A meet-up of acetyl phosphate and c-di-GMP modulates BldD activity for development and antibiotic production.

Actinobacteria are ubiquitous bacteria undergoing complex developmental transitions coinciding with antibiotic production in response to stress or nutrient starvation. This transition is mainly controlled by the interaction between the second messenger c-di-GMP and the master repressor BldD. To date, the upstream factors and the global signal networks that regulate these intriguing cell biological processes remain unknown. In Saccharopolyspora erythraea, we found that acetyl phosphate (AcP) accumulation resulting from environmental nitrogen stress participated in the regulation of BldD activity through cooperation with c-di-GMP. AcP-induced acetylation of BldD at K11 caused the BldD dimer to fall apart and dissociate from the target DNA and disrupted the signal transduction of c-di-GMP, thus governing both developmental transition and antibiotic production. Additionally, practical mutation of BldDK11R bypassing acetylation regulation could enhance the positive effect of BldD on antibiotic production. The study of AcP-dependent acetylation is usually confined to the control of enzyme activity. Our finding represents an entirely different role of the covalent modification caused by AcP, which integrated with c-di-GMP signal in modulating the activity of BldD for development and antibiotic production, coping with environmental stress. This coherent regulatory network might be widespread across actinobacteria, thus has broad implications.

An Orthogonal CRISPR/dCas12a System for RNA Imaging in Live Cells.

CRISPR/Cas technology has made great progress in the field of live-cell imaging beyond genome editing. However, effective and easy-to-use CRISPR systems for labeling multiple RNAs of interest are still needed. Here, we engineered a CRISPR/dCas12a system that enables the specific recognition of the target RNA under the guidance of a PAM-presenting oligonucleotide (PAMmer) to mimic the PAM recognition mechanism for DNA substrates. We demonstrated the feasibility and specificity of this system for specifically visualizing endogenous mRNA. By leveraging dCas12a-mediated precursor CRISPR RNA (pre-crRNA) processing and the orthogonality of dCas12a from different bacteria, we further demonstrated the proposed system as a simple and versatile molecular toolkit for multiplexed imaging of different types of RNA transcripts in live cells with high specificity. This programmable dCas12a system not only broadens the RNA imaging toolbox but also facilitates diverse applications for RNA manipulation.

Universal DNA-Based Sensing Toolbox for Programming Cell Functions.

By recombining natural cell signaling systems and further reprogramming cell functions, use of genetically engineered cells and bacteria as therapies is an innovative emerging concept. However, the inherent properties and structures of the natural signal sensing and response pathways constrain further development. We present a universal DNA-based sensing toolbox on the cell surface to endow new signal sensing abilities for cells, control cell states, and reprogram multiple cell functions. The sensing toolbox contains a triangular-prismatic-shaped DNA origami framework and a sensing core anchored inside the internal confined space to enhance the specificity and efficacy of the toolbox. As a proof of principle, the sensing toolbox uses the customizable sensing core with signal sensing switches and converters to recognize unconventional signal inputs, deliver functional components to cells, and then control cell responses, including specific tumor cell death, immune cell disinhibition and adhesion, and bacterial expression. This work expands the diversity of cell sensing signals and reprograms biological functions by constructing nanomechanical-natural hybrid cells, providing new strategies for engineering cells and bacteria in diagnosis and treatment applications.

RNase H-Driven crRNA Switch Circuits for Rapid and Sensitive Detection of Various Analytical Targets.

The clustered regularly interspaced short palindromic repeats (CRISPR/Cas12a) system has exhibited great promise in the rapid and sensitive molecular diagnostics for its trans-cleavage property. However, most CRISPR/Cas system-based detection methods are designed for nucleic acids and require target preamplification to improve sensitivity and detection limits. Here, we propose a generic crRNA switch circuit-regulated CRISPR/Cas sensor for the sensitive detection of various targets. The crRNA switch is engineered and designed in a blocked state but can be activated in the presence of triggers, which are target-induced association DNA to initiate the trans-cleavage activity of Cas12a for signal reporting. Additionally, RNase H is introduced to specifically hydrolyze RNA duplexed with the DNA trigger, resulting in the regeneration of the trigger to activate more crRNA switches. Such a combination provides a generic and sensitive strategy for the effective sensing of the p53 sequence, thrombin, and adenosine triphosphate. The design is incorporated with nucleic acid nanotechnology and extensively broadens the application scope of the CRISPR technology in biosensing.

