Leveraging DNA-encoded Cell-mimics for Environment-adaptive Transmembrane Channel Release-induced Cell Death.

The advancement of cell-mimic materials, which can forge sophisticated physicochemical dialogues with living cells, has unlocked a realm of intriguing prospects within the fields of synthetic biology and biomedical engineering. Inspired by the evolutionarily acquired ability of T lymphocytes to release perforin and generate transmembrane channels on targeted cells for killing, herein we present a pioneering DNA-encoded artificial T cell mimic model (ARTC) that accurately mimics T-cell-like behavior. ARTC responds to acidic conditions similar to those found in the tumor microenvironment and then selectively releases a G-rich DNA strand (LG4) embedded with C12 lipid and cholesterol molecules. Once released, LG4 effectively integrates into the membranes of neighboring live cells, behaving as an artificial transmembrane channel that selectively transports K+ ions and disrupts cellular homeostasis, ultimately inducing apoptosis. We hope that the emergence of ARTC will usher in new perspectives for revolutionizing future disease treatment and catalyzing the development of advanced biomedical technologies.

Polyfluoroalkyl Tag Decoration Enables Significantly Enhanced Tumor Penetration Ability of a PTK7 Targeting Aptamer.

Aptamers are widely used molecular recognition tools in targeted therapy, but their ability to effectively penetrate deep into solid tumors remains a significant challenge, leading to suboptimal treatment efficacy. Here, we developed a polyfluoroalkyl (PFA) decoration strategy to enhance aptamer recognition, cell internalization, and solid tumor penetration. Our results indicate that PFA with around 11 fluorine atoms significantly improves aptamer internalization both in vitro and in vivo settings. However, we also observed that the use of PFA tags containing 19 and 23 fluorine atoms on aptamers resulted in nonspecific cell anchoring in control cell lines, affecting the specificity of aptamers. Overall, we found that using a chemical modification strategy could enhance the deep tumor penetration ability of aptamers and validate their effectiveness in vivo. This approach has significant practical applications in targeted drug delivery for cancer treatment.

DNA Reaction Circuits to Establish Designated Biological Functions in Multicellular Community.

In multicellular organisms, individual cells are coordinated through complex communication networks to accomplish various physiological tasks. Aiming to establish new biological functions in the multicellular community, we used DNA as the building block to develop a cascade of nongenetic reaction circuits to establish a dynamic cell-cell communication network. Utilizing membrane-anchored amphiphilic DNA tetrahedra (TDN) as the nanoscaffold, reaction circuits were incorporated into three unrelated cells in order to uniquely regulate their sense-and-response behaviors. As a proof-of-concept, this step enabled these cells to simulate significant biological events involved in T cell-mediated anticancer immunity. Such events included cancer-associated antigen recognition and the presentation of antigen-presenting cells (APCs), APC-facilitated T cell activation and dissociation, and T cell-mediated cancer targeting and killing. By combining the excellent programmability and molecular recognition ability of DNA, our cell-surface reaction circuits hold promise for mimicking and manipulating many biological processes.

Neuronal basis and diverse mechanisms of pathogen avoidance in Caenorhabditis elegans.

Pathogen avoidance behaviour has been observed across animal taxa as a vital host-microbe interaction mechanism. The nematode Caenorhabditis elegans has evolved multiple diverse mechanisms for pathogen avoidance under natural selection pressure. We summarise the current knowledge of the stimuli that trigger pathogen avoidance, including alterations in aerotaxis, intestinal bloating, and metabolites. We then survey the neural circuits involved in pathogen avoidance, transgenerational epigenetic inheritance of pathogen avoidance, signalling crosstalk between pathogen avoidance and innate immunity, and C. elegans avoidance of non-Pseudomonas bacteria. In this review, we highlight the latest advances in understanding host-microbe interactions and the gut-brain axis.

A DNA circuit that records molecular events.

Characterizing the relative onset time, strength, and duration of molecular signals is critical for understanding the operation of signal transduction and genetic regulatory networks. However, detecting multiple such molecules as they are produced and then quickly consumed is challenging. A MER can encode information about transient molecular events as stable DNA sequences and are amenable to downstream sequencing or other analysis. Here, we report the development of a de novo molecular event recorder that processes information using a strand displacement reaction network and encodes the information using the primer exchange reaction, which can be decoded and quantified by DNA sequencing. The event recorder was able to classify the order at which different molecular signals appeared in time with 88% accuracy, the concentrations with 100% accuracy, and the duration with 75% accuracy. This simultaneous and highly programmable multiparameter recording could enable the large-scale deciphering of molecular events such as within dynamic reaction environments, living cells, or tissues.

