Construction Of DNA Nanostructures And Their Application In Tumor Markers Imaging Analysis

As one of the most fatal diseases in the world,cancer remains a very serious health problem.Despite the considerable manpower,materials and financial resources have been invested in the development of new cancer treatment methods,the success of the current clinical treatments is limited due to the complexity,diversity and heterogeneity of tumor.Therefore,the early diagnosis and clinical treatment of malignant tumor is still an urgent problem to be solved,and the first problem in cancer treatment is how to achieve accurate diagnosis.On the one hand,in the early stage of tumor onset,the patients do not have obvious discomfort and abnormal phenomenon,thus the best treatment period is missed;After the definite diagnosis,most patients have entered the middle and late stage of tumor development,when tumors have been infiltrated and metastasized,and it is difficult for the current treatment methods to effectively inhibit or kill tumor proliferation in this period.In order to detect and diagnose malignant tumors as early as possible,researchers are trying to develop sensitive image method for tumor biomarkers at the cellular level.Since the concept of tumor marker was put forward in 1978,it has become an important tool for tumor diagnosis.Because the content of markers is far beyond that in healthy persons,the detection of tumor markers can provide effective information for the early diagnosis.There are many methods for the detection of tumor markers,including electrochemical methods,colorimetry,mass spectrometry,chemiluminescence and surface enhanced Raman scattering.These methods have made great progress in the detection of tumor markers,but most of these methods can only detect tumor markers in vitro,and unable used for the tumor markers imaging in living cells.To solve this problem,many nano materials,such as liposomes,silicon nanoparticles,gold nanoparticles,quantum dots,graphene oxide,Mn O₂nanosheets,have been developed for in situ imaging analysis of tumor markers in living cells.These methods have achieved great results,but still have some shortcomings,such as the encapsulation of liposomes is disordered,and the probes delivered into cells are hydrolyzed by enzymatic;besides the fluorescent probes based on inorganic nanomaterials often have the disadvantages of potential biological toxicity and complex synthesis and modification.Therefore,it is of great significance to search for a carrier with good biocompatibility,simple synthesis and good stability for in situ imaging of tumor markers in living cells.In recent years,DNA nanostructures,such as DNA tetrahedron,DNA triangular prism and DNA nanospheres,have been widely used in the imaging analysis of tumor markers in living cells due to their excellent monodispersity,good biocompatibility,simple manipulation of modification and excellent nucleic acid stability.However,most of these probes only imaging one tumor marker,the accuracy of tumor diagnosis is insufficient.Because a tumor can produce a variety of different tumor markers at the same time,and different tumors or different types of the same tumor can produce the same tumor marker,it is often difficult to obtain accurate diagnosis results by single tumor marker imaging analysis method.Therefore,it is important to develop a simultaneous imaging analysis method for multiple tumor markers in living cells to improve the accuracy of tumor diagnosis.Finally,as one of the most widely used methods of cancer treatment,chemotherapy has achieved remarkable results in cancer treatment and prolonging the life of patients,but there are still many problems,one of which is the side effects of chemotherapy.Therefore,through the functional design of drug delivery system,establish the controlled drug delivery system triggered by tumor marker response can not only image and analyze tumor markers in living cells,but also monitor the drug release and tumor treatment process,which is of great significance to improve the tumor targeted therapy efficiency.In order to solve the above problems,this paper mainly developed fluorescent probes for imaging analysis of a variety of tumor markers based on DNA nanostructures,and designed a targeted controlled drug release strategy guided by tumor markers for precise tumor treatment.The main points of our work are sumarized as follows:1.Accurate imaging of double miRNA based on the catalytic hairpin assembly on DNA tetrahedron.The abnormal expression of micro RNA(miRNA)is closely related to the occurrence and development of tumor.Thus,the establishment of miRNA in situ imaging analysis method is of great significance for the early diagnosis of tumors.In this work,we designed a fluorescent probe based on DNA tetrahedron for intracellular miRNA imaging.First,four vertices