Programming Affinity for Precise Tumor Recognition with Allosteric Nanosensing-Circles.

Although many smart probes for precise tumor recognition have been reported, the challenge of "on-target, off-tumor" remains. Therefore, we herein report the fabrication of a series of allosterically tunable DNA nanosensing-circles (NSCs). The recognition affinity of NSCs is programmed through sensitivity to tumor microenvironment (TME) hallmarks such as small molecules, acidity, or oncoproteins. Because of their special programming conditions and active targeting capabilities, NSCs can overcome the obstacles noted above, thus achieving precise tumor recognition. Results from in vitro analysis demonstrated that NSCs obtain their recognition ability through allosteric regulation after sensing TME hallmarks. Furthermore, in vivo imaging indicated that NSCs enable precise tumor imaging. These results demonstrate that our NSCs will be promising tools for precise tumor imaging and therapy.

An Aptamer-Functionalized DNA Circuit to Establish an Artificial Interaction between T Cells and Cancer Cells.

Nongenetic strategies that enable control over the cell-cell interaction network would be highly desired, particularly in T cell-based cancer immunotherapy. In this work, we developed an aptamer-functionalized DNA circuit to modulate the interaction between T cells and cancer cells. This DNA circuit was composed of recognition-then-triggering and aggregation-then-activation modules. Upon recognizing target cancer cells, the triggering strand was released to induce aggregation of immune receptors on the T cell surface, leading to an enhancement of T cell activity for effective cancer eradication. Our results demonstrated the feasibility of this DNA circuit for promoting target cancer cell-directed stimulation of T cells, which, consequently, enhanced their killing effect on cancer cells. This DNA circuit, as a modular strategy to modulate intercellular interactions, could lead to a new paradigm for the development of nongenetic T cell-based immunotherapy.

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.

Aptamer-based expansion microscopy platform enables signal-amplified imaging of dendritic spines.

Super-resolution imaging of dendritic spines (DS) can provide valuable information for mechanistic studies related to synaptic physiology and neural plasticity, but challenged by their small dimension (50-200 nm) below the spatial resolution of conventional optical microscopes. In this work, by combining the molecular recognition specificity of aptamer with high programmability of DNA nanotechnology, we developed an expansion microscopy (ExM) platform for imaging DS with enhanced spatial resolution and amplified signal output. Our results demonstrated that the aptamer probe could specifically bind to DS of primary hippocampal neurons. With physical expansion, the DS structure could be effectively enlarged by 4-5 folds, leading to the generation of more structural information. Meantime, the aptamer binding signal could be readily amplified by the introduction of DNA signal amplification strategy, overcoming the drawback of fluorescence dilution during the ExM treatment. This platform enabled evaluation of ischemia-induced early stroke based on the morphological change of DS, highlighting a promising avenue for studying nanoscale structures in biological systems.

Aptamer-Based Targeted Delivery of Functional Nucleic Acids.

Functional nucleic acid (NA)-based drugs have a broad range of applications since they allow the alteration and control of gene/protein expression patterns in cells. In principle, functional NAs need to be transported precisely and efficiently to target cells to guarantee both functionality and safety. Owing to their negative charges, it is difficult for natural NAs to cross the cell membrane composed of lipid bilayer and enter targeted cells. Worse still, the delivery of undirected functional NAs to nontargeted healthy cells and/or tissues would induce unpredictable adverse effects. Therefore, the precisely targeted delivery of functional NAs to specific cells/organs, particularly in extrahepatic sites, is required. Since aptamers can bind to various proteins on the cell surface with high specificity and selectivity, they can serve as the molecular recognition units to accurately bind target cells and subsequently enable the efficient delivery of cargo. In this perspective, we summarize the original, proof-of-concept aptamer-based strategies for the targeted delivery of functional NAs. A few specific examples are then discussed, followed by our perspectives on some of the challenges and opportunities that lie ahead.

Bioinspired molecular engineering of bivalent aptamers by ligand-induced self-dimerization.

A bioinspired "point-and-shoot" molecular strategy is described for engineering noncovalent aptamers via K+-induced reassembly of a split G-quadruplex sequence at the end of monomeric aptamers. This design compensates for insufficient cell recognition of monovalent aptamers in biological environments and minimizes structure-induced nonspecific cell binding, expending the aptamer toolbox available for complex systems.

X-Ray-triggered Carbon Monoxide and Manganese Dioxide Generation based on Scintillating Nanoparticles for Cascade Cancer Radiosensitization.

