47 results about "Conformational change" patented technology

The invention discloses a deep learning prediction method fused with multi-modal features, which can accurately identify protein hidden binding sites in a ligand-free (apoo) state. The method comprises the following steps of: firstly, constructing a protein graph by taking residues as nodes and taking C alpha distance less than or equal to 14 as edges, wherein node feature sets comprise amino acid one-hot, secondary structures, atomic attributes, protein language model embedding and BLOSUM62 evolutionary information, and edge features comprise distance and angle similarity; then capturing three-dimensional geometric equivariant features by adopting an equivariant graph neural network (EGNN), and modeling a chemical topological relation by using a graph isomorphic network (GINE) with edge features; eGNN and GINE double-branch feature fusion and global dependence integration are realized through gating cross attention and gating multi-head attention; and finally, inputting the fusion features into a Kolmogorov-Arnold network (KAN) classifier, and predicting whether each residue belongs to a hidden binding site or not. The method can adapt to large-scale conformation change without coordinate alignment, AUC and F1 on a standard data set are remarkably superior to those of an existing method, high robustness and generalization are kept for multi-chain protein and complex conformation, and the method can be widely applied to drug target discovery and structure-driven drug design.

The invention provides a method for analyzing interaction between procambarus clarkia arginine kinase and shark source nano antibody. The method comprises the following steps: performing three-dimensional structure prediction on a VNAR sequence, and screening a high-confidence VNAR model by using a Laplace map; docking an amino acid sequence corresponding to the VNAR model with an arginine kinase sequence to obtain an optimal compound model, and analyzing an interaction site of an arginine kinase epitope region and a nanometer antibody complementary determining region CDR3; performing molecular dynamics simulation on the compound model, calculating conformational change indexes and binding free energy through energy optimization, system balance and extended simulation sampling, and outputting a binding stability sequence of the nano antibody and arginine kinase; the method comprises the following steps: screening out a high-stability nano antibody, pre-incubating a fusion protein of the high-stability nano antibody and immobilized arginine kinase to form a compound, adding serum of an allergic patient for competitive binding, quantifying the binding activity of residual IgE through an immunodetection technology, and verifying the inhibition effect of the shark source nano antibody on the sensitization effect of arginine kinase.

The invention discloses a cell membrane in-situ drug membrane protein target screening method based on variable configuration DNA, and belongs to the technical field of biological medicine. Two pairs of functionalized nucleic acid structures are constructed, the first pair is S1S2 semi-complementary DNA double strands, the second pair is S3S4 DNA-RNA hybrid double strands, and the S1S2 structure is covalently coupled to a non-natural amino acid site-directed modified membrane protein through a click chemical reaction in a living cell to realize tagging of the membrane protein; when the candidate drug coupled in S3 interacts with the membrane protein, triggering an allosteric response system to release the tagged chain in S1; the released DNA tag is subjected to PCR amplification and sequencing, and accurate identification and analysis of a membrane protein target are realized according to a tag sequence. The invention provides a high-specificity and non-in-vitro drug membrane protein target screening technology. According to the technology, active compound discovery or accurate screening of drug membrane protein targets in a living cell in-situ environment can be realized.

The invention relates to an antibody-biomacromolecule conjugate. The antibody-biomacromolecule conjugate comprises an antibody targeting module, a complement effect module and a connexon, the antibody targeting module can be specifically combined with a cell membrane surface antigen, and the complement effect module is a key enzyme C1s in a classical complement pathway or a functional structural domain with complement splitting activity of the key enzyme C1s. The linker is used for connecting the antibody targeting module with the complement effect module, and is designed to generate cutting or conformational change on the outer side of a cell membrane or in an extracellular microenvironment, so that the complement effect module is converted from a limited state to a functional accessible state; to promote initiation of a classical complement pathway in the neighborhood of the cell membrane located by the antibody targeting module. The antibody-biomacromolecule conjugate provided by the invention can be used for engineering regulation and control of complement-related reactions.

