Identification of the Binding Site between Aptamer sgc8c and PTK7.

Aptamers are single-stranded RNA or DNA molecules that can specifically bind to targets and have found broad applications in cancer early-stage detection, accurate drug delivery, and precise treatment. Although various aptamer screening methods have been developed over the past several decades, the accurate binding site between the target and the aptamer cannot be characterized during a typical aptamer screening process. In this research, we chose a widely used aptamer screened by our group, sgc8c, and its target protein tyrosine kinase 7 (PTK7) as the model aptamer and target and tried to determine the binding site between aptamer sgc8c and PTK7. Through sequential protein truncation, we confirmed that the exact binding site of sgc8c was within the region of Ig 3 to Ig 4 in the extracellular domain of PTK7. Using in vitro expressed Ig (3-4), we successfully acquired the crystal of an sgc8c-Ig (3-4) binding complex. The possible sgc8c-binding amino acid residues on PTK7 and PTK7-binding nucleotide residues on sgc8c were further identified and simulated by mass spectrometry and molecular dynamics simulation and finally verified by aptamer/protein truncation and mutation.

A de novo strategy to develop NIR precipitating fluorochrome for long-term in situ cell membrane bioimaging.

Cell membrane-targeted bioimaging is a prerequisite for studying the roles of membrane-associated biomolecules in various physiological and pathological processes. However, long-term in situ bioimaging on the cell membrane with conventional fluorescent probes leads to diffusion into cells from the membrane surface. Therefore, we herein proposed a de novo strategy to construct an antidiffusion probe by integrating a fluorochrome characterized by strong hydrophobicity and low lipophilicity, with an enzyme substrate to meet this challenge. This precipitating fluorochrome HYPQ was designed by conjugating the traditionally strong hydrophobic solid-state fluorochrome 6-chloro-2-(2-hydroxyphenyl) quinazolin-4(3H)-one (HPQ) with a 2-(2-methyl-4H-chromen-4-ylidene) malononitrile group to obtain closer stacking to lower lipophilicity and elongate emission to the far-red to near-infrared wavelength. As proof-of-concept, the membrane-associated enzyme γ-glutamyltranspeptidase (GGT) was selected as a model enzyme to design the antidiffusion probe HYPQG. Then, benefiting from the precipitating and stable signal properties of HYPQ, in situ imaging of GGT on the membrane was successfully realized. Moreover, after HYPQG was activated by GGT, the fluorescence signal on the cell membrane remained unchanged, with incubation time even extending to 6 h, which is significant for in situ monitoring of enzymatic activity. In vivo testing subsequently showed that the tumor region could be accurately defined by this probe after long-term in situ imaging of tumor-bearing mice. The excellent performance of HYPQ indicates that it may be an ideal alternative for constructing universal antidiffusion fluorescent probes, potentially providing an efficient tool for accurate imaging-guided surgery in the future.

Self-Powered Biosensor Based on DNA Walkers for Ultrasensitive MicroRNA Detection.

A novel self-powered biosensor is fabricated for ultrasensitive microRNA-21 (miRNA-21) detection, which includes an enzymatic biofuel cell (EBFC), DNA walkers, a digital multimeter (DMM), and a capacitor. As a novel strategy for signal amplification, DNA walkers are designed in the cathode, while the capacitor stores electrochemical energy from the EBFC to further boost the instantaneous current displayed by the DMM. When miRNA-21 is present, the DNA walkers are provoked to walk from as-opened hairpin structures to other hairpin structures, generating double-strand DNA structures, which stimulate [Ru(NH3)6]3+ to be adsorbed on the cathode surface by electrostatic interaction. Afterward, [Ru(NH3)6]3+ is reduced to [Ru(NH3)6]2+, and the open circuit voltage (EOCV) is significantly increased. Depending on the approach of signal amplification from DNA walkers, this biosensor displays an ultrasensitive assay toward miRNA-21 in the range of 0.5 to 104 fM, with a detection limit of 0.15 fM. In addition, this self-powered biosensor displays high selectivity for miRNA-21 assay in human serum samples.

