Methods for the detection of protein -nucleic acid and protein -protein interactions.

摘要

We describe the first general approach for the DNA templated reassembly of proteins, which we term SEquence-Enabled Reassembly or SEER. SEER makes use of dissected signaling domains which are each attached to separate, sequence specific DNA-binding proteins. Described herein is an embodiment of SEER in which DNA catalyzes the reassembly of the green fluorescent protein which leads to a direct fluorescence readout of the corresponding DNA sequence. This strategy has also been extended to the first direct method for the site specific detection of DNA methylation. This mCpG-SEER system is capable of discriminating between methylated versus nonmethylated DNA with a 40-fold increase in fluorescence signal.;In a separate undertaking we tested the efficiency of disulfide bond formation within the context of the ribosome display in vitro selection methodology. We established conditions for the enrichment of a cyclic peptide, which is specific for Neutravidin, by 2 x 106-fold. Using the knowledge gained from the above experiments, we combined the rapid protein expression and folding benefits of cell-free translation systems with a sensitive split-luciferase reassembly assay to yield the most rapid method to date for the detection of protein-nucleic acid and protein-protein interactions. Furthermore, we have shown that these split-luciferase cell-free reassembly systems can be compartmentalized, allowing for future molecular evolution studies.;Lastly, we have applied this rapid cell-free split-luciferase assay system to the direct detection of clinically relevant proteins. We have engineered a system for the rapid characterization of HIV-1 clades utilizing single-chain antibody specificities. We also demonstrate that this platform can be used to determine the relative amounts of HER2 expression in human breast cancer cells, using a homogeneous assay format in which cells and reagents are mixed and luminescence is monitored directly.;We envision that the assay platforms described herein will find applications in the rapid detection of nucleic acid sequences, protein identities, and relative protein abundances in the laboratory and clinic.