G-Quadruplex mRNAs Silencing with Inducible Ribonuclease Targeting Chimera for Precision Tumor Therapy.

Ribonuclease targeting chimera (RIBOTAC) represents an emerging strategy for targeted therapy. However, RIBOTAC that is selectively activated by bio-orthogonal or cell-specific triggers has not been explored. We developed a strategy of inducible RIBOTAC (iRIBOTAC) that enables on-demand degradation of G-quadruplex (G4) RNAs for precision cancer therapy. iRIBOTAC is designed by coupling an RNA G4 binder with a caged ribonuclease recruiter, which can be decaged by a bio-orthogonal reaction, tumor-specific enzyme, or metabolite. A bivalent G4 binder is engineered by conjugating a near-infrared (NIR) fluorescence G4 ligand to a noncompetitive G4 ligand, conferring fluorescence activation on binding G4s with synergistically enhanced affinity. iRIBOTAC is demonstrated to greatly knockdown G4 RNAs upon activation under bio-orthogonal or cell-specific stimulus, with dysregulation of gene expressions involving cell killing, channel regulator activity, and metabolism as revealed by RNA sequencing. This strategy also shows a crucial effect on cell fate with remarkable biochemical hallmarks of apoptosis. Mice model studies demonstrate that iRIBOTAC allows selective imaging and growth suppression of tumors with bio-orthogonal and tumor-specific controls, highlighting G4 RNA targeting and inducible silencing as a valuable RIBOTAC paradigm for cancer therapy.

Whole-genome sequencing of Pseudoalteromonas piscicida 2515 revealed its antibacterial potency against Vibrio anguillarum: a preliminary invitro study.

Pseudoalteromonas piscicida 2515, isolated from Litopenaeus vannamei culture water, is a potential marine probiotic with broad anti-Vibrio properties. However, genomic information on P. piscicida 2515 is scarce. In this study, the general genomic characteristics and probiotic properties of the P. piscicida 2515 strain were analysed. In addition, we determined the antibacterial mechanism of this bacterial strain by scanning electron microscopy (SEM). The results indicated that the whole-genome sequence of P. piscicida 2515 contained one chromosome and one plasmid, including a total length of 5,541,406 bp with a G + C content of 43.24%, and 4679 protein-coding genes were predicted. Various adhesion-related genes, amino acid and vitamin metabolism and biosynthesis genes, and stress-responsive genes were found with genome mining tools. The presence of genes encoding chitin, bromocyclic peptides, lantibiotics, and sactipeptides showed the strong antibacterial activity of the P. piscicida 2515 strain. Moreover, in coculture with Vibrio anguillarum, P. piscicida 2515 displayed vesicle/pilus-like structures located on its surface that possibly participated in its bactericidal activity, representing an antibacterial mechanism. Additionally, 16 haemolytic genes and 3 antibiotic resistance genes, including tetracycline, fluoroquinolone, and carbapenem were annotated, but virulence genes encoding enterotoxin FM (entFM), cereulide (ces), and cytotoxin K were not detected. Further tests should be conducted to confirm the safety characteristics of P. piscicida 2515, including long-term toxicology tests, ecotoxicological assessment, and antibiotic resistance transfer risk assessment. Our results here revealed a new understanding of the probiotic properties and antibacterial mechanism of P. piscicida 2515, in addition to theoretical information for its application in aquaculture.

Lipoproteins and metabolites in diagnosing and predicting Alzheimer's disease using machine learning.

BACKGROUND: Alzheimer's disease (AD) is a chronic neurodegenerative disorder that poses a substantial economic burden. The Random forest algorithm is effective in predicting AD; however, the key factors influencing AD onset remain unclear. This study aimed to analyze the key lipoprotein and metabolite factors influencing AD onset using machine-learning methods. It provides new insights for researchers and medical personnel to understand AD and provides a reference for the early diagnosis, treatment, and early prevention of AD. METHODS: A total of 603 participants, including controls and patients with AD with complete lipoprotein and metabolite data from the Alzheimer's disease Neuroimaging Initiative (ADNI) database between 2005 and 2016, were enrolled. Random forest, Lasso regression, and CatBoost algorithms were employed to rank and filter 213 lipoprotein and metabolite variables. Variables with consistently high importance rankings from any two methods were incorporated into the models. Finally, the variables selected from the three methods, with the participants' age, sex, and marital status, were used to construct a random forest predictive model. RESULTS: Fourteen lipoprotein and metabolite variables were screened using the three methods, and 17 variables were included in the AD prediction model based on age, sex, and marital status of the participants. The optimal random forest modeling was constructed with "mtry" set to 3 and "ntree" set to 300. The model exhibited an accuracy of 71.01%, a sensitivity of 79.59%, a specificity of 65.28%, and an AUC (95%CI) of 0.724 (0.645-0.804). When Mean Decrease Accuracy and Gini were used to rank the proteins, age, phospholipids to total lipids ratio in intermediate-density lipoproteins (IDL_PL_PCT), and creatinine were among the top five variables. CONCLUSIONS: Age, IDL_PL_PCT, and creatinine levels play crucial roles in AD onset. Regular monitoring of lipoproteins and their metabolites in older individuals is significant for early AD diagnosis and prevention.