Ligand Dilution Analysis Facilitates Aptamer Binding Characterization at the Single-Molecule Level.

Cell-specific aptamers offer a powerful tool to study membrane receptors at the single-molecule level. Most target receptors of aptamers are highly expressed on the cell surface, but difficult to analyze in situ because of dense distribution and fast velocity. Therefore, we herein propose a random sampling-based analysis strategy termed ligand dilution analysis (LDA) for easily implemented aptamer-based receptor study. Receptor density on the cell surface can be calculated based on a regression model. By using a synergistic ligand dilution design, colocalization and differentiation of aptamer and monoclonal antibody (mAb) binding on a single receptor can be realized. Once this is accomplished, precise binding site and detailed aptamer-receptor binding mode can be further determined using molecular docking and molecular dynamics simulation. The ligand dilution strategy also sets the stage for an aptamer-based dynamics analysis of two- and three-dimensional motion and fluctuation of highly expressed receptors on the live cell membrane.

Phase Separation of DNA-Encoded Artificial Cells Boosts Signal Amplification for Biosensing.

Life-like hierarchical architecture shows great potential for advancing intelligent biosensing, but modular expansion of its sensitivity and functionality remains a challenge. Drawing inspiration from intracellular liquid-liquid phase separation, we discovered that a DNA-encoded artificial cell with a liquid core (LAC) can enhance peroxidase-like activity of Hemin and its DNA G-quadruplex aptamer complex (DGAH) without substrate-selectivity, unlike its gelled core (GAC) counterpart. The LAC is easily engineered as an ultrasensitive biosensing system, benefiting from DNA's high programmability and unique signal amplification capability mediated by liquid-liquid phase separation. As proof of concept, its versatility was successfully demonstrated by coupling with two molecular recognition elements to monitor tumor-related microRNA and profile cancer cell phenotypes. This scalable design philosophy offers new insights into the design of next generation of artificial cells-based biosensors.

DNA-Based Dynamic Reaction Networks.

Deriving from logical and mechanical interactions between DNA strands and complexes, DNA-based artificial reaction networks (RNs) are attractive for their high programmability, as well as cascading and fan-out ability, which are similar to the basic principles of electronic logic gates. Arising from the dream of creating novel computing mechanisms, researchers have placed high hopes on the development of DNA-based dynamic RNs and have strived to establish the basic theories and operative strategies of these networks. This review starts by looking back on the evolution of DNA dynamic RNs; in particular' the most significant applications in biochemistry occurring in recent years. Finally, we discuss the perspectives of DNA dynamic RNs and give a possible direction for the development of DNA circuits.

Artificial Signal Feedback Network Mimicking Cellular Adaptivity.

Inspired by this elegant system of cellular adaptivity, we herein report the rational design of a dynamic artificial adaptive system able to sense and respond to environmental stresses in a unique sense-and-respond mode. Utilizing DNA nanotechnology, we constructed an artificial signal feedback network and anchored it to the surface membrane of a model giant membrane vesicle (GMV) protocell. Such a system would need to both senses incoming stimuli and emit a feedback response to eliminate the stimuli. To accomplish this mechanistically, our DNA-based artificial signal system, hereinafter termed DASsys, was equipped with a DNA trigger-induced DNA polymer formation and dissociation machinery. Thus, through a sequential cascade of stimulus-induced DNA strand displacement, DASsys could effectively sense and respond to incoming stimuli. Then, by eliminating the stimulus, the membrane surface would return to its initial state, realizing the formation of a cyclical feedback mechanism. Overall, our strategy opens up a route to the construction of artificial signaling system capable of maintaining homeostasis in the cellular micromilieu, and addresses important emerging challenges in bioinspired engineering.

Generalized Preparation of Two-Dimensional Quasi-nanosheets via Self-assembly of Nanoparticles.

