| Aptamers are developed by SELEX（systematic evolution of ligands by exponential enrichment）A short single-stranded DNA or RNA molecule with high affinity and specificity to the target molecule.Since DNA is more stable than RNA,most initial libraries are synthesized from DNA.The oligonucleotide library consists of a central region with random sequences and two primer regions with fixed sequences.Fixed sequences act as primer binding sites during amplification.The capacity of initial screening library used for aptamer screening is generally 1013～1015.Aptamers quickly found their potential in applications ranging from therapy,drug delivery,diagnostics and genomics to biosensing due to various advantages,such as ease of preservation,small batch to batch variation,low immunogenicity,and ease of chemical modification to enhance stability and targeting.However,the current overall success rate for SELEX is less than 30%and remains a major obstacle to aptamer-based research and application.It makes sense to develop stable,low-cost,and simple screening methods.The thesis mainly completed the following three aspects of work:1.Semi-automated instrument was used to realize semi-automated SELEX process,and qPCR was used to monitor the SELEX process.A total of 8 proteins of great significance were selected,and some successes and failures were compared with the results.And the enrichment of library.This work greatly shortens the overall SELEX process by running parallel SELEX on multiple targets simultaneously.At the same time to minimize human interference factors.The introduction of qPCR for real-time detection of SELEX process not only helps us to monitor the state of SELEX,but also facilitates us to timely and accurately adjust the experimental parameters of each round of SELEX.2.Through rigorous monitoring of the SELEX process and library affinity testing,this method successfully selected aptamers targeting 6 proteins.According to the data analysis,SELEX’s failure was mainly due to the fact that the retention rate of Counter SELEX was higher than that of Positive SELEX and remained the same.Unlike ROR1,Positive SELEX retention increases after a single round of pressure adjustment.This method provides feasibility for further exploring the sequence differences obtained by different modification bases on the same target.The large amount of repeatable data available with machines instead of humans is potentially helpful in exploring the SELEX mechanism.3.Using the aptamer ROR1-02 modified Biotin,an efficient sensing analysis method for detecting tumor-specific targets was successfully constructed.The luminescence method amplifying the signal of aptamer is simple and easy to operate.The method can perform preliminary detection and analysis on some important protein targets.The results show that the probe can detect target proteins as low as 5 pg/mL,providing a new method and idea for detecting important proteins in human body. |
