Morphology Dictated Immune Activation with Framework Nucleic Acids.

Framework nucleic acids (FNAs) of various morphologies, designed using the precise and programmable Watson-Crick base pairing, serve as carriers for biomolecule delivery in biology and biomedicine. However, the impact of their shape, size, concentration, and the spatial presentation of cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODNs) on immune activation remains incompletely understood. In this study, representative FNAs with varying morphologies are synthesized to explore their immunological responses. Low concentrations (50 nM) of all FNAs elicited no immunostimulation, while high concentrations of elongated DNA nanostrings and tetrahedrons triggered strong activation due to their larger size and increased cellular uptake, indicating that the innate immune responses of FNAs depend on both dose and morphology. Notably, CpG ODNs' immune response can be programmed by FNAs through regulating the spatial distance, with optimal spacing of 7-8 nm eliciting the highest immunostimulation. These findings demonstrate FNAs' potential as a designable tool to study nucleic acid morphology's impact on biological responses and provide a strategy for future CpG-mediated immune activation carrier design.

Immunomodulation with Nucleic Acid Nanodevices.

The precise regulation of interactions of specific immunological components is crucial for controllable immunomodulation, yet it remains a great challenge. With the assistance of advanced computer design, programmable nucleic acid nanotechnology enables the customization of synthetic nucleic acid nanodevices with unprecedented geometrical and functional precision, which have shown promising potential for precise immunoengineering. Notably, the inherently immunologic functions of nucleic acids endow these nucleic acid-based assemblies with innate advantages in immunomodulatory engagement. In this review, the roles of nucleic acids in innate immunity are discussed, focusing on the definition, immunologic modularity, and enhanced bioavailability of structural nucleic acid nanodevices. In light of this, molecular programming and precise organization of functional modules with nucleic acid nanodevices for immunomodulation are emphatically reviewed. At last, the present challenges and future perspectives of nucleic acid nanodevices for immunomodulation are discussed.

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.

Sequential Therapy of Acute Kidney Injury with a DNA Nanodevice.

The high demand for acute kidney injury (AKI) therapy calls the development of multifunctional nanomedicine for renal management with programmable pharmacokinetics. Here, we developed a renal-accumulating DNA nanodevice with exclusive kidney retention for longitudinal protection of AKI in different stages in a renal ischemia-reperfusion (I/R) model. Due to the prolonged kidney retention time (>12 h), the ROS-sensitive nucleic acids of the nanodevice could effectively alleviate oxidative stress by scavenging ROS in stage I, and then the anticomplement component 5a (aC5a) aptamer loaded nanodevice could sequentially suppress the inflammatory responses by blocking C5a in stage II, which is directly related to the cytokine storm. This sequential therapy provides durable and pathogenic treatment of kidney dysfunction based on successive pathophysiological events induced by I/R, which holds great promise for renal management and the suppression of the cytokine storm in more broad settings including COVID-19.

Programming CircLigase Catalysis for DNA Rings and Topologies.

Circular single-stranded (ss) DNA is an essential element in rolling circle amplification and many DNA nanotechnology constructions. It is commonly synthesized from linear ssDNA by a ligase, which nevertheless suffers from low and inconsistent efficiency due to the simultaneous formation of concatemeric byproducts. Here, we design an intramolecular terminal hybridization strategy to program the ring formation catalytic process of CircLigase, a thermostable RNA ligase 1 that can ligate ssDNA in an intramolecular fashion. With the enthalpy gained from the programmed hybridization to override disfavored entropic factors associated with end coupling, we broke the limit of natural CircLigase on circularization of ssDNA, realizing over 75% yields of byproduct-free monomeric rings on a series of hundred-to-half-kilo-based linear DNAs. We found that this hybridization strategy can be twisted from intra- to intermolecular to also program CircLigase to efficiently and predominantly join one ssDNA strand to another. We focused on DNA rings premade by CircLigase and demonstrated their utility in elevating the preparation, quantity, and quality of DNA topologies. We expect that the new insights on engineering CircLigase will further promote the development of nucleic acid biotechnology and nanotechnology.

Engineering Allosteric Ribozymes to Detect Thiamine Pyrophosphate in Whole Blood.

Thiamine deficiency contributes to several human diseases including Alzheimer's. As its biologically active form, thiamine pyrophosphate (TPP) has been considered as a potential biomarker for Alzheimer's disease (AD) based on several clinical reports that apparently lower blood TPP levels were found in patients with mild cognitive impairment to AD. However, highly sensitive and high-throughput detection of TPP in biological fluids remains an analytical challenge. Here, we report engineering RNA-based sensors to quantitatively measure TPP concentrations in whole blood samples with a detection limit down to a few nM. By fusing a TPP-specific aptamer with the hammerhead ribozyme for in vitro selection, we isolated an allosteric ribozyme with an EC50 value (68 nM) similar to the aptamer's KD value (50 nM) for TPP, which for the first time demonstrates the possibility to maintain the effector binding affinity of the aptamer in such engineered allosteric RNA constructs. Meanwhile, we developed a new blood sample preparation protocol to be compatible with RNA. By coupling the TPP-induced ribozyme cleavage event with isothermal amplification, we achieved fluorescence monitoring of whole blood TPP levels through the "mix-and-read" operation with high-throughput potential. We expect that the engineered TPP-sensing RNAs will facilitate clinical research on AD as well as other thiamine-related diseases.

