Enzyme-Free Dynamic DNA Reaction Networks for On-Demand Bioanalysis and Bioimaging.

ConspectusThe pursuit of in-depth studying the nature and law of life activity has been dominating current research fields, ranging from fundamental biological studies to applications that concern synthetic biology, bioanalysis, and clinical diagnosis. Motivated by this intention, the spatiotemporally controlled and in situ analysis of living cells has been a prospective branch by virtue of high-sensitivity imaging of key biomolecules, such as biomarkers. The past decades have attested that deoxyribonucleic acid (DNA), with biocompatibility, programmability, and customizable features, is a competitive biomaterial for constructing high-performance molecular sensing tools. To conquer the complexity of the wide extracellular-intracellular distribution of biomarkers, it is a meaningful breakthrough to explore high-efficiently amplified DNA circuits, which excel at operating complex yet captivating dynamic reaction networks for various bioapplications. In parallel, the multidimensional performance improvements of nucleic acid circuits, including the availability, detection sensitivity, and reliability, are critical parameters for realizing accurate imaging and cell regulation in bioanalysis.In this Account, we summarize our recent work on enzyme-free dynamic DNA reaction networks for bioanalysis from three main aspects: DNA circuitry functional extension of molecular recognition for epigenetic analysis and regulation, DNA circuitry amplification ability improvement for sensitive biomarker detection, and site-specific activation of DNA circuitry systems for reliable and accurate cell imaging. In the first part, we have designed an epigenetically responsive deoxyribozyme (DNAzyme) circuitry system for intracellular imaging and gene regulation, which enriches the possible analyzed species by chemically modifying conventional DNAzyme. For example, an exquisite N6-methyladenine (m6A)-caged DNAzyme was built for achieving the precise FTO (fat mass and obesity-associated protein)-directed gene regulation. In addition, varieties of DNAzyme-based nanoplatforms with self-sufficient cofactor suppliers were assembled, which subdued the speed-limiting hardness of DNAzyme cofactors in live-cell applications. In the second part, we have developed a series of hierarchically assembled DNA circuitry systems to improve the signal transduction ability of traditional DNA circuits. First, the amplification ability of the DNAzyme circuit has been significantly enhanced via several heterogeneously or homogeneously concatenated circuitry models. Furthermore, a feedback reaction pathway was integrated into these concatenated circuits, thus dramatically increasing the amplification efficiency. Second, considering the complex cellular environment, we have simplified the redundancy of multicomponents or reaction procedures of traditional cascaded circuits, relying on the minimal component complexity and merely one modular catalytic reaction, which guaranteed high cell-delivering uniformity while fostering reaction kinetics and analysis reliability. In the third part, we have constructed in-cell-selective endogenous-stimulated DNA circuitry systems via the multiply guaranteed molecular recognitions, which could not only eliminate the signal leakage, but could also retain its on-site and multiplex signal amplification. Based on the site-specific activation strategy, more circuitry availability in cellular scenarios has been acquired for reliable and precise biological sensing and regulation. These enzyme-free dynamic DNA reaction networks demonstrate the purpose-to-concreteness engineering for tailored multimolecule recognition and multiple signal amplification, achieving high-gain signal transduction and high-reliability targeted imaging in bioanalysis. We envision that the enzyme-free dynamic DNA reaction network can contribute to more bioanalytical layouts, which will facilitate the progression of clinical diagnosis and prognosis.

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.

Spatiotemporally Programmed Disassembly of Multifunctional Integrated DNAzyme Nanoplatfrom for Amplified Intracellular MicroRNA Imaging.

