Stimuli‐Responsive DNA Circuits for High‐Performance Bioimaging Application

Probing endogenous molecules in living entities is significant to help decipher biological functions and exploit novel theranostics. DNA circuits that can recognize molecular inputs of interest and transduce them into readable signal outputs in an isothermal and autonomous manner have been actively pursued as versatile toolkits for intracellular biosensing research. Tremendous efforts are being devoted to developing integrated DNA circuits with high sensitivity, while spatiotemporal selectivity is often overlooked in the construction of functional DNA circuitry systems. This requires the development of stimuli‐responsive DNA circuits that can be activated on‐demand from the initial sensing‐blunt state to the sensing‐ready state under a programmable manner, for achieving precise bioimaging with high spatiotemporal control. In this review, an overview of recent advances in the construction of stimuli‐responsive DNA circuits that respond to particular triggers, including external physical stimuli and endogenous biological cues, and their spatiotemporally controllable molecular bioimaging applications is provided. The current challenges and potential solutions of these stimuli‐responsive DNA circuits for their future developments in this emerging field are also discussed.


Introduction
The unique properties of DNA, including inherent programmability, nanoscale predictability, and rich functionality, have led to an explosion in dynamic DNA nanotechnology that aims to engineer molecular circuits for executing algorithms. The algorithm is directly encoded into DNA circuits whose output is reflected by DOI: 10.1002/adsr.202200102 changes in their particular physical states such as sequence exposure or structure transformation. The key mechanism for DNA circuit operation is toeholdmediated strand displacement. [1][2][3] In this process, an output DNA strand hybridizes with a substrate strand that contains an extended single-stranded toehold domain, and is displaced by a branch migration initiated by the binding of the input strand with a toehold. [4] The exploitation of dynamic DNA circuits was pioneered by Yurke et al., [5] who created a set of DNA tweezers, and was then studied in detail by Winfree et al., [6] Seeman et al., [7] and Pierce et al. [8,9] To date, various DNA circuits have been developed for different purposes such as smart theranostics, [10][11][12][13][14][15] the implementation of chemical reaction networks, [16][17][18] the control of molecular reaction pathways, [8,19] and the execution of molecular computation. [20][21][22] In particular, substantial efforts have been devoted to the design of DNA circuitry systems for the detection and imaging of far-ranging analytes (e.g., nucleic acids, proteins, metal ions, and small molecules) in living entities, [23][24][25][26][27][28][29][30] thus DNA circuits could easily contribute to decipher fundamental pathophysiological mechanism and to facilitate further medical diagnosis.
Albeit substantial progress is accomplished, all current readyto-response DNA circuitry systems have inherent limitations which would inevitably hinder their extensive utilization in precise intracellular imaging. A typical hurdle is the nonspecific strand-crosstalk (signal leakage) that usually occurs in the absence of a target considering that the DNA reactants with defined conformations are thermodynamically metastable. [55][56][57][58] The stability and performance of these DNA reactants are particularly susceptible to the complex physiological microenvironment, which is usually accompanied by fluctuant pH, salt strength, and structurally complicated biomacromolecules. Moreover, DNA circuits that respond to analytes through irreversible mechanisms could be irreversibly turned-on during cellular delivery and uptake processes if these target molecules, for example, metal ions (e.g., Na + , Mg 2+ , and Zn 2+ ) [59] and ATP, [60] are ubiquitously distributed in extracellular-intracellular medium thus leading to undesired false results. Apart from the aforementioned off-site stimulation issues, cell-targeting selectivity is also a significant limitation of current DNA circuitry systems as different types of cells usually share common disease-related biomarkers with subtle variations, and these include proteins and RNAs. [61][62][63][64] This hampers the use of DNA circuits for molecular imaging in specific cells, an ability that could be of considerable medical value in clinical diagnosis. To solve these issues, considerable endeavors have been devoted to the stimuli-responsive DNA circuits that can be activated on-demand from the initial sensing-blunt state to the sensing-ready state under a programmable manner by proceeding in a strict spatially and temporally regulated manner.

External Stimuli-Responsive DNA Circuits for Bioimaging
External stimuli, implemented by artificial physical techniques, show peculiar features of easy manipulation and facile modula-tion that could facilitate the ultrafine spatiotemporal remote control and the programmed control in a noninvasive way. Current external stimuli, explored for DNA circuits include light, ultrasound, and magnetic field.

