Programing Immunogenic Cell Death in Breast Tumors with Designer DNA Frameworks.

The low response rate and serious side effects of cancer treatment pose significant limitations in immunotherapy. Here, we developed a multifunctional tetrahedral DNA framework (TDF) as a drug carrier to recruit chemotherapeutants and trigger immunogenic cell death (ICD) effects, which could turn tumors from cold to hot to boost the efficacy of antitumor immunotherapy. A tumor-targeting peptide RGD was modified on the TDF to increase the delivery efficiency, and the chemotherapeutant doxorubicin (DOX) was loaded to induce ICD effects, which were assisted by the immune adjuvant of CpG immunologic sequences linked on TDF. We demonstrated that the multifunctional TDF could suppress 4T1 breast tumor growth by increasing tumor infiltration of CD8+ T cells, upregulating granzyme B and perforin expressions to twice as much as the control group, and decreasing 30% CD25+ Treg cells. Furthermore, the combination of α-PD-1 could inhibit the growth of distant tumor and suppressed tumor recurrence in a bilateral syngeneic 4T1 mouse model; the distant tumor weight inhibition rate was about 91.6%. Hence, through quantitatively targeting the delivery of DOX to reduce the side effects of chemotherapy and sensitizing the immune response by ICD effects, this multifunctional TDF therapeutic strategy displayed better treatment effect and a promising clinical application prospect.

Sensing gastric cancer exosomes with MoS2-based SERS aptasensor.

Exosomes have been widely used in early cancer diagnosis as promising cancer biomarkers due to their abundant tumor-specific molecular information. In this study, we developed a sensitive and straightforward surface-enhanced Raman scattering (SERS) aptasensor to detect exosomes based on gold nanostars-decorated molybdenum disulfide (MoS2) nanocomposites (MoS2-AuNSs). ROX-labeled aptamers (ROX-Apt) were assembled on MoS2-AuNSs surface as recognition probes that specifically bind with transmembrane protein CD63 (a representative surface marker on exosomes). Thus obvious ROX Raman signals were obtained through the synergistic Raman enhancement effect of AuNSs and MoS2 nanosheet. In presence of exosomes, ROX-Apt is preferentially tethered onto exosomes and released from the surface of nanocomposites, resulting in a decrease of the SERS signal. Expectedly, the as-fabricated SERS aptasensor was capable of detecting exosomes in a wide range from 55 to 5.5 × 105 particles µL-1 with a detection limit of 17 particles µL-1. Moreover, the aptasensor exhibited accepted stability and potential clinical applicability.

Unbiased Enrichment of Circulating Tumor Cells Via DNAzyme-Catalyzed Proximal Protein Biotinylation.

Circulating tumor cells (CTCs) are noninvasive biomarkers with great potential for assessing neoplastic diseases. However, the enrichment bias toward heterogeneous CTCs remains to be minimized. Herein, a DNAzyme-catalyzed proximal protein biotinylation (DPPB) strategy is established for unbiased CTCs enrichment, employing DNA-framework-based, aptamer-coupled DNAzymes that bind to the surface marker of CTCs and subsequently biotinylated membrane proteins in situ. The DNA framework enables the construction of multivalent DNAzyme and serves as steric hindrance to avoid undesired interaction between DNAzymes and aptamer, leading to efficient binding and biotinylation. Compared with a biotinylated-aptamer strategy, fivefold lower bias of cell subpopulations was achieved by DPPB before and after capture, which enabled a 4.6-fold performance for CTCs analysis in clinic blood samples. DPPB is envisioned to offer a new solution for CTC-based cancer diagnostics.

Programming cell communications with pH-responsive DNA nanodevices.

DNA nanoswitches on cell surfaces could respond to changes of pH under physiological conditions by switching from a three-chain structure to a double-chain structure, thus connecting another set of cells modified with complementary single-stranded DNA. This pH-triggered cell communication offers a promising approach for cell-based therapy under a tumor microenvironment.

Responsive optical probes for deep-tissue imaging: Photoacoustics and second near-infrared fluorescence.