of the DNA tetrahedron was linked with the aptamer of nucleolin AS1411,two harpins of catalytic hairpin assembly(CHA),and a capture strand.The capture chain was hybridized with one initiate chain and one lock chain.The fluorescent probe could specifically internalizated into tumor cells due to AS1411,and only when there were both miR-21 and miR-155 in tumor cells,the capture chain could hybridize with the two miRNAs respectively to release the initiate chain,which could further activate the CHA in the DNA tetrahedral,bringing the donor and acceptor molecules modified on the two hairpin DNA close to each other,resulting in a distinct fluorescence resonance energy transfer(FRET)signal that enables sensitive detection and imaging of the two miRNAs in living cells.This design is expected to improve the accuracy of the diagnosis of miRNA-related tumors by combining nucleolin targeting,the CHA circulation in tetrahedral and dual miRNA imaging.2.Recognition of double miRNA by AND logic gate based on DNA nanosheet.The FRET probes were widely used in tumor marker detection,imaging and tumor diagnosis dur to it can simultaneously observe the changes of donor and acceptor,avoiding false positive signals effectively.However,most of the current fluorescent probes are based on studies of the donor and acceptor within 10 nm,limiting their further use over longer distances.In this work,we used DNA nanosheets as a template to study the energy transfer between different donor and receptors with diverse distances and configurations.After rational design,we have prepared cascade long range resonance energy transfer based on Alexa fluor 430/Cy3,Cy3/Cy3,and Cy3/Cy5.Subsequently,an AND logic gate system based on cascading long-range resonance energy transfer was constructed by using BHQ2 to quench the fluorescence of two Cy3 molecules.The AND logic gate system can generate output signals only when the two input signals(miR-21and miR-155)are present simultaneously,so as to realize simultaneous imaging of the two miRNAs in living cells.This work is expected to broaden the application of long-range fluorescence resonance energy transfer in tumor diagnosis.3.Nucleolin targeted and telomerase responsive DNA nanotubes for precise tumor therapy.Accurate diagnosis and treatment of tumor are the important guarantees to improve the curative tumor treatment.We developed a DNA nanotube with nucleolin targeting and FRET indicating telomerase respone to controlled drug release for precise diagnosis and treatment of tumors.First,DNA nanosheets were synthesized by DNA assembly,and the ricin A chain(RTA)was loaded.Subsequently,the DNA nanosheet was converted into DNA nanotubes by binding the Cy3 labeled DNA fastener to the telomerase primer and another single strand modified with Cy5 on both sides of the nanosheet.Due to the proximity of Cy3 and Cy5,a significant FRET signal could be observed.Finally,the two ends of the nanotubes were modified with nucleolin aptamer AS1411.The RTA loaded nanotubes can specifically enter into tumor cells mediated by nucleolin,which can be further reopened into nanosheets under the response of overexpressed telomerase,resulting in significant changes of FRET signals,enabling accurate diagnosis of tumor cells.RTA exposed to the cytoplasm can be further released by nuclease to play a role of tumor therapy.However,even if some nanotubes entered into the normal cells,it could not be opened because of the insufficient telomerase,greatly reducing the side effects.This study is expected to reduce the side effects and increase the therapeutic effect of drugs.In summary,in this study,we constructed FRET probes based on DNA nanostructures for simultaneous detection of double tumor markers.First,DNA tetrahedron was used as carriers to construct targeted and intramolecular CHA FRET probe for simultaneous detection of the two miRNAs,which improved the accuracy of tumor diagnosis.Then,DNA nanosheets were used as templates and explore the cascade long range resonance energy transfer under different donor and receptor pair by modified the fluorescent groups on their surfaces.After that,an AND logic gate was constructed for simultaneous imaging of two tumor biomarkers in cells.Finally,we developed a nucleolin-targeting and FRET-indicated telomerase-responsive drug release DNA nanotube for accurate diagnosis and treatment of tumors.Therefore,this study not only improves the accuracy of miRNA related tumor diagnosis,but also broadens the application of cascaded long-range resonance energy in biochemical analysis.Moreover,a tumor diagnosis and treatment system integrating tumor marker imaging and tumor diagnosis,controlled drug release triggered by tumor markers for precise tumor treatment has been established,which is helpful to improve the curative effect of tumor treatment and reduce the side effects.