Screening of key immune-related gene in Parkinson's disease based on WGCNA and machine learning. / 基于WGCNAå’Œæœºå™¨å­¦ä¹ ç­›é€‰å¸•é‡‘æ£®ç— å ç–«ç›¸å ³å ³é”®åŸºå› .

OBJECTIVES: Abnormal immune system activation and inflammation are crucial in causing Parkinson's disease. However, we still don't fully understand how certain immune-related genes contribute to the disease's development and progression. This study aims to screen key immune-related gene in Parkinson's disease based on weighted gene co-expression network analysis (WGCNA) and machine learning. METHODS: This study downloaded the gene chip data from the Gene Expression Omnibus (GEO) database, and used WGCNA to screen out important gene modules related to Parkinson's disease. Genes from important modules were exported and a Venn diagram of important Parkinson's disease-related genes and immune-related genes was drawn to screen out immune related genes of Parkinson's disease. Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to analyze the the functions of immune-related genes and signaling pathways involved. Immune cell infiltration analysis was performed using the CIBERSORT package of R language. Using bioinformatics method and 3 machine learning methods [least absolute shrinkage and selection operator (LASSO) regression, random forest (RF), and support vector machine (SVM)], the immune-related genes of Parkinson's disease were further screened. A Venn diagram of differentially expressed genes screened using the 4 methods was drawn with the intersection gene being hub nodes (hub) gene. The downstream proteins of the Parkinson's disease hub gene was identified through the STRING database and a protein-protein interaction network diagram was drawn. RESULTS: A total of 218 immune genes related to Parkinson's disease were identified, including 45 upregulated genes and 50 downregulated genes. Enrichment analysis showed that the 218 genes were mainly enriched in immune system response to foreign substances and viral infection pathways. The results of immune infiltration analysis showed that the infiltration percentages of CD4+ T cells, NK cells, CD8+ T cells, and B cells were higher in the samples of Parkinson's disease patients, while resting NK cells and resting CD4+ T cells were significantly infiltrated in the samples of Parkinson's disease patients. ANK1 was screened out as the hub gene. The analysis of the protein-protein interaction network showed that the ANK1 translated and expressed 11 proteins which mainly participated in functions such as signal transduction, iron homeostasis regulation, and immune system activation. CONCLUSIONS: This study identifies the Parkinson's disease immune-related key gene ANK1 via WGCNA and machine learning methods, suggesting its potential as a candidate therapeutic target for Parkinson's disease.

Dual-Salt Electrolyte Additive Enables High Moisture Tolerance and Favorable Electric Double Layer for Lithium Metal Battery.

The carbonate electrolyte chemistry is a primary determinant for the development of high-voltage lithium metal batteries (LMBs). Unfortunately, their implementation is greatly plagued by sluggish electrode interfacial dynamics and insufficient electrolyte thermodynamic stability. Herein, lithium trifluoroacetate-lithium nitrate (LiTFA-LiNO3 ) dual-salt additive-reinforced carbonate electrolyte (LTFAN) is proposed for stabilizing high-voltage LMBs. We reveal that 1) the in situ generated inorganic-rich electrode-electrolyte interphase (EEI) enables rapid interfacial dynamics, 2) TFA- preferentially interacts with moisture over PF6 - to strengthen the moisture tolerance of designed electrolyte, and 3) NO3 - is found to be noticeably enriched at the cathode interface on charging, thus constructing Li+ -enriched, solvent-coordinated, thermodynamically favorable electric double layer (EDL). The superior moisture tolerance of LTFAN and the thermodynamically stable EDL constructed at cathode interface play a decisive role in upgrading the compatibility of carbonate electrolyte with high-voltage cathode. The LMBs with LTFAN realize 4.3 V-NCM523/4.4 V-NCM622 superior cycling reversibility and excellent rate capability, which is the leading level of documented records for carbonate electrode.

