MicroRNA Imaging Research Articles

Localized hybridization chain reaction amplifier utilizing three-dimensional DNA nanostructures for efficient and reliable microRNA imaging in living cells

MicroRNAs (miRNAs) are pivotal in regulating biological processes such as cell proliferation and disease progression. Traditional miRNA detection methods like qRT-PCR and Northern blotting do not allow for monitoring dynamic changes in living cells and typically require invasive sample collection. This research presents a robust, non-enzymatic technique known as the Localized Hybridization Chain Reaction Amplifier (LHCRA), designed for real-time, in vivo miRNA imaging. This method addresses these limitations by providing rapid and sensitive detection of miRNAs, crucial for progressing clinical diagnostics and research. The LHCRA utilizes hairpin chain reaction probes embedded within self-assembled DNA micelles (DM), significantly amplifying miRNA signals. LHCRA demonstrated exceptional performance, with a detection limit of 100 pM and a response time of 10 min. Importantly, it effectively differentiated between cancerous and normal cells using clinical peripheral blood samples, showcasing high specificity and stability under physiological conditions. Additionally, LHCRA maintained consistent performance in complex biological media, including 10 % serum and 2 U/mL DNase I, confirming its broad applicability in various diagnostic scenarios. This performance highlights LHCRA's potential as a transformative tool in miRNA-based diagnostics. The introduction of LHCRA marks a significant advancement in miRNA detection technology. By enabling accurate, non-invasive, and real-time monitoring of miRNA dynamics within living cells, this method offers substantial potential to enhance diagnostic accuracy and deepen our understanding of disease mechanisms, particularly in cancer. Thus, it could greatly improve therapeutic strategies and patient outcomes in clinical environment.

Cell-selective imaging of MicroRNAs via an enzyme-controlled engineered DNA nanodevice

Interest is growing in imaging intracellular microRNAs (miRNAs) because they are considered to be informative biomarkers for the diagnosis of cancers. DNA nanodevice is a desirable tool for the analysis of miRNA profiles in biological systems. Conventional DNA nanodevice, however, is always in an activated state and immediately initiates their sensing function when meeting target molecules, which precludes precise molecular sensing at the desired site. In this study, we construct a DNA nanodevice that can distinguish between two types of miRNA (miR-10b and miR-21) in cancer cells from those in normal cells through an apurinic/apyrimidinic endonuclease 1 (APE1)-controlled strand displacement reaction (SDR). Specifically, the sensing activity of this DNA nanodevice is inhibited (in the silence state) by the incorporation of a blocking sequence harboring an apurinic/apyrimidinic (AP) site during the delivery process, but this sequence can be activated by a cancer-specific APE1 after delivery of cytoplasm localization; thus, two distinct miRNA inputs can consecutively initiate toehold-mediated SDR, resulting in the generation of a fluorescence signal. Notably, the APE1-controlled DNA nanodevice facilitates dual miRNAs imaging with a superior signal-to-background ratio by enabling cancer cell-specific detection and visualization of miRNAs via an enzymatic activation pathway. Furthermore, we envision that our findings offer a straightforward yet versatile methodology with potential applications in the detailed exploration of miRNA-associated biological events, thus providing new insights into their functional significance and pathological implications.

Titanium Carbide-Based Spatiotemporally Selectable-Activated Entropy-Driven DNA Nanoplatform for Amplified MicroRNA Imaging and Photothermal Therapy In Vivo.