Optical imaging has played a vital role in development of biomedicine and image-guided theragnostic. Nevertheless, the clinical translation of optical molecular imaging for deep-tissue visualization is still limited by poor signal-to-background ratio and low penetration depth owing to light scattering and tissue autofluorescence. Hence, to facilitate precise diagnosis and accurate surgery excision in clinical practices, the responsive optical probes (ROPs) are broadly designed for specific reaction with biological analytes or disease biomarkers via chemical/physical interactions for photoacoustic and second near-infrared fluorescence (NIR-II, 900-1700 nm) fluorescence imaging. Herein, the recent advances in the development of ROPs including molecular design principles, activated mechanisms and treatment responses for photoacoustic and NIR-II fluorescence imaging are reviewed. Furthermore, the present challenges and future perspectives of ROPs for deep-tissue imaging are also discussed.

Encapsulation and release of living tumor cells using hydrogels with the hybridization chain reaction.

Circulating tumor cells (CTCs) enable noninvasive liquid biopsy and identification of cancer. Various approaches exist for the capture and release of CTCs, including microfluidic methods and those involving magnetic beads or nanostructured solid interfaces. However, the concomitant cell damage and fragmentation that often occur during capture make it difficult to extensively characterize and analyze living CTCs. Here, we describe an aptamer-trigger-clamped hybridization chain reaction (atcHCR) method for the capture of CTCs by porous 3D DNA hydrogels. The 3D environment of the DNA networks minimizes cell damage, and the CTCs can subsequently be released for live-cell analysis. In this protocol, initiator DNAs with aptamer-toehold biblocks specifically bind to the epithelial cell adhesion molecule (EpCAM) on the surface of CTCs, which triggers the atcHCR and the formation of a DNA hydrogel. The DNA hydrogel cloaks the CTCs, facilitating quantification with minimal cell damage. This method can be used to quantitively identify as few as 10 MCF-7 cells in a 2-µL blood sample. Decloaking of tumor cells via gentle chemical stimulus (ATP) is used to release living tumor cells for subsequent cell culture and live-cell analysis. We also describe how to use the protocol to encapsulate and release cells of cancer cell lines, which can be used in preliminary experiments to model CTCs. The whole protocol takes ~2.5 d to complete, including downstream cell culture and analysis.

Programming Cell-Cell Communications with Engineered Cell Origami Clusters.

Cells existing in the form of clusters often exhibit distinct physiological functions from their monodispersed forms, which have a close association with tissue and organ development, immunoresponses, and cancer metastasis. Nevertheless, the ability to construct artificial cell clusters as in vitro models for probing and manipulating intercellular communications remains limited. Here we design DNA origami nanostructure (DON)-based biomimetic membrane channels to organize cell origami clusters (COCs) with controlled geometric configuration and cell-cell communications. We demonstrate that programmable patterning of homotypic and heterotypic COCs with different configurations can result in three distinct types of intercellular communications: gap junctions, tunneling nanotubes, and immune/tumor cell interactions. In particular, the organization of T cells and cancer cells with a prescribed ratio and geometry can program in vitro immunoresponses, providing a new route to understanding and engineering cancer immunotherapy.

DNA Origami-Enabled Engineering of Ligand-Drug Conjugates for Targeted Drug Delivery.

Effective drug delivery systems that can systematically and selectively transport payloads to disease cells remain a challenge. Here, a targeting ligand-modified DNA origami nanostructure (DON) as an antibody-drug conjugate (ADC)-like carrier for targeted prostate cancer therapy is reported. Specifically, DON of six helical bundles is modified with a ligand 2-[3-(1,3-dicarboxy propyl)-ureido] pentanedioic acid (DUPA) against prostate-specific membrane antigen (PSMA), to serve as the antibody for drug conjugation in ADC. Doxorubicin (Dox) is then loaded to DON through intercalation to dsDNA. This platform features in spatially controllable organization of targeting ligands and high drug loading capacity. With this nanocomposite, selective delivery of Dox to the PSMA+ cancer cell line LNCaP is readily achieved. The consequent therapeutic efficacy is critically dependent on the numbers of targeting ligand assembled on DON. This target-specific and biocompatible drug delivery platform with high maximum tolerated doses shows immense potential for developing novel nanomedicine.

DNA Framework-Programmed Cell Capture via Topology-Engineered Receptor-Ligand Interactions.