A DNA Nanodevice-Based Platform with Diverse Capabilities.

Social biotic colonies often perform intricate tasks by interindividual communication and cooperation. Inspired by these biotic behaviors, a DNA nanodevice community is proposed as a universal and scalable platform. The modular nanodevice as the infrastructure of platform contains a DNA origami triangular prism framework and a hairpin-swing arm machinery core. By coding and decoding a signal domain on the shuttled output strand in different nanodevices, an orthogonal inter-nanodevice communication network is established to connect multi-nanodevices into a functional platform. The nanodevice platform enables implementation of diverse tasks, including signal cascading and feedback, molecular input recording, distributed logic computing, and modeling of simulation for virus transmission. The nanodevice platform with powerful compatibility and programmability presents an elegant example of the combination of the distributed operation of multiple devices and the complicated interdevice communication network, and may become a new generation of intelligent DNA nanosystems.

Nano-Biohybrid DNA Engager That Reprograms the T-Cell Receptor.

Although engineered T cells with transgenic chimeric antigen receptors (CARs) have made a breakthrough in cancer therapeutics, this approach still faces many challenges in the specificity, efficacy, and self-safety of genetic engineering. Here, we developed a nano-biohybrid DNA engager-reprogrammed T-cell receptor (EN-TCR) system to improve the specificity and efficacy, mitigate the excessive activation, and shield against risks from transgenesis, thus achieving a diversiform and precise control of the T-cell response. Utilizing modular assembly, the EN-TCR system can graft different specificities on T cells via antibody assembly. Besides, the designability of DNA hybridization enables precise target recognition by the library of multiantigen cell recognition circuits and allows gradual tuning of the T-cell activation level by the signaling switch and independent control over different types of T cells. Furthermore, we demonstrated the effectiveness of the system in tumor models. Together, this study provides a nongenetic T-cell engineering strategy to overcome major hindrances in T-cell therapy and may be extended to a general and convenient cell engineering strategy.

An Ultrasensitive, One-Pot RNA Detection Method Based on Rationally Engineered Cas9 Nickase-Assisted Isothermal Amplification Reaction.

RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR) have revolutionized molecular diagnostics by offering versatile Cas effectors. We previously developed an isothermal amplification reaction method using Cas9 nickase (Cas9 nAR) to detect genomic DNA. However, slow dissociation of Cas9n from nicked double-stranded DNA (dsDNA) substrates dramatically hampers the cooperation between Cas9n and DNA polymerase, leading to low amplification efficiency. Here, we use structure-guided protein engineering to generate a Cas9n variant with faster kinetics and enhanced targeting specificity, and apply it to develop Cas9 nAR version 2 (Cas9 nAR-v2) by deftly merging reverse transcription with nicking-extension-displacement-based amplification for isothermal, one-pot RNA detection. This assay is validated by detecting Salmonella typhimurium 16S rRNA, Escherichia coli O157:H7 16S rRNA, synthetic SARS-CoV-2 genes, and HIV virus RNA, showing a quantitative analysis over a wide, linear range and a detection limit as low as fewer than ten copies of RNA molecules per reaction (20 µL volume). It also shows an excellent nucleotide-mutation discrimination capability in detecting SARS-CoV-2 variants. Furthermore, Cas9 nAR-v2 is compatible with low-cost point-of-care (POC) tests based on fluorescence and lateral-flow readouts. In summary, this method provides a new paradigm for sensitive, direct RNA detection and would spur the exploration of engineered Cas effectors with improved properties for a wide range of biological applications.

Switching the Activity of CRISPR/Cas12a Using an Allosteric Inhibitory Aptamer for Biosensing.

