Target-Activated Transcription For The Amplified Detection Of Matrix Metalloproteinase-2(MMP-2)

Protease is a type of enzyme that hydrolyzes protein or peptides that plays a key role in many physiological and biological processes,including inflammation,cardiovascular disease,cancer and so on.Therefore,it is widely used as targets in clinical diagnisis.Matrix metalloproteinases(MMPs)enzymes can degrade extracellular matrix and basement membrance,and are closely related to the occurrence and development of tumors,including tumor invasion,metastasis and angiogenesis.In recent years,many MMPs have become the focus of cancer research and are expected to become important targets for tumor therapy.it is of great significance to develop a simple,rapid and highly sensitive method for detecting MMPs activity for medical diagnosis,treatment and prognosis monitoring.Existing MMPs detection methods face two main problems:1)false positive results caused by the inability of antibodies to distinguish proteases from their inactive precursors in immunoassays;2)low sensitivity caused by the lack of signal amplification mechanisms for each proteolysis event.These limitations highlight the need for a sensitive assay giving unambiguous results that reflect the protease activities accurately.As a rising science in biology in the 21^st century,synthetic biology provides many new tools and methods for the implantation of biosensors and the development of signal recognition components.Recently,activatable RNA polymerase(RNAP)elements have attracted much attention.It can respond to signal molucules such as light,small molecules or proteins in the cell,can convert various chemical events in the cell into codable genetic biological information.Inspired by this phenomenon,RNAP was constructed as a protease activatable RNAP(PR)in vitro,which converts proteslytic events into multiple RNA outputs,providing a powerful biometric element for detection of protease.At the same time,the combination of nucleic acid signal and sensor output is used to construct protease biosensors meeting different conditions or requirements.The main points are summarized as follows:(1)Construction of PR elements in response to proteolytic enzyme activation.In this section,the recombinant vector expressing PR was designed and constructed.The protein was expressed,and purified in the prokaryotic expression system,as well as the activity of the PR element was verified.The specific process is as follows:MMP-2specific substrate sequence was used as the linking peptide to connect the two proteins,and obtained the plasmid of PR_MMP-2 by using homology recombination technology.The expression of PR_MMP-2 protein was induced by the expression system of E.coli,and purified by Ni-NTA column and desalination column on AKTA Purifier system to obtain high purity PR protein.At the same time,the specificity and activability of PR element for target recognition were verified by enzyme digestion reaction and transcription step in vitro.In addition,PR elements was constructed for different proteases(thrombin and NS3/4A)by rational design of the ligating peptide,and demonstrated the extensibility of PR elements.(2)Construction of highly sensitive fluorescence sensor of proteolytic enzyme.In this section,the transcribed RNAs were designed to form G-quadruplexes(G4)that can be lit up by specific organic fluorescent dyes(Th T).PR-based assay(PRA)was constructed by combining the T7 RNAP-mediated high efficient transcription with the excellent specificity of G4 RNA–dye interaction.Using the optimal reaction conditions of PRA sensor explored,the detection limit of MMP-2 can reach 0.07 p M.In addition,this method was applied to the monitoring of MMP-2 activity level in cell culture supernatant and clinical samples,providing a highly sensitive general method for monitoring the activity of protease in clinical diagnosis.(3)Construction of colorimetric detection strategy for MMP-2 activity based on the combination of PR and SNAs module.In this section,RNA transduced by PR was designed to trigger the assembly of SNAs through specific base pairing,resulting in visible color change of spherical nucleic acids due to the redshift of wavelength caused by surface plasmon resonance(SPR).This modular combination rationally coupled their signal transduction and amplification capacities,thereby achieving amplified colorimetric analysis of MMP-2 with picomolar sensitivity.Furthermore,by the immobilization of SNAs on glass fiber membrane,a test strip that enables the portable detection of target protease with a smartphone was developed.With the use of a mobile application to capture and process the colorimetric signals,this portable detection system allowed for the sensitive assessment of MMP-2 levels in biological and clinical samples,highlighting its potential in point of care diagnosis of diseases.(4)The construction of MMP-2 point of care detection strategy.In this chapter,based on the PR constructed in the previous chapters,the transcribed RNAs are directly applied to the paper-based platform,and the RNA products form sandwich structure by complementary hybridization with T-line capture probes and Au NPs-labeling probes.In addition,this was applied to detect the activity of MMP-2 in serum samples of tumor-bearing mice and the results were consistent with those obtained by peptide-based assay.The activity level of MMP-2 in human serum samples was further tested,which proved the application of this method in clinical samples detection.