Isolation and characterization of 2'-fluoro-, 2'-amino-, and 2'-fluoro-/amino-modified RNA ligands to human IFN-gamma that inhibit receptor binding.
CD4+ Th cells produce cytokines that play a pivotal role in the induction and regulation of cell-mediated and humoral immunity. Th1 cells, characterized by their secretion of IFN-gamma, induce macrophage cytotoxicity, delayed hypersensitivity, and enhanced cellular immunity. Secretion of IFN-gamma may even suppress Th2-enhanced humoral immunity. A counterproductive Th1 response and concomitant secretion of IFN-gamma may result in inflammatory and autoimmune diseases. IFN-gamma regulation of T cell function has potential for therapeutic intervention. To isolate high affinity oligonucleotide inhibitors of IFN-gamma activity, combinatorial libraries of RNA molecules modified at the 2' position of pyrimidine nucleotides with fluoro (F), amino (NH2), or a mixture of F and NH2 (2'-F/NH2) were screened using the SELEX (systematic evolution of ligands by exponential enrichment) combinatorial chemistry process. Each modified library of RNA molecules provides an expanded repertoire of molecules with increased structural diversity and unique binding properties. This added diversity increases the possibility of isolating molecules with the desired functional properties. These RNAs modified at the 2' position have also been shown to be nuclease resistant. High affinity ligands to human IFN-gamma from each modified library were isolated and characterized. The K(d)s of these ligands were determined and their secondary structures were predicted. The specificity of these ligands for IFN-gamma binding was confirmed, and their ability to inhibit binding of IFN-gamma to its receptor on A549 human lung carcinoma cells was determined. A 2'-NH2-modified ligand (2'-NH2-30) is described that binds IFN-gamma with high affinity and inhibits IFN-gamma-induced expression of MHC class I and ICAM-1 by human myeloid leukemia cells.
Kubik
M F
MF
NeXstar Pharmaceuticals, Boulder, CO 80301, USA. kubik@nexstar.com
Bell
C
C
Fitzwater
T
T
Watson
S R
SR
Tasset
D M
DM
D001483:Base Sequence; D015723:Gene Library; D006801:Humans; D007111:Immunity, Cellular; D008024:Ligands; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000032:analysis / Q000235:genetics; D019151:Peptide Library; D012313:RNA / Q000032:analysis* / Q000235:genetics; D017471:Receptors, Interferon / Q000037:antagonists & inhibitors* / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology; D014407:Tumor Cells, Cultured; D000096743:Interferon gamma Receptor
D008024:Ligands; D009841:Oligonucleotides; D019151:Peptide Library; D012313:RNA; D017471:Receptors, Interferon; D000096743:Interferon gamma Receptor