Engineering an elaborate nanotheranostic platform that can achieve spatiotemporally selective microRNA (miRNA) imaging and imaging-guided therapy in time is critical for precise cancer diagnosis and efficient treatment, yet remains a challenge. Herein, we present an on-site-activatable nanotheranostic platform (Ti3C2-NEDR) that engineers a photothermal-activated entropy-driven strand displacement reaction (NEDR) module on a photothermal conversion module (Ti3C2) for achieving spatiotemporally controlled miRNA-21 imaging in vivo and imaging-guided photothermal therapy only by varying the power of the near-infrared (NIR) laser. The upstream NIR photothermal conversion module, Ti3C2, can act not only as a DNA circuit carrier to deliver the NEDR module but also as a photothermal agent to activate the downstream NEDR module in low-power NIR laser irradiation. Once the NEDR module is activated by the NIR laser, the entropy-driven strand displacement reaction can be innated by intracellular miRNA-21 to generate an amplified fluorescence signal for the spatiotemporally selective imaging of miRNA-21 in vivo. Thereafter, the imaging-guided in vivo photothermal therapy can be achieved in time only by switching to the high-power NIR laser. It is envisioned that this strategy of NIR light-activated spatiotemporally selective miRNA imaging and imaging-guided on-demand therapy may expand the nanotheranostic platform for precise cancer diagnosis and personalized therapy in time, providing a remarkable prospect in biomedical diagnosis and therapy.

Engineering two-in-one DNA nanohybrids to evaluate anti-cancer effects through activatable RNA imaging

The development of DNA-based imaging techniques provides a promising way for theranostic applications. However, the dramatic chemical difference between DNA probes and therapeutics significantly hinders the construction of an ideal theranostic system for evaluation of therapeutic effects via in situ molecular imaging. In this work, we report a simple approach for the construction of a two-in-one DNA nanohybrid via one-step assembly of DNA probes and small molecular drugs, enabling evaluation of therapeutic effects via sensitive imaging of apoptosis-related mRNA. The nanohybrid was self-assembled from a rationally designed molecular beacon (MB) probe, doxorubicin (DOX, a chemotherapeutic drug) and Fe (II) ions through coordination interactions, which possesses anti-cancer effects from chemotherapeutics and the capability of efficient co-delivery of DNA probes without transfection agents. By tracking pro-apoptotic Bax mRNA expression with the MB, this system allows real-time monitoring of cell apoptotic process in response to drug treatment, enabling assessment of therapeutic effects of small molecular drugs. In addition, the approach is extended to the imaging of target microRNA in the drug treatment based on flexible DNA probe design, demonstrating the universality of this strategy. Moreover, the modular design of this DNA nanohybrid allows the introduction of photosensitizers into this system for efficient photodynamic therapy and simultaneous mRNA monitoring. This strategy expands the theranostic toolbox for the in situ evaluation of treatment response and screening anticancer drugs.

RNA-Activated CRISPR/Cas12a Nanorobots Operating in Living Cells.

Active clustered regularly interspaced short palindromic repeats (CRISPR/Cas12a) systems possess both cis-cleavage (targeted) and trans-cleavage (collateral) activities, which are useful for genome engineering and diagnostic applications. Both single- and double-stranded DNA can activate crRNA-Cas12a ribonucleoprotein (RNP) to achieve cis- and trans-cleavage enzymatic activities. However, it is not clear whether RNA can activate the CRISPR/Cas12a system and what is critical to the trans-cleavage activity. We report here that RNA can activate the CRISPR/Cas12a system and trigger its trans-cleavage activity. We reveal that the activated crRNA-Cas12a RNP favors the trans-cleavage of longer sequences than commonly used. These new findings of the RNA-activated trans-cleavage capability of Cas12a provided the foundation for the design and construction of CRISPR nanorobots that operate in living cells. We assembled the crRNA-Cas12a RNP and nucleic acid substrates on gold nanoparticles to form CRISPR nanorobots, which dramatically increased the local effective concentration of the substrate in relation to the RNP and the trans-cleavage kinetics. Binding of the target microRNA to the crRNA-Cas12a RNP activated the nanorobots and their trans-cleavage function. The repeated (multiple-turnover) trans-cleavage of the fluorophore-labeled substrates generated amplified fluorescence signals. Sensitive and real-time imaging of specific microRNA in live cells demonstrated the promising potential of the CRISPR nanorobot system for future applications in monitoring and modulating biological functions within living cells.

