The Construction Of Multifunctional Nucleic Acid Probes And Their Applications In Biological Detection And Imaging

Sensitive detection of tumor markers has important clinical significance for early diagnosis,monitoring and predicting prognosis of cancer.Currently,the detection of tumor markers in clinical liquid samples（urine,plasma or cell lysate）can provide the average expression level of tumor markers,but the detection method is mainly based on the detection of a single type of markers.Many studies have demonstrated that the occurrence and development of tumors are often closely related to the synergistic effect of various markers of different types.Therefore,a single type of tumor marker detection method is easy to cause false positive signals,which is not conducive to clinical application.In addition,in order to further understand the heterogeneous expression and dynamic distribution of intracellular tumor markers,a variety of in-situ detection techniques for intracellular tumor markers have been established.In this regarded,the in situ imaging technology can provide the expression level and dynamic distribution of tumor gene markers in the manner of real-time online,which is regarded as a new strategy for early clinical gene diagnosis of cancer.Although the existing organic/inorganic nanomaterial-based nanoprobe can monitor the expression of tumor markers in living cells,its low efficiency,high cost of fluorescent labeling and the adverse interference with cell activity remains,which leads to inaccurate detection.The excellent biocompatibility and programmability of nucleic acid molecules can provide new ideas for in situ imaging of tumor markers.In summary,a series of multi-functional biological probes based on the combination of nucleic acid probes,metal nanoparticles and signal amplification strategy were constructed in this thesis.It mainly involved in the sensitive and simultaneous detection of multiple tumor markers in clinical liquid samples,in situ imaging of fixed intracellular tumor markers and sensitive imaging detection of nucleic acid tumor markers in living cells as followed:（1）Using telomerase and telomerase RNA（TR）as tumor marker models,combined with deoxyuridine/biotin-containing molecular beacon and bidirectional polymerization-cleavage isothermal amplification reaction,a highly sensitive fluorescence method for simultaneous label-free detection of protein and RNA was constructed.The deoxyuridine acts as a barrier to block DNA extension due to its strong inhibitory effects on DNA polymerization reactions and to make sure that the two chain displacement reactions occurred independently.This method avoided the demands of multiple probe and reduced the experimental cost.With the G-quadruplex binding with ZnPPIX and ssDNA binding with SG for specific fluorescence responses,the label-free multiple detection can be achieved,which have been evaluated as powerful tools for biomedical research and clinical diagnosis.（2）Using fixed intracellular telomerase RNA（TR）as a tumor marker model,combined with tandem rolling circle transcription reaction and fluorescent Spinach RNA signal transduction mechanism,a Spinach RNA aptamer string strategy was constructed for in situ imaging detection of low intracellular TR.A sealed DNA was used lock the resulted Spinach RNA aptamer,guaranteeing its stability in the complex cellular environment.The structurally stable“Spinach RNA aptamer string”can activate the fluorescence of DFHBI fluorophores and enhance the signal-to-noise ratio of the system.In this strategy,a large amount of DFHBI binding sites were generated by tandem rolling circle transcription reaction,which improved the fluorescence yield and sensitivity of the system.In addition,the expression of TR in different cells was sensitively visualized based on the proposed“spinach RNA aptamer string”,offering a powerful tool for in situ label-free imaging of low-abundance biomarker in cells.（3）Using telomerase RNA（TR）in living cells as a tumor marker model,a simple DNA/RNA nanoprobe（NFs）was designed based on the“zipper lock-and-key”to realize the controllable loading and intracellular self-release of multiple nucleic acid probes.First,a long RNA generated by rolling circle transcription acts as both the“smart zipper lock”and the delivery carrier to alternately lock multiple functional DNAs through DNA-RNA base pairing,and the resulting RNA/DNA hybrids self-assemble into packed NFs.After NFs internalization by the cells,the intracellular RNase H acts as the“key”to specifically open the DNA/RNA NFs by cleaving the RNA in the DNA/RNA hybrid,releasing high amounts of H₁and H₂ in a confined space and thereby facilitating the HCR amplification analysis of cytoplasmic TR.With the addition of a DNA-nuclear localization peptide component in the same NF,nuclear TR can also be sensitively detected.Furthermore,the strategy was successfully used to assay the expression levels of TR in different cells,indicating that this method has the potential to sensitively detect low-abundance tumor markers in living cells.