The current CRISPR/Cas12a-based diagnostic techniques focus on designing the crRNA or substrate DNA elements to indirectly switch the trans-cleavage activity of Cas12a responsive to target information. Here, we propose the use of an allosteric DNA probe to directly regulate the trans-cleavage activity of Cas12a and present a method for sensing different types of analytes. An allosteric inhibitor probe is rationally designed to couple the target recognition sequence with the inhibitory aptamer of the CRISPR/Cas12a system and enables binding to a specific target to induce the change of conformation, which leads to the loss of its inhibitory function on Cas12a. As a result, the structure-switchable probe can regulate the degree of activity of Cas12a depending on the dose of target. Scalability of our strategy can be achieved by simply replacing the loop domain with different target recognition sequences. The proposed method was validated by detecting adenosine triphosphate and let-7a, giving the detection limits of 490 nM and 26 pM, respectively, and showing an excellent specificity. We believe that this work exploits a viable approach to use the inhibitory aptamer of Cas12a as a regulatory element for biosensing purposes, enriching the arsenal of CRISPR/Cas12a-based methods for molecular diagnostics and spurring further development and application of aptamers of the CRISPR/Cas system.

Simultaneous imaging of cancer biomarkers in live cells based on DNA-engineered exosomes.

Cancer biomarkers are directly related to the development of cancers. Noninvasive identification of the location and expression levels of these biomarkers in live cancer cells offers great potential for accurate early-stage cancer diagnosis and cancer metastasis monitoring. Herein, we propose a DNA-engineered exosome (DNA-Exo) nanoplatform to image dual cancer biomarkers at the single-cell level, in which DNA probes were modified with the cholesterol group to facilely anchor on the exosomal membrane through hydrophobic interaction. Fluorophore-labeled DNA aptamer and hairpin probes targeting two kinds of cancer biomarkers of transmembrane glycoprotein mucin 1 (MUC1) and cytoplasmic microRNA-21 (miR-21), respectively, were employed for convenient dual-fluorescence imaging of cancer cells. The cellular uptake of DNA-Exos induced the specific recognition of MUC1 and miR-21, allowing the acquisition of the expression levels and spatial distributions of these two biomarkers in three tested cell lines. Our work demonstrated that the proposed DNA-Exos with designable functions have the capacity to visually discriminate different cell types based on the specific recognition of analytes.

Multimachine Communication Network That Mimics the Adaptive Immune Response.

Biological organisms capable of controlling and performing a wide variety of functions have inspired attempts to mimic biological systems with designable intelligence. Here we develop a multimachine communication network (MMCN) to mimic the operation and function of adaptive immune response (AIR) via connecting three kinds of DNA machines built from module-functionalized gold nanoparticles. These machines simulate three critical immune cells, dendritic cells, T and B lymphocytes, and their differentiation and coordinated interaction upon exposure and response to an invading pathogen. MMCN is composed of standard modules with track, movement, and fuel components that allow for the (1) integration and adaptability of a single machine, (2) convenient spatiotemporal control of the sequential activation of a single machine, and (3) rapid reaction rate and high efficiency owing to an enhanced local concentration of interacting species. We show that the proposed network can sense and clear the corresponding pathogen via consecutive activation and connection of the machines, simultaneously forming a memory to respond more rapidly and effectively upon the second invasion of the pathogen. This system may be extended to construct powerful networks to execute more sophisticated tasks and accomplish diverse functions.

Multiple and sensitive SERS detection of cancer-related exosomes based on gold-silver bimetallic nanotrepangs.

Exosomes are endogenous vesicles of cells, and can be used as important biomarkers for cancers. In this work, we developed a sensitive and reliable SERS sensor for simultaneous detection of multiple cancer-related exosomes. The SERS detection probes were made of bimetallic SERS-active nanotags, gold-silver-silver core-shell-shell nanotrepangs (GSSNTs), which were composed of bumpy surface nanorod (gold nanotrepang, GNT) cores and bilayer silver shells, and decorated with linker DNAs, which were complementary to the aptamer targeting exosomes. Three kinds of SERS detection probes were designed via the adoption of different Raman reporter molecules and linker DNAs. The capture probes were prepared by modifying specific aptamers of the target exosomes on magnetic beads (MBs). In the absence of target exosomes, SERS detection probes were coupled with MBs via specific DNA hybridization for use as aptamer-based SERS sensors. In the presence of target exosomes, the aptamer specifically recognized and captured the exosomes, and GSSNTs were subsequently released into the supernatant. Therefore, attenuated SERS signals were detected on the MBs, indicating the presence of target exosomes. The proposed aptamer-based SERS sensor is expected to be a facile and sensitive method for the multiplex detection of cancer biomarkers and has potential future applications in clinical diagnosis.

