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bioRxiv · 10.1101/2024.10.23.619861

DNA Hanger: Surface Free Single-Molecule Blotting Platform

Seol, J.·Kim, B.·Yu, E.-S.·Jeong, C.·Lee, J.-B.
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Abstract

Immunoblotting typically requires protein immobilization onto a membrane surface for quantification. Particularly, in real-time single-molecule blotting, stringent surface passivation is essential to prevent false-positive signals from nonspecific binding. In this study, we introduce a novel surface-free single-molecule blotting platform, termed the DNA Hanger. This platform employs a 3D structure of a quartz slide. Modified{lambda} -phage DNA molecules are randomly biotinylated along their bases and suspended between 4 m-high thin barriers, positioned away from the quartz slide surface. A light sheet, produced within the 3D structure, illuminates fluorophore-conjugated proteins bound to the biotinylated DNA, enabling single-molecule detection. The DNA Hanger assay significantly reduces nonspecific binding and enhances sensitivity to sub-picomolar concentrations, suggesting that this platform provides a novel, surface-condition-independent, and real-time approach in a wide range of single-molecule blotting.

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Seol, J., Kim, B., Yu, E.-S., Jeong, C., Lee, J.-B.. 2024-10-28. DNA Hanger: Surface Free Single-Molecule Blotting Platform. https://doi.org/10.1101/2024.10.23.619861

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