Aptamer-Based Nongenetic Reprogramming of CARs Enables Flexible Modulation of T Cell-Mediated Tumor Immunotherapy.

Innovating the design of chimeric antigen receptors (CARs) beyond conventional structures would be necessary to address the challenges of efficacy, safety, and applicability in T cell-based cancer therapy, whereas excessive genetic modification might complicate CAR design and manufacturing, and increase gene editing risks. In this work, we used aptamers as the antigen-recognition unit to develop a nongenetic CAR engineering strategy for programming the antitumor activity and specificity of CAR T cells. Our results demonstrated that aptamer-functionalized CAR (Apt-CAR) T cells could be directly activated by recognizing target antigens on cancer cells, and then impart a cytotoxic effect for cancer elimination in vitro and in vivo. The designable antigen recognition capability of Apt-CAR T cells allows for easy modulation of their efficacy and specificity. Additionally, multiple features, e.g., tunable antigen-binding avidity and the tumor microenvironment responsiveness, could be readily integrated into Apt-CAR design without T cell re-engineering, offering a new paradigm for developing adaptable immunotherapeutics.

Aptamer Technologies in Neuroscience, Neuro-Diagnostics and Neuro-Medicine Development.

Aptamers developed using in vitro Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technology are single-stranded nucleic acids 10-100 nucleotides in length. Their targets, often with specificity and high affinity, range from ions and small molecules to proteins and other biological molecules as well as larger systems, including cells, tissues, and animals. Aptamers often rival conventional antibodies with improved performance, due to aptamers' unique biophysical and biochemical properties, including small size, synthetic accessibility, facile modification, low production cost, and low immunogenicity. Therefore, there is sustained interest in engineering and adapting aptamers for many applications, including diagnostics and therapeutics. Recently, aptamers have shown promise as early diagnostic biomarkers and in precision medicine for neurodegenerative and neurological diseases. Here, we critically review neuro-targeting aptamers and their potential applications in neuroscience research, neuro-diagnostics, and neuro-medicine. We also discuss challenges that must be overcome, including delivery across the blood-brain barrier, increased affinity, and improved in vivo stability and in vivo pharmacokinetic properties.

Engineering a Dual-Receptor Targeted Multivalent Probe for Enhanced Magnetic Resonance Imaging of Metastatic Cancer.

Noninvasive monitoring of cancer metastasis is essential to improving clinical outcomes. Molecular MRI (mMRI) is a special implementation of noninvasive molecular imaging that promises to offer a powerful means for early detection and analysis of pathological states of cancer by tracking molecular markers. However, this is often hindered by the challenging issue of obtaining transformable mMRI contrast agents with high sensitivity, specificity, and broad applicability, given the high tumor heterogeneity and complex metastatic features. Herein, we present a dual-receptor targeted, multivalent recognition strategy and report a new class of mMRI probes for enhanced imaging of metastatic cancer. This probe is designed by covalently conjugating Gd-chelate with phenylboronic acid and an aptamer via an affordable polymerization chemistry to concurrently target two different cell-membrane receptors that are commonly overexpressed and highly implicated in both tumorigenesis and metastasis. Moreover, the polymerization chemistry allows the probe to contain a bunch of targeting ligands and signal reporters in a single chain, which not only leads to more than 2-fold enhancement in T1 relaxivity at 1.5 T compared to the commercial contrast agent but also enables it to actively target tumor cells in a multivalent recognition manner, contributing to a much higher imaging contrast than single-receptor targeted probes and the commercial agent in mouse models with lung metastases, yet without inducing systemic side effects. We expect this study to offer a useful molecular tool to promote transformable applications of mMRI and a better understanding of molecular mechanisms involved in cancer development.

Enhancing Specific Fluorescence In Situ Hybridization with Quantum Dots for Single-Molecule RNA Imaging in Formalin-Fixed Paraffin-Embedded Tumor Tissues.