Programming nanoparticle valence bonds with single-stranded DNA encoders.

Nature has evolved strategies to encode information within a single biopolymer to program biomolecular interactions with characteristic stoichiometry, orthogonality and reconfigurability. Nevertheless, synthetic approaches for programming molecular reactions or assembly generally rely on the use of multiple polymer chains (for example, patchy particles). Here we demonstrate a method for patterning colloidal gold nanoparticles with valence bond analogues using single-stranded DNA encoders containing polyadenine (polyA). By programming the order, length and sequence of each encoder with alternating polyA/non-polyA domains, we synthesize programmable atom-like nanoparticles (PANs) with n-valence that can be used to assemble a spectrum of low-coordination colloidal molecules with different composition, size, chirality and linearity. Moreover, by exploiting the reconfigurability of PANs, we demonstrate dynamic colloidal bond-breaking and bond-formation reactions, structural rearrangement and even the implementation of Boolean logic operations. This approach may be useful for generating responsive functional materials for distinct technological applications.

Treating Acute Kidney Injury with Antioxidative Black Phosphorus Nanosheets.

Black phosphorus nanosheets (BPNSs) have been actively employed as nanomedicine agents for photothermal and photodynamic therapy by virtue of their unique optical properties. However, their chemical reactivity as a competent biomaterial has not been fully explored yet. Here, we report on the use of BPNSs as reactive oxygen species (ROS) scavengers to cure acute kidney injury (AKI) in mice. Importantly, in vivo analysis in mice revealed that BPNSs were preferably accumulated in kidney. We found that BPNSs alleviated oxidative-pressure-induced cellular apoptosis. In a ROS-triggered acute kidney injury (AKI) model, BPNSs effectively consumed ROS in kidney, demonstrating high efficacy for curing AKI. BPNSs also exhibited excellent biocompatibility and biodegradability, making them promising candidates for therapeutic treatment of AKI and other renal diseases.

Ultrafast DNA Sensors with DNA Framework-Bridged Hybridization Reactions.

Intracellular DNA-based hybridization reactions generally occur under tension rather than in free states, which are spatiotemporally controlled in physiological conditions. However, how nanomechanical forces affect DNA hybridization efficiencies in in-vitro DNA assays, for example, biosensors or biochips, remains largely elusive. Here, we design DNA framework-based nanomechanical handles that can control the stretching states of DNA molecules. Using a pair of tetrahedral DNA framework (TDF) nanostructured handles, we develop bridge DNA sensors that can capture target DNA with ultrafast speed and high efficiency. We find that the rigid TDF handles bind two ends of a single-stranded DNA (ssDNA) and hold it in a stretched state, with an apparent stretching length comparable to its counterpart of double-stranded DNA (dsDNA) via atomic force microscopy measurement. The DNA stretching effect of ssDNA is then monitored using single-molecule fluorescence energy transfer (FRET), resulting in decreased FRET efficiency in the stretched ssDNA. By controlling the stretching state of ssDNA, we obtained significantly improved hybridization kinetics (within 1 min) and hybridization efficiency (∼98%) under the target concentration of 500 nM. The bridge DNA sensors demonstrated high sensitivity (1 fM), high specificity (single mismatch mutation discrimination), and high selectivity (suitable for the detection in serum and blood) under the target concentration of 10 nM. Controlling the stretching state of ssDNA shows great potential in biosensors, bioimaging, and biochips applications.

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 Nanoribbon-Templated Self-Assembly of Ultrasmall Fluorescent Copper Nanoclusters with Enhanced Luminescence.

Fluorescent copper nanoclusters (CuNCs) have been widely used in chemical sensors, biological imaging, and light-emitting devices. However, individual fluorescent CuNCs have limitations in their capabilities arising from poor photostability and weak emission intensities. As one kind of aggregation-induced emission luminogen (AIEgen), the formation of aggregates with high compactness and good order can efficiently improve the emission intensity, stability, and tunability of CuNCs. Here, DNA nanoribbons, containing multiple specific binding sites, serve as a template for in situ synthesis and assembly of ultrasmall CuNCs (0.6 nm). These CuNC self-assemblies exhibit enhanced luminescence and excellent fluorescence stability because of tight and ordered arrangement through DNA nanoribbons templating. Furthermore, the stable and bright CuNC assemblies are demonstrated in the high-sensitivity detection and intracellular fluorescence imaging of biothiols.

Nanoparticle-Assisted Alignment of Carbon Nanotubes on DNA Origami.