Persistent autonomous molecular motion of DNA walker along a single-molecule nano-track for intracellular MicroRNA imaging

While the intracellular imaging of miRNA biomarkers is of significant importance for the diagnosis and treatment of human cancers, DNA assembled nanoprobe has recently attracted considerable attention for imaging intracellular biomolecules. However, the complex construction process, intrinsic vulnerability to nuclease degradation and the limited signal transduction efficiency hamper its widespread application. In this contribution, based on persistent autonomous molecular motion of DNAzyme walker along a nano-substrate track, a DNA nanosphere probe (PNLD) is developed for the sensitive intracellular miR-21 imaging. Specifically, DNA nanosphere (called PN, single-molecule nano-track) is assembled from only one palindromic substrate, into which the locking strand-silenced DNAzymes (LD) are installed in a controlled manner. PNLD (made of PN and LD) can protect all DNA components against nuclease attack and maintain its structural integrity in serum solution over 24 h. Upon the activation by target miRNA, DNAzyme walker can move on the substrate scattered within PNLD (or on the surface) and between different PNLD objects and cleave many DNA substrates, generating an amplified signal. As a result, miR-21 can be detected down to 6.83 pM without the detectable interference from co-existing nontarget miRNAs. Moreover, PNLD system can accurately screen the different expression levels of miR-21 within the same type of cells and different types of cells, which is consistent with gold standard polymerase chain reaction (PCR) assay. Via changing the target recognition sequence, the PNLD system can be suitable for the intracellular imaging of miR-155, exhibiting the desirable universality. In addition, the DNAzyme walker-based PNLD system can be used to distinguish cancer cells from healthy cells, implying the potential application in cancer diagnosis and prognosis.

A self-powered theranostic DNA nanodevice for amplification of both intracellular microRNA imaging and photodynamic therapy

While numerous methods exist for diagnosing tumors through the detection of miRNA within tumor cells, few can simultaneously achieve both tumor diagnosis and treatment. In this study, a novel graphene oxide (GO)-based DNA nanodevice (DND), initiated by miRNA, was developed for fluorescence signal amplification imaging and photodynamic therapy in tumor cells. After entering the cells, tumor-associated miRNA drives DND to Catalyzed hairpin self-assembly (CHA). The CHA reaction generated a multitude of DNA Y-type structures, resulting in a substantial amplification of Ce6 fluorescence release and the generation of numerous singlet oxygen (1O2) species induced by laser irradiation, consequently inducing cell apoptosis. In solution, DND exhibited high selectivity and sensitivity to miRNA-21, with a detection limit of 11.47 pM. Furthermore, DND discriminated between normal and tumor cells via fluorescence imaging and specifically generated O21 species in tumor cells upon laser irradiation, resulting in tumor cells apoptosis. The DND offer a new approach for the early diagnosis and timely treatment of malignant tumors.

Dual-Function DNA Nanowires with Self-Feedback Amplification and Efficient Signal Transduction for Intracellular Imaging of MicroRNA-155.

Intracellular detection and imaging of microRNAs (miRNAs) with low expression usually face the problem of unsatisfactory sensitivity. Herein, a novel dual-function DNA nanowire (DDN) with self-feedback amplification and efficient signal transduction was developed for the sensitive detection and intracellular imaging of microRNA-155 (miRNA-155). Target miRNA-155 triggered catalytic hairpin assembly (CHA) to generate plenty of double-stranded DNA (dsDNA), and a trigger primer exposed in dsDNA initiated a hybridization chain reaction (HCR) between four well-designed hairpins to produce DDN, which was encoded with massive target sequences and DNAzyme. On the one hand, target sequences in DDN acted as self-feedback amplifiers to reactivate cascaded CHA and HCR, achieving exponential signal amplification. On the other hand, DNAzyme encoded in DDN acted as signal transducers, successively cleaving Cy5 and BHQ-2 labeled substrate S to obtain a significantly enhanced fluorescence signal. This efficient signal transduction coupling self-feedback amplification greatly improved the detection sensitivity with a limit of detection of 160 aM for miRNA-155, enabling ultrasensitive imaging of low-abundance miRNA-155 in living cells. The constructed DDN creates a promising fluorescence detection and intracellular imaging platform for low-expressed biomarkers, exhibiting tremendous potential in biomedical studies and clinical diagnosis of diseases.

