Collagen Anchoring Protein-Nucleic Acid Chimeric Probe for In Situ In Vivo Mapping of a Tumor-Specific Protease.

In situ analysis of biomarkers in the tumor microenvironment (TME) is important to reveal their potential roles in tumor progression and early diagnosis of tumors but remains a challenge. In this work, a bottom-up modular assembly strategy was proposed for a multifunctional protein-nucleic chimeric probe (PNCP) for in situ mapping of cancer-specific proteases. PNCP, containing a collagen anchoring module and a target proteolysis-responsive isothermal amplification sensor module, can be anchored in the collagen-rich TME and respond to the target protease in situ and generate amplified signals through rolling cycle amplification of tandem fluorescent RNAs. Taking matrix metalloproteinase 2 (MMP-2), a tumor-associated protease, as the model, the feasibility of PNCP was demonstrated for the in situ detection of MMP-2 activity in 3D tumor spheroids. Moreover, in situ in vivo mapping of MMP-2 activity was also achieved in a metastatic solid tumor model with high sensitivity, providing a useful tool for evaluating tumor metastasis and distinguishing highly aggressive forms of tumors.

Visualization of Deep Tissue G-quadruplexes with a Novel Large Stokes-Shifted Red Fluorescent Benzothiazole Derivative.

G-quadruplex (G4) is a noncanonical nucleic acid secondary structure that has implications for various physiological and pathological processes and is thus essential to exploring new approaches to G4 detection in live cells. However, the deficiency of molecular imaging tools makes it challenging to visualize the G4 in ex vivo tissue samples. In this study, we established a G4 probe design strategy and presented a red fluorescent benzothiazole derivative, ThT-NA, to detect and image G4 structures in living cells and tissue samples. By enhancing the electron-donating group of thioflavin T (ThT) and optimizing molecular structure, ThT-NA shows excellent photophysical properties, including red emission (610 nm), a large Stokes shift (>100 nm), high sensitivity selectivity toward G4s (1600-fold fluorescence turn-on ratio) and robust two-photon fluorescence emission. Therefore, these features enable ThT-NA to reveal the endogenous RNA G4 distribution in living cells and differentiate the cell cycle by monitoring the changes of RNA G4 folding. Significantly, to the best of our knowledge, ThT-NA is the first benzothiazole-derived G4 probe that has been developed for imaging G4s in ex vivo cancer tissue samples by two-photon microscopy techniques.

A ligation-driven CRISPR-Cas biosensing platform for non-nucleic acid target detections.

Herein, we describe a CRISPR-Cas12a sensing platform activated by a DNA ligation reaction for the sensitive detection of non-nucleic acid targets, including NAD+, ATP and polynucleotide kinase (PNK). In this design, the DNA ligation reaction triggered by these biomolecules generates DNA duplexes, which can activate the nuclease activity of Cas12a to produce amplified fluorescence signals. As a result, this work provides an alternative strategy to expand the applicability of the CRISPR-Cas system into the detection of non-nucleic acid biomolecules.

Amplified and label-free electrochemical detection of a protease biomarker by integrating proteolysis-triggered transcription.

Cell-free synthetic biology provides a promising strategy for developing high-performance biosensors by integrating with advanced testing technologies. However, the combination of synthetic biology with electrochemical testing techniques is still underdeveloped. Here, we proposed an electrochemical biosensor for the label-free and ultrasensitive detection of target protease biomarker by coupling a protease-responsive RNA polymerase (PR) for signal amplification. Taking tumor biomarker matrix metalloprotease-2 (MMP-2) as a model protease, we employed PR to transduce each proteolysis reaction mediated by MMP-2 into multiple programmable RNA outputs that can be captured by the DNA probes immobilized on a gold electrode. Moreover, the captured RNAs are designed to contain a guanine-rich sequence that can form G-quadruplex and bind to hemin in the presence of potassium ions. In this scenario, the activity of MMP-2 is converted and amplified into the electrochemical signals of hemin. Under the optimal conditions, this PR-based electrochemical biosensor enabled the sensitive detection of MMP-2 in a wide linear dynamic range from 10 fM to 1.0 nM, with a limit of detection of 7.1 fM. Moreover, the proposed biosensor was further applied in evaluating MMP-2 activities in different cell cultures and human tissue samples, demonstrating its potential in the analysis of protease biomarkers in complex clinical samples.