The invention discloses a neutralizing antibody with an AQP3 channel blocking function as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The neutralizing antibody can be specifically combined with an extracellular domain of AQP3, after combination, a water channel and a small molecule transport function of the AQP3 are blocked through steric hindrance and conformation change, and meanwhile, antigen combination specificity is reserved. The preparation method is realized through'recombinant antigen-immunization-B cell screening culture-recombinant antibody expression and affinity purification-ELISA detection ', and the functions of the antibody are ensured. The antibody can be efficiently combined with AQP3 positive cells, is strong in targeting property and high in specificity (the serum titer reaches 1: 1024 * 10, and the purified antibody titer is greater than or equal to 1: 128 * 10), and can be independently used or combined with a nano-carrier for treating related diseases, so that the technical bottleneck that a common AQP3 antibody can only be combined and cannot efficiently play a blocking role is solved.

The invention discloses a method for detecting the conformational change of an APO-Cascade protein. Through mutation of Cas8 protein amino acid N376 and Cas5 protein K169 in a Casade protein, the obtained Cas8 and Cas5 labeled by non-natural amino acids can maintain functions, and at the same time, the mutant type of the Cas5 / 8 protein can be subjected to fixed-point fluorescence labeling; according to the method, the Cas6 is used as a template, the N end of the Cas6 is further modified with biotin, and a formed Casade compound can be fixed on a biotin-PEG functionalized single-molecule slide, so that the conformation state of the Casade protein can be truly reflected by using single-molecule fluorescence resonance energy transfer, an effective means is provided for in-vitro research on the structure-function relationship of a system in CRISPR / Casade, and the method has a wide application prospect. And the method is of great significance in designing the Cascade protein with optimized properties and enabling the Cascade protein to play new functions.

The invention belongs to the technical field of medicine and pharmacy, and particularly relates to application of an AP1B1 allosteric activator of ethyl gallate. In the invention, AP1B1 is a beta 1 subunit (AP1B1) of a linking factor associated protein complex 1 (AP-1), and is an essential component for assembling a functional AP-1B complex. The ethyl gallate is used as an allosteric activator of AP1B1 and is specifically combined with an allosteric pocket of the AP1B1 to induce the AP1B1 to generate conformational change, so that the vesicle transportation function of an AP1B complex is activated, and the substrate-side membrane protein sorting capacity is recovered. A target dependence experiment verifies that the effect of ethyl gallate on promoting tight junction protein ZO-1 expression and membrane localization in AP1B1 knocked-down Caco-2 cells is remarkably weakened, and it is prompted that the intestinal barrier protection function of ethyl gallate depends on AP1B1. The invention provides a new strategy for developing medicines for treating intestinal barrier injury related diseases.

The invention discloses a cell single-molecule mechanical force sensor for single-molecule mechanical force detection in a cell phagocytosis process as well as a preparation method and application of the cell single-molecule mechanical force sensor. The sensor comprises a microsphere substrate and a plurality of double-stranded DNA mechanical probes modified on the surface of the microsphere substrate, the probe is composed of a first single-stranded DNA and a partially complementary second single-stranded DNA and forms an FRET or fluorescence quenching pair, when a pulling force applied to the probe by a receptor-ligand effect exceeds a preset melting threshold value in a process of swallowing or wrapping microspheres by a cell, the double-stranded DNA generates melting or conformational change and generates fluorescence signal change, and the fluorescence signal change is detected by the first single-stranded DNA and the partially complementary second single-stranded DNA. Therefore, the detection of the monomolecular mechanical force of the phagocytic interface is realized. By adjusting the geometric conformation and sequence design of the probe, a probe library with different melting force thresholds can be constructed, and quantitative analysis and spatial distribution characterization of mechanical force in the phagocytosis process are realized. According to the application, a three-dimensional phagocytic interface can be truly simulated, and an effective tool is provided for revealing a mechanism for regulating and controlling endothelial cell phagocytic behaviors through blood flow mechanics and screening related disease drugs.

