Systematic screening and optimization of single-stranded DNA aptamer specific for N-acetylneuraminic acid: A comparative study

Abstract

Sialic acids (SAs), particularly its most common derivative N-acetylneuraminic acid (Neu5Ac) are involved in many biological processes in vivo. Thus it is of great significance to detect Neu5Ac and trace the sialylation of glycoproteins and other bio-active molecules. Aptamers are molecular elements for bio-recognition with high affinity, which have been widely used in bioanalysis and imaging. However, the ideal aptamers for saccharides have seldom been reported. Magnetic beads (MB)-based SELEX is a commonly used method for screening of aptamers. Herein, three types of MB-based SELEX with different immobilization manners were carried out to systematically study and compare the screening efficiency, which may provide reference for screening of aptamers specific for low-molecular targets. After screening and sequencing, the obtained aptamer sequences were aligned and analyzed, among which three aptamers were selected as candidates. Then, the aptamer ap 3-1 with the highest affinity was optimized according to the results of molecular simulation docking. An optimal aptamer ap 3-1-a with 21 nucleotides (nt) was eventually obtained, with the Kd value of 55.71 ± 12.29 nM. To verify the practicability of the aptamer, a fluorescent biosensor for Neu5Ac was constructed employing graphene oxide (GO)-based fluorescence quenching and Neu5Ac-induced fluorescence restoration. The biosensor showed a linear response range within 20−1000 nM. The specificity was verified and the interferents-resistance was validated by detecting Neu5Ac in actual samples. This study may inspire the efficient and universal strategy for screening and application of robust and practicable aptamers for low-molecular targets.