A CRISPR-Cas autocatalysis-driven feedback amplification network for supersensitive DNA diagnostics.

Artificial nucleic acid circuits with precisely controllable dynamic and function have shown great promise in biosensing, but their utility in molecular diagnostics is still restrained by the inability to process genomic DNA directly and moderate sensitivity. To address this limitation, we present a CRISPR-Cas-powered catalytic nucleic acid circuit, namely, CRISPR-Cas-only amplification network (CONAN), for isothermally amplified detection of genomic DNA. By integrating the stringent target recognition, helicase activity, and trans-cleavage activity of Cas12a, a Cas12a autocatalysis-driven artificial reaction network is programmed to construct a positive feedback circuit with exponential dynamic in CONAN. Consequently, CONAN achieves one-enzyme, one-step, real-time detection of genomic DNA with attomolar sensitivity. Moreover, CONAN increases the intrinsic single-base specificity of Cas12a, and enables the effective detection of hepatitis B virus infection and human bladder cancer-associated single-nucleotide mutation in clinical samples, highlighting its potential as a powerful tool for disease diagnostics.

Proteolysis-Responsive Rolling Circle Transcription Assay Enabling Femtomolar Sensitivity Detection of a Target Protease Biomarker.

Proteases play crucial roles in the malignant progression of tumor and thus have been regarded as biomarkers for many cancers. Although protease assays such as immunoassays and fluorogenic substrate probes have been developed, it remains challenging for them to give consideration to both sensitivity and accuracy. Here, we describe a proteolysis-responsive rolling circle transcription assay (PRCTA) for the ultrasensitive and accurate detection of protease activities by the rational integration of a protease-responsive RNA polymerase and rolling circle transcription. Taking cancer biomarker matrix metalloproteinase-2 (MMP-2) as the model, the PRCTA, which can transduce and amplify each proteolysis event catalyzed by MMP-2 into the output of multiple tandem fluorescent RNAs by in vitro transcription, is constructed for the sensitive analysis of MMP-2 activities. Such a rational integration greatly enhances the signal gain in PRCTA, and it enables the limit of detection of MMP-2 as low as 3 fM. The feasibility of PRCTA has been validated by the sensitive analysis of cellular MMP-2 activities of different cell lines with good accuracy, and the readout can be readily visualized by a fluorescence imaging system. Therefore, PRCTA has achieved the detection of target protease biomarkers with femtomolar sensitivity, exhibiting promising potential in biomedicine research and cancer diagnosis.

Integrating CRISPR-Cas12a with a DNA circuit as a generic sensing platform for amplified detection of microRNA.

CRISPR-based diagnostics (CRISPR-Dx) has shown great promise in molecular diagnostics, but its utility in the sensing of microRNA (miRNA) biomarkers is limited by sensitivity, cost and robustness. Here, we describe a CRISPR-Dx method for the sensitive and cost-effective detection of miRNAs by rationally integrating CRISPR-Cas12a with DNA circuits. In this work, a modular catalytic hairpin assembly (CHA) circuit is designed to convert and amplify each target into multiple programmable DNA duplexes, which serve as triggers to initiate the trans-cleavage activity of CRISPR-Cas12a for further signal amplification. Such rational integration provides a generic assay for the effectively amplified detection of miRNA biomarkers. By simply tuning the variable regions in the CHA modules, this assay achieves sub-femtomolar sensitivity for different miRNA biomarkers, which improves the detection limit of CRISPR-Dx in the analysis of miRNA by 3-4 orders of magnitude. With the usage of the proposed assay, the sensitive assessment of miR-21 levels in different cancer cell lines and clinical serum samples has been achieved, providing a generic method for the sensitive detection of miRNA biomarkers in molecular diagnosis.