Circadian clock protein Bmal1 accelerates acute myeloid leukemia by inhibiting ferroptosis through the EBF3/ALOX15 axis.

Acute myeloid leukemia (AML) is a major leukemia with high mortality. Ferroptosis is an important regulator of cancers. However, the role of ferroptosis and its regulatory mechanisms in AML remain largely unknown. In this study, we reported elevated brain and muscle ARNT-Like protein-1 (Bmal1) expression in AML patients and cell lines, and its upregulation indicated the poor survival of patients. The correlation analysis showed that Bmal1 expression was closely correlated with cytogenetics and the French-American-British subtypes, but was not correlated with age, gender and white blood cells. RSL3 reduced Bmal1 expression in HL-60 and NB4 cells. Malondialdehyde, total iron, Fe2+ , glutathione and lipid peroxidation were examined to evaluate ferroptosis. Overexpression of Bmal1 repressed RSL3-induced ferroptosis in AML cells. Bmal1 recruited Enhancer of zeste homolog 2 (EZH2) to the Early B cell factor 3 (EBF3) promoter and enhanced its methylation, thus suppressing EBF3 expression. Moreover, the knockdown of Bmal1 sensitized AML cells to RSL3-induced ferroptosis, and it was counteracted by EBF3 knockdown. Furthermore, EBF3 bound to the Arachidonate 15-pipoxygenase (ALOX15) promoter to enhance its expression, and overexpression of EBF3 enhanced RSL3-induced ferroptosis dependent on ALOX5. We established a subcutaneous AML xenograft tumor model and reported that knockdown of Bmal1 and overexpression of EBF3 restrained AML growth by promoting ALOX15-mediated ferroptosis in vivo. Collectively, Bmal1 inhibits RSL3-induced ferroptosis by promoting EZH2-mediated EBF3 methylation and suppressing the expression of EBF3 and ALOX15, thus accelerating AML.

Genetically Encoded RNA Sensors for Ratiometric and Multiplexed Imaging of Small Molecules in Living Cells.

Genetically encoded sensors afford powerful tools for studying small molecules and metabolites in live cells. However, genetically encoded sensors with a general design remain to be developed. Here we develop genetically encoded RNA sensors with a modular design for ratiometric and multiplexed imaging of small molecules in live cells. The sensor utilizes aptazyme as a recognition module and the light-up RNA aptamer as a signal reporter. The conformation of light-up aptamers is abrogated by a blocking sequence, and aptazyme-mediated cleavage restores the correct conformation, delivering activated fluorescence for small molecule imaging. We first developed a genetically encoded ratiometric sensor using Mango aptamer as a reference and SRB2 as a reporter. It is shown that the sensor allows quantitative imaging and detection of theophylline in live cells. The generality of the design is further demonstrated for imaging other small molecules by replacing the aptazymes. Its ability for multiplexed imaging of small molecules is further explored via the integration of different small-molecule responsive aptazymes and light-up RNA aptamers. This modular design could offer a versatile platform for imaging diverse molecules in living cells.

In Situ Transformable Pro-nanotheranostic Platform for Activable Photoacoustic Imaging and Synergistic Photothermal/Chemodynamic Cancer Therapy.

Nanotheranostic platforms integrated with diagnostic and therapeutic functions have been widely developed for tumor medicine. However, the "always-on" nanotheranostic platforms suffer from poor tumor specificity, which may largely restrict therapeutic efficacy and prevent precise theranostics. Here, we develop an in situ transformable pro-nanotheranostic platform (ZnS/Cu2O@ZIF-8@PVP) by encapsulating ZnS and Cu2O nanoparticles in a metal-organic framework (MOF) nanomaterial of ZIF-8 that allows activable photoacoustic (PA) imaging and synergistic photothermal/chemodynamic therapy (PTT/CDT) of tumors in vivo. It is shown that the pro-nanotheranostic platform gradually decomposes and releases ZnS nanoparticles and Cu+ ions in acidic conditions, which spontaneously trigger a cation exchange reaction and synthesize Cu2S nanodots in situ with activated PA signals and PTT effects. Moreover, the excessive Cu+ ions function as Fenton-like catalysts and catalyze the production of highly reactive hydroxyl radicals (•OH) for CDT using elevated levels of H2O2 in tumor microenvironments (TMEs). In vivo studies demonstrate that the in situ transformable pro-nanotheranostic platform can specifically image tumors via PA and photothermal imaging and efficiently ablate tumors through synergistic CDT/PTT. Our in situ transformable pro-nanotheranostic platform could provide a new arsenal for precise theranostics in cancer therapy.

