Construction Of FRET-Based Aptamer Probes And Analytical Methods For Highly Sensitive Tumor Detection

It is of great value to develop highly specific molecular recognition probes and highly sensitive analytical methods for early tumor diagnosis,early warning of cancer metastasis and response evaluation of treatment.As a new type of molecular recognition tool,aptamer,which possesses many advantages compared with traditional antibodies,such as high affinity,high specificity,easy synthesis,easy modification,stable properties and flexible design,has shown unique advantages and important application value in molecular diagnosis,targeted therapy and related basic researches of tumors.However,the commonly used aptamer-based analytical methods have the disadvantages of high background,difficult to design and low sensitivity.In addition,the existing tumor cell specific aptamers and target molecules are still very limited,which severely limits the wide application of aptamers.Focusing on the above key issues,this thesis aims to develop novel tumor specific probes and analytical methods by using aptamers as molecular recognition tools and introducing FRET signal conversion mechanism and HCR amplification mechanism,and constructed and developed a series of aptamer fluorescence probes and analytical methods with low-background and high-signal for systematic studies of highly sensitive detection of tumor cells,enhanced imaging of tumor cell-surface specific glycosylation and metastatic tumor cell and tissue-specific imaging.The specific research contents are as follows:1.Design of hairpin fluorescence probes based on split aptamers and its application in tumor cell detectionAiming at the problems of long sequences and design difficulties caused by conventional hairpin aptamer fluorescent probes that depend on an intact aptamer,we designed and developed a hairpin split aptamer fluorescence probe(HSAP)for sensitive tumor cell detection based on FRET and split aptamers.This probe not only has a shorter sequence,but also has a simple design and a low background signal.By using human hepatocellular carcinoma SMMC-7721 cells as the target cells,HSAP could detect 24 cells in 100μL of binding buffer and achieve the quantitative detection of target cells in 50%human serum samples.Meanwhile,HSAP showed great detection selectivity and could specifically recognize target cells in mixed cell samples.Moreover,this probe allowed one-step detection of target cells in 15 min without any washing and pretreatment.This fast,simple,sensitive and specific tumor cell detection probe provides a powerful tool for tumor and medical diagnostic research.2.Highly sensitive detection of tumor cells based on target-induced reassembly of double-stranded split aptamer fluorescence probeThe hairpin aptamer fluorescent probe described above is designed based on intramolecular FRET mode.Although the target binding can induce a conformational change of the probe and generate a signal,the quenching group is still on the probe,and FRET still occurs to some extent,leading to the probe fluorescence fail to fully recover.Aiming at this problem,a double-stranded split aptamer fluorescence probe(DSAP)was designed based on intermolecular FRET for highly detection of tumor cells.In free state,DSAP fluorescence was quenched by FRET.When target cells were presented,target cell binding induced reassembly of DSAP and the sequence labeled with quenching group was dissociated from the double-stranded structure,then DSAP fluorescence restored completely and produced a strong fluorescence signal,and the detection sensitivity is greatly improved.In addition,by labeling different fluorescent molecules on DSAP,dual signal detection of target cells could be achieved,and false positive and false negative signals could be effectively avoided.This method is simple,rapid and highly sensitive with detection signal-to-backgroup ratio up to 40 times,successfully achieved detection of 7 tumor cells in 100μL binding buffer without any washing and separation,and further realized target cell detection in 50%human serum complex samples and mixed cell samples.This method is expected to provide a new highly sensitive technique for tumor clinical diagnosis and research.3.Aptamer-guided imaging of tumor cell-surface specific glycosylation based on FRET and metabolic glycan labelingAbnormal cell-surface glycosylation is usually highly correlated with the early development of tumors.Specific imaging of cell-surface glycosylation is of great value for the early detection of tumors.Due to the non-template synthesis and complex conformation of glycans,as well as the small quantity and insufficient selectivity of traditional glycan targeting molecules,specific glycosylation assays are difficult to achieve.In this work,a novel cell-surface glycosylation specific imaging method was developed by using an aptamer as a targeting molecule and combining with metabolic glycan labeling and FRET.With this method,we successfully achieved SMMC-7711 cell-surface protein-specific imaging of GalNAcylation.This method not only had obvious imaging contrast,but also had excellent selectivity,which provides a new idea for the fluorescence assays of tumor cell-surface glycosylation.4.Enhanced imaging of tumor cell-surface specific glycosylation based on aptamer-triggered HCR amplificationGlycoproteins usually contain plenty of monosaccharide units.The number of FRET donors labeled by metabolic glycan labeling is much higher than the number of acceptors on the targeting molecules.The huge number discrepancy between donors and acceptors is not conducive to obtain high FRET signals.Focusing on this problem,an aptamer-trigger probe was designed for HCR amplification to prepare an aptamer-tethered DNA nanoassembly probe.This nanoassembly probe carried multiple FRET acceptors and induced rational distribution of FRET receptors by changing the length of the HCR building blocks.Then multiple FRET occurred between these acceptors and the donors that labeled with metabolic glycan labeling,which greatly enhanced the FRET signal.By using this method,we successfully achieved enhanced imaging of protein-specific GalNAcylation on the SMMC-7721cell-surface.According to the statistical average value,the obtained FRET signal was approximately two times higher than that of FRET signal with no HCR.In addition,this method possessed excellent versatility,not only could be used for the specific imaging of PTK-7-specific sialylation on the CCRF-CEM cell-surface,but also accurate monitoring of the expression changes of PTK-7-specific sialylation after drug treatment.This enhanced FRET imaging strategy is expected to be used as a new highly sensitive analytical method for the detection of tumor cell-surface specific glycosylation.5.Selection of aptamers against colon cancer LoVo cells and its application in metastatic tumor cell and tissue-specific imagingDevelopment of different aptamer targeting molecules can provide more alternative probes for tumor detection research.Metastatic tumor cells can appear at an early stage of tumorigenesis,and the detection of metastatic tumor cells is of great value for the early diagnosis of tumors.In this study,by using human metastatic colon cancer LoVo cells as target cells and human non-metastatic colon cancer SW480 cells as control cells,four aptamers that binding to LoVo target cells with high affinity were selected through cell-SELEX technique.The K_d values of the four aptamers were all in the nanomolar range.The enzymolysis experiments preliminarily demonstrated that the target type of the aptamers were membrane proteins.It was worth noting that one of the aptamers(J3)not only recognized target LoVo cells,but also binded to a variety of metastatic tumor cell lines.After labeling J3 with Cy5near-infrared fluorescence dye,J3-Cy5 successfully achieved imaging contrast between metastatic and non-metastatic tumor cells.More importantly,J3-Cy5 could specifically identify patients’metastatic colorectal cancer tissue with a high recognition rate of 73.9%,while the recognition rate to non-metastatic colon cancer tissues and paracancerous tissues was low.The obtained J3 aptamer can be used as a new molecular recognition tool for metastatic tumor detection,which possesses great potential in the early warning of metastatic tumors.