Construction Of Aptamer-targeted DNA Assembly For Tumor Cell Detection And Killing Based On TdT Enzyme

Exploring and developing new methods for diagnosis and treatment of tumor are of great significance for improving the cure rate and survival rate of patients.As a novel molecular recognition probe for specifically recognizing tumor cells and tumor markers,aptamer has the advantages of small molecular weight,low immunogenicity,simple synthesis method and flexible modification.It has been applied to the diagnosis and treatment of tumors and related basic research.However,the current aptamer-based tumor analysis detection methods often have shortcomings such as design difficulty and low detection sensitivity,which seriously hinders the broad application prospect of aptamer.In addition,although chemotherapeutic drugs are widely used in cancer treatment,they lack specificity and can induce cytotoxicity in cancer cells and normal cells,causing side effects and greatly reducing the efficiency of treatment.A therapeutic diagnostic platform with targeted and effective drug delivery will address these issues.Based on this,this paper aims to develop a novel sensitive detection probe of tumor and a targeted drug delivery platform,using tumor cell-specific aptamer,by introducing terminal deoxynucleotidyl transferase(TdT)combined with DNA nanotechnology and molecular amplification mechanism.A new type of detection and therapeutic probe was developed using human colon cancer cell lines and breast cancer cell lines,and tumor research was carried out at the molecular and cellular levels.The specific research contents are as follows:1.Terminal deoxynucleotidyl transferase-initiated molecule beacons arrayed aptamer probe for sensitive detection of metastatic colorectal cancer cellsA novel TdT-initiated MBs arrayed fluorescent aptamer probe(TMAP)was first developed for specific detection and imaging of metastatic CRC cells.Two DNA probes were firstly designed.One is the aptamer W3 targeting high-metastatic CRC LoVo cells.The other is the MBs labeled with 6-carboxy fluorescein(6-FAM)at its 5? end and Black Hole Quencher 1(BHQ-1)at its 3? end,while the loop of the MBs is poly T sequence.In the presence of TdT and dATP,the TdT can prolong the aptamer W3 at the 3?-hydroxyl terminus with repeated A bases,providing a long poly A sequence for hybridizing to the lots of MBs.Upon this hybridization,the MBs arrayed fluorescent aptamer probe is activated.TMAP has a simple structure and can be successfully constructed without modifying the aptamer.The results indicate that this method can specifically detect and image highly metastatic CRC LoVo cells from a mixture cell sample.Compared with the adaptor W3 labeled with 6-carboxyfluorescein(6-FAM),TMAP realizing a 4-fold increase in signal-to-background ratio(SBR).Flow cytometry allowed detection of up to 23 LoVo cells in 200 ?L cell culture medium.This simple and sensitive detection method provides a new idea for the diagnosis of tumor.2.Construction of a nucleic acid aptamer integrated DNA nano-assembly system for diagnosis and treatment of tumor based on TdT enzymIn the previous work,we constructed a probe for tumor cell detection by TdT enzyme.In order to further expand the application of the probe in diagnosis and treatment,we proposed a nucleic acid aptamer targeting DNA nano assembly system(TCCP)based on TdT enzyme extension for the diagnosis and treatment of tumors.In this system,in the presence of TdT and dCTP,the aptamer AS-1411 is extended,and its 3' hydroxyl terminus forms a AS-1411-Poly C.At the same time,another G-base-rich DNA strand(CS)can hybridize with Poly C in AS-1411 to form a TCCP nanoprobe.The treatment of tumor can be achieved by constructing a TCCP-Dox system by embedding Dox in CG base pairs.The probe has good stability and drug-loading ability,can achieve high specific drug delivery and targeted induction of in situ drug release,thereby achieving killing of MCF-7 cells.The probe is expected to provide ideas for a new diagnostic probe strategy.