Light-Controlled DNA Circuits for Bioimaging
With the advantages of noninvasive modality and facile manipulation, light has emerged as an attractive tool for the precise regulation of chemical and biological processes in a spatiotemporally controlled manner. [91] In general, the principles of current photoactivation can be divided into photolysis activation and photothermal activation.

Photolysis-Activated DNA Circuits for Molecular Imaging
Photolysis-activated DNA circuits rely on the photosensitive group cleavage-induced release of trigger for activating the subsequent DNA hybridization and strand displacement reaction. Through re-engineering of conventional DNA hairpin by the installation of an o-nitrophenylphosphate ester moiety into the loop region, Willner et al. designed photo-caged hairpin probes for fluorescent and electrochemical detection of mRNA in a single living cell ( Figure 1A). [92] Aptamers are single-stranded nucleic acids that can specifically bind with diverse targets including small molecules, metal ions, and cell surface proteins. [93] The fluorescent probe modified with AS1411 aptamer was passively uptake by target cells and was blocked for target mRNA recognition temporally. Upon UV irradiation, the hairpin was photo-cleaved, leading to the target mRNA binding-mediated strand displacement reaction and associated fluorescence recovery. To prevent false results, a nanoelectrode functionalized with a methylene blue redox-active photo-cleavage hairpin was penetrated into a single cell. Photo-cleavage of this hairpin leads to mRNA bindingmediated depletion of voltammetric response. This parallel sensing strategy provided a less error-prone way for the spatiotemporal detection of mRNA at the single-cell level. After the establishment of the method, it was extended to light-controlled ATP detection in a single living cell. [94] Nevertheless, such photo-activation approaches face predicaments as UV and visible light suffer from limited tissue penetration depth (<1 mm) since most photolabile moieties respond to the light within these wavelength ranges. Near-infrared (NIR) light (>800 nm) represents an ideal alternative as it possesses higher penetration depth (up to 3.2 cm) and less photon scattering. [65] Li et al. constructed a NIR-activated DNA nanodevice for in vivo miRNA imaging by integration of UV lightactivatable MBs and upconversion nanoparticles (UCNPs) (Figure 1B). [95] MBs, single-stranded DNA molecules characterized by stem-loop hairpin structure, are a straightforward tool for the detection of nucleic acids according to the Watson-Crick base pairing principle. [96] The photo-caged MBs were designed through the installation of a photo-cleavable (PC) linker into the loop region. The UCNPs absorbed deep-tissue-penetrable NIR light and produced high-energy UV emission locally, [97] which further photo-cleaved the PC linker and enable the target miRNA binding-mediated strand displacement, leading to fluorescence Figure 1. A) Schematic presentation of the optical and electrochemical detection of mRNA through photo-activated toehold-mediated displacement. Reproduced with permission. [92] Copyright 2018, American Chemical Society. B) Schematic illustration of NIR light-controlled miRNA imaging through the engineering of photocaged molecular beacon and further integration with UCNPs. Reproduced with permission. [95] Copyright 2019, American Chemical Society. C) Schematic showing the NIR light-controlled ATP imaging based on the combination of a UV light-activated aptamer probe with UCNPs. Reproduced with permission. [99] Copyright 2018, American Chemical Society. D) Scheme of the UCL-stimulated DNAzyme for metal ion imaging. Reproduced with permission. [105] Copyright 2018, American Chemical Society. enhancement for bioimaging. This nanodevice allowed remote regulation of its miRNAs imaging activity in live cells and animals. Since most physiological and pathological processes involved complicated interaction between multiple components, this method was extended to multiplex miRNAs imaging based on the upconversion luminescence (UCL)-responsive triangle DNA nano sucker. [98] Except for the aforementioned typical DNA structure, Li et al. employed a structure-switchable duplex DNA to design a photocaged aptamer circuit ( Figure 1C). [99] The aptamer was hybridized with a complementary DNA containing a PC linker and the recognition ability of which was blocked temporally. Then the UCL-activated photo-cleavage of the PC linker reduced the hybridization affinity of the complementary DNA to the aptamer, allowing structure-switch of the aptamer to recognize and detect the target ATP molecule, leading to the recovery of fluo-rescence signals. This aptamer-based duplex DNA circuit provides a versatile platform for controllable molecular imaging in vivo. Later, the Li group employed this aptamer-based DNA circuity for spatiotemporally controlled tumor recognition and targeting photodynamic therapy. [100] In addition, by integrating with mitochondria-targeting function, this structure-switchable duplex design has been applied for imaging of mitochondrial miRNAs and enzyme activity in a controllable manner. [101,102] Since sensing pH with high spatiotemporal selectivity plays a key role in monitoring different