Endogenously Activated and Self‐Reinforced DNA Lipid Nanodevice for Spatial‐Specific and High‐Contrast Imaging of MicroRNA in Cells and Animals

AbstractTumor‐specific microRNA (miRNA) imaging strategies are critical for investigating mechanisms associated with cancer progression; however, nonspecific signal leakage and false‐positive signaling limit their selectivity and efficiency. In this paper, an endogenously activated and self‐reinforced DNA lipid nanodevice (LND) for spatial‐specific and high‐contrast imaging of miRNAs in living cells and animals is presented. The LND is functionalized on cholesterol‐based lipid micelles (CLMs) containing smart response and self‐fueling DNA components that display redox‐activatable and autocatalytic miRNA probing activities, respectively. The LNDs are initially silenced and selectively activated using glutathione, an endogenous microenvironmental factor overexpressed in tumor cells, to prevent nonspecific signal leakage. Subsequently, an autocatalytic DNA circuit is used for tumor‐specific and high‐contrast miRNA imaging. It is demonstrated that robust CLM nanoparticles can be easily assembled from a cholesterol‐conjugated G‐rich DNA sequence by stirring in a buffered solution, enabling the LND to enter cells naturally. In addition, in vitro and in vivo data illustrate that the enhanced biostability of DNA components on the surface of LNDs can be realized for minimizing false‐positive signals, making the system ideal for miRNA imaging in living mice. This smart LND technique has great potential for precise biomedical imaging and clinical diagnosis.

Light-activated CRISPR-Cas12a for amplified imaging of microRNA in cell cycle phases at single-cell levels.

An ortho-nitrobenzyl phosphate ester-caged nucleic acid hairpin structure coupled to the CRISPR-Cas12a complex is introduced as a functional reaction module for the light-induced activation of the CRISPR-Cas12a (LAC12a) machinery toward the amplified fluorescence detection of microRNA-21 (miRNA-21). The LAC12a machinery is applied for the selective, in vitro sensing of miRNA-21 and for the intracellular imaging of miRNA-21 in different cell lines. The LAC12a system is used to image miRNA-21 in different cell cycle phases of MCF-7 cells. Moreover, the LAC12a machinery integrated in cells enables the two-photon laser confocal microscopy-assisted, light-stimulated spatiotemporal, selective activation of the CRISPR-Cas12a miRNA-21 imaging machinery at the single-cell level and the evaluation of relative expression levels of miRNA-21 at distinct cell cycle phases. The method is implemented to map the distribution of cell cycle phases in an array of single cells.

Endogenous Glutathione-Activated Nucleic Acid Molecular Circuitry for Cell-Specific MicroRNA Imaging.

Sensitive and reliable microRNA imaging in living cells has significant implications for clinical diagnosis and monitoring. Catalytic DNA circuits have emerged as potent tools for tracking these intracellular biomarkers and probing the corresponding biochemical processes. However, their utility is hindered by the low resistance to external interference, leading to undesired off-site activation and consequent signal leakage. Therefore, achieving the endogenous control of the DNA circuit's activation is preferable to the reliable target analysis in living cells. In this study, we attempted to address this challenge by engineering a simple yet effective endogenous glutathione (GSH)-regulated hybridization chain reaction (HCR) circuit for acquiring high-contrast miRNA imaging. Initially, the HCR hairpin reactants were blocked by the engineered disulfide-integrated DNA duplex, thus effectively passivating their sensing function. And the precaged HCR hairpin was liberated by the cell-specific GSH molecule, thus initiating the HCR system for selectively amplified detection of microRNA-21 (miR-21). This approach prevented unwanted signal leakage before exposure into target cells, thus ensuring robust miR-21 imaging with high accuracy and reliability in specific tumor cells. Moreover, the endogenously responsive HCR circuit established a link between the small regulatory factors and miRNA, thereby enhancing the signal gain. In summary, the endogenously activatable DNA circuit represents a versatile toolbox for robust bioanalysis and exploration of potential signaling pathways in living cells.