Receptor-ligand interactions (RLIs) that play pivotal roles in living organisms are often depicted with the classic keys-and-locks model. Nevertheless, RLIs on the cell surface are generally highly complex and nonlinear, partially due to the noncontinuous and dynamic distribution of receptors on extracellular membranes. Here, we develop a tetrahedral DNA framework (TDF)-programmed approach to topologically engineer RLIs on the cell membrane, which enables active recruitment-binding of clustered receptors for high-affinity capture of circulating tumor cells (CTCs). The four vertices of a TDF afford orthogonal anchoring of ligands with spatial organization, based on which we synthesized n-simplexes harboring 1-3 aptamers targeting epithelial cell adhesion molecule (EpCAM) that are overexpressed on the membrane of tumor cells. The 2-simplex with three aptamers not only shows increased binding affinity (∼19-fold) but prevents endocytosis by cells. By using 2-simplex as the capture probe, we demonstrate the high-efficiency CTC capture, which is challenged in real clinical breast cancer patient samples. This TDF-programmed platform thus provides a powerful means for studying RLIs in physiological settings and for cancer diagnosis.

Rapid Transmembrane Transport of DNA Nanostructures by Chemically Anchoring Artificial Receptors on Cell Membranes.

Highly efficient transmembrane transport of exogenous reagents into cells is of vital importance for developing drug-delivery systems. Conventionally, transmembrane transport of exogenous reagents was accomplished with the assistance of transfection agents or other artificial means. However, the high toxicity and low transport efficiency of current delivery techniques still remain to be solved. In this work, by anchoring artificial receptors onto cell membranes with a mild chemical reaction, we demonstrated that the exogenous reagent framework nucleic acids, namely tetrahedral DNA nanostructures (TDN) can bind onto cell membranes effectively by the hybridization between single-stranded DNA (ssDNA) and pendant ssDNA of TDN. The transport rate was greatly enhanced, with the endocytosis time could be as fast as 0.5 h. Furthermore, the transport quantity was prominently improved, with around 30 % of TDN endocytosed within 4 h. Owing to its rapid transmembrane transport speed and improved endocytosis quantity, this artificial-receptor-mediated transmembrane transport is a promising tool for achieving rapid and highly efficient transmembrane transport of exogenous reagents.

Poly-adenine-mediated spherical nucleic acids for strand displacement-based DNA/RNA detection.

DNA-gold nanoparticles (AuNPs) conjugate is one of the most versatile bionanomaterials for biomedical and clinical diagnosis. However, to finely tune the hybridization ability and precisely control the orientation and conformation of surface-tethered oligonucleotides on AuNPs remains a hurdle. In this work, we developed a poly adenine-mediated spherical nucleic acid (polyA-mediated SNA) strategy by assembling di-block DNA probes on gold nanoparticles (AuNPs) to spatially control interdistance and hybridization ability of oligonucleotides on AuNPs. By modulating length of poly A bound on the SNA with different degrees of constructing, we presented significant improved biosensing performance including high hybridization efficiency, and expanded dynamic range of analytes with more sensitive detection limit. Furthermore, this polyA design could facilitate the programmable detection for DNA in serum environment and simultaneous multicolor detection of three different microRNAs associated with pancreatic carcinoma. The demonstration of the link between modulation of SNA assembly strategy and biodetection capability will increase the development of high performance diagnostic tools for translational biomedicine.

Concept and Development of Framework Nucleic Acids.

The blooming field of structural DNA nanotechnology harnessing the material properties of nucleic acids has attracted widespread interest. The exploitation of the precise and programmable Watson-Crick base pairing of DNA or RNA has led to the development of exquisite nucleic acid nanostructures from one to three dimensions. The advances of computer-aided tools facilitate automated design of DNA nanostructures with various sizes and shapes. Especially, the construction of shell or skeleton DNA frameworks, or more recently dubbed "framework nucleic acids" (FNAs) provides a means to organize molecules or nanoparticles with nanometer precision. The intrinsic biological properties and tailorable functionalities of FNAs hold great promise for physical, chemical, and biological applications. This Perspective highlights state-of-the-art design and construction, of precisely assembled FNAs, and outlines the challenges and opportunities for exploiting the structural potential of FNAs for translational applications.

Valency-Controlled Framework Nucleic Acid Signal Amplifiers.

