in Escherichia coli (E. coli) for investigating potential advantages of using TrpCF bifunctional enzyme which fuses phosphoribosyl anthranilate isomerase (PRAI) and indoleglycerol phosphate synthase (IGPS). We focused on transporting a product of PRAI, 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate (CdRP), which is also the substrate of in IGPS. We report the average association time and direct binding percentage when the substrate concentration is 10.0 μM as an estimated physiological concentration in cells. Our results suggest that final product synthesis efficiency can be achieved by optimizing the interenzyme distance and by properly aligning the orientation of the active sites. In addition, the intermolecular interactions between the substrate and macromolecule crowders largely affected the transportation of a substrate. The overall rate of product synthesis enhancement in a multienzyme complex depends on the distance between enzymes when the substrate leaking phenomenon exists. Our work highlights the importance of the enzyme spatial organization in regulating the production efficiency of a product in multienzyme complexes, broadens implications in synthesis processes under complex cell environments, and brings biophysical insights into the enzyme-substrate association and understanding of target ligand/substrate kinetics.

Remarkably boosting ethanol upgrading to higher alcohols through cooperative catalysis of surface-interface multiple-active sites on nitrogen-doped carbon decorated copper-based catalysts

DNA nanostructures are among the most fascinating self-assembled nanostructures in diverse areas of science and technology, because of their nanoscale precision in biomolecule and nanoparticle organization. The implementation of dynamic and spatial regulation in structural morphology and hierarchical assembly upon specific external stimuli will greatly expand their applications in biocomputation, clinical diagnosis, and cancer therapy. Recently, noncanonical nucleic acids, particularly DNA triplexes, i-motifs, and G-quadruplexes, have become powerful tools for biosensing and mechanical switching. Developments in incorporating stimuli-responsive noncanonical nucleic acids into DNA nanostructures provide a promising approach to regulating the spatial organization and hierarchical assembly of DNA nanostructures. In this review, we briefly introduce recent progress in constructing DNA nanostructures with dynamic regulation of the structural transformation and programmable assembly pathways at the nanometer scale by noncanonical nucleic acids and discuss their potential applications and challenges.

The development of supramolecular hydrogel scaffolds for the precise positioning of biochemical cues is paramount for applications such as tissue engineering. Nucleic acid engineering allows fabrication of three-dimensional (3D) nanostructures with high variability and nanoscale precision. In this study, aptamers were anisotropically functionalized onto branched DNA nanostructures to control their cell adhesion capability, and their efficiency as biological signal inducers for 3D cell cultivation was investigated. Each arm of the X-shaped DNA nanostructure (X-DNA) was functionalized with photo-cross-linkable or cell adhesion moieties, and the steric hindrance of the 3D DNA nanostructures on a cell was optimized. X-DNA nanostructures with cell-positioning parameters were rapidly photopolymerized to form hybrid hydrogels, and their effects on cell behaviors and positions were investigated. We observed that aptamer-functionalized X-DNA nanostructures exhibited significantly enhanced cell proliferation and provided homogeneous distribution and target-specific adhesion of encapsulated cells within hydrogel matrices. Overall, the anisotropic functionalization of DNA nanostructures provides a controllable function for the advancement of conventional 3D culture platforms.

The high catalytic efficiency of enzyme cascade reaction mainly depends on optimal interenzyme distance regulated by the special scaffolds. In this work, the rigid PtNPs with different sizes were employed as scaffolds to regulate interenzyme distance for efficient enzyme cascade amplification to construct electrochemical biosensor for sensitive detection of matrix metalloproteinases-2 (MMP-2), which overcame the drawbacks of instable construction and sophisticated preparation induced by conventional scaffolds such as metal-organic frameworks (MOFs), DNA nanostructures. Here, cucurbit[7]uril functionalized PtNPs (CB[7]@PtNPs) was utilized to load ferrocene (Fc)-labeled horseradish peroxidase (HRP) and glucose oxidase (GOx) via host-guest interaction between Fc and CB[7], respectively, resulting in the formation of a stable three-dimensional netlike structure containing amounts of enzymes. Interestingly, the enzyme cascade reaction regulated by 10 nm PtNPs as scaffold showed highly catalytic efficiency. Meanwhile, the PtNPs could also serve as catalyst to accelerate the enzyme cascade reaction with further enhanced catalytic efficiency. As a result, the proposed biosensor exhibited excellent sensitivity with a wide linear range of 0.1 pg·mL-1 to 20 ng·mL-1 and a detection limit of 0.03 pg·mL-1 for MMP-2. Such a strategy opened a new avenue for adopting metal nanoparticles to regulate interenzyme distance for efficient enzyme cascade amplification, thus providing a universal and easy operating method for sensitively detecting various targets such as DNA, metal ion, and protein.

