Screening,cloning and structure analysis of aptamer binding to H22 tumor cells

Objective:To obtain the single-stranded DNA aptamer that specially binds H22 tumor cells.Methods:H22 tumor cells were acted as target and rat DC cells were acted as counter-target by using the process of systematic evolution of ligands by exponential enrichment,which was abbreviated as SELEX.Aptamer that specially bind H22 cancer cells were selected in vitro from 80bp random single-stranded DNA library.Aptamer affinity with H22 cancer cell was detected by the fluorescence-labeled primers method.The acquired aptamer was analyzed its primary structure isogenetic sequence comparisonby MACAW v2.05 software,and was predicted its secondary structure by using DNASIS v2.5 Software.Results:Library for this experiment 5′-CGTCGCTGCACATTCCG-(46N)-CGCACAGCTGGGAGTAC-3′ had a higher PCR amplification efficacy,was fit for the selection which act cell as target.After 11 cycles selection,fluorescence intensity of random single-stranded DNA binding with target cells was changed from 1% to 59%,binding curve entered platform stage,i.e.,binding level was steady state,the binding that aptamer with target cells was in saturation state.There were 5 conserved sequenced and the structure analysis revealed that the stem-loops or bulge was the main motif in the interaction between aptamers and H22 cancer cell.Conclusion:Aptamers against H22 cancer cell have been identified by SELEX methods from an 80 bp single-stranded DNA random library.