Protein-Scaffolded DNA Nanostructures for Imaging of Apurinic/Apyrimidinic Endonuclease 1 Activity in Live Cells.

Nucleic acids are valuable tools for intracellular biomarker detection and gene regulation. Here we propose a new type of protein (avidin)-scaffolded DNA nanostructure (ADN) for imaging the activity of apurinic/apyrimidinic endonuclease 1 (APE1) in live cells. ADN is designed by assembling an avidin-displayed abasic site containing DNA strands labeled with a fluorophore or a quencher via a complementary linker strand. ADN is nonemissive due to the close proximity of fluorophores and quenchers. APE1-mediated cleavage separates the fluorophores from the quenchers, delivering activated fluorescence. In vitro assays show that ADN is responsive to APE1 with high sensitivity and high specificity. ADN can efficiently enter the cells, and its capability to visualize and detect intracellular APE1 activities is demonstrated in drug-treated cells and different cell lines. The modular and easy preparation of our nanostructures would afford a valuable platform for imaging and detecting APE1 activities in live cells.

Cell-Specific Degradation of Histone Deacetylase Using Warhead-Caged Proteolysis Targeting Chimeras.

Proteolysis targeting chimeras (PROTACs) have shifted the paradigm for drug development via target protein degradation. However, PROTACs may exhibit systemic toxicity to normal cells due to indiscriminate degradation and the utility of inhibitors as a warhead for protein targeting. Here, we propose a new strategy for developing activatable PROTACs for cell-specific degradation of histone deacetylase (HDAC) with minimal side effects via caging of the warhead. Molecular docking reveals that the hydroxyl group of the HDAC inhibitor is crucial for targeting. An enzyme-activatable PROTAC is designed by caging the hydroxyl group with the substrate for NAD(P)H: quinone oxidoreductase 1 (NQO1) overexpressed in cancer cells. We demonstrate that the caged PROTAC can be converted to its active form in response to NQO1. The enzyme-activatable PROTAC allows the efficient and specific degradation of HDAC6 and exerts antiproliferative activity in NQO1-positive cells. The generalizability of the design is further demonstrated by engineering a H2O2-responsive PROTAC for specific degradation of HDAC6 in cells with elevated H2O2. The strategy of caging the ligand for target proteins would afford a new dimension for developing activatable PROTACs with high specificity and minimal side effects.

Characteristic gene prognostic model of type 1 diabetes mellitus via machine learning strategy.

The present study was designed to detect possible biomarkers associated with Type 1 diabetes mellitus (T1DM) incidence in an effort to develop novel treatments for this condition. Three mRNA expression datasets of peripheral blood mononuclear cells (PBMCs) were obtained from the GEO database. Differentially expressed genes (DEGs) between T1DM patients and healthy controls were identified by Limma package in R, and using the DEGs to conduct GO and DO pathway enrichment. The LASSO-SVM were used to screen the hub genes. We performed immune correlation analysis of hub genes and established a T1DM prognosis model. CIBERSORT algorithm was used to identify the different immune cells in distribution between T1DM and normal samples. The correlation of the hub genes and immune cells was analyzed by Spearman. ROC curves were used to assess the diagnostic value of genes in T1DM. A total of 60 immune related DEGs were obtained from the T1DM and normal samples. Then, DEGs were further screened to obtain 3 hub genes, ANP32A-IT1, ESCO2 and NBPF1. CIBERSORT analysis revealed the percentage of immune cells in each sample, indicating that there was significant difference in monocytes, T cells CD8+, gamma delta T cells, naive CD4+ T cells and activated memory CD4+ T cells between T1DM and normal samples. The area under curve (AUC) of ESCO2, ANP32A-IT1 and NBPF1 were all greater than 0.8, indicating that these three genes have high diagnostic value for T1DM. Together, the findings of these bioinformatics analyses thus identified key hub genes associated with T1DM development.

Type I Photosensitizer Targeting G-Quadruplex RNA Elicits Augmented Immunity for Cancer Ablation.