The invention provides a method for detecting mycotoxin by using a carbon nanotube field effect transistor biosensor based on nucleic acid conformation change, which comprises the following steps: anchoring a modified nucleic acid aptamer on the surface of an ion shielding gold film layer through a gold-sulfur bond by using a CNT-FET biosensor with an ion shielding gold film layer above a channel to construct a sensing interface; the modified nucleic acid aptamer consists of a target mycotoxin original nucleic acid aptamer sequence and an oligomeric thymine deoxynucleotide fragment directionally inserted into the 5'end of the target mycotoxin original nucleic acid aptamer sequence; during detection, mycotoxin in a to-be-detected sample is specifically combined with the modified nucleic acid aptamer, secondary conformation change of the mycotoxin close to the gold film layer is triggered, the CNT-FET biosensor converts an electric field effect caused by conformation displacement into a channel carrier concentration change signal, and quantitative detection is achieved according to the linear relation between the signal and the mycotoxin concentration. According to the method, the nucleic acid aptamer is directionally modified, so that the electrical response after the low-charge-quantity / electrically neutral mycotoxin is combined with the sensor is enhanced, and the detection sensitivity is greatly improved.

This invention discloses a machine learning-enhanced fluorescence detection method for silver nanoclusters containing split nucleic acid aptamers, used for the detection of adenosine triphosphate (ATP) in aquatic products, belonging to the field of analytical chemistry. This detection method uses two split nucleic acid aptamers of ATP as templates to synthesize DNA-AgNCs. In the presence of the target ATP, a conformational change is induced, resulting in a decrease in fluorescence intensity. Quantitative analysis of ATP is achieved by detecting this fluorescence change. To further improve the detection effect, machine learning is introduced to preprocess the fluorescence spectrum. A recursive feature elimination algorithm is used to screen characteristic wavelengths, and a multi-dimensional feature set is constructed using maximum fluorescence intensity, peak area, and full width at half maximum (FWHM). The optimal classification model is obtained through cross-validation optimization. This invention combines aptamer recognition, DNA-AgNCs, and machine learning to construct a detection method where fluorescence dynamically decreases with increasing ATP concentration. It has advantages such as low cost, simple operation, and good biocompatibility, effectively improving detection sensitivity and stability, and has promising application prospects in the qualitative and quantitative analysis of ATP in aquatic products.

The embodiment of the invention generally relates to the technical field of bioengineering, in particular to a nucleotide construct, an adeno-associated virus vector, and a system and a method for detecting neural activity. The nucleotide construct comprises an open reading frame, and the open reading frame comprises a voltage response domain coding sequence and a sound wave modulation domain coding sequence. The nucleotide construct is configured to be expressed on an astrocyte membrane under the control of a promoter so as to provide a recombinant protein with a voltage response domain and an acoustic wave modulation domain, so that the recombinant protein can generate corresponding reversible conformation change in response to membrane potential change of the astrocyte, the acoustic characteristics of the recombinant protein can be changed by the reversible conformation change. In this way, the recombinant protein can sense changes in membrane potential and modulate the reflection characteristics to ultrasonic waves.

The invention discloses an enzyme functionalized sensor for detecting nano plastic DPV in a biological matrix as well as a preparation method and a detection method of the enzyme functionalized sensor, and belongs to the field of nano plastic detection. The sensor comprises an electrode and CAT enzyme, wherein the surface of the electrode is modified with positively charged nano-gold particles, and the CAT enzyme is fixed through positive and negative electrostatic interaction. According to the sensor, the CAT enzyme and the nano plastic are combined to generate conformational change to change the activity of the CAT enzyme so as to cause DPV signal change, the sensitivity is high, the sensor has similar DPV signal response to different types of nano plastic, and a unified calibration model can be utilized to detect a complex biological matrix system so as to realize total amount determination of various nano plastic. And the electrochemical detection method does not need complex instruments and pretreatment, and provides a rapid, economic and extensible detection scheme for nano plastic exposure evaluation and environmental monitoring.