Weak ligand-receptor recognition events are often amplified by recruiting multiple regulatory biomolecules to the action site in biological systems. However, signal amplification in in vitro biomimetic systems generally lack the spatiotemporal regulation in vivo. Herein we report a framework nucleic acid (FNA)-programmed strategy to develop valence-controlled signal amplifiers with high modularity for ultrasensitive biosensing. We demonstrated that the FNA-programmed signal amplifiers could recruit nucleic acids, proteins, and inorganic nanoparticles in a stoichiometric manner. The valence-controlled signal amplifier enhanced the quantification ability of electrochemical biosensors, and enabled ultrasensitive detection of tumor-relevant circulating free DNA (cfDNA) with sensitivity enhancement of 3-5 orders of magnitude and improved dynamic range.

Framework Nucleic Acid-Mediated Pull-Down MicroRNA Detection with Hybridization Chain Reaction Amplification.

Gastric cancer remains a disease of high mortality worldwide due to its poor prognosis. Previous studies have shown that microRNAs (miRNAs) are effective biomarkers for early diagnosis of gastric cancer. To realize sensitive detection of related miRNAs for improved early diagnosis, classification, and survival prognosis of gastric cancer, herein we developed a framework nucleic acid (FNA)-mediated microarray for quantitative analysis of multiple miRNAs. By rationally designing FNA with different sizes, we systematically modulated the surface density and lateral interactions of DNA probes, which provides an effective means for programmable tailoring of the hybridization efficiency and kinetics of the biosensing interface. We found that the hybridization efficiency was increased along with the size of the FNA and was optimum for FNA-17. In combination with the hybridization chain reaction amplification strategy, this established FNA microarray can serve as an ultrasensitive and selective analytical platform for simultaneous multiplexed detection of miRNA (e.g., FNA-miR-652, FNA-miR-627, and FNA-miR-629) biomarkers in gastric cancer.

Epitope Binning Assay Using an Electron Transfer-Modulated Aptamer Sensor.

Surface plasmon resonance and quartz crystal microbalance are workhorses of protein-DNA interaction research for over 20 years, providing ways to quantitatively determine the protein-DNA binding. However, the cost, necessary technical expertise, and severe nonspecific adsorption poses barriers to their use. Convenient and effective techniques for the measurement of protein-DNA binding affinity and the epitope binning between DNA and proteins for developing highly sensitive detection platform remain challenging. Here, we develop a binding-induced alteration in electron transfer kinetics of the redox reporter labeled (methylene blue) on DNA aptamer to measure the binding affinity between prostate-specific antigen (PSA) and aptamer. We demonstrate that the binding of PSA to aptamer decreases the electron transfer rate of methylene blue for ∼45%. Further, we identify the best pairwise selection of aptamers for developing sandwich assay by sorting from 10 pairwise modes with the PSA detection limit of 500 ng/mL. Our study provides promising ways to analyze the binding affinity between ligand and receptor and to sort pairwise between aptamers or antibodies for the development of highly sensitive sandwich immunoassays.

Carbon nanotubes multifunctionalized by rolling circle amplification and their application for highly sensitive detection of cancer markers.

There are still challenges for the development of multifunctional carbon nanotubes (CNTs). Here, a multiwalled carbon nanotube (MWCNT)-based rolling circle amplification system (CRCAS) is reported which allows in situ rolling circle replication of DNA primer on the surface of MWCNTs to create a long single-strand DNA (ssDNA) where a large number of nanoparticles or proteins could be loaded, forming a nano-biohybridized 3D structure with a powerful signal amplification ability. In this strategy, the binding ability of proteins, hybridization, replication ability of DNA, and the catalytical ability of enzymes are integrated on a single carbon nanotube. The CRCAS is then used to develop colorimetric and chemiluminescent assays for the highly sensitive and specific detection of cancer protein markers, alpha-fetoprotein (AFP) and prostate specific antigen (PSA). The colorimetric CRCAS assay is 4000 times more sensitive than a conventional enzyme-linked immunosorbent assay (ELISA), and its concentration range is 10,000 times wider. Control experiments show that as low as 10 pg mL⁻¹ AFP or PSA could be detected even in the presence of interfering protein markers with a more than 105-fold greater concentration in the sample, demonstrating the high specificity of the CRCAS assay. The limit of detection of the chemiluminescent CRCAS assays for AFP and PSA are 5 fg mL⁻¹ (70 aM) and 10 fg mL⁻¹ (0.29 fM), respectively, indicating that the sensitivity is much higher than that of the colorimetric CRCAS assay. Importantly, CRCAS works well with real biological samples.