G-quadruplex 2′-F-modified RNA aptamers targeting hemoglobin: Structure studies and colorimetric assays

Biosensors that rely on aptamers as analyte-recognizing elements (also known as aptasensors) are gaining in popularity during recent years for analytical and biomedical applications. Among them, colorimetric ELISA-like systems seem very promising for biomarker detection in medical diagnostics. For their development, one should thoroughly consider the characteristics of the aptamers, with a particular focus on the secondary structure. In this study, we performed an in-depth structural study of previously selected hemoglobin-binding 2′-F-RNA aptamers using CD spectroscopy, enzymatic probing, and specific fluorophore binding. Only a combination of different assays allowed us to prove G-quadruplex formation for anti-hemoglobin 2′-F-RNA aptamers. We also demonstrated a possible application of these 2′-F-RNA aptamers for microplate colorimetric detection of human hemoglobin in both direct and sandwich formats. [Display omitted] •Confirmed formation of quadruplexes for three hemoglobin-specific 2'-F-RNA aptamers using a combination of experimental assays.•Establsihed an aptamer-based microplate colorimetric detection of human hemoglobin both in direct and sandwich assays.•Demonstrated that hemoglobin detection system based on two aptamers could generate a nonspecific colorimetric signal.