Described herein are compositions and methods for bioluminescent analyte detection. In some embodiments, a recombinant bioluminescent polypeptide sensor is disclosed comprising a luminescent signaling domain, an analyte binding domain, and one or more peptide linkers, wherein the luminescent signaling domain is oriented in relation to the analyte binding domain such that binding of an analyte to the analyte binding domain induces a conformational change in the luminescent signaling domain to generate a luminescent signal.

The invention provides a traditional Chinese medicine disease prevention risk prediction method, device and system and a storage medium, and relates to the technical field of biomedical engineering. The traditional Chinese medicine disease-free risk prediction method comprises the following steps: performing enhanced sampling molecular dynamics simulation on a target protein related to a disease to obtain a simulation track; performing clustering analysis on the basis of the simulated trajectory to divide metastable states representing different conformations; calculating a conformation entropy change rate based on the probability distribution and the transition time of the metastable state; and outputting a prediction result according to the conformation entropy change rate. According to the method, a key dynamic conformation change of protein is simulated and captured by enhancing sampling molecular dynamics, and a conformation entropy change rate is introduced as a quantitative index, so that a critical point of disease occurrence is accurately identified on a molecular level. The timeliness and accuracy of'disease prevention 'risk prediction can be remarkably improved, an early warning window is greatly advanced to 18-24 months, and a reliable basis is provided for implementing early and accurate intervention measures.

This invention relates to de novo-designed voltage-gated anion channel proteins and their applications. The de novo-designed voltage-gated anion channel proteins (dVGACs) have a funnel-shaped pentamer structure, wherein each monomer in the pentamer consists of three transmembrane helices, and the amino acid sequence of each monomer includes SEQ ID No.:1 or a mutant thereof, wherein the amino acid sequence of the mutant is mutated to at least arginine or aspartic acid at positions 43, 47, 51, and 55 of SEQ ID No.:1. The dVGACs of this invention are selective for chloride ions and exhibit voltage-responsive conformational changes, leading to the opening of the central pore at membrane potentials of approximately +40 mV or +20 mV and above. Due to these properties, they have broad application prospects in the treatment of neurological diseases, regulation of cell behavior, and the development of biosensors.

The invention belongs to the field of biological medicine, particularly relates to a pancreatitis TMPRSS15 abnormal activation mechanism research system, and aims to solve the technical bottleneck that conformational change, enzyme activity and signal interaction of TMPRSS15 in pancreatitis are difficult to dynamically analyze in the prior art. The system comprises an organoid culture module, a micro-fluidic inflammation simulation module, a conformation-active coupling sensing module, a multi-omics integration module and a dynamic regulation and control feedback module, real-time monitoring, causal inference and closed-loop intervention of a TMPRSS15 activation process are realized through a three-dimensional human-derived organoid and a controllable inflammation microenvironment, and a new platform is provided for pancreatitis mechanism research and precise treatment.

The invention relates to the technical field of analytical chemistry, and particularly discloses a double-signal reverse change ratio type up-conversion nanoprobe for lead ion detection as well as a preparation method and application of the double-signal reverse change ratio type up-conversion nanoprobe. The double-signal reverse change ratio type up-conversion nanoprobe for lead ion detection comprises an up-conversion nanoparticle coated with polyacrylic acid and a DNA (Deoxyribose Nucleic Acid) aptamer modified by Cy3, wherein the Cy3 modified DNA aptamer is used as a specific recognition unit of Pb < 2 + >, the Cy3 is used as an energy receptor, and the polyacrylic acid coated up-conversion nanoparticles are used as an energy donor. According to the double-signal reverse change ratio type up-conversion nanoprobe, the DNA aptamer is induced to generate conformational change through specific binding of the DNA aptamer and Pb < 2 + >, the distance between up-conversion nanoparticles and Cy3 is shortened, and a ratio type detection signal is constructed through quenching of an emission peak of the up-conversion nanoparticles at 540 nm and enhancement of an emission peak of Cy3 at 565 nm; finally, high-sensitivity and high-selectivity detection on trace Pb < 2 + > is realized, and the detection sensitivity is improved.