Two-dimensional (2D) nanomaterials are attracting increasing research interest because of their unique properties and promising applications. Here, we report a facile method to manipulate the assembly of nanoparticles (NPs) to fabricate free-standing 2D quasi-nanosheets. The as-generated 2D products are composed of few-layer NPs; that is, their thicknesses are only tens of nanometers but lateral dimensions could be up to several micrometers. Therefore, the novel structure was denoted as 2D "quasi-nanosheets (QNS)". Specifically, several types of building blocks could be assembled into 2D unary, binary, ternary, and even quaternary QNS by a universal procedure. The entire assembly process is carried out in solution and mediated simply by tuning the concentration of ligands surrounding the NPs. In contrast to traditional assembly techniques, even without any substrate or template, these QNS showed exceptionally high stability. They can remain intact for several days without any disassembly regardless of the solvent environment (e.g., water, ethanol, methanol, and hexane). In general, our method has effectively tackled several limitations associated with traditional assembly techniques and allows more freedom in manipulating assembly of NPs, which may hold great potential for future fabrication of 2D devices with rich functionalities.

Engineering a 3D DNA-Logic Gate Nanomachine for Bispecific Recognition and Computing on Target Cell Surfaces.

Among the vast number of recognition molecules, DNA aptamers generated from cell-SELEX exhibit unique properties for identifying cell membrane biomarkers, in particular protein receptors on cancer cells. To integrate all recognition and computing modules within a single structure, a three-dimensional (3D) DNA-based logic gate nanomachine was constructed to target overexpressed cancer cell biomarkers with bispecific recognition. Thus, when the Boolean operator "AND" returns a true value, it is followed by an "ON" signal when the specific cell type is presented. Compared with freely dispersed double-stranded DNA (dsDNA)-based molecular circuits, this 3D DNA nanostructure, termed DNA-logic gate triangular prism (TP), showed better identification performance, enabling, in turn, better molecular targeting and fabrication of recognition nanorobotics.

Identification and Characterization of DNA Aptamers Specific for Phosphorylation Epitopes of Tau Protein.

Tau proteins are proteins that stabilize microtubules, but their hyperphosphorylation can result in the formation of protein aggregates and, over time, neurodegeneration. This phenomenon, termed tauopathy, is pathologically involved in several neurodegenerative disorders. DNA aptamers are single-stranded oligonucleotides capable of specific binding to target molecules. Using tau epitopes predisposed for phosphorylation, we identified six distinct aptamers that bind to tau at two phosphorylatable epitopes (Thr-231 and Ser-202) and to full-length Tau441 proteins with nanomolar affinity. In addition, several of these aptamers also inhibit tau phosphorylation (IT4, IT5, IT6) and tau oligomerization (IT3, IT4, IT5, IT6). This is the first report to identify tau epitope-specific aptamers. Such tau aptamers can be used to detect tau in biofluids and uncover the mechanism of tauopathy. They can be further developed into novel therapeutic agents in mitigating tauopathy-associated neurodegenerative disorders.

Constructing Smart Protocells with Built-In DNA Computational Core to Eliminate Exogenous Challenge.

A DNA reaction network is like a biological algorithm that can respond to "molecular input signals", such as biological molecules, while the artificial cell is like a microrobot whose function is powered by the encapsulated DNA reaction network. In this work, we describe the feasibility of using a DNA reaction network as the computational core of a protocell, which will perform an artificial immune response in a concise way to eliminate a mimicked pathogenic challenge. Such a DNA reaction network (RN)-powered protocell can realize the connection of logical computation and biological recognition due to the natural programmability and biological properties of DNA. Thus, the biological input molecules can be easily involved in the molecular computation and the computation process can be spatially isolated and protected by artificial bilayer membrane. We believe the strategy proposed in the current paper, i.e., using DNA RN to power artificial cells, will lay the groundwork for understanding the basic design principles of DNA algorithm-based nanodevices which will, in turn, inspire the construction of artificial cells, or protocells, that will find a place in future biomedical research.

Portable and Label-Free Detection of Blood Bilirubin with Graphene-Isolated-Au-Nanocrystals Paper Strip.

