Use of protein assemblies to record cellular events
AssigneePresident and Fellows of Harvard College
InventorsAdam Ezra Cohen, Dingchang Lin, Xiuyuan E. Li, Doyeon Kim
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Abstract
The present disclosure provides methods for profiling cellular events in a cell over time using protein assemblies. Methods for profiling the effect of a stimulus, such as a candidate therapeutic agent, are also provided by the present disclosure. Proteins, polynucleotides (e.g., vectors), and pairs of polynucleotides for use in the methods described herein, as well as systems and kits comprising such proteins, polynucleotides, and pairs of polynucleotides, are also provided herein.
Claims27
A method of profiling one or more cellular events in a cell over time, the method comprising:
- incubating a cell for a period of time, wherein the cell comprises:
- a first polynucleotide encoding a first protein assembly monomer under control of a first promoter;
- and
- a second polynucleotide encoding a second protein assembly monomer under control of a second promoter;
- wherein a cellular event leads to expression of the first and/or second protein assembly monomer or a visualizable change or modification of the first and/or second protein assembly monomer in a protein assembly, and
- wherein activation of the second promoter is dependent on a cellular event being profiled.
A method for profiling a cellular event over time comprising:
- a) incubating a cell, wherein the cell comprises:
- a first polynucleotide encoding a protein assembly monomer fused to a visualizable tag under control of a first promoter;
- and
- a second polynucleotide encoding a protein assembly monomer fused to a visualizable protein under control of a second promoter, wherein activation of the second promoter is associated with an occurrence of the cellular event being profiled;
- for a period of time t1 in the presence of a first fluorescent dye that binds to the visualizable tag, thereby allowing the protein assembly monomers expressed by the first and second polynucleotides to assemble into a protein assembly;
- b) further incubating the cell for a period of time t2 in the presence of a second fluorescent dye that binds to the visualizable tag, thereby allowing the protein assembly monomers expressed by the first and second polynucleotides to further assemble onto the protein assembly;
- and
- c) imaging the protein assembly in the cell to determine the location of the protein assembly monomer fused to the visualizable protein relative to the locations of the first and the second fluorescent dyes bound to the visualizable tag, thereby determining the times at which the cellular event occurs.
Dependencies are inferred from the claim text.