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

Abstract

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.

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

“…In contrast, nearly no detectable fluorescence response appeared in normal human lung fibroblast (MRC-5) and MCF-10A cells, implying a relatively higher expression of miR-21 in tumour cells than in normal cells, which was consistent with previous research. [41][42][43][44][45] Meanwhile, the performance of our designed LCC system to distinguish the varied miR-21 expression levels in different cells was furtherly verified by a quantitative flow cytometry assay (Figure 6B). In addition, the shortest characteristic diffusion time was exhibited in MCF-10A and MRC-5 cells, followed by HeLa cells, while the slowest molecular diffusion signal was shown in MCF-7 cells (Figure 6C).…”

“…In contrast, nearly no detectable uorescence response appeared in normal human lung broblast (MRC-5) and MCF-10A cells, implying a relatively higher expression of miR-21 in tumour cells than that in normal cells, which was consistent with previous research. [41][42][43][44][45] Meanwhile, the performance of our designed LCC system to distinguish the varied miR-21 expression levels in different cells was further veried by a quantitative ow cytometry assay (Fig. 6B).…”

An intelligent, autocatalytic, DNAzyme biocircuit for amplified imaging of intracellular microRNAs

“…In our previous study, we have reported DNAzyme-MnO 2 nanosystems for imaging miRNAs in living cells by target-triggered, self-powered DNAzyme-catalyzed reactions. 42,43 Recently, many CHA-DNAzyme circuits for intracellular microRNA imaging have been widely reported; [44][45][46] to address the problems of limited signal amplification capability and an additional supply of cofactors of DNAzyme-based bioassays, it is crucial to design a strategy that integrates DNAzyme biocircuits containing an intact feedback mechanism with nanomaterials that can provide catalytic capabilities.…”

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

“…Owing to the endogenous low abundance of PNK, the stacked configuration format still exhibited inadequate signal amplification depth. Positive feedback circuits with exponential dynamics are a promising method for advancing the amplification efficiency, which could achieve the explosive-like autocatalytic generation of the initiator. − CDA could produce short duplex DNA products, − where is featured on facile design and rapid reaction kinetics, thus making it an ideal candidate to integrate with the HCA circuit to build an autocatalysis-driven positive feedback system. The initiator-regeneration strategy could improve the biosensing performance, facilitating the development of self-sustainable nucleic acid circuits for in situ monitoring intracellular PNK that are still seldom explored.…”