Identification and Characterization of Aptamers Targeting Ovarian Cancer Biomarker Human Epididymis Protein 4 for the Application in Urine

SIMPLE SUMMARY: Ovarian cancer is the deadliest gynecological cancer. Due to the lack of effective diagnostic methods and the non-specific symptoms of the disease, late diagnosis remains a main factor of the poor prognosis. Therefore, development of novel diagnostic approaches are needed. Recently, urine has become an interesting non-invasive source of cancer biomarkers. Human epididymis protein 4 (HE4) is a protein overexpressed in ovarian cancer, but not in healthy or benign conditions. In urine, HE4 stands as a biomarker with high stability and diagnostic value for detection of ovarian cancers. Recently, aptamers emerged as inexpensive detection probes for cancer detection. Aptamers are single-stranded oligonucleotides that bind with high affinity to target molecules. Here, we selected, identified and characterized DNA aptamers targeting human HE4 in urine, with the affinities in the nanomolar range. Therefore, they could represent a promising tool for application in diagnostics and future development of urine tests or biosensors for ovarian cancer. ABSTRACT: Ovarian cancer is the deadliest gynecological cancer. With non-specific symptoms of the disease and the lack of effective diagnostic methods, late diagnosis remains the crucial hurdle of the poor prognosis. Therefore, development of novel diagnostic approaches are needed. The purpose of this study is to develop DNA-based aptamers as potential diagnostic probes to detect ovarian cancer biomarker Human epididymis protein 4 (HE4) in urine. HE4 is a protein overexpressed in ovarian cancer, but not in healthy or benign conditions. With high stability and diagnostic value for detection of ovarian cancer, urine HE4 appears as an attractive non-invasive biomarker. The high-affinity anti-HE4 DNA aptamers were selected through 10 cycles of High Fidelity Systematic Evolution of Ligands by EXponential enrichment (Hi-Fi SELEX), a method for aptamer selection based on digital droplet PCR. The anti-HE4 aptamers were identified using DNA sequencing and bioinformatics analysis. The candidate aptamer probes were characterized in urine for binding to HE4 protein using thermofluorimetry. Two anti-HE4 aptamers, AHE1 and AHE3, displayed binding to HE4 protein in urine, with a constant of dissociation in the nanomolar range, with K(d) (AHE1) = 87 ± 9 nM and K(d) (AHE3) aptamer of 127 ± 28 nM. Therefore, these aptamers could be promising tools for application in diagnostics and future development of urine tests or biosensors for ovarian cancer.