A Cas12a-mediated cascade amplification method for microRNA detection.

MicroRNAs (miRNAs) play a vital role in various biological processes and act as important biomarkers for clinical cancer diagnosis, prognosis, and therapy. Here, we took advantage of Cas12a trans-cleavage activity to develop an enzyme-assisted cascade amplification method for isothermal miRNA detection. A target miRNA-initiated ligation reaction would allow for the production of transcription templates that triggered the transcriptional amplification of RNA strands. These RNA strands were cleaved by the 8-17E DNAzyme to generate crRNAs and recycled RNAs which have the same sequence as the target miRNA. The amplified abundant crRNAs bound to Cas12a and dsDNA activators to form the complex, which trans-cleaved the ssDNA reporters to generate a fluorescence signal for miRNA quantitative analysis. The proposed method exhibits a femtomolar limit of detection and a good specificity in distinguishing the homologous sequences of miRNAs. Its practical application ability was further tested in different cell lines.

Construction of a DNA-AuNP-based satellite network for exosome analysis.

As exosomes have been playing an increasingly important role in the diagnosis, treatment and prognosis of diseases, the analysis of exosome contents becomes more crucial. Therefore, the development of a cost-effective and simple exosome separation method that achieves high purity is urgently needed, and it is vital for further research in cancer. In this work, we constructed a DNA-AuNP-based satellite network which integrates low-speed centrifugal exosome isolation, detection and protein analysis. The rolling circle amplification (RCA) reaction is used to produce a long-chain DNA hairpin structure comprising a plurality of functional domains, such as CD63 aptamer sequences, linker sequences, and spacer sequences with complementary base pairs to form a hairpin structure. When the CD63 aptamers bind to exosomes, the hairpin structure changes its conformation, exposing the linker sequences (AuNP binding sequence). Then the probe on the surface of AuNPs combines with the long-chain DNA by the toehold-mediated strand displacement reaction, releasing the fluorescent labeled complementary probe as the detection signal and simultaneously forming the DNA-AuNP-based satellite network. Thus, exosomes can be isolated by low-speed centrifugation. The formation of the DNA-AuNP-based satellite network was confirmed by transmission electron microscopy and confocal fluorescence microscopy. In addition, we established a standard curve for exosome detection which showed good linearity of the fluorescence ratio vs. log(exosome concentration). LC/MS for protein profiling of the captured exosomes demonstrated that our method has potential application in the field of exosome research.

A dual signal amplification method for exosome detection based on DNA dendrimer self-assembly.

An increasing number of studies have found that circulating exosomes play a vital role in the occurrence and metastasis of cancer. Therefore, a direct, sensitive and specific method for detection of tumor exosomes will contribute to the diagnosis and prognosis of cancer. In this work, we take advantage of the facile adaptability of aptamers to design an exosome quantitative method, which converts an exosome capture event to nucleic acid detection. With the help of a hairpin DNA cascade reaction (HDCR) and easy accessibility of DNA dendrimer self-assembly, dual signal amplification was achieved. A CD63 aptamer linked via a DNA probe to magnetic beads acts as the capture component. In the presence of target exosomes, aptamers identify and combine with exosomes, releasing the DNA probe as a trigger to initiate the HDCR (the first signal amplification process) by opening hairpin DNA (HP1) bound to gold nanoparticles (AuNPs). Fluorescently-labeled DNA dendrimers concatenate with HP1 as the second signal amplification stage to increase the signal-to-noise ratio. Under the optimal conditions, our method achieved a good linear response for HepG2 cell-derived exosomes in a concentration range from 1.75 × 103 to 7.0 × 106 particles per µL with a detection limit of 1.16 × 103 particles per µL. It also shows a good performance for detection of exosomes in biological samples.