Single-molecule fluorescence in situ hybridization (smFISH) represents a promising approach for the quantitative analysis of nucleic acid biomarkers in clinical tissue samples. However, low signal intensity and high background noise are complications that arise from diagnostic pathology when performed with smFISH-based RNA imaging in formalin-fixed paraffin-embedded (FFPE) tissue specimens. Moreover, the associated complex procedures can produce uncertain results and poor image quality. Herein, by combining the high specificity of split DNA probes with the high signal readout of ZnCdSe/ZnS quantum dot (QD) labeling, we introduce QD split-FISH, a high-brightness smFISH technology, to quantify the expression of mRNA in both cell lines and clinical FFPE tissue samples of breast cancer and lung squamous carcinoma. Owing to its high signal-to-noise ratio, QD split-FISH is a fast, inexpensive, and sensitive method for quantifying mRNA expression in FFPE tumor tissues, making it suitable for biomarker imaging and diagnostic pathology.

Domain-Targeted Membrane Partitioning of Specific Proteins with DNA Nanodevices.

The cell membrane exhibits a remarkable complexity of lipids and proteins that dynamically segregate into distinct domains to coordinate various cellular functions. The ability to manipulate the partitioning of specific membrane proteins without involving genetic modification is essential for decoding various cellular processes but highly challenging. In this work, by conjugating cholesterols or tocopherols at the three bottom vertices of the DNA tetrahedron, we develop two sets of nanodevices for the selective targeting of lipid-order (Lo) and lipid-disorder (Ld) domains on the live cell membrane. By incorporation of protein-recognition ligands, such as aptamers or antibodies, through toehold-mediated strand displacement, these DNA nanodevices enable dynamic translocation of target proteins between these two domains. We first used PTK7 as a protein model and demonstrated, for the first time, that the accumulation of PTK7 to the Lo domains could promote tumor cell migration, while sequestering it in the Ld domains would inhibit the movement of the cells. Next, based on their modular nature, these DNA nanodevices were extended to regulate the process of T cell activation through manipulating the translocation of CD45 between the Lo and the Ld domains. Thus, our work is expected to provide deep insight into the study of membrane structure and molecular interactions within diverse cell signaling processes.

Enzyme Core Spherical Nucleic Acid That Enables Enhanced Cuproptosis and Antitumor Immune Response through Alleviating Tumor Hypoxia.

Cuproptosis, a copper-dependent cell death process, has been confirmed to further activate the immune response and mediate the immune resistance. However, hypoxic tumor microenvironment hampers cuproptosis sensitivity and suppresses the body's antitumor immune response. Herein, we have successfully immobilized and functionalized catalase (CAT) with long single-stranded DNA containing polyvalent CpG sequences through rolling circle amplification (RCA) techniques, obtaining an enzyme-cored spherical nucleic acid nanoplatform (CAT-ecSNA-Cu) to deliver copper ions for cuproptosis. The presence of long-stranded DNA-protected CAT enhances mitochondrial respiration by catalyzing the conversion of H2O2 to O2, thereby sensitizing cuproptosis. Meanwhile, increased tumor oxygenation suppresses the expression of the hypoxia-inducible factor-1 (HIF-1) protein, resulting in the alleviation of the immunosuppressive tumor microenvironment. Of note, cuproptosis induces immunogenic cell death (ICD), which facilitates dendritic cell (DC) maturation and enhances antigen presentation through polyCpG-supported Toll-like receptor 9 (TLR9) activation. Furthermore, cuproptosis-induced PD-L1 upregulation in tumor cells complements checkpoint blockers (αPD-L1), enhancing antitumor immunity. The strategy of enhancing cuproptosis-mediated antitumor immune responses by alleviating hypoxia effectively promotes the activation and proliferation of effector T cells, ultimately leading to long-term immunity against cancer.

Multivalent Aptamer-Based Lysosome-Targeting Chimeras (LYTACs) Platform for Mono- or Dual-Targeted Proteins Degradation on Cell Surface.

Selective protein degradation platforms have opened novel avenues in therapeutic development and biological inquiry. Antibody-based lysosome-targeting chimeras (LYTACs) have emerged as a promising technology that extends the scope of targeted protein degradation to extracellular targets. Aptamers offer an advantageous alternative owing to their potential for modification and manipulation toward a multivalent state. In this study, a chemically engineered platform of multivalent aptamer-based LYTACs (AptLYTACs) is established for the targeted degradation of either single or dual protein targets. Leveraging the biotin-streptavidin system as a molecular scaffold, this investigation reveals that trivalently mono-targeted AptLYTACs demonstrate optimum efficiency in degrading membrane proteins. The development of this multivalent AptLYTACs platform provides a principle of concept for mono-/dual-targets degradation, expanding the possibilities of targeted protein degradation.

