Method for identification of polynucleotides capable of cleaving target mRNA sequences
View Patent ↗Provided are methods for identifying a polynucleotide that can reduce the level of a target mRNA. The method can be performed by providing cells that express an RNA polynucleotide that contains a target mRNA sequence, an internal ribosome entry sequence (IRES) and a sequence encoding a secreted reporter protein; introducing to the cells a test polynucleotide, and measuring activity of the secreted reporter protein. A reduction in secreted reporter protein activity relative to a control cell into which the test polynucleotide has not been introduced is indicative that the test polynucleotide is capable of reducing the level of the target mRNA in the cells. The method is adaptable for high throughput screening methods and is suited for identifying polynucleotides that can catalyze cleavage of target mRNA and/or act on target mRNA through and antisense or RNAi mechanism.
1. A method for identifying a test polynucleotide that can reduce the level of a target mRNA comprising the steps of:
i) providing cells expressing an RNA polynucleotide comprising a target mRNA sequence, a single internal ribosome entry sequence (IRES) and a sequence encoding a secreted reporter protein, wherein the single IRES is 3′ to the target mRNA sequence and 5′ to the sequence encoding the secreted reporter protein;
ii) introducing to the cell a test polynucleotide, and
iii) measuring activity of the secreted reporter protein;
wherein a reduction in secreted reporter protein activity relative to a control cell expressing an RNA polynucleotide comprising the target mRNA sequence, the single IRES and the sequence encoding the secreted reporter protein into which the test polynucleotide has not been introduced is indicative that the test polynucleotide is capable of reducing the level of the target mRNA in the cells.
2. The method of claim 1 , wherein the test polynucleotide is selected from the group consisting of a hammerhead ribozyme (hhRz), an antisense RNA, a small interfering RNA (siRNA), a DNAzyme, a hairpin ribozyme, and a Hepatitis Delta Virus ribozyme.
3. The method of claim 1 , wherein the RNA polynucleotide comprising the target mRNA sequence, the IRES and the sequence encoding the secreted reporter protein is expressed from a DNA expression vector that has been transfected into the cell.
4. The method of claim 2 , wherein the hhRz, antisense RNA or the siRNA is expressed from a DNA expression vector that has been transfected into the cell.
5. The method of claim 1 , wherein the secreted reporter protein is selected from the group consisting of chloramphenicol acetyl transferase, β-galactosidase, Green Fluorescent Protein (GFP), Enhanced Green Fluorescent Protein, luciferase, a luciferase-GFP fusion protein, and secreted placental alkaline phosphatase (SEAP).
6. The method of claim 5 , wherein the secreted reporter protein is SEAP.
7. The method of claim 1 , wherein the single IRES is an Encephalomyocarditis virus IRES.
8. The method of claim 1 , further comprising prior to performing step ii) analyzing the nucleotide sequence of the target mRNA to predict a location of a single stranded region of the target mRNA.
9. The method of claim 8 , wherein analyzing the nucleotide sequence of the target mRNA is performed using a computer program.
10. The method of claim 8 , wherein the single stranded region of the target mRNA comprises a hammerhead ribozyme cleavage site.
11. The method of claim 1 , wherein the cells are divided into a plurality of reactions, and wherein steps ii) and iii) are performed with a distinct test polynucleotide in each reaction chamber.
12. The method of claim 1 , wherein the target mRNA sequence comprises a sequence encoding a complete protein.