<p indent="0mm">Arranging biomacromolecules in a specific order and orientation on the nanoscale has long been a prominent research topic. Significant progress has been made in understanding intermolecular interactions within bionetworks and elucidating biosystem mechanisms. DNA nanostructures, serving as ideal scaffolds, have been extensively employed to achieve precise arrangement of diverse biomolecules at the nanoscale due to their well-defined geometry, programmability, addressability at the nanometer scale, functionalization capabilities and excellent biocompatibility. The advent of DNA origami has further enhanced the ability to design intricate and accurate DNA frameworks. This technique utilizes numerous short DNA strands, called staples, to fold a longer DNA strand, known as the scaffold, enabling the creation of more structurally complex and precise DNA architectures. This review is mainly structured into four main sections. Firstly, we provide a comprehensive overview of the various methods employed for the three key steps involved in assembling DNA nanostructures with biomolecules. These steps encompass the conjugation of oligonucleotides and biomolecules, the hybridization of DNA nanostructures with biomolecules, and the purification of DNA nanostructures–biomolecule complexes. Secondly, we briefly summarize the relevant applications of DNA nanostructures–biomolecules self-assembly, with a primary focus on the construction of enzyme cascade reaction systems. The combination of enzyme cascades and addressable DNA assembly has proven highly effective in creating stable artificial multi-enzyme complexes. This integration allows for precise control over the position, shape, and composition of the complexes, enabling efficient manipulation of enzyme distance, substrate channeling, and compartmentalization. Thirdly, we focus on the application of DNA nanostructures in diverse imaging techniques, including atomic force microscopy (AFM), fluorescence imaging, and cryo-electron microscopy (cryo-EM). DNA nanostructure offers distinct advantages in advancing bioimaging modalities, such as enabling multiplexed imaging, facilitating logical responsive imaging, and enabling protein structural analysis. While AFM has been extensively employed for the characterization of various biomolecules, it still has limitations in direct observation and comparison of freely dispersed molecules. However, by utilizing DNA nanostructures as a platform for loading molecules, it becomes possible to realize the visualization of dynamic intermolecular interactions using AFM. Besides, through precisely manipulating the number and combination of selected fluorophores, DNA nanostructures provide a means to mitigate the issue of spectral overlap among fluorophore colors. This precise control allows for tuning the overall color of probes, effectively overcoming spectrum overlapping and enabling the generation of distinct and distinguishable colors. Moreover, DNA nanostructures show potential in assisting the determination of protein orientation during cryo-electron microscopy (cryo-EM) imaging and potentially mitigating issues such as protein unfolding or nonrandom absorption at the water-air interface. The final section of this review discusses the technical challenges that need to be addressed to enhance the utilization of DNA nanoscaffolds, for example achieving precise orientation control of biomolecules on DNA scaffolds, optimizing the stability of the overall complexes to achieve <italic>in vivo</italic> functionality and overcoming technological bottlenecks that impede the application of DNA nanoscaffolds in cryo-EM imaging.

Single turnover measurements of a fluorogenic reaction at the surface of a nanoparticle provide a detailed view of reaction dynamics at a catalyst with multiple heterogeneous active sites. This picture must be extracted from a fluorescence trajectory of one particle, which records individual reaction and desorption events. We have previously proposed analyzing fluorescence trajectories with constrained mean dwell times in either light or dark states, which are averaged over a subensemble of events in which the dwell time in the previous state satisfies a criterion of being less than or greater than a specified time. We have shown that these quantities can be used to distinguish between correlated and independent fluctuations at multiple active sites. Here we show that this analysis is complementary to calculating dwell time correlation functions, whose decay with turnover index quantifies dynamical disorder in the underlying kinetics. We analyze a measured fluorescence trajectory from a gold nanoparticle in terms of both constrained mean dwell times and dwell time correlation functions. The analysis demonstrates that the minimal kinetic model with discrete states that is qualitatively consistent with the data allows active sites to fluctuate among at least three substates with distinct adsorption and reaction rates.

