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Determination of Aptamer Binding Affinity to Fallopian Tube Biomarkers Using Fluorescence Anisotropy
Samarakoon, Bhagya
Samarakoon, Bhagya
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Abstract
High grade serous ovarian carcinoma (HGSOC) has been discovered to originate from Fallopian tube (FT) epithelium. Fallopian tube biomarkers are therefore a promising new class of targets that may enable development of a novel screening tool for the early detection of HGSOC, which is not accessible with currently available ovarian-derived biomarkers. Integrin β3 (ITGB3) is one of the FT biomarkers that have been identified. A DNA aptamer (s10hy2) recognizing ITGB3 has been reported, but binding of this aptamer to its intended target and possible off-target proteins has not been thoroughly characterized in the sample environments relevant to clinical applications, specifically blood products (serum and plasma). In this study we have validated the binding of Texas Red-labeled s10yh2 to ITGB3 using fluorescence anisotropy and found that the affinity of this interaction is in the low nanomolar range (Kd = 50 ± 27 nM). The assay was performed by preparing replicate samples each containing 75 nM aptamer and varied ITGB3, with concentration ranging from 0 to 440 nM. The binding affinity of the aptamer with the heterodimer αIIbβ3 was also investigated. The Kd values were consistent, indicating that the presence of the αIIb subunit did not affect the binding of the aptamer with the β3 subunit. The binding affinity experiments were done in the absence of bovine serum albumin (BSA), which is often used to prevent nonspecific binding in fluorescence anisotropy experiments. BSA was found to interfere with the binding between the aptamer and the integrin since the aptamer also binds to BSA with a Kd of 371 ± 94 nM. Human serum has a Kd of 47 ± 32 nM with the aptamer, which could be attributed to binding between albumin in human serum with the aptamer. Further experiments are being carried out to characterize the binding between human serum albumin and the aptamer since this could be an interference when employing the aptamer as an ovarian cancer screening tool.
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This poster was presented at ACS- Midwest Regional Meeting on 10/14/2024.
Date
2024-10-14
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University of Kansas
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BhagyaS_2025.pdf
Adobe PDF, 1.57 MB
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Keywords
Ovarian cancer, Fallopian tube biomarkers, Fluorescence anisotropy, Aptamers, Binding affinity