physiological processes and disease states, Li et al. reported a spatially and temporally controlled pH sensing in tumor-bearing mice through the reengineering of duplex DNA with i-motif sequence. [103] DNAzymes are single-stranded DNA molecules that have been used as effective signal transducers for biosensing based on the unique cofactor-dependent and sequence-specific catalytic Figure 2. A) Schematic illustration of a NIR light-controlled HCR for amplified imaging of mRNA. Reproduced with permission. [114] Copyright 2019, Wiley-VCH. B) Schematic presentation of UCL-stimulated CHA for amplified imaging of miRNA. Reproduced with permission. [118] Copyright 2020, American Chemical Society. C) Schematic illustration of a NIR light-controllable DNA walker driven by endogenous ATP for intracellular miRNA imaging. Reproduced with permission. [121] Copyright 2021, American Chemical Society. D) Schematic presentation of UCL-stimulated DNA walker propelled by DNAzyme for intracellular miRNA imaging. Reproduced with permission. [122] Copyright 2020, Royal Society of Chemistry.
features. [104] Through reengineering of the conventional adenosine ribonucleotide (rA) scissile position of DNAzyme with 2′-O-nitrobenzyl adenosine, Lu et al. synthesized a photo-caged DNAzyme, rendering the DNAzyme inactive. By conjugating photo-caged DNAzymes with UCNP, they further constructed a UCL-activated nanodevice that allowed for optical metal ion imaging in live cells and zebrafish ( Figure 1D). [105] In this design, the metal ion-sensing function of the engineered DNAzymes was blocked until the UCL-mediated photo-dissociation of the caged group. Except for UV light activation, Xiang et al. also developed wavelength-selective activation of photo-caged DNAzymes for metal ion sensing in live cells based on phosphorothioate chemistry. [106] The aforementioned DNA circuits based on a signal outputto-input ratio of 1:1 suffer from insufficient sensitivity for the detection of low-abundance target molecules in the biological milieu. Thus, various catalytic DNA circuits, including HCR, [107,108] CHA, [109] EDR, [110] as well as DNAzyme, [111][112][113] have been developed for achieving signal amplification. Li et al. designed a photo-activated DNA amplicon through the re-programming of HCR and further integration with UCNP (Figure 2A). [114] HCR, developed by Dirks and Pierce, [9] involves a target-initiated self-sustained cross-opening of two hairpins and generates long double-strand DNA (dsDNA) polymers, has been widely used for amplified sensing and imaging. [47,115] In their design, the toehold of one hairpin was caged by a short DNA strand linked by a PC linker which hindered the reaction activity of HCR toward initiator mRNA. Upon photolysis of the PC group by UCL, the released toehold allowed for target mRNA-initiated successive hybridization between the two hairpin reactants, leading to photo-regulated amplification of fluorescence signal and sensitive imaging of mRNA in vivo. To achieve spatiotemporally controlled probe delivery and molecular sensing, HCR hairpins were directly conjugated on nanocarriers through a PC linker. By controllably releasing HCR hairpins with high local concentrations to efficiently trigger HCR and further recruiting the long dsDNA polymers, this method contributed to highly sensitive and accurate miRNA imaging in living cells. [116,117] CHA, as a typical enzyme-free amplification technique, promotes catalyzed hybridization of hairpins and produces numerous short dsDNA products. [8,48] By extending the stem of a conventional DNA hairpin to form a dumbbell structure and introducing a PC linker into the extended segment, Luo et al. designed a UV light-responsive CHA amplicon ( Figure 2B). [118] Combining this pre-locked DNA dumbbell with UCNPs, they demonstrated NIR light-activated CHA for sensitive miRNA  [86] Copyright 2017, Wiley-VCH. B) Schematic illustration of photothermally activated strand displacement reaction for miRNA imaging and guided tumor ablation. Reproduced with permission. [126] Copyright 2021, Springer Nature. detection in live cells. In addition, the CHA circuit was coupled with photo-caged DNAzyme to amplify its signal gain for a trace amount of intracellular metal ions. [119] DNA walkers, propelled by strand displacement or DNAzyme, have been widely used for enhancing signal transduction and amplifying molecular imaging signals. According to the former principle, Huang et al. designed a UV light-controlled DNA walker powered by intracellular mRNA for precise and sensitive miRNA imaging in live cells. [120] After cooperating with upconversion nanotechnology, Xian et al. developed a NIR light controllable DNA walker driven by endogenous ATP for spatiotemporally amplified imaging of intracellular miRNA ( Figure 2C). [121] In this design, a PC linker was inserted into the loop region for the DNA hairpin, which was anchored on the UCNPs. UCL-induced photo-cleavage of the PC linker enabled the cleaved hairpin to recognize the target miRNA and released an initiator strand for the downstream amplification of AuNPs. The hairpin-stable walking strand on the AuNPs was unfolded by the initiator and was recycled by ATP recognition-mediated strand displacement, resulting in substantial fluorescence enhancement.