Unlocking the Potential of Pterostilbene: A Pharmaceutical Element for Aptamer-Based Nanomedicine.

Natural compounds like pterostilbene (PTE) have gained recognition for their various biological activities and potential health benefits. However, challenges related to bioavailability and limited clinical efficacy have prompted efforts to strengthen their therapeutic potential. To meet these challenges, we herein rationally designed and successfully synthesized a pharmaceutical phosphoramidite that allows for the programmable incorporation of PTE into oligonucleotides. The resultant aptamer-PTE conjugate can selectively bind to cancer cells, leading to a specific internalization and drug release. Moreover, compared with free PTE, the conjugate exhibits superior cytotoxicity in cancer cells. Specifically, in a zebrafish xenograft model, the nanomedicine effectively inhibits tumor growth and neovascularization, highlighting its potential for targeted antitumor therapy. This approach presents a promising avenue for harnessing the therapeutic potential of natural compounds via a nanomedicine solution.

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.

Artificial DNA Framework Channel Modulates Antiapoptotic Behavior in Ischemia-Stressed Cells via Destabilizing Promoter G-Quadruplex.

Regulating folding/unfolding of gene promoter G-quadruplexes (G4s) is important for understanding the topological changes in genomic DNAs and the biological effects of such changes on important cellular events. Although many G4-stabilizing ligands have been screened out, effective G4-destabilizing ligands are extremely rare, posing a great challenge for illustrating how G4 destabilization affects gene function in living cells under stress, a long-standing question in neuroscience. Herein, we report a distinct methodology able to destabilize gene promoter G4s in ischemia-stressed neural cells by mitigating the ischemia-induced accumulation of intracellular K+ with an artificial membrane-spanning DNA framework channel (DFC). We also show that ischemia-triggered K+ influx is positively correlated to anomalous stabilization of promoter G4s and downregulation of Bcl-2, an antiapoptotic gene with neuroprotective effects against ischemic injury. Intriguingly, the DFC enables rapid transmembrane transport of excessive K+ mediated by the internal G4 filter, leading to the destabilization of endogenous promoter G4 in Bcl-2 and subsequent turnover of gene expression at both transcription and translation levels under ischemia. Consequently, this work enriches our understanding of the biological roles of endogenous G4s and may offer important clues to study the cellular behaviors in response to stress.

Toxicological evaluation and concentration of airborne PM0.1 in high air pollution period in Guangzhou, China.

The emissions and exposure limits for airborne PM0.1 are lacking, with limited scientific data for toxicity. Therefore, we continuously monitored and calculated the number and mass concentrations of airborne PM0.1 in December 2017, January 2018 and March 2018 during the high pollution period in Guangzhou. We collected PM0.1 from the same period and analyzed their chemical components. A549, THP-1 and A549/THP-1 co-cultured cells were selected for exposure to PM0.1, and evaluated for toxicological responses. Our aims are to 1) measure and analyze the number and mass concentrations, and chemical components of PM0.1; 2) evaluate and compare PM0.1 toxicity to different airway cells models at different time points. Guangzhou had the highest mass concentration of PM0.1 in December 2017, while the number concentration was the lowest. Chemical components in PM0.1 vary significantly at different time periods, and the correlation between the chemical composition or source of PM0.1 and the mass and number concentration of PM0.1 was dissimilar. Exposure to PM0.1 disrupted cell membranes, impaired mitochondrial function, promoted the expression of inflammatory mediators, and interfered with DNA replication in the cell cycle. The damage caused by exposure to PM0.1 at different times exhibited variations across different types of cells. PM0.1 in March 2018 stimulated co-cultured cells to secrete more inflammatory mediators, and CMA was significantly related to the expression of them. Our study indicates that it is essential to monitor both the mass and number concentrations of PM0.1 throughout all seasons annually, as conventional toxicological experiments and the internal components of PM0.1 may not effectively reveal the health damages caused by elevated number levels of PM0.1.

Exosome Heterogeneity Affects the Distal "Barrier-Crossing" Trafficking of Exosome Encapsulated Quantum Dots.

