Fine-tuning of aptamer complementary DNA for fluorescence resonance energy transfer assay

• We established a ratio-based FRET analysis method for an OTA-oriented aptamer. • Here, we combined circular dichroism spectroscopy with molecular dynamic simulation. • We provide a reference for the use of aptamers and cDNA to determine analytes. When developing a fluorescence resonance energy transfer (FRET) method to detect target analytes, nucleic acid aptamers with high affinity and specificity are often used as capture probes, and complementary DNA (cDNA) competing with the target analyte for binding with the aptamers is used to detect the analyte. During the development of such methods, the position, length, and base sequence of cDNA play a key role in improving detection sensitivity. Based on relevant research and understanding of the conformation and binding site of nucleic acid aptamers, we established a ratio-based FRET analysis method for an ochratoxin A (OTA)-oriented aptamer and its cDNAs by combining circular dichroism spectroscopy with molecular dynamic simulation. The influence of the position, length, and base sequence of cDNA on the fluorescence response ratio was explored, and a cDNA sequence was fine-tuned, designed, and optimized. Compared with the unoptimized cDNA, the fluorescence response ratio increased 13-fold, and the detection limit was as low as 3.9 nM. This study provides a general reference for the use of aptamers and cDNA to establish a method to determine analytes.