Heterogeneous high-entropy catalyst nanoparticles for oxygen evolution reaction: Impact of oxygen and fluorine introduction

MOF-derived CoP-nitrogen-doped carbon@NiFeP nanoflakes as an efficient and durable electrocatalyst with multiple catalytically active sites for OER, HER, ORR and rechargeable zinc-air batteries

Synthesis of magnetic bifunctional monomer molecularly imprinted polymers for highly selective separation of paclitaxel from Taxus cuspidata

Ionic liquids with multiple active sites supported by SBA-15 for catalyzing conversion of CO2 into cyclic carbonates

Increased anticoagulant activity of thrombin-binding DNA aptamers by nanoscale organization on DNA nanostructures

&lt;p dir="ltr"&gt;The physical and chemical properties of DNA, including its structure predictability thanks to Watson-Crick base pairing, make it into an obvious polymer of choice to use as a biomaterial for the fabrication of complex three dimensional nanostructures. These nanostructures are produced by the technique of DNA origami, where a long single stranded circular DNA, called scaffold, is folded into a pre-designed shape by the hybridization of partially complementary oligonucleotides, the staples. The programmability of DNA origami, i.e. the specific control, by design, of the location of every DNA sequence within the structure, can be harnessed for the positioning of molecules with nanoscale precision. In the present thesis, we have explored different branches of the DNA origami technology, from its functionalization with proteins, its structural characterization and its application for method development, targeted treatment and study of molecular processes taking place at the nanoscale.&lt;/p&gt;&lt;p dir="ltr"&gt;In Paper I, we develop an approach to quantify the incorporation of proteins in DNA origami using DNA-PAINT, a multiplexed super-resolution imaging method that allows the characterization of structures with single-molecule resolution and high reliability. Even as protein-decorated DNA nanostructures become increasingly important tools for biomedical sciences, the existing strategies to study and maximize protein incorporation provide limited insight into functionalization. With DNA-PAINT, we were able to explore factors influencing incorporation rate such as oligonucleotide quality, protein size, or purification method, rank their impact, and model their combined effects. Thus, we were able to offer a comprehensive view of functionalization efficiency and precise parameters for yield optimization, which can have significant implications for the application of DNA origami in fields beyond academia.&lt;/p&gt;&lt;p dir="ltr"&gt;In Paper II, we introduce the new method PLASTIQ for assessing DNA origami structural integrity in vivo with a detection sensitivity of 0.01 femtomolar. Despite the potential of DNA origami in therapeutics, the lack of structural assessment methods for in vivo use hampers its clinical initiation. Sampling only 1 ul of blood, PLASTIQ allowed us to monitor the degradation patterns of nanostructures over time. We examined the protective effects of PEGylation and obtained the pharmacokinetic profiles of different origamis. Additionally, we were able to observe differential degradation of structural regions depending on how exposed they were to the environment. Altogether, PLASTIQ is an accurate tool for assessing structural stability, offering valuable insights for advancing DNA origami-based drug development.&lt;/p&gt;&lt;p dir="ltr"&gt;In Paper III, we present PANMAP, a method based on antigen patterning on DNA nanostructures to measure antibody affinity while taking multivalency into account. Multivalency is fundamental in many biological systems, especially in antibody-antigen interactions where multiple binding sites improve affinity and specificity. However, conventional affinity assays such as ELISA provide only a limited perspective on binding characterization and do not address multivalency. With PANMAP overcoming these limitations, we found that antibody binding equilibrium is influenced by antigen spacing, leading to competitive exclusion at close distances, optimal bivalent binding at intermediate distances, and a monovalent regime at longer distances. Thus, PANMAP enabled a complete profile of multivalency and the binding states that constitute it, potentially providing useful insights into biological processes and engineering applications.&lt;/p&gt;&lt;p dir="ltr"&gt;In Paper IV, we apply DNA origami displaying Jag1 ligands to stimulate neuroepithelial stem-like cells to study the molecular mechanism leading to Notch receptor activation. The Notch pathway is a highly evolutionarily conserved signaling system that plays a key role in embryonic and nervous system development. However, it is unclear how the activation unravels, with the leading hypothesis being force-driven conformational changes. Here, we demonstrate that Notch triggering can occur without pulling forces and that it instead proceeds upon extended binding. These findings suggest an alternative molecular mechanism for receptor activation, suggesting potential for the design of soluble agonists.&lt;/p&gt;&lt;p dir="ltr"&gt;In Paper V, we engineer a pH-responsive DNA robotic switch that selectively displays death receptor ligands only in acidic tumor microenvironments to induce apoptosis of cancer cells. As these receptors are responsible for initiating cell death but are ubiquitously expressed on the membranes of most cells, a targeted approach for their use in tumor therapies is desired. The DNA robotic switch hides the ligands, arranged in a hexagonal pattern inside a cavity while at neutral pH, until encountering pH 6.5 where the ligands are revealed, leading to clustering of DR and triggering apoptosis of breast cancer cells. Our results probe the functionality of the nanodevice in vitro and shows the significant tumor volume reduction in mice with human breast cancer xenografts. Overall, this work highlights the potential for targeted cancer treatment using DNA origami.&lt;/p&gt;&lt;h3&gt;List of scientific papers&lt;/h3&gt;&lt;p dir="ltr"&gt;1. &lt;b&gt;Iris Rocamonde-Lago&lt;/b&gt;, Ferenc Fördös, Cagla Sahin, Ian T. Hoffecker &amp; Björn Högberg. Exploring DNA origami protein functionalization using super resolution imaging. [Manuscript]&lt;/p&gt;&lt;p dir="ltr"&gt;II. Yang Wang*, &lt;b&gt;Iris Rocamonde-Lago&lt;/b&gt;*, Janine Waldvogel, Shuya Zang, Igor Baars, Alexander Kloosterman, Boxuan Shen, Ian T. Hoffecker, Qin He &amp; Björn Högberg. DNA origami structural integrity tracked in vivo using proximity ligation. [Manuscript]&lt;/p&gt;&lt;p dir="ltr"&gt;III. &lt;b&gt;Iris Rocamonde-Lago&lt;/b&gt;, Ieva Berzina, Ian T. Hoffecker &amp; Björn Högberg. Profiling of multivalent binding with DNA origami reveals spatial determinants of antigen-antibody interactions. [Manuscript]&lt;/p&gt;&lt;p dir="ltr"&gt;IV. Ioanna Smyrlaki, Ferenc Fördös, &lt;b&gt;Iris Rocamonde-Lago&lt;/b&gt;, Yang Wang, Boxuan Shen, Antonio Lentini, Vincent C. Luca, Björn Reinius, Ana I. Teixeira &amp; Björn Högberg. Soluble and multivalent Jag1 DNA origami nanopatterns activate Notch without pulling force. Nature Communications, 15(1), 465. &lt;a href="https://doi.org/10.1038/s41467-023-44059-4" rel="noreferrer" target="_blank"&gt;https://doi.org/10.1038/s41467-023-44059-4&lt;/a&gt;&lt;/p&gt;&lt;p dir="ltr"&gt;V. Yang Wang, Igor Baars, Ieva Berzina, &lt;b&gt;Iris Rocamonde-Lago&lt;/b&gt;, Boxuan Shen, Yunshi Yang, Marco Lolaico, Janine Waldvogel, Ioanna Smyrlaki, Keying Zhu, Robert A. Harris &amp; Björn Högberg. A DNA robotic switch with regulated autonomous display of cytotoxic ligand nanopatterns. Nature Nanotechnology, 19(9), 1366-1374. &lt;a href="https://doi.org/10.1038/s41565-024-01676-4" rel="noreferrer" target="_blank"&gt;https://doi.org/10.1038/s41565-024-01676-4&lt;/a&gt;&lt;/p&gt;&lt;p dir="ltr"&gt;*Shared first authorship&lt;/p&gt;