According to the later principle, Ju et al. developed a UCLactivated DNAzyme walker for precise miRNA imaging in living cells ( Figure 2D). [122] The miRNA recognition strand was caged into a hairpin structure which was installed with a PC linker in the loop region. The DNAzyme walker strand was designed to hybridize to the toehold of the photo-caged hairpin. The UCL-activated miRNA recognition liberated the DNAzyme walker to cyclically cleavage the substrate on the UCNPs and thus generated an amplified fluorescence signal. In addition, several enzymatic catalytic DNA circuits have been integrated with photocaging technologies to improve the sensitivity and accuracy of molecular detection, [123,124] their application in living entities still encounters a significant challenge because the cellular delivery of exogenous enzymes may lead to unnecessary damage to cells and unexpected intracellular molecule fluctuations.

Photothermal-Activated DNA Circuits for Molecular Imaging
Besides the photolysis strategy, photothermal activation represents an ideal alternative approach to control the sensing capability of DNA circuits by virtue of their facile manipulation and precise addressability. Zhu et al. constructed a NIR photothermally activated DNAzyme by using gold nanoshells (AuNS) as a light-to-heat transducer ( Figure 3A). [86] They designed a three-strand DNAzyme precursor, in which the catalytic core of DNAzyme was blocked by a complementary strand that linked to the AuNS. Upon NIR irradiation, the locally increased temperature around AuNS facilitated de-hybridization of the DNAzyme and linker strand, rending the DNAzyme active. Then the activated DNAzymes carried out metal ion-dependent substrates cleavage, resulting in fluorescence recovery. This strategy  [70] Copyright 2022, American Chemical Society. B) Schematic of magnetic field-activated DNAzyme for mRNA sensing in live cells. Reproduced with permission. [68] Copyright 2017, American Chemical Society.
has been successfully applied for photothermal-controllable Zn 2+ imaging in living cells. After that, they took this photothermalactivatable DNAzyme circuit for monitoring and regulating dysfunctional islet -cells. [125] Zhang et al. designed a thermal-activatable nanodevice for intracellular miRNA imaging by assembling trimeric DNA hybrids into mesoporous polydopamine nanoparticles ( Figure 3B). [126] The trimeric DNA hybrid featured a heteroduplex DNA where a miRNA-sensing strand was hybridized to a short switching strand to form a "shielded" internal toehold and a duplex structure of moderate melting temperature, as well as a linker strand that embedded in the mesoporous of polydopamine nanoparticles. Then NIR irradiation-induced local heat triggered the thermal-responsive exposure of the miRNA recognition segment and subsequent toehold-mediated strand displacement reactions, providing spatiotemporally controlled miRNA imaging and further guided tumor ablation.

Ultrasound-Responsive DNA Circuit for Bioimaging
Ultrasound possesses a deep tissue penetration and shows frequency-dependent biological effects. Recently, Lu and coworkers developed a high-intensity focused ultrasound (HIFU)controlled DNAzyme through spatiotemporally resolved thermal activation ( Figure 4A). [70] In the design, DNAzyme was inactivated by a protector strand to block the formation of the catalytic enzyme structure. Upon HIFU treatment, the local temperature increasement induced the dissociation of DNAzyme for fur-ther substrate cleavage in the presence of target Zn 2+ . With this design, noninvasive and spatiotemporal control of Zn 2+ -specific imaging in vivo has been realized using ultrasound stimuli.