Carbon monoxide (CO) is an endogenous signaling molecule with broad therapeutic effects. Here, a multifunctional X-ray-triggered carbon monoxide (CO) and manganese dioxide (MnO2 ) generation nanoplatform based on metal carbonyl and scintillating nanoparticles (SCNPs) is reported. Attributed to the radioluminescent characteristic of SCNPs, UV-responsive Mn2 (CO)10 is not only indirectly activated to release CO by X-ray but can also be degraded into MnO2 . A high dose of CO can be used as a glycolytic inhibitor for tumor suppression; it will also sensitize tumor cells to radiotherapy. Meanwhile MnO2 , as the photolytic byproduct of Mn2 (CO)10 , has both glutathione (GSH) depletion and Fenton-like Mn2+ delivery properties to produce highly toxic hydroxyl radical (⋅OH) in tumors. Thus, this strategy can realize X-ray-activated CO release, GSH depletion, and ⋅OH generation for cascade cancer radiosensitization. Furthermore, X-ray-activated Mn2+ in vivo demonstrates an MRI contrast effect, making it a potential theranostic nanoplatform.

Aptamer-Based Targeted Protein Degradation.

The selective removal of misfolded, aggregated, or aberrantly overexpressed protein plays an essential role in maintaining protein-dominated biological processes. In parallel, the precise knockout of abnormal proteins is inseparable from the accurate identification of proteins within complex environments. Guided by these precepts, small molecules, or antibodies, are commonly used as protein recognition tools for developing targeted protein degradation (TPD) technology. Indeed, TPD has shown tremendous prospects in chronic diseases, rare diseases, cancer research, and other fields. Meanwhile, aptamers are short RNA or DNA oligonucleotides that can bind to target proteins with high specificity and strong affinity. Accordingly, aptamers are actively used in designing and constructing TPD technology. In this perspective, we provide a brief introduction to TPD technology in its current progress, and we summarize its application challenges. Recent advances in aptamer-based TPD technology are reviewed, together with corresponding challenges and outlooks.

Molecular elements: novel approaches for molecular building.

Classically, a molecular element (ME) is a pure substance composed of two or more atoms of the same element. However, MEs, in the context of this review, can be any molecules as elements bonded together into the backbone of synthetic oligonucleotides (ONs) with designed sequences and functions, including natural A, T, C, G, U, and unnatural bases. The use of MEs can facilitate the synthesis of designer molecules and smart materials. In particular, we discuss the landmarks associated with DNA structure and related technologies, as well as the extensive application of ONs, the ideal type of molecules for intervention therapy aimed at correcting disease-causing genetic errors (indels). It is herein concluded that the discovery of ON therapeutics and the fabrication of designer molecules or nanostructures depend on the ME concept that we previously published. Accordingly, ME will be our focal point as we discuss related research directions and perspectives in making molecules and materials. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.

Artificial Nucleotide Aptamer-Based Field-Effect Transistor for Ultrasensitive Detection of Hepatoma Exosomes.

An aptamer-based field-effect transistor (Apta-FET) is a well-developed assay method with high selectivity and sensitivity. Due to the limited information density that natural nucleotide library holds, the Apta-FET faces fundamental restriction in universality to detect various types of analytes. Herein, we demonstrate a type of Apta-FET sensors based on an artificial nucleotide aptamer (AN-Apta-FET). The introduction of an artificial nucleotide increases the diversity of the potential aptamer structure and expands the analyte category of the Apta-FET. The AN-Apta-FET specifically detects hepatoma exosomes, which traditional Apta-FET fails to discriminate from other tumor-derived exosomes, with a limit of detection down to 242 particles mL-1. The AN-Apta-FET distinguishes serum samples of hepatocellular carcinoma patients within 9 min from those of healthy people, showing the potential as a comprehensive assay tool in future disease diagnosis.

Bispecific Aptamer-Based Recognition-then-Conjugation Strategy for PD1/PDL1 Axis Blockade and Enhanced Immunotherapy.

