Screening Aptamer of Apo-transferrin via Capillary Electrophoresis-Systematic Evolution of Ligands by Exponential Enrichment and Environmental Factors Analysis

Abstract

Aptamer is oligonucleotide with high affinity and specificity toward the target which is obtained via systematic evolution of ligands by exponential enrichment (SELEX). However, due to the complexity of environmental factors and the unknown influence on the selection and application of aptamers, the optimization of multiple environmental factors through conventional experiments requires a huge amount of work and samples, so there are few systematic studies. Capillary electrophoresis (CE)-SELEX has become a highly efficient technique for aptamers screening because of the advantages of CE, e.g. microscale, high efficiency and low cost. In this work, human apo-transferrin (A-TF) was used as a model to study the effects of factors based on CE, including length of ssDNA library, incubation temperature, type and pH value of incubation buffer, metal ions etc. The results showed that shorter ssDNA library had higher affinity with the target protein. Incubation temperature, type and pH value of incubation buffer affected the formation of complex. Low concentrations of K+, Ca2+ and Mg2+ metal ions promoted the formation of complexes. The A-TF aptamer Seq A3 obtained by three rounds of selection had a dissociation constant (KD) of 0.476 μmol/L, and its affinity and specificity were verified via CE-laser induced fluorescence in human serum. The results indicated that Seq A3 could specifically recognize A-TF in serum.