Magnetic-Responsive DNA Circuit for Bioimaging
With excellent tissue penetration capabilities, external magnetic field gradients have been used to regulate DNA circuits. Katz et al. developed a magnetic field-activated binary DNAzyme for mRNA sensing in living cells ( Figure 4B). [68] The binary DNAzyme consisted of two split DNAzyme subunits that served as analyte binding arms and a fluorogenic reporter substrate. Two species of magnetic beads were respectively conjugated with a split DNAzyme subunit and a hook strand that was complementary to the substrate. Target mRNA-induced formation of DNAzyme core and magnetic field-induced substrate hybridization corporately activated the catalytic activity of DNAzyme, leading to substrate cleavage and amplification of fluorescent signals. Through the magnetic-controlled 3D motion and hybridization, this DNAzyme circuit achieved specific mRNA imaging in living cells.

Internal Stimuli-Responsive DNA Circuits for Bioimaging
Internal stimuli, implemented by innate biological cues, could avoid native state fluctuation of live entities by minimizing the latent side effect of exogenous stimuli, thus providing more precise on-site activation and eventually cell-specific circuitry sensing. Current internal stimuli explored for DNA circuits mainly include pH, enzymes, nucleic acids, redox, and small molecules.

pH-Responsive DNA Circuit for Bioimaging
pH values vary in the disease environment (cancer or inflammatory sites) and intracellular compartments (endosomes or lysosomes (pH 4-6.5)). [127,128] These environmental pH differences could facilitate the selective activation of DNA circuits. Li et al. designed a pH-regulated framework nucleic acid (FNA) platform consisting of two tetrahedral DNA nanostructures (TDNs) with different branched vertexes that carried split i-motif and ATP aptamer for subcellular imaging (Figure 5A). [129] Once the TDNs entered lysosomes in cells, they assembled into a dimeric structure through the pH-triggered reconfiguration of i-motif. Then, ATP is allowed to bind the split ATP aptamer via an interstructural manner, generating a larger-size FNA nanoplatform where subcellular ATP imaging was achieved.
Zhang et al. engineered a pH-actuated Y-shaped DNA nanodevice to probe intracellular miRNA by ATP-powered cascade amplification ( Figure 5B). [130] The Y-DNA circuit was formed by hybridizing an acidic-sensitive i-motif sequence, ATP-specific aptamer, and a target miRNA-binding strand. The Y-motif DNA structures modified on the CuS@SiO 2 NPs underwent a triplex DNA structure switch thereby exposing the target miRNA recognition domain in an acidic microenvironment after uptake by cells. The endogenous specific miRNA further initiated the Figure 5. A) Schematic illustration of pH-controlled reconfiguration of framework nucleic acid for subcellular imaging. Reproduced with permission. [129] Copyright 2019, Wiley-VCH. B) Schematic illustration of pH-stimulated Y-motif DNA circuit for amplified miRNA imaging through ATP self-powered strand-displacement cascade amplification. Reproduced with permission. [130] Copyright 2018, Royal Society of Chemistry. C) Schematic presentation of pH-controlled DNA walker propelled by DNAzyme for intracellular miRNA imaging. Reproduced with permission. [132] Copyright 2022, American Chemical Society. strand displacement amplification fueled by intracellular ATP, producing a dramatic fluorescence enhancement for miRNA imaging. A similar Y-motif DNA circuit incorporating with mere ATP aptamer has also been applied for real-time monitoring of ATP in lysosomes. [131] Very recently, Chao et al. designed a pH-responsive DNAzyme walker that enabled multilayer DNA cascades for precise sensing of intracellular biomolecules ( Figure 5C). [132] The four-stranded DNAzyme walker precursor was anchored on the surface of AuNP decorated with substrate strands. The DNAzyme and imotif domains in the walker strand were respectively blocked by two locking strands. Once the DNA walker was uptake by cells and passed through lysosomes with acidic pH, the precursor changed its configuration to triplex and became active toward miRNA. Then, miRNA hybridization-induced liberation of DNAzyme triggered the following catalytic substrate cleavage, allowing amplified intracellular miRNA imaging. Taking advantage of the metal-dependent cleavage of DNAzyme, the DNAzyme circuit with an acidic DNA switch has also been developed for multiple metal ions imaging in living cells. [133] In addition, the i-motif and aptamer-installed DNAzyme molecular machine was designed for dynamic cellular regulation. [134]

