In vitro selection of human cerebrospinal fluid-specific aptamers using clinical samples.

Abstract

Cerebrospinal fluid (CSF) leaks may occur due to numerous etiologies and are associated with severe morbidity. Currently in the U.S., confirming the presence of a CSF leak requires protein electrophoresis testing, oftentimes involving specialized processing, and there exists no point-of-care (POC) device for CSF detection. We aimed to discover a single-stranded deoxyribonucleic acid (ssDNA) aptamer capable of selectively binding to CSF-specific biomarkers, with the future goal of developing an aptamer-based POC CSF detection device. To identify a candidate aptamer, we performed Systematic Evolution of Ligands by EXponential enrichment (SELEX) using a DNA library containing a randomized 63-nucleotide (nt) stretch flanked by 2 primer-binding sites. Quantitative polymerase chain reaction (qPCR) and fluorescence anisotropy (FA) assessed aptamer binding affinity and kinetics. Following 14 SELEX cycles, 2 dominant and functionally viable 98-nt ssDNA sequences (C2 and C3) were found. C2 and C3 demonstrated ~586x and ~82x higher affinity for CSF compared to serum, respectively. Increases in FA upon aptamer exposure to higher CSF concentrations demonstrated a K1⁄2 of 5.0% and 14.1% for C2 and C3, respectively. In vitro selection of a diverse pool of ssDNA sequences yielded 2 aptamers with high selectivity for CSF-specific biomarkers, with potential for integration into a rapid POC electrochemical diagnostic system.