Type I photodynamic therapy (PDT) represents a promising treatment modality for tumors with intrinsic hypoxia. However, type I photosensitizers (PSs), especially ones with near infrared (NIR) absorption, are limited and their efficacy needs improvement via new targeting tactics. We develop a NIR type I PS by engineering acridinium derived donor-π-acceptor systems. The PS exhibits an exclusive type I PDT mechanism due to effective intersystem crossing and disfavored energy transfer to O2 , and shows selective binding to G-quadruplexes (G4s) via hydrogen bonds identified by a molecular docking study. Moreover, it enables fluorogenic detection of G4s and efficient O2 ⋅- production in hypoxic conditions, leading to immunogenic cell death and substantial variations of gene expression in RNA sequencing. Our strategy demonstrates augmented antitumor immunity for effective ablation of immunogenic cold tumor, highlighting its potential of RNA-targeted type I PDT in precision cancer therapy.

Genetically Encoded Light-Up RNA Amplifier Dissecting MicroRNA Activity in Live Cells.

Live cell dissection of microRNA activities is crucial for basic and translational medicine, but current hybridization-based strategies may fail to dissect surrounding-dependent activities. Here, we develop a genetically encoded miRNA-induced light-up RNA amplifier (iLAMP) that enables fast-activated, signal-amplified, fluorogenic imaging of miRNA activities in live cells. iLAMP responds to miRNA targets in the mode of "activation upon cleavage", in which the light-up RNA aptamer restores its fluorescence rapidly upon cleavage by the RNA-induced silencing complex. We demonstrate that iLAMP affords substantial signal amplification of ∼100-fold and high specificity in single nucleotide discrimination because of the miRNA-mediated cyclic cleavage. Combined with a Mango RNA aptamer reference module and a pseudoknot terminal stabilizer, iLAMP is shown for quantitative ratiometric imaging and dynamic monitoring of miRNA activities under exogenous stimulations. iLAMP is featured by a modular "plug and play" design and can be readily adapted to the detection of other miRNAs, highlighting its potential in tracking cell differentiation and screening miRNA therapeutics.

Chromogranin A-derived peptide CGA47-66 protects against septic brain injury by reducing blood-brain barrier damage through the PI3K/AKT pathway.

CGA47-66 (Chromofungin, CHR), is a peptide derived from the N-terminus of chromogranin A (CgA), has been proven to inhibit the lipopolysaccharide (LPS)-induced brain injury. However, the underlying mechanism is still unknown. We found that CGA47-66 exerted a protective effect on cognitive impairment by inhibiting the destruction of the blood-brain barrier (BBB) in the LPS-induced sepsis mice model. In addition, the hCMEC/D3 cell line was used to establish an in vitro BBB model. Under LPS stimulation, CGA47-66 could significantly alleviate the hyperpermeability of the BBB, the destruction of tight junction proteins, and the rearrangement of F-actin. To investigate the underlying mechanism, we used LY294002, a PI3K inhibitor, which partially reduced the protective effect of CGA47-66 on the integrity of BBB. Indicating that the PI3K/AKT pathway plays a vital role in the brain-protective function of CGA47-66, which might be a potential therapeutic target for septic brain injury.

A Reactivity-Tunable Self-Immolative Design Enables Histone Deacetylase-Targeted Imaging and Prodrug Activation.

Histone deacetylase (HDAC)-targeted probes and prodrugs are crucial for cancer theranostics. We developed a self-immolative design that enables in vivo activatable near-infrared fluorescence (NIRF) and photoacoustic (PA) imaging and prodrug release in response to HDAC. This design comprises a phenyl ester linker with tunable reactivity, facilitating efficient release of caged fluorophores/drugs upon deacetylation. We engineered a new fluorophore using a spirocyclic xanthene scaffold with ring-open property, affording NIRF/PA detection with high contrast. We showed that a nitro-substituted self-immolative linker allows sensitive NIRF/PA in vivo imaging of HDAC with minimal interference. A highly efficient prodrug system was further developed for targeted therapy in HDAC-overexpressed triple negative breast tumors in mice. Our study provides a valuable paradigm for HDAC-targeted NIRF/PA imaging and prodrug release in vivo, highlighting its potential for bioimaging and drug development.

Genetically Encoded Sensor Enables Endogenous RNA Imaging with Conformation-Switching Induced Fluorogenic Proteins.

