Assay for identifying compounds which affect stability of mrna

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.

1 - 9 . (canceled) 
     
     
         10 . A method of screening for one or more compound which affect mRNA stability comprising the steps of: i) contacting a DNA expression system with at least one test compound under conditions whereby, in the absence of the test compound, said DNA expression system expresses a protein having a detectable signal, wherein the mRNA which is transcribed from said expression system and encodes said protein comprises at least one copy of a heterologous mRNA instability sequence; (ii) measuring said detectable signal; and (iii) 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. 
     
     
         11 . The method according to  claim 10 , wherein said control comprises measuring the detectable signal from the DNA expression system in the absence of said test compound. 
     
     
         12 . The method according to  claim 10 , wherein said control comprises contacting a control expression system capable of expressing a second protein having a second detectable signal with the test compound and measuring said second detectable signal. 
     
     
         13 . The method according to  claim 10 , wherein said compounds are being screened for their ability to induce mRNA degradation, and wherein a decrease in the measured detectable signal compared to said control indicates a compound that induces mRNA degradation. 
     
     
         14 . A method for comparing the extent of mRNA degradation induced by two or more compounds comprising the steps of: (i) contacting a DNA expression system separately with two or more test compounds under conditions whereby, in the absence of the test compounds, said DNA expression system expresses a protein having a detectable signal, wherein the mRNA which is transcribed from said expression system and encodes said protein comprises at least one copy of a heterologous mRNA instability sequence; (ii) measuring said detectable signal in the presence of each test compound; and (iii) comparing the measured detectable signals; wherein a lower measured detectable signal indicates a greater extent of mRNA degradation. 
     
     
         15 - 17 . (canceled) 
     
     
         18 . A high throughput method for screening libraries of compounds to identify compounds that affect the stability of mRNA comprising: (i) inoculating wells of one or more multi-well plates comprising a growth medium with a stably transfected cell line comprising a DNA expression vector, which in the absence of a test compound expresses 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) 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; (iii) contacting the cells with one or more test compound; (iv) measuring said detectable signal; and (v) 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. 
     
     
         19 - 22 . (canceled) 
     
     
         23 . The method of  claim 10 , wherein said heterologous mRNA instability sequence is derived from a naturally occurring gene. 
     
     
         24 . The method of  claim 23 , wherein said expression system comprises an instability sequence DNA comprising said heterologous mRNA instability RNA sequence and DNA corresponding to sequences that flank said mRNA instability sequence in the naturally occurring gene. 
     
     
         25 . The method of  claim 24 , wherein said instability sequence DNA comprises DNA corresponding to the whole, or a substantial part, of the 3′UTR from said naturally occurring gene. 
     
     
         26 . The method of  claim 24 , wherein said instability sequence DNA comprises one or more coding region determinant (CRD). 
     
     
         27 . The method of  claim 23 , wherein said naturally occurring gene is selected from the group consisting of a gene encoding a cytokine, a gene encoding a chemokine, a gene encoding a nuclear transcription factor, a gene encoding an oxygenase, a proto-oncogene, an immediate early gene, a cell cycle controlling gene, and a gene involved in apoptosis. 
     
     
         28 . The method of  claim 10 , wherein said expression system comprises one or more 3′UTR sequence that is heterologous to said heterologous mRNA instability sequence. 
     
     
         29 . The method of  claim 28 , wherein said heterologous mRNA instability sequence is inserted into said 3′UTR sequence. 
     
     
         30 . The method of  claim 14 , wherein said heterologous mRNA instability sequence is derived from a naturally occurring gene. 
     
     
         31 . The method of  claim 30 , wherein said expression system further comprises an instability sequence DNA comprising said heterologous mRNA instability RNA sequence and DNA corresponding to sequences that flank said mRNA instability sequence in the naturally occurring gene. 
     
     
         32 . The method of  claim 31 , wherein said instability sequence DNA comprises DNA corresponding to the whole, or a substantial part, of the 3′UTR from said naturally occurring gene. 
     
     
         33 . The method of  claim 31 , wherein said instability sequence DNA comprises one or more coding region determinant (CRD). 
     
     
         34 . The method of  claim 30 , wherein said naturally occurring gene is selected from the group consisting of a gene encoding a cytokine, a gene encoding a chemokine, a gene encoding a nuclear transcription factor, a gene encoding an oxygenase, a proto-oncogene, an immediate early gene, a cell cycle controlling gene, and a gene involved in apoptosis. 
     
     
         35 . The method of  claim 14 , wherein said expression system comprises one or more 3′UTR sequence that is heterologous to said heterologous mRNA instability sequence. 
     
     
         36 . The method of  claim 35 , wherein said heterologous mRNA instability sequence is inserted into said 3′UTR sequence. 
     
     
         37 . The method according to  claim 18 , wherein said control comprises measuring the detectable signal from the DNA expression vector in the absence of a test compound. 
     
     
         38 . The method according to  claim 18 , wherein said compounds are being screened for their ability to induce mRNA degradation, and wherein a decrease in the measured detectable signal compared to said control indicates a compound that induces mRNA degradation. 
     
     
         39 . The method of  claim 18 , wherein said heterologous mRNA instability sequence is derived from a naturally occurring gene. 
     
     
         40 . The method of  claim 39 , wherein said expression vector comprises an instability sequence DNA comprising said heterologous mRNA instability RNA sequence and DNA corresponding to sequences that flank said mRNA instability sequence in the naturally occurring gene. 
     
     
         41 . The method of  claim 40 , wherein said instability sequence DNA comprises DNA corresponding to the whole, or a substantial part, of the 3′UTR from said naturally occurring gene.