MONOCLONAL ANTIBODY PROBES FOR A, B, AND Z HELICES

提出的研究目标是产生和表征单克隆

The goal of the proposed research is to produce and characterize monoclonal antibodies against antigenic determinants on biologically relevant double-stranded DNA and RNa conformations and sequences. The primary research objective is to use mouse monoclonal antibody probes to distinguish unique as well as common structural determinants on A, B, and Z double-stranded nucleic acid helices. These structures include antigenic features of the grooves, phosphate backbones, or bases in A, B, or Z helices. This immunochemical analysis of DNA and RNA helices is of fundamental importance in understanding features of double-helices present in physiological, environmentally stressed, or chemical carcinogen modified sequences recognized by enzymes or proteins. The specific aims are: (1) to prepare and characterize new monoclonal antibodies against antigens of the synthetic linear Z-poly[d(A-C) d(G-T)] family which we have recently stabilized in a Z state under physiological ionic conditions, (2) to continue studying common and unique antigenic determinants on left-handed Z DNA and Z RNA sequences using monoclonal antibodies prepared against Z RNA (e.g. br-poly[r(G-C)] immunogens), (3) to continue the immunological analysis of B DNA and A RNA sequences using enzymatically and/or chemically synthesized sequences as immunogens for in vitro and in vivo immunizations and as substrates for immunoassay, (4) to determine the effects of cations, drugs, and carcinogens (e.g. N-acetoxy-2-acetylaminofluorene) on antibody recognized Z conformations and (5) as a longer-term aim to introduce these monoclonal antibodies to A, B, and Z helices into living cells (e.g. by microinjection) and determine their various distributions and potential functions.