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Due to its excellent programmability and biocompatibility, DNA molecule has unique advantages in cell surface engineering. Recent progresses provide a reliable and feasible way to engineer cell surfaces with diverse DNA molecules and DNA nanostructures. The abundant form of DNA nanostructures has greatly expanded the toolbox of DNA-based cell surface engineering and gave rise to a variety of novel and fascinating applications. In this review, we summarize recent advances in DNA-based cell surface engineering and its biological applications. We first introduce some widely used methods of immobilizing DNA molecules on cell surfaces and their application features. Then we discuss the approaches of employing DNA nanostructures and dynamic DNA nanotechnology as elements for creating functional cell surfaces. Finally, we review the extensive biological applications of DNA-based cell surface engineering and discuss the challenges and prospects of DNA-based cell surface engineering., (© 2022 Wiley-VCH GmbH.)

DNA is well-documented to stimulate immune response. However, the nature of the DNA to activate immune surveillance is less understood. Here, we show that the activation of cyclic GMP-AMP synthase (cGAS) depends on DNA mechanical flexibility, which is controlled by DNA-sequence, -damage and -length. Consistently, DNA-sequence was shown to control cGAS activation. Structural analyses revealed that a conserved cGAS residue (mouse R222 or human R236) contributed to the DNA-flexibility detection. And the residue substitution neutralised the flexibility-controlled DNA-potential to activate cGAS, and relaxed the DNA-length specificity of cGAS. Moreover, low dose radiation was shown to mount cGAS-mediated acute immune surveillance (AIS) via repairable (reusable) DNAs in hrs. Loss of cGAS-mediated AIS decreased the regression of local and abscopal tumours in the context of focal radiation and immune checkpoint blockade. Our results build a direct link between immunosurveillance and DNA mechanical feature., (© 2022. The Author(s).)

Manipulation of cell-cell interactions via cell surface engineering has potential biomedical applications in tissue engineering and cell therapy. However, manipulation of the comprehensive and multiple intercellular interactions remains a challenge and missing elements. Herein, utilizing a DNA triangular prism (TP) and a branched polymer (BP) as functional modules, we fabricate tunable DNA scaffold networks on the cell surface. The responsiveness of cell-cell recognition, aggregation and dissociation could be modulated by aptamer-functionalized DNA scaffold networks with high accuracy and specificity. By regulating the DNA scaffold networks coated on the cell surface, controlled intercellular molecular transportation is achieved. Our tunable network provides a simple and extendible strategy which addresses a current need in cell surface engineering to precisely manipulate cell-cell interactions and shows promise as a general tool for controllable cell behavior., (© 2021 Wiley-VCH GmbH.)

Engineering cell-derived nanovesicles with active-targeting ligands is an important strategy to enhance the targeting efficiency. However, the enhanced binding capability to targeting cells also leads to the binding with nontarget cells that share the same biomarkers. DNA-based logic gate is a kind of molecular system that responds to chemical inputs by generating output signals, and the relationship between the input and the output is based on a certain logic. Thus, the DNA-based logic gate could provide a new approach to improve the delivery efficiency of the nanovesicle. In this work, we developed a DNA logic-gated module that coupled two tumor cell-targeting factors (e.g., low pH and a tumor cell biomarker) in a Boolean manner. Immobilization of this module on the surface of the nanovesicle enables the nanovesicle to sense tumor cell-targeting factors and regard these cues as inputs AND logic gate. With the guide of DNA-based logic gate, gold carbon dots (GCDs) encapsulated within nanovesicles were delivered into target cells, and then the intracellular redox status variation was reflected by fluorescence change of GCDs. Overall, we developed DNA logic-gated nanovesicles that contract different targeting factors into a unique tag for target cells. This facile functionalization strategy can pave the way for constructing smart nanovesicles and would broaden their application in the field of precision medicine and personalized treatment.