CsPbBr3 Perovskite Quantum Dots Encapsulated by a Polymer Matrix for Ultrasensitive Dynamic Imaging of Intracellular MicroRNA.

Herein, CsPbBr3 perovskite quantum dots (CPB PQDs)@poly(methyl methacrylate) (PMMA) (CPB@PMMA) nanospheres were used as energy donors with high Förster resonance energy transfer (FRET) efficiency and exceptional biocompatibility for ultrasensitive dynamic imaging of tiny amounts of microRNAs in living cells. Impressively, compared with traditional homogeneous single QDs as energy donors, CPB@PMMA obtained by encapsulating numerous CPB PQDs into PMMA as energy donors could not only significantly increase the efficiency of FRET via improving the local concentration of CPB PQDs but also distinctly avoid the problem of cytotoxicity caused by divulged heavy metal ions entering living cells. Most importantly, in the presence of target miRNA-21, DNA dendrimer-like nanostructures labeled with 6-carboxy-tetramethylrhodamine (TAMRA) were generated by the exposed tether interhybridization of the Y-shape structure, which could wrap around the surface of CPB@PMMA nanospheres to remarkably bridge the distance of FRET and increase the opportunity for effective energy transfer, resulting in excellent precision and accuracy for ultrasensitive and dynamic imaging of miRNAs. As proof of concept, the proposed strategy exhibited ultrahigh sensitivity with a detection limit of 45.3 aM and distinctly distinguished drug-irritative miRNA concentration abnormalities with living cells. Hence, the proposed enzyme-free CPB@PMMA biosensor provides convincing evidence for supplying accurate information, which could be expected to be a powerful tool for bioanalysis, diagnosis, and prognosis of human diseases.

Ultrahigh-Speed 3D DNA Walker with Dual Self-Protected DNAzymes for Ultrasensitive Fluorescence Detection and Intracellular Imaging of microRNA.

Herein, a dual self-protected DNAzyme-based 3D DNA walker (dSPD walker), composed of activated dual self-protected walking particles (ac-dSPWPs) and track particles (TPs), was constructed for ultrasensitive and ultrahigh-speed fluorescence detection and imaging of microRNAs (miRNAs) in living cells. Impressively, compared with the defect that "one" target miRNA only initiates "one" walking arm of the conventional single self-protected DNAzyme walker, the dSPD walker benefits from the secondary amplification and spatial confinement effect and could guide "one" target miRNA to generate "n" secondary targets, thereby initiating "n" nearby walking strands immediately, realizing the initial rate over one-magnitude-order faster than that of the conventional one. Moreover, in the process of relative motion between ac-dSPWPs and TPs, the ac-dSPWPs could cleave multiple substrate strands simultaneously to speed up movement and reduce the derailment rate, as well as combine with successive TPs to facilitate a large amount of continuous signal accumulation, achieving an ultrafast detection of miRNA-221 within 10 min in vitro and high sensitivity with a low detection limit of 0.84 pM. In addition, the DNA nanospheres obtained by the rolling circle amplification reaction can capture the Cy5 fluorescence dispersed in liquids, which achieves the high-contrast imaging of miRNA-221, resulting in further ultrasensitive imaging of miRNA-221 in cancer cells. The proposed strategy has made a bold innovation in the rapid and sensitive detection as well as intracellular imaging of low-abundance biomarkers, offering promising application in early diagnosis and relevant research of cancer and tumors.

Endogenous Enzyme-Driven Amplified DNA Nanocage Probe for Selective and Sensitive Imaging of Mature MicroRNAs in Living Cancer Cells.