Aligning carbon nanotubes (CNTs) is a key challenge for fabricating CNT-based electronic devices. Herein, we report a spherical nucleic acid (SNA) mediated approach for the highly precise alignment of CNTs at prescribed sites on DNA origami. We find that the cooperative DNA hybridization occurring at the interface of SNA and DNA-coated CNTs leads to an approximately five-fold improvement of the positioning efficiency. By combining this with the intrinsic positioning addressability of DNA origami, CNTs can be aligned in parallel with an extremely small angular variation of within 10°. Moreover, we demonstrate that the parallel alignment of CNTs prevents incorrect logic functionality originating from stray conducting paths formed by misaligned CNTs. This SNA-mediated method thus holds great potential for fabricating scalable CNT arrays for nanoelectronics.

Fractal Nanoplasmonic Labels for Supermultiplex Imaging in Single Cells.

The rapidly increasing need for systems biology stimulates the development of supermultiplex (SM) methods for simultaneously labeling multiple biomolecules/cells with distinct colors. Here we report the development of DNA-engineered fractal nanoplasmonic labels with ultrahigh brightness and photostability for SM imaging in single cells. These color-resolvable nanoplasmonic labels have a uniform size of ∼50 nm with an inner hollow gap of ∼1 nm. The outer shell morphology is highly tunable with the tip aspect ratio covering the range of δ = 0.29-1.66, which supports SM plasmonic imaging exceeding the conventional fluorescence multiplexing limit. We demonstrate the use of these SM labels for quantitative imaging of receptor-mediated endocytosis and intracellular transport of multiple protein-NP structures in a single cell in real time. This SM-plasmonic method sheds light on elucidating complex interactions among protein-NPs in nanotoxicology and facilitates the development of novel nanomedicines for diagnosis and therapy.

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.

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.

Programming Cell Adhesion for On-Chip Sequential Boolean Logic Functions.

Programmable remodelling of cell surfaces enables high-precision regulation of cell behavior. In this work, we developed in vitro constructed DNA-based chemical reaction networks (CRNs) to program on-chip cell adhesion. We found that the RGD-functionalized DNA CRNs are entirely noninvasive when interfaced with the fluidic mosaic membrane of living cells. DNA toehold with different lengths could tunably alter the release kinetics of cells, which shows rapid release in minutes with the use of a 6-base toehold. We further demonstrated the realization of Boolean logic functions by using DNA strand displacement reactions, which include multi-input and sequential cell logic gates (AND, OR, XOR, and AND-OR). This study provides a highly generic tool for self-organization of biological systems.

Hybridization chain reaction amplification of microRNA detection with a tetrahedral DNA nanostructure-based electrochemical biosensor.

There remains a great challenge in the sensitive detection of microRNA because of the short length and low abundance of microRNAs in cells. Here, we have demonstrated an ultrasensitive detection platform for microRNA by combining the tetrahedral DNA nanostructure probes and hybridization chain reaction (HCR) amplification. The detection limits for DNA and microRNA are 100 aM and 10 aM (corresponding to 600 microRNAs in a 100 µL sample), respectively. Compared to the widely used supersandwich amplification, the detection limits are improved by 3 orders of magnitude. The uncontrolled surface immobilization and consumption of target molecules that limit the amplification efficiency of supersandwich are eliminated in our platform. Taking advantage of DNA nanotechnology, we employed three-dimensional tetrahedral DNA nanostructure as the scaffold to immobilize DNA recognition probes to increase the reactivity and accessibility, while DNA nanowire tentacles are used for efficient signal amplification by capturing multiple catalytic enzymes in a highly ordered way. The synergetic effect of DNA tetrahedron and nanowire tentacles have proven to greatly improve sensitivity for both DNA and microRNA detection.

DNA Framework-Enabled 3D Organization of Antiarrhythmic Drugs for Radiofrequency Catheter Ablation.

Preorganizing molecular drugs within a microenvironment is crucial for the development of efficient and controllable therapeutic systems. Here, the use of tetrahedral DNA framework (TDF) is reported to preorganize antiarrhythmic drugs (herein doxorubicin, Dox) in 3D for catheter ablation, a minimally invasive treatment for fast heartbeats, aiming to address potential complications linked to collateral tissue damage and the post-ablation atrial fibrillation (AF) recurrence resulting from incomplete ablation. Dox preorganization within TDF transforms its random distribution into a confined, regular spatial arrangement governed by DNA. This, combined with the high affinity between Dox and DNA, significantly increases local Dox concentration. The exceptional capacity of TDF for cellular internalization leads to a 5.5-fold increase in intracellular Dox amount within cardiomyocytes, effectively promoting cellular apoptosis. In vivo investigations demonstrate that administering TDF-Dox reduces the recurrence rate of electrical conduction after radiofrequency catheter ablation (RFCA) to 37.5%, compared with the 77.8% recurrence rate in the free Dox-treated group. Notably, the employed Dox dosage exhibits negligible adverse effects in vivo. This study presents a promising treatment paradigm that strengthens the efficacy of catheter ablation and opens a new avenue for reconciling the paradox of ablation efficacy and collateral damage.