Cell-cell communication plays a vital role in biological activities; in particular, membrane-protein interactions are profoundly significant. In order to explore the underlying mechanism of intercellular signaling pathways, a full range of artificial systems have been explored. However, many of them are complicated and uncontrollable. Herein we designed an artificial signal transduction system able to control the influx of environmental ions by triggering the activation of synthetic transmembrane channels immobilized on giant membrane vesicles (GMVs). A membrane protein-like stimulator from one GMV community (GMV B ) stimulates a receptor on another GMV community (GMV A ) to release ssDNA messengers, resulting in the activation of synthetic transmembrane channels to enable the influx of ions. This event, in turn, triggers signal responses encapsulated in the GMV A protocell model. By mimicking natural signal transduction pathways, this novel prototype provides a workable tool for investigating cell-cell communication and expands biological signaling systems in general as well as explores useful platforms for addressing scientific problems which involve materials science, chemistry, and medicine.

In order to maintain tissue homeostasis, cells communicate with the outside environment by receiving molecular signals, transmitting them, and responding accordingly with signaling pathways. Thus, one key challenge in engineering molecular signaling systems involves the design and construction of different modules into a rationally integrated system that mimics the cascade of molecular events. Herein, we rationally design a DNA-based artificial molecular signaling system that uses the confined microenvironment of a giant vesicle, derived from a living cell. This system consists of two main components. First, we build an adenosine triphosphate (ATP)-driven DNA nanogatekeeper. Second, we encapsulate a signaling network in the biomimetic vesicle, consisting of distinct modules, able to sequentially initiate a series of downstream reactions playing the roles of reception, transduction and response. Operationally, in the presence of ATP, nanogatekeeper switches from the closed to open state. The open state then triggers the sequential activation of confined downstream signaling modules.

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.

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.

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.

DNA molecular machines show great promise in fields such as biomarker discovery and biological activity regulation, but operating DNA machines with specific functions within living systems remains extremely challenging. Although DNA machines have been engineered with exact molecular-level specifications, some intrinsic imperfections such as poor cell permeation and fragility in complex cytoplasmic milieu persist due to the well-established character of nucleic acid molecules. To circumvent these problems, we herein report a molecularly engineered, entropy-driven three-dimensional DNA amplifier (EDTD) that can operate inside living cells in response to a specific mRNA target. In particular, mRNA target/EDTD interaction can specifically initiate an autonomous DNA circuit inside living cells owing to the exclusive entropy-driven force, thus providing enormous signal amplification for ultrasensitive detection of the mRNA. Moreover, owing to molecular engineering of a unique DNA tetrahedral framework into the DNA amplifier, EDTD exhibits significantly enhanced biostability and cellular uptake efficiency, which are prerequisites for DNA machines used for in vivo applications. This programmable DNA machine presents a simple and modular amplification mechanism for the detection of intracellular biomarkers. Moreover, this study provides a potentially valuable molecular tool for understanding the chemistry of cellular systems and offers a design blueprint for further expansion of DNA nanotechnology in living systems.

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.

In contrast to small molecules, DNA and RNA macromolecules can be accurately formulated with base "elements" abbreviated as A, T, U, C, and G. However, the development of functionally artificial bases can result in the generation of new biomaterials with unique properties and applications. Therefore, we herein report the design and synthesis of a photoresponsive base as a new functional or molecular "element" for constructing DNA nanomolecules. The new base is made by fusion of an azobenzene with a natural T base (zT). zT, a new molecular element, is not only the most size-expanded T analogue but also a photoresponsive base capable of specific self-assembly through hydrogen bonding. Our results showed that stable and selective self-assembly of double-stranded DNAs occurred through zT-A base pairing, but it could still be efficiently dissociated by light irradiation. The photoresponsive DNA bases will provide the versatility required for constructing desired DNA nanomolecules and nanodevices.

We describe a comprehensive protocol for the preparation of multifunctional DNA nanostructures termed nanoflowers (NFs), which are self-assembled from long DNA building blocks generated via rolling-circle replication (RCR) of a designed template. NF assembly is driven by liquid crystallization and dense packaging of building blocks, which eliminates the need for conventional Watson-Crick base pairing. As a result of dense DNA packaging, NFs are resistant to nuclease degradation, denaturation or dissociation at extremely low concentrations. By manually changing the template sequence, many different functional moieties including aptamers, bioimaging agents and drug-loading sites could be easily integrated into NF particles, making NFs ideal candidates for a variety of applications in biomedicine. In this protocol, the preparation of multifunctional DNA NFs with highly tunable sizes is described for applications in cell targeting, intracellular imaging and drug delivery. Preparation and characterization of functional DNA NFs takes ∼5 d; the following biomedical applications take ∼10 d.