Cytotoxic T cells initiate antitumor effects mainly through direct interactions with tumor cells. As a counter to this, tumor cells can put the brakes on such T-cell activity via specific linkage between programmed death ligand 1 (PDL1) and its receptor programmed cell death protein 1 (PD1). Bispecific inhibitors that enabled synchronous blockade of PD1 and PDL1, thereby releasing the brakes on T-cell antitumor activity, should significantly improve the efficacy of immune checkpoint blockade (ICB) therapy. In this work, we identified a DNA aptamer, Ap3, that could specifically recognize PDL1 on tumor cells and competed with the binding of PD1. By integrating Ap3 with an anti-PD1 aptamer, the bispecific aptamer Ap3-7c was constructed, and it showed promise for improving the T-cell immune response. We further designed a dibenzocyclooctyne (DBCO)-labeled bispecific aptamer, D-Ap3-7c, allowing covalent conjugation of aptamers onto PD1 and PDL1 after specific cell recognition. Our in vivo studies showed that this recognition-then-conjugation strategy could induce a potent immunological effect against tumors. This work is expected to provide clues for antitumor immunotherapy.

A pH-Responsive Covalent Nanoscale Device Enhancing Temporal and Force Stability for Specific Tumor Imaging.

Approaches to DNA probe-mediated precision medicine have been extensively explored for the diagnosis and treatment of diverse types of cancer. Despite this, simple nanoscale devices with the required recognition specificity and sensitivity for clinical application have remained elusive until now. Here, we report a pH-driven covalent nanoscale device that integrates pH-responsive, switchable structure and proximity-driven covalent cross-linking. A tumor acidic, pH-driven mechanism eliminates "on-target, off-tumor" nonspecific recognition. By manipulating covalent binding to target molecule on the cell surface, this nanodevice avoids binding-then-shedding to improve the sensitivity of tumor recognition. We envision that this pH-driven covalent nanoscale device will inspire more clinical applications toward specific, long-term tumor imaging in the cancer microenvironment.

Oral antibiotics relieve allergic asthma in post-weaning mice via reducing iNKT cells and function of ADRB2.

The role of normal gut microbiota in asthma or ovalbumin (OVA)-induced asthma tolerance (OT) remains unclear. Here, we established mouse models of asthma and OT followed by 2 weeks of antibiotic treatment, to clear the gut microbiota. Antibiotic treatment was found to alleviate allergic asthma accompanied with a reduction of invariant natural killer (iNKT) cells. By RNA-seq analysis, we found that ß-adrenergic receptor (ADRB) genes, including Adrb1, Adrb2, and Adrb3, were downregulated in asthmatic lungs, but these changes were reversed in OT lungs. Moreover, Adrb2 and Adrb3 were significantly upregulated in asthmatic lungs after antibiotic treatment. Surprisingly, blocking ADRB with propranolol relieved allergic asthma while reducing T helper 2 (Th2) and Treg cell numbers. Further analyses using flow cytometry and immunofluorescence showed that the protein expression level of ADRB2 was higher in asthmatic lungs than that in the control and OT lungs. Notably, dendritic cells (DCs), especially the ADRB2+ DCs, were increased in asthmatic lungs compared to that in the control and OT lungs. In addition, ADRB2+ DCs were significantly reduced following the administration of the ADRB2-specific antagonist ICI118551. Our findings suggest that antibiotic treatment can alleviate OVA-induced allergic asthma via reducing the frequency of iNKT cells and function of ADRB2.

Aptamer-Functionalized Nanodevices for Dynamic Manipulation of Membrane Receptor Signaling in Living Cells.

The capacity to regulate the signaling amplitude of membrane receptors in a user-defined manner would open various opportunities for precise biological study and therapy. While partial agonists enabled downtuning of cellular responses, they required esoteric optimization of the ligand-receptor interface, limiting their practical applications. Herein, we developed an aptamer-functionalized, tweezer-like nanodevice to dynamically modulate the cellular behavior through control over the distance between receptors in the dimer with no need to involve complicated structural analysis. By combining a reversible conformation switch with aptamer-based molecular recognition, this nanodevice showed excellent performance on dynamic regulation of CD28 receptor-mediated T cell immunity. With the modular design, this nanodevice could be extended to dynamically modulate the activity of other membrane receptors (e.g., c-Met), expecting to offer a new paradigm for precise study and manipulation of specific molecular events in complex biological systems.

Engineering Aptamers with Selectively Enhanced Biostability in the Tumor Microenvironment.

Aptamers are emerging as promising molecular tools in cancer-targeted theranostics. Improving their in vivo stability has been a critical issue in promoting clinical translation, but such efforts could lead to more serious side effects resulting from prolonged retention in healthy organs. To address this problem, we developed an environment-responsive stabilization strategy for the selective enhancement of aptamer biostability in the tumor microenvironment (TME). Briefly, by means of the end extension of an ATP-responsive protection (ARP) module, the designed aptamer could be protected from nuclease degradation through the specific incorporation of ATP. Based on our in vivo results, this ARP-aptamer probe was effectively accumulated in tumors via aptamer-based molecular recognition. It showed selectively prolonged tumor retention time, but rapid digestion in healthy organs. Our strategy should provide a new paradigm for the development of organ-specific nucleic acid-based imaging and therapeutic agents.

