Fig. 2. Homogenous EcoRI-HF SELEX to generate structure-switching aptamers. (a) FAM-labeled DNA library having an N₄₀ random region was hybridized to capture strand and the complex digested with EcoRI-HF. The cleaved product (70 nt) was gel purified and full-length product (90 nt) was recovered through split ligation by T4 DNA ligase. The re-ligated library members were hybridized to free capture strand and incubated with the target. Active biosensor sequences were enriched by EcoRI-HF digestion followed by PCR amplification. ssDNA library was purified by 10% denaturing PAGE and carried on to subsequent selection rounds. (b) Chemical structure of kanamycin A. (c) Progression of EcoRI-HF SELEX to generate structure-switching aptamers to kanamycin A. Following digestion, cleavage products were monitored by 10% denaturing PAGE. Band intensity was used to quantify percent uncleaved.