Biosensors based on nanomaterials have been used for detection of various biological molecules with high sensitivity and selectivity. Herein, we developed a simple and ultrasensitive electrochemical DNA biosensor using long-range self-assembled DNA nanostructures as carriers for signal amplification, which can achieve an impressive detection limit of 5 aM human immunodeficiency virus (HIV) DNA even in complex biological samples. In this study, we designed two auxiliary probes. A cascade of hybridization events between the two auxiliary probes can lead to long-range self-assembly and form micrometer-long one-dimensional DNA nanostructures. In the presence of target DNA, each copy of the target can act as a trigger to connect a DNA nanostructure to a capture probe on the electrode surface. Then, a great amount of redox indicator [Ru(NH(3))(6)](3+) can be electrostatically bound to the DNA nanostructures and eventually result in significantly amplified electrochemical signals.

Recent synthetic advances led to the development of new catalytic particles with well-defined atomic structures and multiple active sites, which are called nanocatalysts. Experimental studies of processes at nanocatalysts uncovered a variety of surprising effects, but the molecular mechanisms of these phenomena remain not well understood. We propose a theoretical method to investigate the dynamics of chemical reactions on catalytic particles with multiple active sites. It is based on a discrete-state stochastic description that allows us to explicitly evaluate dynamic properties of the system. It is found that for independently occurring chemical reactions, the mean turnover times are inversely proportional to the number of active sites, showing no stochastic effects. However, the molecular details of reactions and the number of active sites influence the higher moments of reaction times. Our theoretical method provides a way to quantify the molecular mechanisms of processes at nanocatalysts.