The application provides a single-molecule protein sequencing device based on a quantum tunneling electrode, a method and application thereof. First, a tunneling electrode pair with a gap of less than 5 nanometers is prepared, and then an exopeptidase is bridged between the electrodes in a directional manner through asymmetric modification. When the exopeptidase cuts amino acids from the end of a substrate peptide chain, the conformational change of the exopeptidase modulates the tunneling current. The current signal is collected in real time, and the amino acid sequence can be analyzed by using a machine learning algorithm to analyze characteristic fingerprints. Based on this, in the future, the method can be developed into a label-free and real-time single-molecule protein sequencing technology, which can be used for low-abundance proteome analysis, post-translational modification identification and single-cell protein research.

The invention discloses a preparation method and application of an electrochemical biosensor based on a self-protection palindromic DNA (deoxyribonucleic acid) nanosphere. The electrochemical biosensor comprises an HP-trigger, a target miRNA (micro ribonucleic acid) and a self-protection DNA palindromic nanosphere core structure. The DNA nanosphere is of a three-dimensional spherical structure formed by four single-stranded DNAs through complementary base pairing, the three single strands of SEP-Sa, SEP-Sb and SEP-Sc contain 12 nt palindromic cohesive ends and 15 nt single-stranded arms, the single strand of HP-Sc contains 35 nt prolonged hairpin structures, the structural stability is enhanced through a self-protection mechanism of a palindromic sequence, and structural dissociation triggered by target miRNA is achieved. The target miRNA is combined with an HP-Sc hairpin structure to trigger conformational change, release complementary chains and dissociate palindromic nanospheres, and exposed DNA sites are combined with MB to generate electrochemical signals, so that a novel tool is provided for early diagnosis of tumors.

The invention discloses a comprehensive evaluation method based on principal component analysis (PCA), and aims to systematically evaluate the inhibition effect of candidate active substances on protein non-enzymatic glycosylation and conformational change and aggregation induced by the protein non-enzymatic glycosylation. The method comprises the following steps: firstly, establishing an in-vitro non-enzymatic glycosylation model of a protein-dicarbonyl compound, and under the action of active substances, detecting multi-dimensional key indexes such as formation of advanced glycation end products (advanced glycation end products (AGEs)), conformational change of protein, aggregation level and the like. Then, PCA is adopted to carry out dimension reduction processing on multiple indexes, a weighted comprehensive scoring function is constructed, objective quantification and sorting of candidate active substance intervention effects are realized, and a comprehensive scoring result is further verified in a cell model. According to the evaluation method, multi-dimensional information is effectively integrated, the limitation of single index evaluation is overcome, and an efficient, systematic and quantitative technical means is provided for drug research and development of non-enzymatic glycosylation related diseases and drug effect screening of active substances.

This invention belongs to the field of biomaterials, specifically relating to a bivalent nucleic acid aptamer and its design method. The purpose of this invention is to provide a bivalent nucleic acid aptamer that solves the problem of drastic conformational changes during the binding of nucleic acid aptamers to receptors, leading to a significant reduction in conformational freedom, entropy, and affinity. The bivalent nucleic acid aptamer of this invention includes a monovalent aptamer A and a monovalent aptamer B, wherein monovalent aptamer A and monovalent aptamer B may be the same or different monovalent aptamers; the double-stranded ends of monovalent aptamer A are extended to form a double-stranded extension arm A, and the double-stranded ends of monovalent aptamer B are extended to form a double-stranded extension arm B, with extension arms A and B connected by a double-stranded intermediate bridge; the extension arm A, the intermediate bridge, and the extension arm B form a U-shaped secondary structure connecting portion, which connects and assembles monovalent aptamer A and monovalent aptamer B.

