Proximity Ligation for Protein Detection and Protein-compound Interaction
Open Access
- Author:
- Mccaffrey, Jennifer Lauren
- Area of Honors:
- Bioengineering
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- Dr. William O Hancock, Thesis Supervisor
Peter J Butler, Thesis Honors Advisor - Keywords:
- proximity ligation
encoded library technology
protein-compound interaction - Abstract:
- The ability to generate compounds to treat or cure a disease is central to the pharmaceutical industry. GlaxoSmithKline (GSK) has developed Encoded Library Technology (ELT) for the synthesis and selection of DNA-encoded libraries (DELs) containing millions to billions of potential drug candidates. There is a need for an efficient method to detect the interactions between proteins and compounds in cells and tissues. Proximity ligation is a relatively new method for highly specific and sensitive detection of proteins in solution, cell culture, or localized tissue. Modifying proximity ligation to quantify the interactions between proteins and compounds using DNA- encoded compounds will prove to be beneficial to drug discovery. Applied Biosystems produces a TaqMan Assay kit that uses the method of proximity ligation with a TaqMan probe to detect protein levels. A modification of the TaqMan Assay was made by replacing an antibody of the probe pair with a DNA-encoded compound in order to quantify protein-compound interactions. These experiments were not accurately characterized with a nonlinear regression, one- site specific binding model. A new model was generated to quantify protein-compound interactions. Although the assay produced a protein dependent signal with both purified protein and cell lysate, there were discrepancies between the experimental data and the theoretical model. The probe concentrations, oligonucleotide lengths, and the connector length need to be optimized in order to quantify the interactions between proteins and potential drug candidates.