Study Of Online Reaction For Aptamers Selection Based On Capillary Electrophoresis

Aptamers are single-stranded DNA or RNA,which are selected from a random synthesized oligonucleotides library through the process of Systematic Evolution of Ligands by Exponential Enrichment(SELEX),With high affinity and specificity when binding targets,easy preparation and modification paths,as well as a widely distributed target molecules,aptamers have a great potential in biology,medicine,molecular recognition and detection,etc.Now multiple apatamer slection methods have been developed,such as affinity chromatography,membrane filtration,magnetic bead separation,microfluidic chip and capillary electrophoresis,The application of capillary electrophoresis on aptamer selection(CE-SELEX)can reduce the selection cycle to one third of the traditional SELEX technology,greatly accelerating the selection efficiency and reducing the experiment cost.In this paper,CE online reaction was used for SELEX.This paper mainly includes the following parts:(1)The characteristics of aptamers were summarized.Introduce the common separation methods of SELEX,such as affinity chromatography,membrane filtration,magnetic bead separation,microfluidic chip and capillary electrophoresis.Finally,the common CE online reaction were summarized.(2)We presented the ppKCE application,name it online CE-SELEX.With protein and ssDNA models,in coated capillary with neglected EOF,ssDNA passed through protein zone under negative voltage.During the process,protein and ssDNA binding occurs,the online binding in capillary can be an alternative to the incubation vial offline capillary.Using human thrombin(H-Thr)and FAM labled Apt29(F-Apt29)as model,the effect of borate buffer concentration,pH,metal cations concentration in buffer and reactant ratio on complex formation are investigated.The online reaction of F-Apt15 and H-Thr is also compared with F-Apt29 and H-Thr.In order to demonstrate this method,H-Thr with ssDNA library online reaction,collecting the complex for PCR amplification,the PCR products were identified by Qsep100 DNA Fragment Analyzer.The online CE-SELEX doesn't need incubation.Our results show the online CE-SELEX is a quick,convenient and high efficiency universal method for high affinity aptamers selection.(3)We established the double online CE-SELEX based on online CE-SELEX.With double protein-ssDNA models or protein-double ssDNA models,in coated capillary with neglected EOF,ssDNA passed through double protein zones in sequence,or protein react with double ssDNA in sequence.Using H-Thr and its two aptamers as model,double online CE-SELEX is established.Then,using nucleic acid libraries and several common proteins as model,the method is feasible.The double online CE-SELEX simplifys the experimental steps and improves the screening efficiency..