Ultrasensitive SERS detection of specific oligonucleotides based on Au@AgAg bimetallic nanorods.

We synthesized a novel and sensitive Au/Ag bimetallic SERS-active nanotag, Au-Ag-Ag core-shell-shell nanorod (Au@AgAgNR). The Au@AgAgNR nanotag exhibited a strong SERS signal and was easily assembled from bilayer silver shells on an Au nanorod (AuNR) core with embedded Raman reporter molecules in the core-shell-shell gaps. The SERS activity of the nanotags was investigated with 2-mercaptopyridine (2-Mpy) as a Raman reporter, which could form pyridine/Ag+ coordination complexes to mediate the formation of silver shells. Specific enhancement of Raman signals was observed in the following order: AuNR < Au@AgNR < Au@AgAgNR. Then, Au@AgAgNR nanotags were coupled with magnetic beads (MBs) via specific DNA hybridization as a SERS sensor with a detection limit of 1 fM for a segment of the gene HPV-16. Factors affecting sensitivity and selectivity were investigated, including Raman dye concentration, silver nitrate dosage and the response to similar oligonucleotides. The proposed SERS sensor is expected to be a facile and sensitive method for specific gene detection.

Sirtuin-dependent reversible lysine acetylation of glutamine synthetases reveals an autofeedback loop in nitrogen metabolism.

In cells of all domains of life, reversible lysine acetylation modulates the function of proteins involved in central cellular processes such as metabolism. In this study, we demonstrate that the nitrogen regulator GlnR of the actinomycete Saccharopolyspora erythraea directly regulates transcription of the acuA gene (SACE_5148), which encodes a Gcn5-type lysine acetyltransferase. We found that AcuA acetylates two glutamine synthetases (GlnA1 and GlnA4) and that this lysine acetylation inactivated GlnA4 (GSII) but had no significant effect on GlnA1 (GSI-ß) activity under the conditions tested. Instead, acetylation of GlnA1 led to a gain-of-function that modulated its interaction with the GlnR regulator and enhanced GlnR-DNA binding. It was observed that this regulatory function of acetylated GSI-ß enzymes is highly conserved across actinomycetes. In turn, GlnR controls the catalytic and regulatory activities (intracellular acetylation levels) of glutamine synthetases at the transcriptional and posttranslational levels, indicating an autofeedback loop that regulates nitrogen metabolism in response to environmental change. Thus, this GlnR-mediated acetylation pathway provides a signaling cascade that acts from nutrient sensing to acetylation of proteins to feedback regulation. This work presents significant new insights at the molecular level into the mechanisms underlying the regulation of protein acetylation and nitrogen metabolism in actinomycetes.

An RNA-Guided Cas9 Nickase-Based Method for Universal Isothermal DNA Amplification.

We have developed an ingenious method, termed Cas9 nickase-based amplification reaction (Cas9nAR), to amplify a target fragment from genomic DNA at a constant temperature of 37 °C. Cas9nAR employs a sgRNA:Cas9n complex with a single-strand nicking property, a strand-displacing DNA polymerase, and two primers bearing the cleavage sequence of Cas9n, to promote cycles of DNA replication through priming, extension, nicking, and displacement reaction steps. Cas9nAR exhibits a zeptomolar limit of detection (2 copies in 20 µL of reaction system) within 60 min and a single-base discrimination capability. More importantly, the underlying principle of Cas9nAR offers simplicity in primer design and universality in application. Considering the superior sensitivity and specificity, as well as the simple-to-implement, rapid, and isothermal features, Cas9nAR holds great potential to become a routine assay for the quantitative detection of nucleic acids in basic and applied studies.

Quantification of Exosome Based on a Copper-Mediated Signal Amplification Strategy.

