Our goals are to continue to produce and characterize specific monoclonal antibodies (Mabs) that recognize unique structures or conformations on double-helical RNA or DNA sequences and to use these Mabs as experimental immunochemical proves to study duplex nucleic acid structures and functions. We will investigate nucleolar, nuclear, and cytoplasmic double- stranded nucleic acids and study the synthesis, transport, stability, and functions of double-stranded RNa in living cells. Microinjected IgGs directed against RNA or DNA duplexes that specifically bind nucleic acids in vivo will continue to be used by our group to probe nucleic acid metabolism and functions in living human cells (e.g., immune antibody inhibition of cell multiplication).
The specific aims are to: (1) Generate and characterize new high-affinity mouse Mabs against unique determinants on biologically important double-stranded RNAs and DNAs. Emphasis will be placed on A-RNA and B-DNA duplexes as well as on N-AcO-AFF adducted sequences. (2) Use these new and existing experimental and autoimmune anti-nucleic-acid Mabs to quantitate the in situ distribution of unique determinants on RNA and DNA double helices, including N-AcO-AAF adducted sequences in fixed cells, primarily by quantitative fluorescence laser microscopy. Also, we propose to localize and identify the Mab reactive targets by immunoelectronmicroscopy. (3) Study double-stranded RNA and DNA functions in living cells. Cytoplasmic and/or nuclear microinjection of these anti-nucleic-acid Mab probes will be used to determine their effects on nucleic acid metabolism and cell functions. These analyses are fundamental to understanding the in situ distribution and recognition of unique natural and/or chemical-carcinogen-modified duplex sequences. The results of these immunocytological studies will significantly advance our knowledge of structure-function relationships of double helices.