Enzyme-Responsive DNA Circuit for Bioimaging
Alternation of enzyme expressions often occurs in pathological conditions. Particularly, diverse proteases are found overex-pressed in cancer cells and have been recognized as biomarkers of malignancies, [135,136] making them an appealing choice to engineer tumor-specific DNA circuits for accurate and reliable bioimaging application. Recently, Li et al. reported the design of a protease-activatable aptamer circuit for tumor-specific molecular imaging (Figure 6A). [137] The circuit was built on a triple-stranded structure in which the structure-switchable aptamer was hybridized with a rationally designed PNA-peptide-PNA (PpP) triblock copolymer and a short complementary DNA strand. Protease-induced proteolysis of the peptide facilitated the activation of aptamer toward ATP. Then the ATP binding led to the dissociation of aptamer and fluorescence output. Considering diverse biomolecules involved in tumor metastasis, such as ATP and matrix metalloproteinases, they further extended this strategy to the detection of pro-metastatic targets in the extracellular microenvironment. [138] Other than a triple-stranded hybrid, the typical doublestranded DNA structure has also been applied for the construction of an enzyme-responsive circuit. Li et al. constructed an enzymatically controlled nanoflare via the engineering of structure-switchable aptamers with the incorporation of an apurinic/apyrimidinic (AP) site in the anti-aptamer strand and further conjugation on AuNPs ( Figure 6B). [139] The APE1, an essential enzyme co-expressed in the cytoplasm of many cancer cells rather than in the nucleus of normal cells, [140,141] specifically recognized and cleaved AP site in tumor cells, leading to the disruption of the duplex DNA and activation of aptamer toward target molecules, thereby enabling cancer-specific sensing in vivo Figure 6. A) Schematic illustration of the protease-activatable aptamer sensor for tumor cell-specific molecular imaging. Reproduced with permission. [137] Copyright 2021, Wiley-VCH. B) Schematic illustration of the enzyme-triggered nanoflare for intracellular ATP bioimaging. Reproduced with permission. [139] Copyright 2022, American Chemical Society.
with improved tumor specificity. This aptamer-based, enzymeactivatable double-stranded DNA structure has been extended to apoptosis-related dual protein imaging in real-time. [142] In addition, after displacing the aptamer with a telomerase (TE) primerbearing strand, the DNA duplex-based sensor was constructed for correlated enzymatic activities imaging through APE1-mediated specific cleavage and TE-induced DNA elongation. [143] In addition to the above-mentioned DNA circuits without amplification, various catalytic DNA circuits have also been used to construct enzyme-activatable DNA sensors for amplified molecular imaging. Zhao et al. fabricated a nanozyme via the rational design of catalytic DNAzyme complementarily hybridized with a damaged bases-containing excision strand that was adsorbed on a MnO 2 nanosheet (Figure 7A). [144] These DNA reactants were rapidly released by intracellular reduction of MnO 2 nanosheets, and the resulting Mn 2+ conversely served as DNAzyme cofactors. The damaged bases in excision/DNAzyme duplexes were recognized and excised by corresponding DNA base-excision repair enzymes, thus generating AP sites in situ. Then, APE1 recognized and cleaved the AP sites, leading to the dissociation of DNAzyme and the subsequent substrate cleavage-associated fluorescence recovery. The proposed DNAzyme circuit offered at least 40-fold amplified signals for base-excision repair activity in living cells.
By rational engineering of DNAzyme with a blocking strand containing the recognition site of a specific endogenous enzyme, DNAzyme re-configuration-mediated enzyme-responsive circuit has also been established for distinguishing metal-ion signals in tumor cells from those in normal cells. [145] Apart from the configuration regulation, Wang et al. developed an epigenetic regulation strategy for constructing an enzyme-responsive DNAzyme catalytic circuit ( Figure 7B). [146] The circuit was pre-pared by modifying the DNAzyme with an m 6 A methylation group that could deactivate the DNAzyme, while the cell-specific demethylase-mediated removal of methylation modification efficiently restored the initial catalytic DNAzyme for highly reliable metal-ion imaging in live cells.