Dual-Product Synergistically Enhanced Colorimetric Assay for Sensitive Detection of Lipid Transferase Activity.

Lipid transferase-catalyzed protein lipidation plays critical roles in many physiological processes and it has been an increasingly attractive therapeutic target from cancer to neurodegeneration, while sensitive detection of lipid transferase activity in biological samples remains challenging. Here, we presented an AuNP-based colorimetric method with dual-product synergistically enhanced sensitivity for convenient detection of lipid transferase activity. Homo sapiens N-myristoyltransferase 1 (HsNMT1), a key lipid transferase, was selected as the model. Accordingly, positively charged substrate peptides (Pep) of HsNMT1 can induce the aggregation of AuNPs through disrupting their electrostatic repulsion, while the HsNMT1-catalyzed lipid modification generates aggregated lipidated peptides (C14-Pep) and negatively charged HS-CoA, which will eliminate the disruption and stabilize the AuNPs by the formation of Au-S bonds, respectively. Consequently, charge reversal of the biomolecules and the formation of Au-S bonds synergistically contribute to the stability of AuNPs in the presence of HsNMT1. Therefore, the HsNMT1 activity can be visually detected by the naked eye through the color change of the AuNPs originated from the change in their distance-dependent surface plasmon resonance absorptions. Here, the A520/A610 ratio can sensitively reflect the activity of HsNMT1 in the linear range of 2-75 nM with a low detection limit of 0.56 nM. Moreover, the method was successfully applied for probing the HsNMT1 activities in different cell lysates and inhibitor screening. Furthermore, given the replaceability of the substrate peptide, the proposed assay is promising for universal application to other lipid transferases and exhibits great potential in lipid transferase-targeted drug development.

Fluorometric and Colorimetric Dual-Readout Assay for Histone Demethylase Activity Based on Formaldehyde Inhibition of Ag+-Triggered Oxidation of O-Phenylenediamine.

Histone demethylases (HDMs) are vital players in epigenetic regulation and important targets in cancer treatment, but effective molecular tools for analyzing HDMs activity are still limited. Interestingly, we found that the process of Ag+-triggered oxidation of O-phenylenediamine (OPD) to 2,3-diaminophenazine (OPDox) could be efficiently inhibited by formaldehyde (HCHO), with the decrease of fluorescent and colorimetric signals from OPDox. Accordingly, we developed a novel label-free fluorescent and colorimetric dual-readout assay for HDMs activity based on direct quantitation of HCHO liberated in the demethylation process. On the basis of the excellent performance of the Ag+-OPD-based method for HCHO quantitation, lysine-specific demethylase 1(LSD1) activity was not only successfully detected with a low detection limit of 0.3 nM (fluorescence) and 0.5 nM (colorimetric) but also observed by the naked eye. Moreover, the feasibility of the proposed assay was further expanded to assess the LSD1 activity in cancer cell lysate and its inhibition through a mix-and-readout procedure. This label-free, cost-effective, and highly sensitive dual-readout assay presents a valuable tool for epigenetics research and drug discovery.

Enzyme-activated anchoring of peptide probes onto plasma membranes for selectively lighting up target cells.

In a cellular microenvironment, numerous biomolecules are involved in various physiological and pathological processes. However, for the in-depth and comprehensive understanding of their roles at the molecular level, there is still a lack of detection techniques for the in situ tracking of these biomolecules in a local environment. Herein, we engineered a membrane insertion peptide (MIP) as an enzyme-activated membrane insertion peptide probe (eaMIP) that allowed the in situ tracking of the activity of target enzymes in living cells. In this strategy, the membrane insertion capacity of the MIP motif in each eaMIP was caged by appending a chemical moiety. In the presence of target enzymes, the caging moiety in each eaMIP was removed by enzymatic decaging, leading to the generation of active MIPs. The versatility of this design was demonstrated by lighting up different tumor cells with distinct fluorescence signal patterns, affording an alternative tool for clinical diagnostics, biochemical research and membrane engineering.