Genetically encoded molecular tools are crucial for live cell RNA imaging, and few are available for endogenous RNA imaging. We develop a new genetically encoded sensor using conformation switching RNA induced fluorogenic proteins that enable multicolor and signal-amplified imaging of endogenous RNAs. The sensor system is designed with an RNA sensing module and a degron-fused fluorescent protein reporter. Target RNA induces conformation switching of the RNA sensing module to form RNA aptamers that stabilize the degron-fused protein for fluorogenic imaging. This sensor is demonstrated for high-contrast imaging of survivin mRNA abundance and dynamics in live cells. Moreover, the sensor system is extended to a multicolor palette by screening fluorogenic proteins of distinct colors, and engineered into a signal amplifier using the split fluorescent protein design. The sensor is further exploited for imaging lncRNA MALAT-1 and its translocation dynamics during mitosis. Our sensor system can afford a valuable platform for RNA imaging in biomedical research and clinical theranostics.

Genetically Encoded Dual-Color Light-Up RNA Sensor Enabled Ratiometric Imaging of MicroRNA.

MicroRNAs (miRNAs) play essential roles in regulating gene expression and cell fate. However, it remains a great challenge to image miRNAs with high accuracy in living cells. Here, we report a novel genetically encoded dual-color light-up RNA sensor for ratiometric imaging of miRNAs using Mango as an internal reference and SRB2 as the sensor module. This genetically encoded sensor is designed by expressing a splittable fusion of the internal reference and sensor module under a single promoter. This design strategy allows synchronous expression of the two modules with negligible interference. Live cell imaging studies reveal that the genetically encoded ratiometric RNA sensor responds specifically to mir-224. Moreover, the sensor-to-Mango fluorescence ratios are linearly correlated with the concentrations of mir-224, confirming their capability of determining mir-224 concentrations in living cells. Our genetically encoded light-up RNA sensor also enables ratiometric imaging of mir-224 in different cell lines. This strategy could provide a versatile approach for ratiometric imaging of intracellular RNAs, affording powerful tools for interrogating RNA functions and abundance in living cells.

Inducible CRISPR-dCas9 Transcriptional Systems for Sensing and Genome Regulation.

The clustered, regularly interspaced short palindromic repeats-associated protein 9 endonuclease (CRISPR-Cas9) and the nuclease-deactivated Cas9 (dCas9) systems have revolutionized our ability to precisely engineer and regulate genomes. Inducible CRISPR-dCas9-based transcriptional systems have been rapidly developed to conditionally control genetic manipulation. Current strategies mainly focus on conditional control of gRNA function and dCas9 protein using exogenous and endogenous triggers, including external light, small molecules, synthetic and intracellular oligonucleotides. These strategies have established novel platforms for the spatiotemporal regulation of genome activation and repression, epigenome editing, and so on. Herein, we summarize the recent progress in conditionally controlling CRISPR-dCas9 transcriptional systems through gRNA modulation and dCas9 protein engineering.

X-ray diffractive imaging of controlled gas-phase molecules: Toward imaging of dynamics in the molecular frame.

We report experimental results on the diffractive imaging of three-dimensionally aligned 2,5-diiodothiophene molecules. The molecules were aligned by chirped near-infrared laser pulses, and their structure was probed at a photon energy of 9.5 keV (λ ≈ 130 pm) provided by the Linac Coherent Light Source. Diffracted photons were recorded on the Cornell-SLAC pixel array detector, and a two-dimensional diffraction pattern of the equilibrium structure of 2,5-diiodothiophene was recorded. The retrieved distance between the two iodine atoms agrees with the quantum-chemically calculated molecular structure to be within 5%. The experimental approach allows for the imaging of intrinsic molecular dynamics in the molecular frame, albeit this requires more experimental data, which should be readily available at upcoming high-repetition-rate facilities.

Activatable CRISPR Transcriptional Circuits Generate Functional RNA for mRNA Sensing and Silencing.

CRISPR-dCas9 systems that are precisely activated by cell-specific information facilitate the development of smart sensors or therapeutic strategies. We report the development of an activatable dCas9 transcriptional circuit that enables sensing and silencing of mRNA in living cells using hybridization-mediated structure switching for gRNA activation. The gRNA is designed with the spacer sequence blocked by a hairpin structure, and mRNA hybridization induces gRNA structure switching and activates the transcription of reporter RNA. An mRNA sensor developed using a light-up RNA reporter shows high sensitivity and fast-response imaging of survivin mRNA in cells under drug treatments and different cell lines. Furthermore, a feedback circuit is engineered by incorporating a small hairpin RNA in the reporter RNA, demonstrating a smart strategy for dynamic sensing and silencing of survivin with induced tumor cell apoptosis. This circuit illustrates a broadly applicable platform for the development of cell-specific sensing and therapeutic strategies.