In Vitro Selection Of Aptamer To Taxinol Resletane Cells And Basic Application Of Aptamer

Aptamer is single strand DNA or RNA sequence selected by SELEX(Systematic evolution of ligands by exponential enrichment technology).Aptamer is also named chemical antibody for its same recognize mechanism as protein antibody.And aptamer has some advantages over protein antibody:could be chemical synthesized directly and modified by order;has high affinity to target with nanomolar dissociation constant;has stable structure and easy to preserve.Cell-SELEX use in vivo tumor cell as target to select aptamer,which could recognize,diagnose and treat cancer cell with unknown target molecule.This article use cell-SELEX technology to select aptamers targeting to tumor cell,analyze the structures and characters of the selected aptamers and try to make use of these aptamers to develop new ways to tumor diagnosis and therapy,as follows.1)Selection and characterization of aptamers targeting drug resistant ovarian cancer cells.We selected aptamers HF3 and HA5 using taxinol resistant ovarian cancer cells A2780T as positive target and wild ovarian cancer cells A2780 as negative target.Then we characterized and optimized the sequencs and got HF3-58 and HA5-68.Further characterization indicated that HF3-58 and HA5-68 had good resistant to temperature,divalent ion had no influence to their affinity and their targets may be glycoprotein.2)Basic application of HF3-58 and HA5-68.The functions of aptamers selected by cell-SELEX should be investigated if we want to use them to real application.For example,wether aptamer could be degraded in human serum and human plasma simulation;wether aptamer affinity could be influenced by temperature and wether aptamer has cytotoxicity.By series experiments,we verified that HF3-58 and HA5-68 were stable in human serum supplemented with heparin or EDTA,and almost had no cytotoxicity to cells that indicated HF3-58 and HA5-68 had good bio-application prospect.3)Application of targeting drug carried aptamer.We elongated and plus a short sequence to aptamer.The short sequence induced two stem-loop short single strand DNA to hybridize and formed a long double strand DNA,which made one aptamer molecule could load many doxorubicin molecules.Due to the specific recognition of aptamer to target tumor cells,this way could reduce serious side work of doxorubicin.Aptamer HG1-7 target to transferrin receptor,which expressed in tumor cell surface much higher than normal cells.We use this aptamer to set up long double strand DNA to load doxorubicin molecules and target tumor cells.