The invention discloses a three-dimensional Gaussian modeling method for analyzing biomacromolecule components and conformation heterogeneity, and belongs to the technical field of cryoelectron microscope image processing. According to the method, the component and conformation heterogeneity can be directly represented through the change of Gaussian parameters, and an intuitive and explainable structure change description is provided; the consistency and integrity of the local structure along the transformation trajectory are ensured through geometric constraint; modeling of tens of thousands of Gaussian models on a pseudo-atom level is supported; gaussian variation is converted into an atomic conformation landscape through natural support, and a direct bridge is provided between density-based analysis and pseudo-atom level interpretation; the method is realized through an efficient Gaussian projection algorithm and parallel computing, and the processing capacity of a large-scale data set is remarkably improved. According to the method, the component and conformation heterogeneity can be effectively processed at the same time, the continuous conformation change is visually represented, the local structure consistency is kept, the density model and the atomic model are naturally connected, and the defects in the prior art are overcome.

The invention discloses a method for detecting malachite green based on a fluorescence resonance energy transfer principle, a biosensor and a kit, and belongs to the technical field of aquatic product quality safety detection. According to the method, near-infrared light excited lanthanide doped up-conversion nanoparticles (UCNPs) are used as an energy donor, and a nucleic acid aptamer for specifically recognizing malachite green is covalently linked to the surface of the UCNPs to form the nano probe. When the malachite green does not exist in the sample, the aptamer is in a flexible conformation, and the fluorescence of the UCNPs is quenched due to the FRET effect. When malachite green exists in a sample, the aptamer is specifically combined with the malachite green and is subjected to conformational change, so that the spatial distance between the nanoprobe and a quenching agent is increased, the FRET process is blocked, and UCNPs fluorescence is recovered. The qualitative and quantitative analysis of malachite green can be realized by detecting the change of a fluorescence signal. The method has the advantages of high sensitivity, strong specificity, simple operation and rapid response, and is suitable for on-site rapid detection of malachite green residues in aquatic products and aquatic water.

This invention discloses a dynamic multidimensional detection system for DNA oxidative damage interfaces based on the synergistic use of fiber optic SPR and electrochemical dual signals. By simultaneously acquiring electrochemical and SPR signals through a fiber optic SPR probe that also functions as a working electrode, it can distinguish between early-stage non-fracture conformational damage and late-stage strand breakage damage. This system is label-free, highly sensitive, and suitable for fields such as toxicology assessment and antioxidant screening. This invention integrates the advantages of two sensing technologies. It captures interfacial charge transfer changes caused by early DNA base modifications and conformational changes through electrochemical signals, and identifies interfacial refractive index changes caused by late-stage DNA strand breakage and desorption through SPR signals. It achieves in-situ dynamic monitoring and staged analysis of the entire damage cycle without complex sample pretreatment, solving the pain points of traditional offline detection methods, such as sampling lag and inability to distinguish damage stages. This system can be extended to applications such as early disease screening and reactive oxygen species toxicity assessment.

The invention discloses a method for screening a high-activity selenium-enriched yeast strain based on a protein conformation index, yeast protein and application. Relates to the technical field of food processing. Comprising the following steps: S1, providing a plurality of different yeast strains; s2, respectively culturing the yeast strains in a selenium-containing culture medium and a control selenium-free culture medium to obtain selenium-enriched yeast and control yeast; s3, respectively extracting proteins from the selenium-enriched yeast and the control yeast to obtain selenium-enriched yeast proteins and control yeast proteins; s4, representing and comparing conformational changes of the selenium-enriched yeast protein and the control yeast protein, wherein the conformational changes comprise beta-folding content and surface hydrophobicity; and S5, based on the conformational change, screening out the selenium-enriched yeast protein. According to the method provided by the invention, the problem of screening blindness caused by pure dependence on selenium content indexes in the prior art is solved, and a new technical path is provided for development of high-activity selenium-enriched yeast products.

The invention discloses an aptamer regulation and control electrochemical sensor based on an Au (at) Ti3C2MXene composite material, and a preparation method and application of the aptamer regulation and control electrochemical sensor based on the Au (at) Ti3C2MXene composite material. A glassy carbon electrode is used as a working electrode, and the surface of the electrode is modified by an Au-coated Ti3C2MXene composite material prepared by an in-situ reduction method so as to enhance the electron transfer efficiency; a locking compound is formed by a specific aptamer of the lactobacillus acidophilus and an activation chain, and signal regulation and control are realized by combining the trans-cleavage activity of CRISPR-Cas12a. During detection, specific binding of the lactobacillus acidophilus and the aptamer causes conformational change, an activation chain is released to trigger the MB labeled DNA probe on the surface of the Cas12a cutting electrode, and quantification is realized through DPV detection signal change. The detection limit of the method is as low as 4 CFU / mL, the linear range is 10-106 CFU / mL, and the method is suitable for accurate detection of lactobacillus acidophilus in food and probiotic preparations.