The sensing performance of DNAzymes in live cells is tremendously hampered by the inefficient and inhomogeneous delivery of DNAzyme probes and their incontrollable off-site activation, originating from their susceptibility to nuclease digestion. This requires the development of a more compact and robust DNAzyme-delivering system with site-specific DNAzyme activation property. Herein, a highly compact and robust Zn@DDz nanoplatform is constructed by integrating the unimolecular microRNA-responsive DNA-cleaving DNAzyme (DDz) probe with the requisite DNAzyme Zn2+ -ion cofactors, and the amplified intracellular imaging of microRNA via the spatiotemporally programmed disassembly of Zn@DDz nanoparticles is achieved. The multifunctional Zn@DDz nanoplatform is simply composed of a structurally blocked self-hydrolysis DDz probe and the inorganic Zn2+ -ion bridge, with high loading capacity, and can effectively deliver the initially catalytic inert DDz probe and Zn2+ into living cells with enhanced stabilities. Upon their entry into the acidic microenvironment of living cells, the self-sufficient Zn@DDz nanoparticle is disassembled to release DDz probe and simultaneously supply Zn2+ -ion cofactors. Then, endogenous microRNA-21 catalyzes the reconfiguration and activation of DDz for generating the amplified readout signal with multiply guaranteed imaging performance. Thus, this work paves an effective way for promoting DNAzyme-based biosensing systems in living cells, and shows great promise in clinical diagnosis.

Bioorthogonal Disassembly of Hierarchical DNAzyme Nanogel for High-Performance Intracellular microRNA Imaging.

Rolling circle amplification (RCA) enables the facile construction of compact and versatile DNA nanoassemblies which are yet rarely explored for intracellular analysis. This is might be ascribed to the uncontrollable and inefficient probe integration/activation. Herein, by encoding with tandem allosteric deoxyribozyme (DNA-cleaving DNAzyme), a multifunctional RCA nanogel was established for realizing the efficient intracellular microRNA imaging via the successive activation of the RCA-disassembly module and signal amplification module. The endogenous microRNA stimulates the precise degradation of DNA nanocarriers, thus leading to the efficient exposure of RCA-entrapped DNAzyme biocatalyst for an amplified readout signal. Our bioorthogonal DNAzyme disassembly strategy achieved the robust analysis of intracellular biomolecules, thus showing more prospects in clinical diagnosis.

Tailoring a Minimal Self-Replicate DNA Circuit for Highly Efficient Intracellular Imaging of microRNA.

Trace analyte detection in complex intracellular environment requires the development of simple yet robust self-sufficient molecular circuits with high signal-gain and anti-interference features. Herein, a minimal non-enzymatic self-replicate DNA circuitry (SDC) system is proposed with high-signal-gain for highly efficient biosensing in living cells. It is facilely engineered through the self-stacking of only one elementary cascade hybridization reaction (CHR), thus is encoding with more economic yet effective amplification pathways and reactants. Trigger (T) stimulates the activation of CHR for producing numerous T replica that reversely motivate new CHR reaction cycles, thus achieving the successive self-replication of CHR system with an exponentially magnified readout signal. The intrinsic self-replicate circuity design and the self-accelerated reaction format of SDC system is experimentally demonstrated and theoretically simulated. With simple circuitry configuration and low reactant complexity, the SDC amplifier enables the high-contrast and accurate visualization of microRNA (miRNA), ascribing to its robust molecular recognition and self-sufficient signal amplification, thus offering a promising strategy for monitoring these clinically significant analytes.

A Smart Deoxyribozyme-Programmable Catalytic DNA Circuit for High-Contrast MicroRNA Imaging.

Synthetic catalytic DNA circuits have been recognized as a promising signal amplification toolbox for sensitive intracellular imaging, yet their selectivity and efficiency are always constrained by uncontrolled off-site signal leakage and inefficient on-site circuitry activation. Thus, the endogenously controllable on-site exposure/activation of DNA circuits is highly desirable for achieving the selective imaging of live cells. Herein, an endogenously activated DNAzyme strategy was facilely integrated with a catalytic DNA circuit for guiding the selective and efficient microRNA imaging in vivo. To prevent the off-site activation, the circuitry constitute was initially caged without sensing functions, which could be selectively liberated by DNAzyme amplifier to guarantee the high-contrast microRNA imaging in target cells. This intelligent on-site modulation strategy can tremendously expand these molecularly engineered circuits in biological systems.

An Isothermal Autocatalytic Hybridization Reaction Circuit for Sensitive Detection of DNA Methyltransferase and Inhibitors Assay.

