Generation of Stable 2’-F/OMe-RNA Aptasensors for <i>Bacillus cereus</i> 5/B/6 Metallo-β-Lactamase

Aptamer-based sensors (aptasensors) have been extensively employed in biosensing and diagnostics. However, the broader application of aptasensors fabricated with DNA and RNA aptamers has been limited due to the intolerance of DNA and RNA to nucleases in biological fluids. A majority of sugar-modified nucleic acids, in particular 2'-F and 2'-OMe-modified nucleic acids, demonstrate a remarkable resistance to nuclease degradation and thus hold great potential in the development of more stable aptamers and aptasensors. In this work, employing enzymes including a laboratory-evolved polymerase for 2'-modified nucleic acids, we carried out systematic evolution of ligands by exponential enrichment (SELEX) experiments to obtain 2'-F/OMe-RNA aptamers for <i>Bacillus cereus</i> 5/B/6 metallo-β-lactamase (<i>B. cereus</i> 5/B/6 MBL), a crucial marker of many penicillin-resistant strains. Three aptamers composed of 2'-OMe-A, 2'-F-U, 2'-F-C, and 2'-OMe-G nucleotides (Apt-FUC1, Apt-FUC2, and Apt-FUC3) have been obtained and systematically characterized. All of them exhibited excellent target binding affinities, with equilibrium dissociation constant (<i>K</i>_d ) values of 95.9, 70.0, and 81.3 nM, and outstanding biological stability. Combining FAM-labeled Apt-FUC1, Apt-FUC2, and Apt-FUC3 with graphene oxide (GO), which could quench the fluorescence of FAM, we successfully constructed three sensitive and biologically stable 2'-F/OMe-RNA aptasensors for <i>B. cereus</i> 5/B/6 MBL, with the limit of detection (LOD) values of 0.38, 0.27, and 0.29 nM. Moreover, these three aptasensors all demonstrated good specificity for target detection in biological samples. This work has established a streamlined strategy for generating stable 2'-F/OMe-RNA aptamers and aptasensors, which should broaden the application of sugar-modified nucleic acids in biosensing and diagnostics.