The present application relates to the technical field of polypeptide nanomaterials, and particularly relates to a polypeptide nanomaterial for binding NLRP3, a preparation method and application thereof, the polypeptide nanomaterial for binding NLRP3 comprising a polypeptide with an amino acid sequence as shown in SEQ ID NO:1, and a hydrophobic fluorescent group connected to a terminal lysine of the polypeptide through an amide bond. The polypeptide nanomaterial can provide hydrophilic-hydrophobic interaction self-assembly to form spherical nanoparticles in a physiological environment, and can bind to NLRP3 protein in inflammatory or tumor cells based on a specific domain of the polypeptide nanomaterial targeting NLRP3, and then induce a conformational change of the polypeptide through “protein-ligand” specific interaction, and assemble into a nanofiber network structure in situ in a cytoplasm, can be long-term retained in diseased cells, and can aggregate intracellular NLRP3 protein in the fiber network, thereby long-time and high-efficiently inhibiting assembly and activation of NLRP3 inflammasome.

The application discloses an exonuclease fusing an NTPase domain and application thereof, and belongs to the technical field of bioengineering. The exonuclease is a Ppl protein, which comprises a PHP domain at the N terminal and an NTPase domain at the C terminal, wherein the PHP domain has 3'-5' ssDNA exonuclease activity. The NTPase domain of the application regulates the exonuclease activity of the PHP domain through conformational change. Under the condition of high NTP concentration, the NTPase domain inhibits the activity of the PHP domain; when the NTP concentration decreases, the inhibition is released, and the exonuclease activity is significantly activated. The activated PHP domain shows specific cleavage activity to DNA substrates with 3'-hydroxyl overhang. The unique 'NTP concentration sensing and enzyme activity regulation' characteristics make it a core module for developing biological sensors, gene circuits and controllable nucleic acid tools.

The invention discloses a method for detecting conformational change when protein recognizes a target site. The Cys at the 19 / 53 / 127 / 242 / 252 / 284 / 458 sites of the CasA protein in the Casade protein is mutated into Ser amino acid, and meanwhile, the 336 / 401 sites are mutated into the Cys, so that the obtained Cys-mini CasA keeps the function, and the mutant type of the CasA protein can be subjected to FRET fluorescence pair labeling, and therefore, the conformation state of the CasA can be truly reflected on the basis of single-molecule fluorescence resonance energy transfer smFRET, and the CasA can be used for detecting the CasA conformation. An effective means is provided for in-vitro research on the structure-function relationship of a system in CRISPR / Cascade, and the method has important significance for designing Cascade protein with optimized properties and enabling the Cascade protein to exert new functions.

The invention discloses a Cas12a allosteric regulation-based portable hypersensitive detection kit for mycoplasma pneumoniae and application, the kit comprises an RAA amplification system, a Cas12a recognition system and a free nucleic acid probe system, when a mycoplasma pneumoniae target exists, an amplification product firstly activates a classical Cas12a / crRNA compound, Cas12a generates conformational change, and the Cas12a recognition system and the free nucleic acid probe system generate allosteric regulation; then the free split-CrRNA probe competitively binds and continuously activates Cas12a, multiple rounds of reporter probe shearing are achieved, so that a detection signal is greatly amplified, and the sheared reporter probe can be dropwise added to detection test paper due to the fact that the sheared reporter probe carries a Biotin group to achieve portable visual detection. The method has the advantages of extremely high sensitivity, simplicity and convenience in operation, instant on-site detection and the like, and is particularly suitable for primary medical treatment and rapid on-site screening.