Chimeric Peptides Self-Assembling on Titanium Carbide MXenes as Biosensing Interfaces for Activity Assay of Post-translational Modification Enzymes.

Post-translational modifications (PTMs) refer to the chemical modifications of proteins coordinated by PTM enzymes, and they play a key role in numerous physiological and pathological processes. Herein, chimeric peptide-functionalized titanium carbide MXenes (Pep-Ti3C2) were devised for the activity assay of PTM enzymes by integration with carboxypeptidase Y (CPY)-mediated peptide cleavage. The Pep-Ti3C2 is fabricated by self-assembly of chimeric peptide probes on the surface of phospholipid-coated Ti3C2 MXenes and works as the fluorescent nanoprobe for the sensing of PTM enzymes. In the presence of a target PTM enzyme, the modification groups in the peptide probes are removed along with the digestion of the peptides by CPY, thereby leading to the release of labeled fluorophores. Consequently, fluorescent analysis of PTM enzymes, including deacetylase sirtuin-1 and protein phosphatase 2C at low-nanomolar concentrations was achieved. Furthermore, the versatility of the nanoprobes was also demonstrated in simultaneous profiling of the activities of the two PTM enzymes in different cells, as well as in evaluation of the inhibition on PTMs by small molecules in complicated biological samples. Therefore, this work deploys peptide-functionalized MXenes as a generic biosensing interface for the activity assay of PTM enzymes, providing a useful tool for biochemical research and clinical diagnosis.

Protein@Inorganic Nanodumpling System for High-Loading Protein Delivery with Activatable Fluorescence and Magnetic Resonance Bimodal Imaging Capabilities.

Efficient protein delivery into the target cell is highly desirable for protein therapeutics. Current approaches for protein delivery commonly suffer from low-loading protein capacity, poor specificity for target cells, and invisible protein release. Herein, we report a protein@inorganic nanodumpling (ND) system as an intracellular protein delivery platform. Similar to a traditional Chinese food, the dumpling, ND consists of a protein complex "filling" formed by metal-ion-directed self-assembly of protein cargos fused to histidine-rich green fluorescent proteins (H39GFPs), which are further encapsulated by an external surface "wrapper" of manganese dioxide (MnO2) via in situ biomineralization. This ND structure allows for a high loading capacity (>63 wt %) for protein cargos with enhanced stability. NDs can be targeted and internalized into cancer cells specifically through folic acid receptors by surface-tailored folic acid. The protein cargo release is in a bistimuli-responsive manner, triggered by an either reductive or acidic intracellular microenvironment. Moreover, the MnO2 nanowrapper is an efficient fluorescence quencher for inner fused GFPs and also a "switch-on" magnetic resonance imaging (MRI) agent via triggered release of Mn2+ ions, which enables activatable fluorescence/MRI bimodal imaging of protein release. Finally, the ND is highly potent and specific to deliver functional protein ribonuclease A (RNase A) into cultured target cells and the tumor site in a xenografted mouse model, eliminating the tumor cells with high therapeutic efficacy. Our approach provides a promising alternative to advance protein-based cancer therapeutics.

Target-activated transcription for the amplified sensing of protease biomarkers.

Signal amplification is an effective way to achieve sensitive analysis of biomarkers, exhibiting great promise in biomedical research and clinical diagnosis. Inspired by the transcription process, here we present a versatile strategy that enables effective amplification of proteolysis into nucleic acid signal outputs in a homogeneous system. In this strategy, a protease-activatable T7 RNA polymerase is engineered as the signal amplifier and achieves 3 orders of magnitude amplification in signal gain. The versatility of this strategy has been demonstrated by the development of sensitive and selective assays for protease biomarkers, such as matrix metalloproteinase-2 (MMP-2) and thrombin, with sub-picomole sensitivity, which is 4.3 × 103-fold lower than that of the standard peptide-based method. Moreover, the proposed assay has been further applied in the detection of MMP-2 secreted by cancer cells, as well as in the assessment of MMP-2 levels in osteosarcoma tissue samples, providing a general approach for the monitoring of protease biomarkers in clinical diagnosis.