Abnormal DNA methylation contributes to the annoying tumorigenesis and the elevated expression of methylation-related methyltransferase (MTase) is associated with many diseases. Hence DNA MTase could serve as a promising biomarker for cancer-specific diagnosis as well as a potential therapeutic target. Herein, we developed an isothermal autocatalytic hybridization reaction (AHR) circuit for the sensitive detection of MTase and its inhibitors by integrating the catalytic hairpin assembly (CHA) converter with the hybridization chain reaction (HCR) amplifier. The initiator-mediated HCR amplifier could generate amplified fluorescent readout, as well as numerous newly activated triggers for motivating the CHA converter. The CHA converter is designed to expose the identical sequence of HCR initiators that reversely powered the HCR amplifier. Thus, the trace amount of target could produce exponentially amplified fluorescent readout by the autocatalytic feedback cycle between HCR and CHA systems. Then an auxiliary hairpin was introduced to mediate the assay of Dam MTase via the well-established AHR circuit. The Dam MTase-catalyzed methylation of auxiliary hairpin leads to its subsequent efficient cleavage by DpnI endonuclease, thus resulting in the release of HCR initiators to initiate the AHR circuit. The programmable nature of the auxiliary hairpin allows its easy adaption into other MTase assay by simply changing the recognition site. This proposed AHR circuit permits a sensitive, robust, and versatile analysis of MTase with the limit of detection (LOD) of 0.011 U/mL. Lastly, the AHR circuit could be utilized for MTase analysis in real complex samples and for evaluating the cell-cycle-dependent expression of MTase. This developed MTase-sensing strategy holds promising potential for biomedical analysis and clinical diagnosis.

Sensitive Autocatalytic Hybridization Circuit for Reliable In Situ Intracellular Polynucleotide Kinase Imaging.

Exploring the characteristic functions of polynucleotide kinase (PNK) could substantially promote the elucidation of PNK-related mechanistic pathways. Yet, the sensitive and reliable detection of intracellular PNK still presents a challenging goal. Herein, we propose a simple autocatalytic hybridization circuit (AHC) for in situ intracellular imaging of PNK with high reliability. The AHC amplifier consists of two mutually activated hybridization chain reaction (HCR) modules for magnified signal transduction. The PNK is transduced into initiator I by phosphorylation and cleavage of mediator Hp. Initiator I activates the initial HCR-1 module, leading to the formation of long dsDNA nanowires that carry numerous initiator T. Then, T-initiated feedback HCR-2 module generates branched products that contain plentiful initiator I, thus realizing an autocatalytic HCR amplification reaction. Simultaneously, the HCR-2 module is also assembled as a versatile signal transduction unit for generating the amplified readout. Based on the mutually sustained accumulation of two initiators for the reciprocal activation of two reaction modules, continuous signal amplification and assembly of high-molecular-weight copolymers endow the AHC system with high sensitivity and robustness for the PNK assay. Moreover, the PNK-sensing AHC system achieves reliable imaging of intracellular PNK, thus showing great potential to decipher the correlation between PNK and related diseases.

Modular Assembly of a Concatenated DNA Circuit for In Vivo Amplified Aptasensing.

Probing endogenous molecular profiles in living entities is of fundamental significance to decipher biological functions and exploit novel theranostics. Despite programmable nucleic acid-based aptasensing systems across the breadth of molecular imaging, an aptasensing system enabling in vivo imaging with high sensitivity, accuracy, and adaptability is highly required yet is still in its infancy. Artificial catalytic DNA circuits that can modularly integrate to generate multiple outputs from a single input in an isothermal autonomous manner, have supplemented powerful toolkits for intracellular biosensing research. Herein, a multilayer nonenzymatic catalytic DNA circuits-based aptasensing system is devised for in situ imaging of a bioactive molecule in living mice by assembling branched DNA copolymers with high-molecular-weight and high-signal-gain based on avalanche-mimicking hybridization chain reactions (HCRs). The HCRs aptasensing circuit performs as a general and powerful sensing platform for precise analysis of a series of bioactive molecules due to its inherent rich recognition repertoire and hierarchical reaction accelerations. With tumor-targeting capsule encapsulation, the HCRs aptasensing circuit is specifically delivered into tumor cells and allowed the high-contrast imaging of intracellular adenosine triphosphate in living mice, highlighting its potential for visualizing these clinically important biomolecules and for studying the associated physiological processes.

