Selection Of Highly Invasive Aptamers For Targeting Malignant Melanoma Based On Cell-SELEX

Objective:Aptamers are DNA or RNA oligonucleotides capable of binding different classes of targets with high affinity and selectivtity.Aptamers are often selected from random single-stranded oligonucleotide libraries synthesized artificially by SELEX technology. SELEX (systematic evolution of ligands by exponential enrichment) is a new combinatorial chemistry techniques. Aptamers are no immunogenicity and stable.It is easy to prepare in vitro and the cost is low.They have high specificity and high affinity with their target molecules.Thus aptamers become the focus of screening for tumor biomarkers and molecular targeted therapy.Malignant melanoma is in high mortality of malignant tumor and the high incidence disease of yunnan area.Invasion and metastasis are the main death reasons of malignant melanoma. In this paper,we adopted cell-SELEX,with highly invasive melanoma cell A2058 as target cell for rapid s creening of aptamers that can target metastasis associated moleculars of malignant melanoma and achieve the objective that provide new candidate targets for the molecular diagnosis and targeted therapy of malignant melanoma.Methods:Based on cell-SELEX technology, human highly invasive malignant melanoma cell line A2058 cell was used as target cell line, and human immortalized epidermal cell line Hacat cell was used as control cell line. After twelve rounds of repeated screening,we screened out the aptamers that can specifically recognize malignant melanoma cell line A2058. Then the subordinated library was tested with flow cytometry and observed for the combining ability with highly invasive malignant melanoma cell line A2058. At last the subordinated library was sequenced and assayed. We used DNAMEN and oligoanalyzer softwares to predict the structure of the selected aptamer sequences.We synthesized ten representive sequences from the subordinated library.Then we used flow cytometry to verify the binding ability with A2058.Results:1. Through the cell-SELEX platform,we enriched the aptamer library● Determined optimized annealing temperature by PCR amplification, we finally decided 50℃ as the best annealing temperatureAccording to the Tm of the primers, we set the temperature ranging fr om 50℃ to 65℃. Based on the brightness, clarity, dispersity and no nspecific situation of objective stripe during the PCR amplification,w e chose the best annealing temperature.Through RT-PCR,we found that at 50℃ annealing temperature, we found that objective stripe had th e maximum brightness with no dispersion or obvious nonspecific bandi ng.So we chose 50℃ as the best annealing temperature.● Determined optimized primer’s concentration by PCR amplification, we finally determined the 5 uM as the best primer’s concentrationUnder the same PCR reaction system,we set different concentration gra dient of the primer as follows:1uM,2uM,3uM,4uM,5uM,6uM, 7uM,8uM,9uM, 10uM。Based on the brightness, clarity, dispersity a nd nonspecific situation of objective stripe during the PCR amplificati on,we chose the best primer’s concentration.When the primer concentr ation was 5 uM, that objective stripe had the maximum brightness wit h no dispersion or obvious nonspecific banding. So we chose 5uM as t he best primer’s concentration.● Optimize the screening condition,we enriched the best aptamer libraryBy adjusting the screening condition, we aims to increase the specificit y and affinity of the aptamer from the screening.At the same time,w e used RT-PCR to determine the optimum number of cycles for PCR. After twelve rounds of screening, we screened out an enriched pool th at targeted human malignant melanoma cell line A2058. And then flow cytometry experiments confirmed that the subordinated library had a b etter affinity with malignant melanoma cell line A2058,and hardly reco gnized the control cell Hacat.2. High throughput sequencing and analysis of nucleic acid aptamer sequencesTo sequence ssDNA library of the twelfth round,it has a good enrichm ent. From the analysis of the DNAMEN and oligoanalyzer softwares a bout the structures of the twenty sequences,we found that the sequence s can be divided into four parts according to homology and secondary structure is given priority to stem loop structure.3. Flow cytometry has shown the combination characterization of aptamer and cellsAfter flow cytometry verification, we screened out three aptamer seque nces that can identify human malignant melanoma cell line A2058 as f ollows:Seq7,Seq8,SeqlO.The study found that Seq7,Seq8,Seq10 can spec ific recognize A2058 cell,but the affinity is low.Conclusion:Through the cell-SELEX technology,we screened out aptamers that specifi cally target malignant melanoma cell line A2058. Seq7,Seq8,Seq10 can specific ally recognize A2058 cell.