A novel fluorescence aptasensor based on DNA charge transport for sensitive protein detection has been developed. A 15nt DNA aptamer against thrombin was used as a model system. The aptamer was integrated into a double strand DNA (dsDNA) that was labeled with a hole injector, naphthalimide (NI), and a fluorophore, Alexa532, at its two ends. After irradiation by UV light, the fluorescence of Alexa532 was bleached due to the oxidization of Alexa532 by the positive charge transported from naphthalimide through the dsDNA. In the presence of thrombin, the binding of thrombin to the aptamer resulted in the unwinding of the dsDNA into ssDNA, which led to the blocking of charge transfer and the strong fluorescence emission of Alexa532. By monitoring the fluorescence signal change, we were able to detect thrombin in homogeneous solutions with high selectivity and high sensitivity down to 1.2 pM. Moreover, as DNA charge transfer is resistant to interferences from biological contexts, the aptasensor can be used directly in undiluted serum with similar sensitivity as that in buffer. This new sensing strategy is expected to promote the exploitation of aptamer-based biosensors for protein assays in complex biological matrixes.

Manipulation of cell–cell interactions via cell surface engineering has potential biomedical applications in tissue engineering and cell therapy. However, manipulation of the comprehensive and multiple intercellular interactions remains a challenge and missing elements. Herein, utilizing a DNA triangular prism (TP) and a branched polymer (BP) as functional modules, we fabricate tunable DNA scaffold networks on the cell surface. The responsiveness of cell–cell recognition, aggregation and dissociation could be modulated by aptamer‐functionalized DNA scaffold networks with high accuracy and specificity. By regulating the DNA scaffold networks coated on the cell surface, controlled intercellular molecular transportation is achieved. Our tunable network provides a simple and extendible strategy which addresses a current need in cell surface engineering to precisely manipulate cell–cell interactions and shows promise as a general tool for controllable cell behavior. [ABSTRACT FROM AUTHOR]

In the past two decades, the study of cancer therapy has gradually advanced to the 'nano' era. Numerous novel nanomaterials armed with unique physical properties have been introduced into biomedical research. At the same time, functional nucleic acid molecules, especially aptamers, have aroused broad attention from the biomedical community. Benefiting from the advancement of molecular engineering strategies, it is now feasible to combine the cancer-specific recognition capability of aptamers with various other special functions of nanomaterials to develop cancer-specific drugs at the nanoscale. Nanodrugs are now offering an unprecedented opportunity to achieve the goal of efficient targeted delivery as well as controlled release. This review highlights some achievements made in multiple aptamer-based nanodrug systems that have emerged in recent years, including studies in the infant stage of 'proof-of-concept'. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Self-nonself discrimination is vital for the immune system to mount responses against pathogens while maintaining tolerance toward the host and innocuous commensals during homeostasis. Here, we investigated how indiscriminate DNA sensors, such as cyclic GMP-AMP synthase (cGAS), make this self-nonself distinction. Screening of a small-molecule library revealed that spermine, a well-known DNA condenser associated with viral DNA, markedly elevates cGAS activation. Mechanistically, spermine condenses DNA to enhance and stabilize cGAS-DNA binding, optimizing cGAS and downstream antiviral signaling. Spermine promotes condensation of viral, but not host nucleosome, DNA. Deletion of viral DNA-associated spermine, by propagating virus in spermine-deficient cells, reduced cGAS activation. Spermine depletion subsequently attenuated cGAS-mediated antiviral and anticancer immunity. Collectively, our results reveal a pathogenic DNA-associated molecular pattern that facilitates nonself recognition, linking metabolism and pathogen recognition. [Display omitted] • Spermine can act as a pathogenic DNA-associated molecular pattern • Spermine condenses naked DNA (such as viral DNA) but not nucleosomal DNA • Spermine condenses DNA to optimize cGAS-DNA binding and activation • Spermine depletion reduces cGAS-mediated antiviral and antitumor surveillance Self-nonself discrimination is central to effective immunity, but how intracellular DNA is discriminated remains elusive. Here, Wang et al. show that spermine condenses naked DNA (including viral DNA) but not nucleosomal DNA to promote cGAS binding and downstream immune activation, thus providing a mechanism for self-nonself recognition. [ABSTRACT FROM AUTHOR]