CHARACTERIZATION OF AN ANTI-RNA RECOMBINANT AUTOANTIBODY FRAGMENT (SCFV) ISOLATED FROM A PHAGE DISPLAY LIBRARY AND DETAILED ANALYSIS OF ITSBINDING-SITE ON U1 SNRNA

This is the first study in which the complex of a monoclonal autoantib ody fragment and its target, stem loop II of U1 snRNA, was investigate d with enzymatic and chemical probing. A phage display antibody librar y derived from bone marrow cells of an SLE patient was used for select ion of scFvs specific for stem loop II. The scFv specificity was teste d by RNA immunoprecipitation and nitrocellulose filter binding competi tion experiments. Immunofluorescence data and immunoprecipitation of U 1 snRNPs containing U1A protein, pointed to an scFv binding site diffe rent from the U1A binding site. The scFv binding site on stem loop II was determined by footprinting experiments using RNase A, RNase V1, an d hydroxyl radicals. The results show that the binding site covers thr ee sequence elements on the RNA, one on the 5' strand of the stem and two on the 3' strand. Hypersensitivity of three loop nucleotides sugge sts a conformational change of the RNA upon antibody binding. A three- dimensional representation of stem loop II reveals a juxtapositioning of the three protected regions on one side of the helix, spanning appr oximately one helical turn. The location of the scFv binding site on s tem loop II is in full agreement with the finding that both the U1A pr otein and the scFv are able to bind stem loop II simultaneously. As a consequence, this recombinant monoclonal anti-U1 snRNA scFv might be v ery useful in studies on U1 snRNPs and its involvement in cellular pro cesses like splicing.