Universal DNA-Based Sensing Toolbox for Programming Cell Functions.

By recombining natural cell signaling systems and further reprogramming cell functions, use of genetically engineered cells and bacteria as therapies is an innovative emerging concept. However, the inherent properties and structures of the natural signal sensing and response pathways constrain further development. We present a universal DNA-based sensing toolbox on the cell surface to endow new signal sensing abilities for cells, control cell states, and reprogram multiple cell functions. The sensing toolbox contains a triangular-prismatic-shaped DNA origami framework and a sensing core anchored inside the internal confined space to enhance the specificity and efficacy of the toolbox. As a proof of principle, the sensing toolbox uses the customizable sensing core with signal sensing switches and converters to recognize unconventional signal inputs, deliver functional components to cells, and then control cell responses, including specific tumor cell death, immune cell disinhibition and adhesion, and bacterial expression. This work expands the diversity of cell sensing signals and reprograms biological functions by constructing nanomechanical-natural hybrid cells, providing new strategies for engineering cells and bacteria in diagnosis and treatment applications.

A DNA Nanodevice-Based Platform with Diverse Capabilities.

Social biotic colonies often perform intricate tasks by interindividual communication and cooperation. Inspired by these biotic behaviors, a DNA nanodevice community is proposed as a universal and scalable platform. The modular nanodevice as the infrastructure of platform contains a DNA origami triangular prism framework and a hairpin-swing arm machinery core. By coding and decoding a signal domain on the shuttled output strand in different nanodevices, an orthogonal inter-nanodevice communication network is established to connect multi-nanodevices into a functional platform. The nanodevice platform enables implementation of diverse tasks, including signal cascading and feedback, molecular input recording, distributed logic computing, and modeling of simulation for virus transmission. The nanodevice platform with powerful compatibility and programmability presents an elegant example of the combination of the distributed operation of multiple devices and the complicated interdevice communication network, and may become a new generation of intelligent DNA nanosystems.

An miRISC-initiated DNA nanomachine for monitoring MicroRNA activity in living cells.

MicroRNAs (miRNAs) play an important role in post-transcriptional regulation of gene expression. However, methods to accurately detect miRNA activity in living cells are still limited. Here we developed a DNA nanomachine initiated by a miRNA-induced silencing complex (miRISC) for imaging miRNA activity in living cells. miRISC-mediated RNA cleavage reaction activated the DNA nanomachine by the specific cleavage of an RNA strand on the machine, resulting in autonomous movement of the walking leg around the AuNP surface with the release of a large number of fluorescently labeled DNA strands. The DNA nanomachine was successfully applied to detect miR-21 activity in three cell lines with different miR-21 expression profiles. We also demonstrated that terminal uridylyltransferase Tut4 knockdown by siRNA significantly increased the activity of let-7b miRNA, which further verifies the versatility of our DNA nanomachine. This new nanomachine has distinct advantages compared with reported methods for detecting miRNA activity, including simple operating procedures, short analysis time and sensitive signal output. Collectively, this work not only expands the application of the DNA nanomachine in the detection of miRNA activity, but also provides a promising tool for basic research in cell biology and development of clinical biomedicine.

Nano-Biohybrid DNA Engager That Reprograms the T-Cell Receptor.

Although engineered T cells with transgenic chimeric antigen receptors (CARs) have made a breakthrough in cancer therapeutics, this approach still faces many challenges in the specificity, efficacy, and self-safety of genetic engineering. Here, we developed a nano-biohybrid DNA engager-reprogrammed T-cell receptor (EN-TCR) system to improve the specificity and efficacy, mitigate the excessive activation, and shield against risks from transgenesis, thus achieving a diversiform and precise control of the T-cell response. Utilizing modular assembly, the EN-TCR system can graft different specificities on T cells via antibody assembly. Besides, the designability of DNA hybridization enables precise target recognition by the library of multiantigen cell recognition circuits and allows gradual tuning of the T-cell activation level by the signaling switch and independent control over different types of T cells. Furthermore, we demonstrated the effectiveness of the system in tumor models. Together, this study provides a nongenetic T-cell engineering strategy to overcome major hindrances in T-cell therapy and may be extended to a general and convenient cell engineering strategy.

