Discovery of a Novel Cerebrospinal Fluid-Specific DNA Aptamer

Background: Cerebrospinal fluid (CSF) leaks present a significant problem for neurosurgical, otolaryngologic, trauma, and emergency medicine patients. However, determining the presence of a CSF leak currently depends largely on clinical suspicion since gold standard testing for CSF via immunofixation electrophoresis can be time-consuming and typically only available in specialized, centralized laboratories. With the goal of developing a bedside electrochemical-based diagnostic tool for CSF detection, we hereby aimed to develop a novel single-stranded deoxyribonucleic acid (ssDNA) aptamer capable of selectively binding to CSF-specific biomarkers.

Methods: To identify a candidate aptamer, we performed Systematic Evolution of Ligands by EXponential enrichment (SELEX) using a large and diverse random sequence DNA library. This pool of DNA sequences contained a 63-nucleotide (nt) stretch of random sequences flanked by two primer-binding sites, which were further modified to possess methylene blue (MB) at their 5′ terminus to serve as a redox indicator in electrochemical measurements. Quantitative polymerase chain reaction (qPCR) was used to evaluate aptamer binding affinity.

Results: Fourteen cycles of SELEX were performed, after which high-resolution sequencing of the resulting DNA pool revealed 2 dominant and functionally viable 98-nt ssDNA (C2 and C3). Processing of the synthesized ssDNA using complex samples of CSF or plasma, followed by qPCR analysis, demonstrated that C2 and C3 expressed ∼586× and ∼82× higher affinity for CSF compared to plasma (both p < 0.001).

Conclusion: In vitro selection of a diverse pool of ssDNA sequences revealed two aptamers with high selectivity for CSF-specific biomarkers. Integration of these aptamers into an electrochemical platform enables the development of a point-of-care diagnostic device for detecting CSF leaks.

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Artikel online veröffentlicht:
07. Februar 2025