Click-Type Protein-DNA Conjugation for Mn2+ Imaging in Living Cells.

A click-type protein-DNA conjugation, named as MnDDC (Mn2+-activated DCV-DNA conjunction), is presented, where DCV (rep protein of duck circovirus) and its target DNA work as the modular blocks to rapidly and effectively generate Mn2+-dependent and site-specific protein-DNA linkage. On the basis of MnDCC, a fluorescent Mn2+ biosensor composed of DCV and a molecular beacon, was developed for rapid sensing of Mn2+ within 2 min with nanomolar sensitivity. Using the proposed biosensor, not only analysis of Mn2+ in real samples (e.g., serum and food), but also wash-free fluorescent imaging of Mn2+ in extracellular environment and cytoplasm have been achieved. Moreover, the MnDDC-based sensor was proved to be a powerful tool for visualization of Mn2+ during exploration of the associated cytotoxicity in living neural cells, which is helpful to reveal the cellular responses toward the disordered homeostasis of Mn2+ in both extracellular and intracellular microenvironments.

Functional Titanium Carbide MXenes-Loaded Entropy-Driven RNA Explorer for Long Noncoding RNA PCA3 Imaging in Live Cells.

The visualization of the long noncoding RNA of prostate cancer gene 3 (lncRNA PCA3), a specific biomarker for androgen receptor-positive prostate cancer, in living cells not only directly reflects the gene expression and localization but also offers better insight into its roles in the pathological processes. Here, we loaded an entropy-driven RNA explorer (EDRE) on the TAT peptide-functionalized titanium carbide MXenes (Ti3C2-TAT) for the imaging of nuclear lncRNA PCA3 in live cells. The EDRE was condensed on the Ti3C2-TAT (Ti3C2-TAT@EDRE) by electrostatic interaction. Ti3C2-TAT@EDRE enables the entering of cells and release of TAT peptides and EDRE in the cytoplasm by the glutathione (GSH)-triggered cleavage of the disulfide bonds in Ti3C2-TAT. The released EDRE is delivered into the nucleus by the nucleus-targeted guidance of TAT peptides, and initiated by the target lncRNA PCA3, subsequently leading to the continuous accumulation of fluorescence signals. Consequently, fluorescence analysis of lncRNA PCA3 at low-picomolar concentrations in vitro as well as sensitive live cell imaging of lncRNA PCA3 in the nucleus of androgen receptor-positive LNCaP prostate cancer cells were achieved, providing a versatile strategy for the monitoring of nucleic acid biomarkers in the nucleus of living cells.

A semisynthetic fluorescent protein assembly-based FRET probe for real-time profiling of cell membrane protease functions in situ.

A semisynthetic fluorescent protein assembly-based FRET probe (sFPAP) was proposed for cell membrane protease function assay. Here, the probe was anchored on a living cell membrane through a membrane inserting peptide (MIP) and was successfully utilized for in situ imaging of furin activity on the living cell membrane in real time.

Chimeric DNA-Functionalized Titanium Carbide MXenes for Simultaneous Mapping of Dual Cancer Biomarkers in Living Cells.

