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Development of High Throughput Methods for Probing Biomacromolecular Interactions

  • Author(s): Ashby, Jonathan
  • Advisor(s): Zhong, Wenwan
  • et al.
Abstract

Within living organisms, there exists a large variety of macromolecular interactions, such as protein-protein, nucleic acid-protein, or lipid-protein. Also known as the interactome, these interactions are essential to a variety of functions, such as gene regulation and internal/external cell signaling. Engineered nanomaterials (ENMs) have gained increased popularity in recent years as a tool for biosensing and bioanalysis. However, little is known about how these ENMs could adversely affect the interactome, either through intentional or accidental exposure to ENMs.

A powerful tool for probing macromolecular interactions is flow field-flow fractionation (F4), and its successor, asymmetrical F4 (AF4). (A)F4 is an open channel separation technique that fractionates analytes based purely on their size. Similarly to other open channel techniques like size exclusion chromatography and capillary electrophoresis, (A)F4 is well suited to preserving the native structure of the macromolecular complex.

F4 was used to differentiate between the stable and transient protein coronas surrounding ENMs as they enter a biological matrix. Analysis of the isolated ENM-protein coronas has revealed correlations between the nature of the surface coating (hydropathy, charge density) and the composition of the stable and transient corona.

Due to the batch-to-batch variability of ENM synthesis, tools are also needed to rapidly assess how changes in the size, shape or surface coating may adversely affect the behavior of ENMs within the body. A fluorescence assay was designed to take advantage of changes in a protein's tertiary structure upon interaction with a nanoparticle to determine the effects of changes in an ENM's physiochemical parameters.

Finally, an AF4 method was designed for the fractionation of micro RNA (miRNA) into fractions such as small protein-bound, lipoprotein complex-bound, and exosomal. Changes in miRNA levels within the body can be used for early disease detection. By correlating up and down-regulation of miRNA with increased/decreased concentrations of particular proteins, identification of associated protein biomarkers can be done. If these protein markers exist in higher concentrations within the body, changes in this protein level could be used as a guide to direct additional testing for specific miRNA biomarkers.

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