Exosomes, a class of small extracellular vesicles, play important roles in various physiological and pathological processes by serving as vehicles for transferring and delivering membrane and cytosolic molecules between cells. Since exosomes widely exist in various body fluids and carry molecular information on their originating cells, they are being regarded as potential noninvasive biomarkers. Nevertheless, the development of convenient and quantitative exosome analysis methods is still technically challenging. Here, we present a low-cost assay for direct capture and rapid detection of exosomes based on a copper-mediated signal amplification strategy. The assay involves three steps. First, bulk nanovesicles are magnetically captured by cholesterol-modified magnetic beads (MB) via hydrophobic interaction between cholesterol moieties and lipid membranes. Second, bead-binding nanovesicles of exosomes with a specific membrane protein are anchored with aptamer-modified copper oxide nanoparticles (CuO NPs) to form sandwich complexes (MB-exosome-CuO NP). Third, the resultant sandwich complexes are dissolved by acidolysis to turn CuO NP into copper(II) ions (Cu2+), which can be reduced to fluorescent copper nanoparticles (CuNPs) by sodium ascorbate in the presence of poly(thymine). The fluorescence emission of CuNPs increases with the increase of Cu2+ concentration, which is directly proportional to the concentration of exosomes. Our method allows quantitative analysis of exosomes in the range of 7.5 × 104 to 1.5 × 107 particles/µL with a detection of limit of 4.8 × 104 particles/µL in biological sample. The total working time is about 2 h. The assay has the potential to be a simple and cost-effective method for routine exosome analysis in biological samples.

Highly sensitive detection of exosomes by SERS using gold nanostar@Raman reporter@nanoshell structures modified with a bivalent cholesterol-labeled DNA anchor.

Exosomes, as important signal transmitters, play a key role in intercellular communication, especially in cancer metastasis. There is considerable evidence that exosomes can be used as an indicator of cancer. However, convenient and sensitive methods for detecting exosomes are still technically challenging. Here, we present a convenient and highly sensitive surface-enhanced Raman scattering (SERS) based method by combining immunoaffinity, SERS nanoprobes, and portable Raman devices for specific isolation and accurate quantification of exosomes. To construct the SERS-based biosensor, the surfaces of gold nanostar@4-mercaptobenzoic acid@nanoshell structures (AuNS@4-MBA@Au) are modified with a bivalent cholesterol (B-Chol)-labeled DNA anchor to prepare SERS nanoprobes. Exosomes are specifically captured by immunomagnetic beads, and then SERS nanoprobes are fixed on the surface of exosomes by hydrophobic interactions between cholesterol and lipid membranes, thus forming a sandwich-type immunocomplex. The immunocomplex can be magnetically captured and produce enhanced SERS signals. In the absence of exosomes, the sandwich-type immunocomplex cannot be formed, and thus negligible SERS signals are detected. The degree of immunocomplex assembly and the corresponding SERS signals are positively correlated with the exosome concentration over a wide linear range of 40 to 4 × 107 particles per µL and the limit of detection is as low as 27 particles per µL. Consequently, a sensitive and simple strategy for detection of exosomes is successfully constructed. We believe that our biosensor has considerable potential as a convenient and highly sensitive quantification tool to detect exosomes in biological samples.

Simultaneous Surface-Enhanced Raman Spectroscopy Detection of Multiplexed MicroRNA Biomarkers.

Simultaneous detection of cancer biomarkers holds great promise for the early diagnosis of cancer. In the present work, an ultrasensitive and reliable surface-enhanced Raman scattering (SERS) sensor has been developed for simultaneous detection of multiple liver cancer related microRNA (miRNA) biomarkers. We first proposed a novel strategy for the synthesis of nanogap-based SERS nanotags by modifying gold nanoparticles (AuNPs) with thiolated DNA and nonfluorescent small encoding molecules. We also explored a simple approach to a green synthesis of hollow silver microspheres (Ag-HMSs) with bacteria as templates. On the basis of the sandwich hybridization assay, probe DNA-conjugated SERS nanotags used as SERS nanoprobes and capture DNA-conjugated Ag-HMSs used as capture substrates were developed for the detection of target miRNA with a detection limit of 10 fM. Multiplexing capability for simultaneous detection of the three liver cancer related miRNAs with the high sensitivity and specificity was demonstrated using the proposed SERS sensor. Furthermore, the practicability of the SERS sensor was supported by the successful determination of target miRNA in cancer cells. The experimental results indicated that the proposed strategy holds significant potential for multiplex detection of cancer biomarkers and offers the opportunity for future applications in clinical diagnosis.