As a typical catalytic DNA circuit, CHA has been integrated with diverse external stimuli technologies. By caging the hairpin reactant with an elongated double strand in conventional CHA and introducing abasic sites into the caged segments, Wang et al. designed an apurinic/apyrimidinic endonuclease 1 (HAP1)-responsive DNA probe, which allowed for endogenous enzyme-activated CHA and highly robust miRNA imaging in vivo ( Figure 7C). [147] Meanwhile, they reported another enzymecontrolled catalytic circuit for intracellular miRNA imaging by engineering an HCR circuit with a similar hairpin pre-caging technology. [83] In addition to these singlet catalytic circuits, an autocatalytic CHA-DNAzyme circuit has also been developed for endogenous DNA repair enzyme-activated uracil DNA glycosylase imaging. [148]

Nucleic Acid-Responsive DNA Circuit for Bioimaging
The intracellular distribution of mRNA represents an appealing stimulus for favoring the selective and on-site circuity activation in live cells. Wang and coworkers developed a GAPDH mRNApowered nanowalker for intracellular miRNA imaging ( Figure  8A). [149] The GAPDH gene is a housekeeping gene that is moderately abundant expressed in almost all cell types and is generally constant in the same cell type. The nanowalker was designed on the basis of AuNPs, which was functionalized with two  [144] Copyright 2017, WILEY-VCH. B) Illustration of demethylase-activated DNAzyme for intracellular metal ions imaging. Reproduced with permission. [146] Copyright 2021, Royal Society of Chemistry. C) Schematic illustration of endogenous enzyme-activated CHA for miRNA imaging in vivo. Reproduced with permission. [147] Copyright 2022, Elsevier.
types of DNA duplexes, R/S and F*/R*. The sequence of R, designed to recognize target miRNA, was hybridized to S, which was a dyes-labeled reporter strand. The sequence of F*, designed to recognize endogenous mRNA, was hybridized to R*, which could conversely displace target miRNA. In this design, the endogenous mRNA was employed as a fuel molecule to drive the autonomous motion of the nanowalker through the continuous recycling of target miRNA and the liberation of multiple reporter strands from AuNP, thus realizing a trace amount of miRNA probing in a live cell.
Wang et al. developed an orthogonally controlled catalytic DNA circuit for high-fidelity in vivo miRNA imaging by integrating the endogenous mRNA-driven CHA with EDR ( Figure 8B). [90] The auxiliary CHA circuit consisted of three hairpin reactants, in which two hairpins were grafted with split fuel subunits for the EDR circuit. While the chief EDR circuit was composed of a triplex substrate labeled with fluorophore/quencher pair. In target cells, endogenous mRNA initiated the CHA-amplified assembly of fuel strands, which could further activate the EDR circuit to facilitate the sensitive and reliable detection of miRNA. By displacing the auxiliary CHA circuit with the HCR circuit, in which the HCR hairpins were also re-engineered with a split fuel strand for the EDR circuit, they constructed another orthogonal activated catalytic DNA circuit for self-reinforced in vivo miRNA imaging. [75] On the other hand, taking the product of the EDR circuit as an initiator for the downstream circuit, an endogenous miRNA-unlocked and metal ion-responsive EDR-DNAzyme circuit has also been designed for specific tumor imaging in living mice. [150]

Redox-Responsive DNA Circuit for Bioimaging
Aberrant expression of redox species is revealed in distinct pathological conditions. [151] The exploitation of novel nanomaterials and chemical modifications that can be responsive to redox stimuli have also enriched the on-site delivery and activation of DNA circuits. Recently, Li et al. reported a GSH-responsive aptamer sensor for correlating imaging of ATP and GSH in mitochondria ( Figure 9A). [82] The aptamer strand was designed to hybridize with a complementary DNA strand that contained a chemically modified disulfide bond in the middle to form a duplex DNA. Upon disulfide cleavage by GSH, the binding affinity of the duplex was reduced, resulting in the recovery of the structureswitching capability of the aptamer toward ATP and the associated fluorescence signals.
Zhu and coworkers constructed a GSH-activatable nanowalker propelled by DNAzyme for high-fidelity intracellular miRNA imaging ( Figure 9B). [152] In the design, the catalytic DNAzyme was split into two subunits connected with the substrate strand, Figure 8. A) Illustration of the working principle of the miRNA-triggered, mRNA-powered nanowalker for amplified imaging of intracellular miRNA. Reproduced with permission. [149] Copyright 2020, Wiley-VCH. B) Schematic illustration of endogenous mRNA-controlled catalytic DNA circuit for the on-site amplified miRNA sensing in vivo. Reproduced with permission. [90] Copyright 2022, Wiley-VCH. Figure 9. A) Schematic illustration of the redox-controlled aptasensor for mitochondrial molecular imaging. Reproduced with permission. [82] Copyright 2021, American Chemical Society. B) Schematic illustration of redox-stimulated DNA walker propelled by DNAzyme for amplified miRNA imaging in vivo. Reproduced with permission. [152] Copyright 2021, Elsevier.