Point-of-care testing (POCT) devices represent a growing field that aims to develop low-cost, rapid, sensitive diagnostic testing platforms that are portable and self-contained. Surface-enhanced Raman spectroscopy (SERS) is an approach has shown high potential in POCT technology. However, the specificity or ability to uniquely detect a desired biomarker in complex biological samples is a key factor for translating SERS technologies to POCT. Herein, we fabricated cellulose SERS strips (CS) decorated with novel plasmonic nanoparticles, termed graphene-isolated-Au-nanocrystals (GIANs), for the portable detection of complex biological samples. This CS@GIANs SERS strip was used to detect free bilirubin (BR) in the blood of newborns, a biomarker of jaundice, without sample labeling or prepreparation. CS@GIANs showed superior affinity to hydrophobic BR molecules compared to typical SERS substrate, which reduced the steric hindrance effect from the nonspecific binding of BR with serum albumin in blood and improved sensitivity. Meanwhile, with the separation property of cellulose chromatography papers, CS@GIANs showed superior anti-interference to other biomolecules that had been previously adsorbed on the SERS strip. Moreover, the SERS signal from the graphitic shell of GIANs could be used as a stable internal calibration standard, which improved the reproducibility and accuracy of Raman analysis. Such a cellulose SERS strip holds high potential for enhancing current efforts in the development of rapid and low-cost point-of-care diagnostic testing.

Comprehensive Regression Model for Dissociation Equilibria of Cell-Specific Aptamers.

A comprehensive nonlinear regression model for dissociation equilibria of cell-specific aptamers is proposed by considering the effect of receptor expression level. Benefiting from the global regression of simultaneous equations, the fitted parameters reach a very significant level, indicating the statistical validity of this updated model. According to the fitting results, we found that dissociation constants fitted using the previous model are obviously larger than the updated values, which can be explained by the effect of receptor number on curve fitting. In addition, equivalent receptor density can be estimated using the updated model, which may lead to some new judgments about reported results of cell-SELEX.

Cross-Linked Aptamer-Lipid Micelles for Excellent Stability and Specificity in Target-Cell Recognition.

The specific binding ability of DNA-lipid micelles (DLMs) can be increased by the introduction of an aptamer. However, supramolecular micellar structures based on self-assemblies of amphiphilic DLMs are expected to demonstrate low stability when interacting with cell membranes under certain conditions, which could lead to a reduction in selectivity for targeting cancer cells. We herein report a straightforward cross-linking strategy that relies on a methacrylamide branch to link aptamer and lipid segments. By an efficient photoinduced polymerization process, covalently linked aptamer-lipid units help stabilize the micelle structure and enhance aptamer probe stability, further improving the targeting ability of the resulting nanoassembly. Besides the development of a facile cross-linking method, this study clarifies the relationship between aptamer-lipid concentration and the corresponding binding ability.

Facile Assembly/Disassembly of DNA Nanostructures Anchored on Cell-Mimicking Giant Vesicles.

DNA nanostructures assembled on living cell membranes have become powerful research tools. Synthetic lipid membranes have been used as a membrane model to study the dynamic behavior of DNA nanostructures on fluid soft lipid bilayers, but without the inherent complexity of natural membranes. Herein, we report the assembly and disassembly of DNA nanoprisms on cell-mimicking micrometer-scale giant membrane vesicles derived from living mammalian cells. Three-dimensional DNA nanoprisms with a DNA arm and a cholesterol anchor were efficiently localized on the membrane surface. The assembly and disassembly of DNA nanoprisms were dynamically manipulated by DNA strand hybridization and toehold-mediated strand displacement. Furthermore, the heterogeneity of reversible assembly/disassembly of DNA nanoprisms was monitored by Förster resonance energy transfer. This study suggests the feasibility of DNA-mediated functional biomolecular assembly on cell membranes for biomimetics studies and delivery systems.

DNA-Based Molecular Machines: Controlling Mechanisms and Biosensing Applications.

The rise of DNA nanotechnology has driven the development of DNA-based molecular machines, which are capable of performing specific operations and tasks at the nanoscale. Benefitting from the programmability of DNA molecules and the predictability of DNA hybridization and strand displacement, DNA-based molecular machines can be designed with various structures and dynamic behaviors and have been implemented for wide applications in the field of biosensing due to their unique advantages. This review summarizes the reported controlling mechanisms of DNA-based molecular machines and introduces biosensing applications of DNA-based molecular machines in amplified detection, multiplex detection, real-time monitoring, spatial recognition detection, and single-molecule detection of biomarkers. The challenges and future directions of DNA-based molecular machines in biosensing are also discussed.

