Self-Stacking Autocatalytic Molecular Circuit with Minimal Catalytic DNA Assembly.

Isothermal autocatalytic DNA circuits have been proven to be versatile and powerful biocomputing platforms by virtue of their self-sustainable and self-accelerating reaction profiles, yet they are currently constrained by their complicated designs, severe signal leakages, and unclear reaction mechanisms. Herein, we developed a simpler-yet-efficient autocatalytic assembly circuit (AAC) for highly robust bioimaging in live cells and mice. The scalable and sustainable AAC system was composed of a mere catalytic DNA assembly reaction with minimal strand complexity and, upon specific stimulation, could reproduce numerous new triggers to expedite the whole reaction. Through in-depth theoretical simulations and systematic experimental demonstrations, the catalytic efficiency of these reproduced triggers was found to play a vital role in the autocatalytic profile and thus could be facilely improved to achieve more efficient and characteristic autocatalytic signal amplification. Due to its exponentially high signal amplification and minimal reaction components, our self-stacking AAC facilitated the efficient detection of trace biomolecules with low signal leakage, thus providing great clinical diagnosis and therapeutic assessment potential.

Monitoring and modulating a catalytic hybridization circuit for self-adaptive bioorthogonal DNA assembly.

Constructing artificial domino nanoarchitectures, especially dynamic DNA circuits associated with the actuation of biological functions inside live cells, represents a versatile and powerful strategy to regulate the behaviors and fate of various living entities. However, the stepwise operation of conventional DNA circuits always relies on freely diffusing reactants, which substantially slows down their operation rate and efficiency. Herein, a self-adaptive localized catalytic circuit (LCC) is developed to execute the self-sustained bioorthogonal assembly of DNA nanosponges within a crowded intracellular environment. The LCC-generated DNA scaffolds are utilized as versatile templates for realizing the proximity confinement of LCC reactants. Single-molecule-detecting fluorescence correlation spectroscopy (FCS) is used to explore the reaction acceleration of the catalytic circuit. This self-adaptive DNA circuit facilitates the bioorthogonal assembly of highly branched DNA networks for robust and accurate monitoring of miRNA targets. Based on its intriguing and modular design, the LCC system provides a pivotal molecular toolbox for future applications in early disease diagnosis.

An Autocatalytic DNA Circuit Based on Hybridization Chain Assembly for Intracellular Imaging of Polynucleotide Kinase.

Polynucleotide kinase (PNK) plays an essential role in various cellular events by regulating phosphorylation processes, and abnormal homeostasis of PNK could cause many human diseases. Herein, we proposed an autocatalytic hybridization system (AHS) through the elaborate integration of hybridization chain assembly (HCA) and catalytic DNA assembly (CDA) that enables a highly efficient positive feedback amplification. The PNK-targeting AHS biosensor is composed of three modules: a recognition module, an HCA amplification module, and a CDA autocatalytic module. In the presence of PNK, the recognition module could transform the PNK input into an exposed nucleic acid initiator (I). Then the initiator strand I could trigger the autonomous HCA process in the amplification module, and the resulted HCA products could reassemble the split CDA trigger strand T, subsequently inducing the CDA process in the autocatalytic module to form abundant DNA duplex products. Consequently, the embedded initiator strand I was liberated from the CDA duplex product to autonomously trigger the new rounds of HCA circuit. The rational integration and cooperative cross-activation between the HCA and CDA module could prominently accelerate the reaction and realize the exponential amplification efficiency by initiator regeneration. As a result, the self-sustainable AHS amplifier could implement the sensitive detection of PNK in vitro and in biological samples and further fulfill accurate monitoring of the intracellular PNK activity and the effective screening of PNK inhibitors. This work paves a way for exploiting highly efficient artificial DNA circuits to analyze low-abundance biomarkers, holding great potential in biochemical research and clinical diagnosis.

A Deoxyribozyme-Initiated Self-Catalytic DNA Machine for Amplified Live-Cell Imaging of MicroRNA.

Functional DNA nanostructures have been widely used in various bioassay fields. Yet, the programmable assembly of functional DNA nanostructures in living cells still represents a challenging goal for guaranteeing the sensitive and specific biosensing utility. In this work, we report a self-catalytic DNA assembly (SDA) machine by using a feedback deoxyribozyme (DNAzyme)-amplified branched DNA assembly. This SDA system consists of catalytic self-assembly (CSA) and DNAzyme amplification modules for recognizing and amplifying the target analyte. The analyte initiates the CSA reaction, leading to the formation of Y-shaped DNA that carries two RNA-cleaving DNAzymes. One DNAzyme can then successively cleave the corresponding substrate and generate numerous additional inputs to activate new CSA reactions, thus realizing a self-catalytic amplification reaction. Simultaneously, the other DNAzyme is assembled as a versatile signal transducer for cleaving the fluorophore/quencher-modified substrate, leading to the generation of an amplified fluorescence readout. By incorporating a flexible auxiliary sensing module, the SDA system can be converted into a universal sensing platform for detecting cancerous biomarkers, e.g., a well-known oncogene microRNA-21 (miR-21). Moreover, the SDA system realized the precise intracellular miR-21 imaging in living cells, which is attributed to the reciprocal amplification property between CSA reactions and DNAzyme biocatalysis. This compact SDA amplifier machine provides a universal and facile toolbox for the highly efficient identification of cancerous biomarkers and thus holds great potential for early cancer diagnosis.

Orthogonal Demethylase-Activated Deoxyribozyme for Intracellular Imaging and Gene Regulation.

The epigenetic modification of nucleic acids represents a versatile approach for achieving high-efficient control over gene expression and transcription and could dramatically expand their biosensing and therapeutic applications. Demethylase-involved removal of N6-methyladenine (m6A) represents one of the vital epigenetic reprogramming events, yet its direct intracellular evaluation and as-guided gene regulation are extremely rare. The endonuclease-mimicking deoxyribozyme (DNAzyme) is a catalytically active DNA that enables the site-specific cleavage of the RNA substrate, and several strategies have imparted the magnificent responsiveness to DNAzyme by using chemical and light stimuli. However, the epigenetic regulation of DNAzyme has remained largely unexplored, leaving a significant gap in responsive DNA nanotechnology. Herein, we reported an epigenetically responsive DNAzyme system through the in vitro selection of an exquisite m6A-caged DNAzyme that could be specifically activated by FTO (fat mass and obesity-associated protein) demethylation for precise intracellular imaging-directed gene regulation. Based on a systematic investigation, the active DNAzyme configuration was potently disrupted by the site-specific incorporation of m6A modification and subsequently restored into the intact DNAzyme structure via the tunable FTO-specific removal of m6A-caging groups under a variety of conditions. This orthogonal demethylase-activated DNAzyme amplifier enables the robust and accurate monitoring of FTO and its inhibitors in live cells. Moreover, the simple demethylase-activated DNAzyme facilitates the assembly of an intelligent self-adaptive gene regulation platform for knocking down demethylase with the ultimate apoptosis of tumor cells. As a straightforward and scarless m6A removal strategy, the demethylase-activated DNAzyme system offers a versatile toolbox for programmable gene regulation in synthetic biology.