Multiply Guaranteed and Successively Amplified Activation of a Catalytic DNA Machine for Highly Efficient Intracellular Imaging of MicroRNA.

DNA amplification machines show great promise for intracellular imaging, yet are always constrained by off-site machinery activation or signal leakage, originating from the inherent thermodynamically driven hybridization between machinery substrates. Herein, an entropy-driven catalytic DNA amplification machine is integrated with the on-site amplified substrate exposure procedure to realize the high-contrast in vivo imaging of microRNA (miRNA). The key machinery substrate (fuel strands) is initially split into substrate subunits that are respectively grafted into an auxiliary DNA polymerization amplification accessory for eliminating the undesired signal leakage. Meanwhile, in target cells, the auxiliary polymerization accessory can be motivated by cell-specific mRNA for successively restoring their intact machine-propelling functions for guaranteeing the on-site amplified imaging of miRNA with high specificity. This intelligent on-site multiply guaranteed machinery can improve the specificity of catalytic DNA machines for discriminating different cell types and, thus, can provide a remarkable prospect in biomedical 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.

Construction of an Enzyme-Free Initiator-Replicated Hybridization Chain Reaction Circuit for Amplified Methyltransferase Evaluation and Inhibitor Assay.

The enzyme-free nucleic acid amplification circuit, for example, hybridization chain reaction (HCR), has paved a broad avenue for evaluating various enzyme-involved biotransformations, including DNA methyltransferases (MTases). The nonenzymatic MTase-sensing platform has supplemented a versatile toolbox for monitoring aberrant methylation in intricate biological samples, yet their amplification efficiency is always constrained by the initiator-depletion paradigm. Herein, the autonomously initiator-replicated HCR (IR-HCR) was developed as a versatile amplification system for detecting MTase with ∼100-fold sensitivity of the conventional HCR system. The initiator I-triggered HCR leads the assembly of a tandem DNAzyme concatemer that cleaves its substrate. This leads to the cyclic replication of a new initiator I for reversely motivating the initial HCR circuit, resulting in a dramatic Förster resonance energy transfer (FRET) readout. Without M.SssI MTase, hairpin HM can be recognized and digested by restriction endonuclease HpaII to release initiator I for stimulating a high FRET signal. While the M.SssI-methylated HM prohibits the HpaII-mediated cleavage of HM, the caged initiator I fails to trigger the IR-HCR circuit. Based on a systematic investigation, the IR-HCR circuit readily achieves selective and sensitive analysis of M.SssI MTase and its inhibitors. As a general MTase-sensing platform, the IR-HCR principle was further applied to analyze another MTase (Dam) by redesigning HM with the Dam recognition sequence. Overall, the versatile homogeneous MTase sensing platform was achieved via an efficient and robust initiator replication amplification circuit and may have enormous potential for early disease diagnosis.

Real-Time Investigation of Intracellular Polynucleotide Kinase Using a Cascaded Amplification Circuit.

Polynucleotide kinase (PNK) shows an in-depth correlationship with DNA repair and metabolism processes. The in situ visualization of intracellular PNK revealed an extremely biological significance in supplementing reliable and quantitative information on its spatiotemporal distribution in live cells. Herein, we developed a versatile cascaded DNA amplification circuit through the integration of catalytic DNA assembly and hybridization chain reaction circuits and realized the accurate evaluation of intracellular PNK activity via the Förster resonance energy transfer (FRET) principle. Initially, without PNK, trigger T was firmly caged in the PNK-recognizing hairpin HT, resulting in no disturbance of the concatenated circuit. However, with the introduction of PNK, the 5'-OH terminal of PNK-addressing HT was phosphorylated, then the phosphorylated HT could be subsequently digested by λ exonuclease (λ Exo) to produce trigger T of the cascaded DNA circuit. As a result, the integrated circuit was stimulated to produce an amplified FRET signal for quantitatively monitoring the activity of PNK. Due to the λ Exo-specific digestion of 5'-phosphate DNA and the high signal gain of the cascade circuit, our proposed strategy enables the sensitive analysis of PNK activity in vitro and in complex biological samples. Furthermore, our PNK-sensing platform was extensively explored in HeLa cells for realizing reliable intracellular PNK imaging and thus showed high potential in the future diagnosis and treatment of kinase-related diseases.