Aptasensors for Cancerous Exosome Detection.

Cancerous exosomes that carry multiple biomarkers are attractive targets for the early diagnosis and therapy of cancer. As one of the powerful molecular recognition tools, aptamers with excellent binding affinity and specificity toward biomarkers have been exploited to construct various aptamer-based biosensors (aptasensors) for exosome detection. Here, we review recent advances in aptasensors for the detection of cancerous exosomes. We first discuss the importance and potential of cancerous exosomes in cancer diagnosis and then summarize some conventional aptasensors from the perspective of biomarker recognition and signal collection strategies. Finally, we comment on the outlook for aptasensor research and new directions for cancerous exosome detection.

Functional Aptamer-Embedded Nanomaterials for Diagnostics and Therapeutics.

In the past decades, various nanomaterials with unique properties have been explored for bioapplications. Meanwhile, aptamers, generated from the systematic evolution of ligands by exponential enrichment technology, are becoming an indispensable element in the design of functional nanomaterials because of their small size, high stability, and convenient modification, especially endowing nanomaterials with recognition capability to specific targets. Therefore, the incorporation of aptamers into nanomaterials offers an unprecedented opportunity in the research fields of diagnostics and therapeutics. Here, we focus on recent advances in aptamer-embedded nanomaterials for bioapplications. First, we briefly introduce the properties of nanomaterials that can be functionalized with aptamers. Then, the applications of aptamer-embedded nanomaterials in cellular analysis, imaging, targeted drug delivery, gene editing, and cancer diagnosis/therapy are discussed. Finally, we provide some perspectives on the challenges and opportunities that have arisen from this promising area.

Decoding the Complex Free Radical Cascade by Using a DNA Framework-Based Artificial DNA Encoder.

DNA-based molecular communications (DMC) are critical for regulating biological networks to maintain stable organismic functions. However, the complicated, time-consuming information transmission process involved in genome-coded DMC and the limited, vulnerable decoding activity generally lead to communication impairment or failure, in response to external stimuli. Herein, we present a conceptually innovative DMC strategy mediated by the DNA framework-based artificial DNA encoder. With the free-radical cascade as a proof-of-concept study, the artificial DNA encoder shows active sensing and real-time actuation, in situ and broad free radical-decoding efficacy, as well as robust resistance to environmental noise. It can also block undesirable short-to-medium-range communications between free radicals and inflammatory networks, leading to a synergistic anti-obesity effect. The artificial DNA encoder-based DMC may be generalized to other communication systems for a variety of applications.

Transducing Complex Biomolecular Interactions by Temperature-Output Artificial DNA Signaling Networks.

The requirement of special expensive instruments for quantitative information readout has significantly restricted sustainable development, from ideation to execution, of advanced artificial networks. Here we present a step toward a paradigm of evolutionary signaling networks that enable translating complex signaling information into easy-to-read temperature output. Combining DNA molecular engineering with basic optical mechanisms, a DNA/Hemin complex-derived versatile temperature-output transducer is established, which can be coupled with other functional modules to fabricate diverse portable DNA signaling networks by dynamic programming of DNA chemical reactions. Its versatility is successfully demonstrated by constructing self-amplified and logic-circuit-based DNA signaling networks to monitor trace and multibit nucleic acid interactions using a thermometer. This affordable yet powerful DNA signaling network design may portend an era of point-of-care signaling network methodology.

Construction of Bispecific Aptamer-Drug Conjugate by a Hybrid Chemical and Biological Approach.

Bispecific aptamer-drug conjugates (BsApDC) may improve the efficacy of drugs by enhancing cellular internalization and targeted delivery. Nevertheless, the synthesis of single-molecular BsApDC has not yet been reported, and it could be thwarted by synthetic challenges. Herein we report a general approach to synthesize a BsApDC hybridized chemical and biological method. Primers incorporated with 5-Fluorouracil (5-FU), 10-Hydroxycamptothecin, and Maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl-monomethyl auristatin E(vcMMAE) were prepared by chemical synthesis, which were converted to corresponding ApDCs efficiently by enzymatic reaction. Biological studies revealed that BsApDC binds with target cells with enhanced internalization and better inhibitory activity, demonstrating the potential of BsApDCs for targeted tumor therapy.