Dual-responsive 3D DNA nanomachines cascaded hybridization chain reactions for novel self-powered flexible microRNA-detecting platform.

The microRNA-21 is closely related to chromatin remodeling and epigenetic regulation. In this work, an efficient double-response 3D DNA nanomachine (DRDN) was assembled by co-immobilizing two different lengths of hairpin DNA on the surface of gold nanoparticles (AuNPs) to capture microRNA-21 (miRNA-21), recycle miRNA-21, and trigger hybridization chain reactions (HCR). This work reports the fabrication of a laser-scribed graphene (LSG) electrode with excellent flexibility and electrical conductivity by laser-scribing commercial polyimide films (PI). The as-proposed self-powered biosensing platform presents significantly increased instantaneous current to in real-time monitor miRNA-21 by a capacitor. The biosensing platform exhibited highly sensitive detection of miRNA-21 with a detection limit of 0.142 fM in the range of 0.5 fM to 1 × 104 fM, and demonstrated high efficiency in the analysis of the tumor markers.

Membrane-anchored DNA nanojunctions enable closer antigen-presenting cell-T-cell contact in elevated T-cell receptor triggering.

How the engagement of a T-cell receptor to antigenic peptide-loaded major histocompatibility complex on antigen-presenting cells (APCs) initiates intracellular signalling cascades in T cells is not well understood. In particular, the dimension of the cellular contact zone is regarded as a determinant, but its influence remains controversial. This is due to the need for appropriate strategies for manipulating intermembrane spacing between the APC-T-cell interfaces without involving protein modification. Here we describe a membrane-anchored DNA nanojunction with distinct sizes to extend, maintain and shorten the APC-T-cell interface down to 10 nm. Our results suggest that the axial distance of the contact zone is critical in T-cell activation, presumably by modulating protein reorganization and mechanical force. Notably, we observe the promotion of T-cell signalling by shortening the intermembrane distance.

A Dynamic Control Center Based on a DNA Reaction Network for Programmable Building of DNA Nanostructures.

DNA-based nanostructures allow for complex self-assembly with nanometer precision through the specificity of Watson-Crick base pairing, but network behavior-directed control of the kinetic process is less studied. Here we show how the DNA reaction network (DRN), which has emerged as a reliable and programmable way to implement artificial network dynamics, can be built as the control center of programmable nanostructures, allowing spatiotemporal control over the dynamic behavior of DNA nanotubes. We chose a common network motif in biological control systems, the feed-forward loop, as the model network and demonstrated that dynamic behaviors, such as self-tuning control and multilayer hierarchical assembly, could be programmed by constructing an inhibition network and an excitation network, separately, in buffer solution and inside protocells.

Robust Covalent Aptamer Strategy Enables Sensitive Detection and Enhanced Inhibition of SARS-CoV-2 Proteins.

Aptamer-based detection and therapy have made substantial progress with cost control and easy modification. However, the conformation lability of an aptamer typically causes the dissociation of aptamer-target complexes during harsh washes and other environmental stresses, resulting in only moderate detection sensitivity and a decreasing therapeutic effect. Herein, we report a robust covalent aptamer strategy to sensitively detect nucleocapsid protein and potently neutralize spike protein receptor binding domain (RBD), two of the most important proteins of SARS-CoV-2, after testing different cross-link electrophilic groups via integrating the specificity and efficiency. Covalent aptamers can specifically convert aptamer-protein complexes from the dynamic equilibrium state to stable and irreversible covalent complexes even in harsh environments. Covalent aptamer-based ELISA detection of nucleocapsid protein can surpass the gold standard, antibody-based sandwich ELISA. Further, covalent aptamer performs enhanced functional inhibition to RBD protein even in a blood vessel-mimicking flowing circulation system. The robust covalent aptamer-based strategy is expected to inspire more applications in accurate molecular modification, disease biomarker discovery, and other theranostic fields.