Selective and sensitive imaging of intracellular mature microRNAs (miRNAs) is of great importance for biological process study and medical diagnostics. However, this goal remains challenging because of the interference of precursor miRNAs (pre-miRNAs) and the low abundance of mature miRNAs. Herein, we develop an endogenous enzyme-driven amplified DNA nanocage probe (Acage) for the selective and sensitive imaging of mature miRNAs in living cells. The Acage consists of a microRNA-responsive probe, an endogenous enzyme-driven fuel strand, and a DNA nanocage framework with an inner cavity. Benefiting from the size selectivity of DNA nanocage, smaller mature miRNAs rather than larger pre-miRNAs are allowed to enter the cavity of DNA nanocage for molecular recognition; thus, Acage can significantly reduce the signal interference of pre-miRNAs. Moreover, with the driving force of an endogenous enzyme apurinic/apyrimidinic endonuclease 1 (APE1) for efficient signal amplification, Acage enables sensitive intracellular miRNA imaging without an additional external intervention. With these features, Acage was successfully applied for intracellular imaging of mature miRNAs during drug treatment. We believe that this strategy provides a promising pathway for better understanding the functions of mature microRNAs in biological processes and medical diagnostics.

A Methylation-Gated DNAzyme Circuit for Spatially Controlled Imaging of MicroRNA in Cells and Animals.

Epigenetic modification plays an indispensable role in regulating routine molecular signaling pathways, yet it is rarely used to modulate molecular self-assembly networks. Herein, we constructed a bioorthogonal demethylase-stimulated DNA circuitry (DSC) system for high-fidelity imaging of microRNA (miRNA) in live cells and mice by eliminating undesired off-site signal leakage. The simple and robust DSC system is composed of a primary cell-specific circuitry regulation (CR) module and an ultimate signal-transducing amplifier (SA) module. After the modularly designed DSC system was delivered into target live cells, the DNAzyme of the CR module was site-specifically activated by endogenous demethylase to produce fuel strands for the subsequent miRNA-targeting SA module. Through the on-site and multiply guaranteed molecular recognitions, the lucid yet efficient DSC system realized the reliably amplified in vivo miRNA sensing and enabled the in-depth exploration of the demethylase-involved signal pathway with miRNA in live cells. Our bioorthogonally on-site-activated DSC system represents a universal and versatile biomolecular sensing platform via various demethylase regulations and shows more prospects for more different personalized theragnostics.

A GSH/APE1-controlled framework nucleic acids based entropy-driven DNA circuit for high-contrast miRNAs imaging

Catalytic DNA circuits show great potential for precise intracellular imaging. However, their selectivity and efficiency are often hindered by low anti-interference performance in the cytoplasm’s complex environment. Thus, designing DNA circuits that exhibit enhanced stability and specific activation is crucial for the accurate intracellular biomolecule imaging. Herein, we proposed a framework nucleic acids based endogenously activated entropy-driven catalytic (EDC) circuit, FEED, for in vivo microRNA imaging with enhanced selectivity and efficiency. To mitigate undesired signal leakage before reaching the target cells, the dominating EDC circuitry fuel strand was initially blocked with a disulfide bond modified DNA strand, and the target recognition site of sensing module was also closed by apurinic/apyrimidinic (AP) sites. This configuration allowed selective activation of the EDC by endogenous GSH and human apurinic/apyrimidinic endonuclease 1 (APE1) in cancer cells, facilitating high-contrast miRNA imaging. Additionally, the integrating a distinctive DNA tetrahedral structure into the EDC circuit enhances both biostability and cellular reaction efficacy. This in-site activation FEED circuit offers a programmable and modular amplification strategy for biomarker detection in live cells and mice, providing a potentially valuable molecular tool for living systems.

Catalytic hairpin assembly-based AIEgen/graphene oxide nanocomposite for fluorescence-enhanced and high-precision spatiotemporal imaging of microRNA in living cells

The low abundance, heterogeneous expression, and temporal changes of miRNA in different cellular locations pose significant challenges for both the detection sensitivity of miRNA liquid biopsy and intracellular imaging. In this work, we report an intelligently assembled biosensor based on catalytic hairpin assembly (CHA) and aggregation-induced emission (AIE), named as catalytic hairpin aggregation-induced emission (CHAIE), for the ultrasensitive detection and intracellular imaging of miRNA-155. To achieve such goal, tetraphenylethylene-N3 (TPE-N3) is used as AIE luminogen (AIEgen), while graphene oxide is introduced to quench the fluorescence. When the target miRNA is present, CHA reaction is triggered, causing the AIEgen to self-assemble with the hairpin DNA. This will restrict the intramolecular rotation of the AIEgen and produce a strong AIE fluorescence. Interestingly, CHAIE does not require any enzyme or expensive thermal cycling equipment, and therefore provides a rapid detection. Under optimal conditions, the proposed biosensor can determine miRNA in the concentration range from 2 pM to 200 nM within 30 min, with the detection limit of 0.42 pM. The proposed CHAIE biosensor in this work offers a low background signal and high sensitivity, making it applicable for highly precise spatiotemporal imaging of target miRNA in living cells.

