Skip to main content
eScholarship
Open Access Publications from the University of California

UC San Diego

UC San Diego Electronic Theses and Dissertations bannerUC San Diego

High Throughput Methods for Cell-Type Specific Elucidation of Protein Interactions

  • Author(s): Johnson, Kara
  • Advisor(s): Zhong, Sheng
  • et al.
No data is associated with this publication.
Abstract

While it is widely understood that proteins are the functional tools of a cell, there are still no methods that efficiently illuminate the cell-wide networks of protein-protein and protein-RNA interactions through which proteins can act. Existing methods of detecting protein interactions primarily take one of two forms, they are either highly parallelized ‘one-by-one’ assays or several ‘one-by-many’ assays. However, these efforts are expensive and time intensive when attempting to address a cell-wide network.

This dissertation describes the development and validation of three technologies, a protein barcoding technology which generates a library of proteins labeled with specific nucleic acid sequences (SMART-Display), a high throughput proximity ligation technology that elucidates protein-protein interactions (PROPER-Seq), and a high throughput proximity ligation technology that elucidates protein-RNA interactions (PRIM). Leveraging these technologies, protein-protein and protein-RNA interactions can be assessed for a given cell type with an “all-vs-all” approach. The PRIM and PROPER-Seq workflows are characterized by a drastic increase in the number of interactions assayed in a single experiment relative to existing techniques, minimal labor, cost, and time, suitability for automation, and accessibility to any benchtop scientist, as they are devoid of the need for specialized technology or equipment.

SMART-Display produces highly complex protein libraries that closely reflect the mRNA population of the cell-type they are derived from, and which contain protein-specific DNA barcodes that can be used for proxy identification of the proteins themselves. This enables the application of PRIM and PROPER-Seq with libraries containing tens of thousands of uniquely labeled protein interactors. Precision and sensitivity analyses indicate that PROPER-Seq technique demonstrates a detection of interactions that is similar to, or better than, the levels demonstrated by gold-standard techniques such as yeast-2-hybrid. Several novel PROPER-Seq interactions were validated in vivo. Preliminary PRIM data indicates sufficient quality for precision and sensitivity analysis and both literature and in vivo validation.

Main Content

This item is under embargo until July 10, 2022.