T5 Exonuclease Improves the Identification of Aptamers Specific for Small Molecules from the Early Screening Library

Aptamers are usually obtained by the SELEX process; however, it is time-consuming to perform repetitive work. Therefore, a new method was designed to rapidly obtain high-affinity aptamers from an early screening library. Target-binding aptamers were isolated from early library based on the function of T5 exonuclease (Exo), which could digest nonaffinity aptamers and retain the complete sequence of affinity aptamers. Meanwhile, enzymatic electrophoresis, label-free dye displacement assay, q-PCR, and isothermal titration calorimetry were used to characterize aptamer affinity. The ninth ssDNA library (9R) of 5-hydroxymethylfurfural (5-HMF) was digested by T5 Exo, and high-throughput sequencing was performed on the digested library (E-9R). 20 sequences with the highest multiplicity were selected for characterization. The results showed that there were 8 affinity aptamers in the general screening library and 15 in the digested library, which were always more than in the general screening library when the target concentration decreased. Subsequently, the aptamers from enzymatic-electrophoresis experiments were characterized by the label-free dye displacement assay and q-PCR to distinguish the binding strength to 5-HMF. It was found that 9R-e1 from E-9R exhibited the best binding affinity, with the <i>K</i>_d value of 8.8 μM. The same enzymatic conditions were used for the patulin (PAT) screening library to verify this method's universal applicability. Similarly, affinity aptamers were always more abundant in the enzymatic library, and an aptamer with a <i>K</i>_d value of 6.40 μM was found in PAT-E6R. The enzymatic method ensures high-affinity aptamer availability while reducing the number of selection rounds, significantly shortening the selection time and reducing the cost.