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US 2007/0190532 A1Published application
Assay for identifying compounds which affect stability of mrna
AssigneeNovation Pharmaceuticals Inc.
InventorsTania Katelic, Dominique Cheneval
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Abstract
The present invention relates to an assay for the identification of biologically active compounds, in particular to a reporter gene assay for the identification of compounds, which have an effect on mRNA stability. More particularly, the present invention relates to a reporter gene expression system and cell lines comprising said expression system. The invention further relates to compounds which destabilise mRNA.
Claims22
A DNA expression vector comprising:
- i) a first DNA sequence comprising the coding sequence for one or more protein having a detectable signal;
- ii) one or more 3′ UTR sequence and one or more expression control sequence operatively associated with said coding sequence, and
- iii) a heterologous instability sequence DNA inserted into said 3′ UTR sequence comprising a second DNA sequence corresponding to one or more mRNA instability sequence derived from one or more naturally occurring genes.
A stably transfected cell line comprising:
- i) a DNA expression vector comprising a first DNA sequence encoding a first protein having a detectable signal, one or more 3′ UTR sequence and one or more expression control sequence operatively associated with said first DNA sequence, and a heterologous instability sequence DNA inserted into said 3′ UTR sequences, said instability sequence DNA comprising a second DNA sequence corresponding to one or more mRNA instability sequence derived from one or more naturally occurring genes;
- and
- ii) a control DNA expression vector comprising a control DNA sequence encoding a second protein having a detectable signal, and one or more 3′ UTR sequence and one or more expression control sequence operatively associated with said control DNA sequence.
A method of screening for one or more compound which affect mRNA stability comprising the steps of:
- i) providing a DNA expression vector, which in the absence of a test compound is capable of expressing a protein having a detectable signal, wherein the mRNA which is transcribed from said expression vector and encodes said protein comprises at least one copy of a heterologous mRNA instability sequence;
- (ii) contacting said DNA expression vector with at least one test compound under conditions whereby, in the absence of the test compound, said DNA expression system is capable of expressing said protein having a detectable signal;
- (iii) measuring said detectable signal;
- and
- (iv) comparing the measured detectable signal with a control,
- wherein a decrease in the measured detectable signal compared to said control indicates a compound that decreases mRNA stability and an increase in the measured detectable signal compared to said control indicates a compound that increases mRNA stability.
3 more elements
A method for comparing the extent of mRNA degradation induced by two or more compounds comprising the steps of:
- (i) providing a DNA expression vector, which in the absence of a test compound is capable of expressing a protein having a detectable signal, wherein the mRNA which is transcribed from said expression vector and encodes said protein comprises at least one copy of a heterologous mRNA instability sequence;
- (ii) contacting said DNA expression vector separately with two or more test compounds under conditions whereby, in the absence of the test compounds, said DNA expression system is capable of expressing said protein having a detectable signal;
- (iii) measuring said detectable signal in the presence of each test compound;
- and
- (iv) comparing the measured detectable signals;
- wherein a lower measured detectable signal indicates a greater extent of mRNA degradation.
3 more elements
An assay system for screening for compounds which destabilise mRNA comprising:
- (i) a DNA expression vector comprising a first DNA sequence encoding a first protein having a detectable signal, one or more 3′ UTR sequence and one or more expression control sequence operatively associated with said DNA sequence, and a heterologous instability sequence DNA inserted into said 3′ UTR sequence, said instability sequence DNA comprising a second DNA sequence corresponding to one or more mRNA instability sequence derived from one or more naturally occurring genes;
- and
- (ii) a control DNA expression vector comprising a control DNA sequence encoding a control protein having a detectable signal, and one or more 3′ UTR sequence and one or more expression control sequence operatively associated with said control DNA sequence.
A high throughput method for screening libraries of compounds to identify compounds that affect the stability of mRNA comprising:
- (i) providing a stably transfected cell line comprising a DNA expression vector, which in the absence of a test compound is capable of expressing a protein having a detectable signal, wherein the mRNA which is transcribed from said expression vector and encodes said protein comprises at least one copy of a heterologous mRNA instability sequence;
- (ii) inoculating wells of one or more multi-well plates comprising a growth medium with said cell line;
- (iii) maintaining said one or more multi-well plates under conditions that allow cells of said cell line to grow and express said protein having a detectable signal;
- (iv) contacting the cells with one or more test compound;
- (v) measuring said detectable signal;
- and
- (vi) comparing the measured detectable signal with a control;
- wherein a decrease in the measured detectable signal compared to said control indicates a compound that decreases mRNA stability and an increase in the measured detectable signal compared to said control indicates a compound that increases mRNA stability.
5 more elements
A kit comprising an assay system for screening for compounds which destabilize mRNA, said assay system comprising:
- (i) one or more DNA expression vector comprising a first DNA sequence encoding a protein having a detectable signal, one or more 3′ UTR sequence and one or more expression control sequence operatively associated with said first DNA sequence, and a heterologous instability sequence DNA inserted into said 3′ UTR sequence, said instability sequence DNA comprising a second DNA sequence corresponding to one or more mRNA instability sequence derived from one or more naturally occurring genes;
- and
- (ii) a control DNA expression vector comprising a control DNA sequence encoding a second protein having a detectable signal, one or more 3′ UTR sequence and one or more expression control sequence operatively associated with said control DNA sequence;
- and optionally
- (iii) instructions for use.
2 more elements
Dependencies are inferred from the claim text.