We propose to further develop a novel method which couples and amplifies the inhibitory effects of 2',5'-oligoadenylates (2-5A) and antisense on gene expression. The goal is to specifically ablate mRNA species for factors involved in interferon- and dsRNA-signaling pathways. The strategy involves the 2-5A-dependent RNase, and endoribonuclease which mediates inhibitory effects of interferon on virus infection. To direct 2-5A-dependent RNase to cleave unique RNA sequences, 2-5A is covalently linked to antisense oligonucleotide (2-5A-antisense). The antisense oligonucleotide component of 2-5A-antisense binds a specific RNA sequence while the accompanying 2-5A component activates 2-5A-dependent RNase thereby causing the cleavage of the RNA in a region proximal to the targeted sequence. The catalytic degradation on mRNA for dsRNA-dependent protein kinase (PKR) will be measured in reactions containing 2-5A- antisense and homogeneous, recombinant human 2-5A-dependent RNase. The effects of antisense length and sequence, chemical modifications, and hybrid mismatches on the turnover number, kcat, and the Km of the reactions will be determined. the role of PKR in relaying dsRNA generated signals will be studied in cells depleted of PKR with 2-5A- antisense. Similarly, we will deplete cells of the protein tyrosine kinases, JAK-1 and JAK-2, the ISRE-binding protein, IBF-1 and cytosolic phospholipase A2 to determine their functions in regulating interferon- stimulated genes. Because of its specificity, versatility and potency, 2-5A-antisense is a promising approach to the control of gene expression through targeted RNA degradation.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Program Projects (P01)
Project #
5P01CA062220-05
Application #
6269633
Study Section
Project Start
1998-06-01
Project End
1999-05-31
Budget Start
1997-10-01
Budget End
1998-09-30
Support Year
5
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Cleveland Clinic Lerner
Department
Type
DUNS #
017730458
City
Cleveland
State
OH
Country
United States
Zip Code
44195
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