A Self-Catabolic Multifunctional DNAzyme Nanosponge for Programmable Drug Delivery and Efficient Gene Silencing.

DNAzyme-based gene therapy holds immense prospects for effectively treating severe diseases, yet is constrained with inefficient delivery and unconditional activation. Herein, we designed a bioinspired self-catabolic DNA nanocapsule for sustaining tumor-specific cascade activation of therapeutic DNAzyme. The exquisite DNAzyme was temporarily masked by the self-excising DNAzyme in the hierarchical rolling circle replication (RCR) nanostructures, thus stayed in an inactive state in physiological fluids. Through the multivalent tumor-anchoring aptamer strands, the RCR nanocapsule was specifically accumulated in cancer cells and was sequentially activated for motivating the ultimate DNAzyme-mediated gene silencing via the intelligent stimuli-responsive cascade DNAzyme activation. By virtue of the programmable RCR assembly strategy, our compact DNAzyme nanoplatform shows great promise for developing versatile smart gene therapeutics and personalized nanomedicines.

Construction of an Exonuclease III-Propelled Integrated DNAzyme Amplifier for Highly Efficient microRNA Detection and Intracellular Imaging with Ultralow Background.

DNAzyme amplifiers show great potential in bioanalysis but their operation in living cells still remains a challenge because of the intrinsic low-abundance analytes and the undesired background interference. Herein, we constructed a simple yet versatile exonuclease III (Exo-III)-powered cascade DNAzyme amplifier with an ultralow background for highly sensitive and selective microRNA assay in vitro and even in living cells. The present DNAzyme amplifier relies on only one DNAzyme-functionalized hairpin (HP-Dz) probe that is grafted with two exposed subunits of an analyte recognition strand, through which false enzymatic digestion and DNAzyme leakage could be substantially expelled. These protruding ssDNA strands could cooperatively recognize and efficiently bind with the miR-21 analyte, releasing the blunt 3'-terminus for Exo-III digestion and then regenerating miR-21 for a new round of HP-Dz activation. This leads to the production of numerous DNAzyme units for catalyzing the cleavage of the fluorophore/quencher-tethered substrate and yielding an enormously amplified fluorescence readout. The successive Exo-III-mediated analyte regeneration and DNAzyme-involved signal amplification facilitate their ultrasensitive miR-21 assay in vitro and intracellular miR-21 imaging. Note that the present DNAzyme module could be facilely substituted with another versatile HRP-mimicking DNAzyme, thus enabling the colorimetric assay of miR-21 with naked eye observation. Overall, this robust Exo-III-propelled cascaded DNAzyme amplifier provides more general and versatile approaches for understanding miRNA functions of related biological events.

Construction of an Autonomous Nonlinear Hybridization Chain Reaction for Extracellular Vesicles-Associated MicroRNAs Discrimination.

Extracellular vesicles (EVs) have emerged as promising tumor biomarkers for early cancer diagnosis, as primary tumor-secreted EVs carry characteristic molecular information on parent cells. It is thus desirable to realize the efficient discrimination of the signatured EVs-associated microRNAs (miRNAs) with low expression and subtle variation. Here, we introduce an autonomous nonlinear enzyme-free signal amplification paradigm for EVs discrimination through a highly sensitive and selective detection of their inherent miRNAs in situ. Our proposed amplifier consists of a modularized DNAzyme-amplified two-stage cascaded hybridization chain reaction (CHCR-DNAzyme) circuit, where the analyte-generated output of the preceding hybridization chain reaction (HCR1) stage serves as input to motivate the following hybridization chain reaction (HCR2) stage and the concomitant assembly of numerous DNAzyme biocatalysts. By incorporating a flexibly configurable sensing module, this modular CHCR-DNAzyme circuit can further extend to "plug-and-play" sensing mode that enables the miRNA assay with high specificity. The sophisticated design and the detecting performance of our CHCR-DNAzyme scheme were systematically investigated in vitro. The optimized CHCR-DNAzyme system was further applied for distinguishing EVs derived from different cells through the amplified detection of a putative miRNA biomarker in EVs. This compact CHCR-DNAzyme amplifier provides a universal and facile toolbox for highly efficient identification of multiple miRNAs-involved EVs and thus holds great potential for early cancer diagnosis.