while the proximity-based trigger strand for DNAzyme was caged into a hairpin structure that was modified with a disulfide bond in the stem region. The DNA hybrids were conjugated on the surface of AuNPs through strong adsorption of polyA tails. Upon disulfide cleavage by endogenous GSH, the hairpin was unlocked for target miRNA recognition. Then the DNAzyme was activated by miRNA-unlocked loop region through proximity ligation to execute cyclic substrate cleavage, leading to fluorescence recovery. In the meanwhile, the unlocked loop further executed another cycle of the activation of the DNAzyme, resulting in dramatic fluorescence enhancement. In addition to DNAzyme, other catalytic circuits, such as CHA, have also been developed for GSH-activatable DNA circuits for precise miRNA imaging in live cells. [80,153] Apart from the aforementioned internal stimulus, cellular endogenous molecules (such as ATP) recently have been employed to control DNA circuits for precise bioimaging purposes. [154,155] The driving force of the DNA circuits comes from the intracellular aptamer/ATP binding. Essentially, DNA logic gates with multiple inputs manipulated in an "AND" Boolean logic manner also offer paradigms of stimuli-responsive DNA circuits. [156,157] Details on the operational principles and bioimaging applications of logic DNA circuits have been extensively detailed elsewhere. [61,158]

Conclusion and Perspectives
In this review, we summarized the recent progress of stimuliactivatable DNA circuits for spatiotemporally controllable bioimaging according to different activation modes and different designs of DNA formulation. These current stimuli can be divided into two main categories: the external physical stimuli that include light, temperature, magnetic field, and ultrasound, and the internal molecular stimuli that include pH, enzyme, nucleic acid, redox, and small molecules. Various DNA circuits depend on basic structure transformation driven by DNA hybridization reaction or advanced catalytic self-assembly driven by strand displacement have been rationally engineered. Accordingly, a series of stimuli-responsive DNA circuits have been conceived for remotely controlled imaging of diverse molecular targets, such as RNA, proteins, metal ions, and small molecules, and even correlate imaging of multiple biomolecules.
Despite progress made, this field is still in its infancy. More challenges and opportunities still remain in this area: 1) Most external stimuli strategies rely on photoactivation. Even though NIR light has obvious advantages over UV and visible light, its penetration is still limited to superficial tissues. The introduction of a magnetic field or ultrasound as an external controllable medium could improve the activation efficiency in deep tissue. The exploration of more stimuli strategies could be envisioned to improve their clinical potential according to these mature medical imaging techniques such as X-ray. 2) Diverse innate biological cues-activation methods were proposed for realizing the on-site imaging, yet were restrained by the limited disease-related endogenous molecules. Screening and leveraging diseases-specific biomarkers as endogenous stimuli for DNA circuits could offer spatiotemporal controllable molecular imaging with high selectivity. 3) Although DNA circuits based on the structural transformation or singlet amplicon have been proposed for spatiotemporally controllable imaging, extensive application of these strategies is constrained by limited sensitivity for low quantities of analytes due to their inherent low amplification efficiency. The rational engineering of a cascade or autocatalytic DNA circuits with stimuli-responsive moieties could contribute to on-site imaging with high sensitivity. 4) As DNA products of most stimuli-responsive reactions are low-molecule-weight with high mobility, the location accuracy is another problem as the products could not exactly reflect the target molecules originally generated. Therefore, the development of novel catalytic DNA circuits or integration with AIE technology, which could lead to hyperbranched DNA assembly or molecular aggregation, would help to acquire in situ information accurately. 5) Although selfcalibrate signal readout (e.g., FRET signal) has been proposed to avoid external interference (delivery efficiency, instrumentation variables, etc.), these fluorophores still suffer from inherent defects of photobleaching and environmental susceptibility. The introduction of fluorescence lifetime imaging technology that depends on the instinct properties of fluorophores or the combination of multiple-mode analysis techniques may provide accessible ways for preventing false results, in order to ensure the sensitivity and reliability of current stimuli-responsive DNA circuits.