Acquiring multilayer information on diverse biomarkers with different spatial distributions at the cellular level is crucial for monitoring the progression of cancers. Herein, a dual-signal-tagged chimeric DNA-functionalized titanium carbide MXenes nanoprobe (dcDNA-Ti3C2) that responds to biomarkers with different cellular locations from plasma membrane to cytoplasm was designed toward this end. In the presence of cancer biomarkers, including transmembrane glycoprotein mucin 1 (MUC1) and cytoplasmic microRNA-21 (miR-21), the recognition between MUC1 and its aptamer in the dcDNA-Ti3C2 probe induces the separation of TAMRA-MUC1 aptamer from Ti3C2 MXenes, thereby resulting in an increase in red fluorescence; and the hybridization of miR-21 with the hairpin probe triggers the increase of green fluorescence. As a result, dual analysis of MUC1 and miR-21 at low-nanomolar concentrations in vitro, as well as in situ simultaneous imaging of the biomarkers within MCF-7 breast cancer cells, was achieved. The feasibility of the nanoprobe was further demonstrated by monitoring the expression changes of both the biomarkers in cancer cells under different inhibitor combinations. Therefore, this strategy allows us to acquire the expression levels and spatial distributions of different biomarkers in living cells, providing a helpful tool for reliable diagnosis of cancers and basic understanding their progression.

Cell-Surface-Anchored Ratiometric DNA Tweezer for Real-Time Monitoring of Extracellular and Apoplastic pH.

Precise and dynamic imaging of extracellular pH is one crucial yet challenging task for studying cell physiological and pathological processes. Here, we construct a DNA tweezer to dynamically monitor pH changes of cellular microenvironments. The DNA tweezer contains three key elements: a three-strand ssDNA-frame labeled with cholesterol to anchor it on the cell membrane, a pH-sensitive i-motif sequence in the middle to dynamically control the switch between the "open" and "closed" states of the DNA tweezer, and a pair of FRET fluorophores (rhodamine green and rhodamine red) on the two arms of the tweezer to reflect its state. With cholesterol, a natural component of cell membranes, as an anchoring element, the sensor exhibited high cell-membrane-insertion efficiency and low cytotoxicity. Using the i-motif as a sensing element, it can quickly and reversibly respond to extracellular pH in the pH range of 5.0-7.5 and further perform real-time imaging of cell-surface-pH changes with excellent spatial and temporal resolution. Moreover, apoplastic-pH change during the alkalization process of plant roots caused by rapid-alkalinization factor (RALF1) was directly detected by the sensor, demonstrating the potential applications of the sensor in cell biology, biomedical research, and plant-tissue engineering.

Charge designable and tunable GFP as a target pH-responsive carrier for intracellular functional protein delivery and tracing.

A charge designable and tunable green fluorescent protein (GFP)-based protein delivery strategy was proposed. The acquired His29GFP selectively permeates the cell membrane at a target pH of 6.5 and escapes from the endosome efficiently. The delivered RNase A caused substantial mRNA degradation in HeLa cells, and proliferation inhibition in different cell lines and a 3D tumor model at pH 6.5.

An enzymatic polymerization-activated silver nanocluster probe for in situ apoptosis assay.

As an emerging category of fluorophores, nucleic acid-stabilized silver nanoclusters (DNA/AgNCs) have attracted a great deal of interest and have been widely applied for interdisciplinary research. In this work, we have constructed a novel DNA/AgNC probe for cell apoptosis detection and imaging based on an enzyme-polymerized polyadenylic acid (poly-dA) DNA chain and a toehold strand displacement reaction. This method can effectively "tag" intracellular genomic DNA fragments, a biochemical hallmark of apoptosis, with poly-dA DNA chains up to 400-bases produced by terminal deoxynucleotidyl transferase (TdT)-activated polymerization. The strand displacement initiated by the target poly-dA DNA chain releases the quencher labeled-DNA from the DNA/AgNC probe, leading to a significant fluorescence lighting-up of DNA/AgNCs for the sensitive detection of cell apoptosis, with a high signal-to-background ratio (S/B = 58). Using the DNA/AgNC-based assay, as few as 20 apoptotic cells can be detected in vitro. Furthermore, the feasibility of our approach was demonstrated by the in situ quantitative analysis of apoptosis in HepG2 cells without the need for tedious washing and separation steps.