Construction Of A Modular Probe Targeting Tumor Extracellular Matrix Based On Protein-nucleic Acid Chimera

Extra cellular matrix(ECM)is a precise and ordered network structure composed of a variety of fibrins(collagen,elastin,etc.)and polysaccharides(glycosaminoglycan).It not only plays a skeleton support for cells,but also serves as a major binding site for cell adhesion,migration and communication.During the process of tumor formation,the dynamic structural changes of tumor ECM greatly enhance the interaction between the cells and ECM in tumor,thus promoting tumor development and playing an important role in the process of tumor metastasis and drug resistance.Tumor ECM has become an important target for tumor therapy.At present,specific techniques have been developed for staining major components of tumor ECM,such as collagen and elastin,and collagen can be analyzed by second harmonic imaging.Here,ECM was treated like tissue and can achieve high-resolution imaging,not the key chemical information but the structure and composition information of ECM can be obtained.Therefore,it is of great significance to develop a tool to visualize the chemical information in tumor ECM for study of the tumor pathological process as well as for therapy.Protein-nucleic acid chimeric,which combines the rich functional characteristics of protein and the highly programmable nature of nucleic acid,has excited widely attention in academic research.By using protein-nucleic acid chimera,the detection targets and functional applications of biosensors can be expanded,which is of great significance in the fields of biosensors and modular assembly.Collagen,as the main component of ECM,is rarely exposed in normal tissues and does not contact with blood.Due to the high permeability of blood vessels in tumor tissue,molecules in the blood get the opportunity to contact and interact with the exposed tumor matrix collagen,which provides a target for design of tumor ECM targeted biosensor.Accordingly,we used molecular biology techniques to recombine collagen-binding domain A3(CBD A3)with duck circovirus protein(DCV),and green fluorescent protein(GFP).Among them,CBD A3 has the ability to specifically interact with collagen,GFP works as a fluorescence indicator,and DCV can be specifically covalently linked with DNA.Using DCV as the connecting module,we can bind different DNA to CBD A3 protein,where the designed DNA modular can recognize the target and produce signal.Thus,the modular probes can achieve visual analysis of biochemical signals occurring in ECM of tumor,and providing methods for the study and treatment of tumor pathological process.Specific research is as follows:(1)Construction,expression and characterization of CBD-EGFP-DCV.We first constructed the recombinant plasmid p ET28a-cbd-egfp and obtained the recombinant protein CBD-EGFP after in vitro expression and purification.The fluorescence spectra and collagen-binding properties of CBD-EGFP were verified by spectroscopic analysis and fluorescence imaging,which indicated that CBD-EGFP could not only emit green fluorescence,but also have the specific binding ability with collagen.We further constructed and purified the recombinant protein CBD-EGFP-DCV.CBD-EGFP-DCV has the ability of recognizing and linking specific DNA,collagen binding,and fluorescence tracing,which lays a foundation for the subsequent application.(2)Construction,expression and characterization of CBD-m Cherry-DCV and CBD-m IFP-DCV.Near infrared(NIR)imaging window has weak tissue absorption and low background scattering and fluorescence,which is of better for in vivo sensing and imaging.Hence,we replaced and optimized the fluorescence module of the recombinant protein CBD-EGFP-DCV with red and NIR florescence,and successfully constructed and purified the recombinant proteins CBD-m Cherry-DCV and CBDm IFP-DCV.It was found that the replacement of the fluorescent module does not affect the specific binding ability of the recombinant protein to collagen and DNA,and a clear imaging can be obtained for 3D tumor ball imaging.This indicated that the recombinant protein tool constructed here is a modular one and has potential for in situ sensing.(3)Construction of a modular probe targeting tumor ECM based on proteinnucleic acid chimeric.In order to realize the detection of chemical signals in tumor ECM,we selected Platelet derived growth factor(PDGF)as the model protein.DNA labelled with quenching group was linked with the recognition sequence of DCV was hybridization with the aptamer of PDGF labelled with fluorescence group,and further with CBD-m IFP-DCV to obtain the chimeric probe CBD-m IFP-DCV-DNA.Due to the distance between fluorescent group and the quenching group,the fluorescence quenching phenomenon would not occur.In the presence of PDGF,the conformational change of aptamer will lead to the proximity of the fluorescence group and the quenching group,and further the reduction of fluorescence signal,to realize the detection of PDGF.After the experimental conditions were optimized,and the quantitative analysis of PDGF was the detection linear range of 5-40 n M.In addition,the interfacial imaging showed that CBD-m IFP-DCV-DNA can not only target to collagen,but also respond to the concentration change of PDGF.Our work demonstrates that the constructed chimeric tool has the potential to selectively target to tumor ECM,and to sensitively detect different target substances by redesigning the nucleic acid sequence,which has great application in biological and therapeutic and research.