An Autonomous Nonenzymatic Concatenated DNA Circuit for Amplified Imaging of Intracellular ATP.

A robust ATP aptasensor has been successfully constructed for intracellular imaging via the autonomous nonenzymatic cascaded hybridization chain reaction (Ca-HCR) circuit. This compact aptasensor is easily assembled by integrating the sensing module and amplification module, and is furtherly introduced for selective adenosine triphosphate (ATP) assay and for the sensitive tracking of varied ATP expressions in living cells. The ATP-targeting aptamer-encoded sensing module can specifically recognize ATP and release the initiator strand for successively motivating the two-layered HCR (hybridization chain reaction) circuit via the FRET transduction mechanism. The synergistic reaction acceleration of the two HCRs contributes to the high signal gain (amplification efficiency of N2). The whole reaction process was modeled and simulated by MATLAB to deeply explore the underlying molecular reaction mechanism, implying that the cascade HCR is sufficient enough to guarantee the ATP-recognition and amplification processes. The Ca-HCR-amplified aptasensor shows high sensitivity and selectivity for in vitro ATP assay, and can monitor these varied ATP expressions in living cells via intracellular imaging technique. Furthermore, the present aptasensor can be easily extended for monitoring other low-abundance biomarkers, which is especially important for precisely understanding these related biological processes.

The long noncoding RNA PCAT-1 links the microRNA miR-215 to oncogene CRKL-mediated signaling in hepatocellular carcinoma.

The long non-coding RNA (lncRNA) PCAT-1 resides in the chromosome 8q24 cancer-risk locus and acts as a vital oncogene during tumorigenesis and progression. However, how PCAT-1 is post-transcriptionally regulated, for example, by small ncRNAs, such as microRNAs (miRNAs) is largely unknown. Here, we report how miRNAs regulate PCAT-1 expression and also investigate the biological significance of this regulation in hepatocellular carcinoma (HCC). We found that miR-215, a P53-inducible miRNA, is a key regulator of PCAT-1 expression in HCC and identified an interaction between miR-215 and PCAT-1 in dual luciferase reporter gene assays. We also found that post-transcriptional silencing of PCAT-1 by miR-215 or PCAT-1 siRNAs significantly inhibited proliferation of HCC cells and, conversely, that inhibition of endogenous miR-215 up-regulated PCAT-1 expression and promoted cell viability. The tumor-suppressing role of miR-215 was further confirmed in an in vivo mouse HCC xenograft model. Of note, gene profiling assays suggested that the kinase CRK-like proto-oncogene, adaptor protein (CRKL), is a potential downstream target of the miR-215-PCAT-1 axis in HCC, and we demonstrated that CRKL silencing significantly suppresses cell proliferation. Taken together and considering the essential role of CRKL in cancer cells, we propose that the TP53-miR-215-PCAT-1-CRKL axis might represent an important regulatory pathway in HCC. In summary, our results highlight the involvement of several ncRNAs in HCC and thus provide critical insights into the molecular pathways operating in this malignancy.

Rational Engineering of a Multifunctional DNA Assembly for Enhanced Antibacterial Efficacy and Accelerated Wound Healing.

DNA-based assemblies hold immense prospects for antibacterial application, yet are constrained by their poor specificity and deficient antibacterial delivery. Herein, the fabrication of a versatile rolling circle amplification (RCA)-sustained DNA assembly is reported, encoding simultaneously with multivalent aptamers and tandem antibacterial agents, for target-specific and efficient antibacterial application. In the compact RCA-sustained antibacterial platform, the facilely organized multivalent aptamers guarantee the target bacteria-specific delivery of sufficient antibacterial agents which is assembled through DNA-stabilizing silver nanostructures. It is shown that the biocompatible DNA system could enhance bacteria elimination and simultaneously facilitate wound healing in vivo. By virtue of the programmable RCA assembly, the present RCA-sustained system provides a highly modular and scalable approach to design versatile multifunctional therapeutic systems.