Multimachine Communication Network That Mimics the Adaptive Immune Response.

Biological organisms capable of controlling and performing a wide variety of functions have inspired attempts to mimic biological systems with designable intelligence. Here we develop a multimachine communication network (MMCN) to mimic the operation and function of adaptive immune response (AIR) via connecting three kinds of DNA machines built from module-functionalized gold nanoparticles. These machines simulate three critical immune cells, dendritic cells, T and B lymphocytes, and their differentiation and coordinated interaction upon exposure and response to an invading pathogen. MMCN is composed of standard modules with track, movement, and fuel components that allow for the (1) integration and adaptability of a single machine, (2) convenient spatiotemporal control of the sequential activation of a single machine, and (3) rapid reaction rate and high efficiency owing to an enhanced local concentration of interacting species. We show that the proposed network can sense and clear the corresponding pathogen via consecutive activation and connection of the machines, simultaneously forming a memory to respond more rapidly and effectively upon the second invasion of the pathogen. This system may be extended to construct powerful networks to execute more sophisticated tasks and accomplish diverse functions.

Catalytic-Hairpin-Assembly-Assisted DNA Tetrahedron Nanoprobe for Intracellular MicroRNA Imaging.

The aberrant expression levels of microRNAs (miRNAs) are tightly linked with the initiation and development of various diseases and genetic disorders. Here, we reported a catalytic-hairpin-assembly-assisted DNA tetrahedron nanoprobe for intracellular miRNA detection. The target miRNA initiated the catalytic-hairpin-assembly reaction between the tetrahedron nanoprobes to generate large tetrahedron clusters with an enhanced fluorescence resonance energy transfer between the Cy3 and Cy5 dyes. The proposed nanoprobes were capable of visualizing the expression levels and spatial distributions of miRNA in living cells, providing a feasible approach in designing versatile nanostructures for reliable bioanalysis and clinical diagnosis.

A highly integrated DNA nanomachine operating in living cells powered by an endogenous stimulus.

Synthetic molecular machines have received increasing attention because of their great ability to mimic natural biological motors and create novel modes of motion. However, very few examples have been implemented with real autonomous movement inside living cells, due to the challenges of the driving force and highly integrated system design. In this work, we report an elegant, highly integrated DNA nanomachine that can be powered by endogenous ATP molecules and autonomously operated inside living cells without any auxiliary additives. It assembles all components on a single gold nanoparticle (AuNP) including a hairpin-locked swing arm encoding a start triggered by an intracellular target molecule and a two-stranded DNA track responding to the motion of the swing arm. When the intracellular target activates the nanomachine via the unlocking swing arm, the machine autonomously and progressively operates on the established DNA track via intramolecular toehold-mediated strand migration and internal ATP binding. This paper also demonstrates the machine's bioanalytical application for specific microRNA (miRNA) imaging in living cells.

Rational Engineering of a Dynamic, Entropy-Driven DNA Nanomachine for Intracellular MicroRNA Imaging.

We rationally engineered an elegant entropy-driven DNA nanomachine with three-dimensional track and applied it for intracellular miRNAs imaging. The proposed nanomachine is activated by target miRNA binding to drive a walking leg tethered to gold nanoparticle with a high density of DNA substrates. The autonomous and progressive walk on the DNA track via the entropy-driven catalytic reaction of intramolecular toehold-mediated strand migration leads to continuous disassembly of DNA substrates, accompanied by the recovery of fluorescence signal due to the specific release of a dye-labeled substrate from DNA track. Our nanomachine outperforms the conventional intermolecular reaction-based gold nanoparticle design in the context of an improved sensitivity and kinetics, attributed to the enhanced local effective concentrations of working DNA components from the proximity-induced intramolecular reaction. Moreover, the nanomachine was applied for miRNA imaging inside living cells.