The biological activities of nanoparticles (NPs), which include endocytosis by macrophages and subsequent intracellular degradation and/or release, transfer to other cells, or translocation across tissue barriers, highly depend on their fate in living organisms. Yet, translocation across barriers, especially the distal "barrier-crossing" trafficking of NPs, is still unclear. The exosome (Exo) plays a crucial role in intercellular communication and biological barrier trafficking. Here, we report that ZnCdSe@ZnS quantum dots (QDs), as a representation of NPs in biomedical applications, could cross the blood-brain barrier and approach the mouse brain via active Exo encapsulation. By employing multiple techniques, we demonstrated that QDs were internalized by macrophages (J774A.1) and tumor cells (HeLa) and then released to the extracellular environment along with Exo. Exo encapsulation facilitates the distal barrier-crossing trafficking of QDs in vivo, while Exo biogenesis inhibitor GW4869 suppressed the QDs enriched in the brains of mice with a 4T1-Luc breast cancer xenograft. Interestingly, Exo heterogeneity affects the distal trafficking of enveloped QDs. Exo derived from tumorous HeLa cells, not macrophages, that were enriched in functional proteins with cell adhesion, cell migration, axon guidance, and cell motility, showed a better capacity for the remote trafficking of QDs. This study proposes Exo as a vehicle to deliver exogenous NPs to translocate across the distal barrier and provides further information for biomedical application and the risk assessment of NPs.

Aptamer-Based Multiparameter Analysis for Molecular Profiling of Hematological Malignancies.

The subtypes of hematological malignancies (HM) with minimal molecular profile differences display an extremely heterogeneous clinical course and a discrepant response to certain treatment regimens. Profiling the surface protein markers offers a potent solution for precision diagnosis of HM by differentiating among the subtypes of cancer cells. Herein, we report the use of Cell-SELEX technology to generate a panel of high-affinity aptamer probes that are able to discriminate subtle differences among surface protein profiles between different HM cells. Experimental results show that these aptamers with apparent dissociation constants (Kd) below 10 nM display a unique recognition pattern on different HM subtypes. By combining a machine learning model on the basis of partial least-squares discriminant analysis, 100% accuracy was achieved for the classification of different HM cells. Furthermore, we preliminarily validated the effectiveness of the aptamer-based multiparameter analysis strategy from a clinical perspective by accurately classifying complex clinical samples, thus providing a promising molecular tool for precise HM phenotyping.

Functional Selection of Tau Oligomerization-Inhibiting Aptamers.

Pathological hyperphosphorylation and aggregation of microtubule-associated Tau protein contribute to Alzheimer's Disease (AD) and other related tauopathies. Currently, no cure exists for Alzheimer's Disease. Aptamers offer significant potential as next-generation therapeutics in biotechnology and the treatment of neurological disorders. Traditional aptamer selection methods for Tau protein focus on binding affinity rather than interference with pathological Tau. In this study, we developed a new selection strategy to enrich DNA aptamers that bind to surviving monomeric Tau protein under conditions that would typically promote Tau aggregation. Employing this approach, we identified a set of aptamer candidates. Notably, BW1c demonstrates a high binding affinity (Kd=6.6 nM) to Tau protein and effectively inhibits arachidonic acid (AA)-induced Tau protein oligomerization and aggregation. Additionally, it inhibits GSK3ß-mediated Tau hyperphosphorylation in cell-free systems and okadaic acid-mediated Tau hyperphosphorylation in cellular milieu. Lastly, retro-orbital injection of BW1c tau aptamer shows the ability to cross the blood brain barrier and gain access to neuronal cell body. Through further refinement and development, these Tau aptamers may pave the way for a first-in-class neurotherapeutic to mitigate tauopathy-associated neurodegenerative disorders.

Serum Protein Fishing for Machine Learning-Boosted Diagnostic Classification of Small Nodules of Lung.

Diagnosis of benign and malignant small nodules of the lung remains an unmet clinical problem which is leading to serious false positive diagnosis and overtreatment. Here, we developed a serum protein fishing-based spectral library (ProteoFish) for data independent acquisition analysis and a machine learning-boosted protein panel for diagnosis of early Non-Small Cell Lung Cancer (NSCLC) and classification of benign and malignant small nodules. We established an extensive NSCLC protein bank consisting of 297 clinical subjects. After testing 5 feature extraction algorithms and six machine learning models, the Lasso algorithm for a 15-key protein panel selection and Random Forest was chosen for diagnostic classification. Our random forest classifier achieved 91.38% accuracy in benign and malignant small nodule diagnosis, which is superior to the existing clinical assays. By integrating with machine learning, the 15-key protein panel may provide insights to multiplexed protein biomarker fishing from serum for facile cancer screening and tackling the current clinical challenge in prospective diagnostic classification of small nodules of the lung.