Construction of an exogenously and endogenously Co-activated DNA logic amplifier for highly reliable intracellular MicroRNA imaging

DNA-based molecular amplifiers offer significant promise for molecular-level disease diagnosis and treatment, yet tailoring their activation for precise timing and localization remains a challenge. Herein, we’ve pioneered a dual activation strategy harnessing external light and internal ATP to create a highly controlled DNA logic amplifier (FDLA) for accurate miRNA monitoring in cancer cells. The FDLA was constructed by tethered the two functionalized catalytic hairpin assembly (CHA) hairpin modules (ATP aptamer sealed hairpin aH1 and photocleavable (PC-linker) sites modified hairpin pH2) to DNA tetrahedron (DTN). The FDLA system incorporates ATP aptamers and PC-linkers as logic control units, allowing them to respond to both exogenous UV light and endogenous ATP present within cancer cells. This response triggers the release of CHA hairpin modules, enabling amplified FRET miRNA imaging through an AND-AND gate. The DTN structure could improve the stability of FDLA and accelerate the kinetics of the strand displacement reaction. It is noteworthy that the UV and ATP co-gated DNA circuit can control the DNA bio-computing at specific time and location, offering spatial and temporal capabilities that can be harnessed for miRNA imaging. Furthermore, the miRNA-sensing FDLA amplifier demonstrates reliable imaging of intracellular miRNA with minimal background noise and false-positive signals. This highlights the feasibility of utilizing both exogenous and endogenous regulatory strategies to achieve spatial and temporal control of DNA molecular circuits within living cancer cells. Such advancements hold immense potential for unraveling the correlation between miRNA and associated diseases.

Scaling up of a Self-Confined Catalytic Hybridization Circuit for Robust microRNA Imaging.

The precise regulation of cellular behaviors within a confined, crowded intracellular environment is highly amenable in diagnostics and therapeutics. While synthetic circuitry system through a concatenated chemical reaction network has rarely been reported to mimic dynamic self-assembly system. Herein, a catalytic self-defined circuit (CSC) for the hierarchically concatenated assembly of DNA domino nanostructures is engineered. By incorporating pre-sealed symmetrical fragments into the preying hairpin reactants, the CSC system allows the hierarchical DNA self-assembly via a microRNA (miRNA)-powered self-sorting catalytic hybridization reaction. With minimal strand complexity, this self-sustainable CSC system streamlined the circuit component and achieved localization-intensified cascaded signal amplification. Profiting from the self-adaptively concatenated hybridization reaction, a reliable and robust method has been achieved for discriminating carcinoma tissues from the corresponding para-carcinoma tissues. The CSC-sustained self-assembly strategy provides a comprehensive and smart toolbox for organizing various hierarchical DNA nanostructures, which may facilitate more insights for clinical diagnosis and therapeutic assessment.

Light Responsive DNA Nanomaterials and Their Biomedical Applications.

DNA nanomaterials have been widely employed for various biomedical applications. With rapid development of chemical modification of nucleic acid, serials of stimuli-responsive elements are included in the multifunctional DNA nanomaterials. In this review, we summarize the recent advances in light responsive DNA nanomaterials based on photocleavage/photodecage, photoisomerization, and photocrosslinking for efficient bioimaging (including imaging of small molecule, microRNA, and protein) and drug delivery (including delivery of small molecule, nucleic acid, and gene editing system). We also discuss the remaining challenges and future perspectives of the light responsive DNA nanomaterials in biomedical applications.