Use of intrinsic reporters of cell signaling for high content drug profiling and toxicity screening
AssigneeMount Sinai School of Medicine
InventorsStuart Sealfon, Elisa Wurmbach, Tony Yuen
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Abstract
The present invention identifies essentially all of the members of a specific group of genes that are preferentially transcribed upon the initialization of a signal transduction pathway. The present invention also discloses methods for detecting and/or quantifying the transcription of these specific genes. The present invention further discloses methods of using this information to characterize the effect of potential drugs on a cell. Solid supports comprising nucleic acids that can hybridize with the transcripts from this specific group of genes are also described.
Claims26
A solid substrate, comprising a plurality of polymers coupled to the solid substrate in a different known location, wherein each polymer of the plurality of polymers has a different sequence, and wherein at least two copies of each polymer are present on the solid substrate.
3 The solid substrate of claim 1, wherein the plurality of polymers is a plurality of different nucleic acids, and wherein each different nucleic acid hybridizes to at least a portion of an RNA transcript, such that a plurality of different nucleic acids hybridize to a plurality of RNA transcripts.
The solid substrate of claim 3, wherein at least 50% of the plurality of RNA transcripts are encoded by different intrinsic reporters of cell signaling.
A solid substrate comprising 50 to 1000 different nucleic acids coupled to the solid substrate in a different known location;
- wherein each of the 50 to 1000 different nucleic acids hybridizes with at least a portion of an RNA transcript, wherein the plurality of different nucleic acids hybridize to a plurality of RNA transcripts;
- and wherein at least 50% of the plurality of RNA transcripts are encoded by a different early response gene.
A method of identifying a gene that is up-regulated by an agent comprising:
- (a) contacting the agent with a cell for a time period sufficient to allow transcription of a gene to generate an RNA;
- (b) isolating RNA from the cell, whereby an isolated RNA preparation is obtained which comprises individual RNA transcripts generated by the cell;
- (c) hybridizing the isolated RNA preparation to said plurality of different nucleic acids of the solid substrate of claim 3 under conditions wherein an individual RNA transcript of the isolated RNA preparation hybridizes to a specific nucleic acid;
- and
- (d) determining which of the specific nucleic acids of the plurality of different nucleic acids hybridizes to an RNA transcript is increased in amount in the presence of the agent relative to the absence of the agent, wherein the determining is performed in conjunction with statistical analysis;
- and wherein when the amount of the individual RNA transcript has increased in a statistically significant manner in the presence of the agent relative to in its absence, a gene that is up-regulated by the agent is identified.
A method of identifying genes that are up-regulated by an agent comprising:
- (a) contacting an agent with a cell for a sufficient time to allow transcription of a gene to generate an RNA, wherein the cell comprises a set of genes comprising at least 80% of the intrinsic reporters of cell signaling listed in Table 1, or orthologs thereof;
- (b) determining the individual rate of transcription and/or the amount of transcription of the set of genes;
- and
- (c) identifying the genes within said set of genes that have an increased relative level of expression in the presence of the agent relative to in the absence of the agent;
- wherein the genes are identified that have an increased relative level of expression in the presence of the agent.
14 The method of claim 13, wherein the genes that are identified have an increased relative level of expression in the presence of the agent that is 1.5 fold or greater.
The method of claim 14, wherein the contacting time is 1 hour at 37° C.
The method of claim 14, wherein the determining step includes the use of a data analysis algorithm based on thresholding by fold-change and t-test value determination.
A method of selecting a candidate compound comprising:
- (a) contacting an agent with a cell;
- wherein said contacting is for a time period sufficient to allow transcription of a group of selected IRCs to generate RNAs;
- (b) determining the EC50 and maximal response for each IRC;
- and
- (c) comparing the EC50 and maximal response for each IRC determined in step (b) with the corresponding EC50 and maximal response for each IRC determined in the presence of a reference compound;
- wherein the reference compound has a known desirable and/or undesirable activity;
- and wherein the compound is selected as a candidate compound when the compound is determined to be more desirable than the reference compound.
Dependencies are inferred from the claim text.