Characterization of an anti-RNA recombinant autoantibody fragment (scFv) isolated from a phage display library and detailed analysis of its binding site on U1 snRNA

التفاصيل البيبلوغرافية
العنوان: Characterization of an anti-RNA recombinant autoantibody fragment (scFv) isolated from a phage display library and detailed analysis of its binding site on U1 snRNA
المؤلفون: M.H.W. Stassen, G.J.M. Pruijn, Rene Hoet, S.W.M. Teunissen, W. J. Van Venrooij
المصدر: RNA. 4:1124-1133
بيانات النشر: Cold Spring Harbor Laboratory, 1998.
سنة النشر: 1998
مصطلحات موضوعية: Models, Molecular, Phage display, Protein Conformation, RNase P, Molecular Sequence Data, Biology, Antibody Specificity, RNA, Small Nuclear, Humans, Lupus Erythematosus, Systemic, Single-chain variable fragment, Bacteriophages, snRNP, Binding site, Immunoglobulin Fragments, Molecular Biology, Autoantibodies, Gene Library, Binding Sites, Base Sequence, Antibodies, Monoclonal, RNA, Stem-loop, Molecular biology, Recombinant Proteins, Nucleic Acid Conformation, Small nuclear RNA, Research Article
الوصف: This is the first study in which the complex of a monoclonal autoantibody fragment and its target, stem loop II of U1 snRNA, was investigated with enzymatic and chemical probing. A phage display antibody library derived from bone marrow cells of an SLE patient was used for selection of scFvs specific for stem loop II. The scFv specificity was tested by RNA immunoprecipitation and nitrocellulose filter binding competition experiments. Immunofluorescence data and immunoprecipitation of U1 snRNPs containing U1A protein, pointed to an scFv binding site different from the U1A binding site. The scFv binding site on stem loop II was determined by footprinting experiments using RNase A, RNase V1, and hydroxyl radicals. The results show that the binding site covers three sequence elements on the RNA, one on the 5' strand of the stem and two on the 3' strand. Hypersensitivity of three loop nucleotides suggests 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 approximately one helical turn. The location of the scFv binding site on stem loop II is in full agreement with the finding that both the U1A protein and the scFv are able to bind stem loop II simultaneously. As a consequence, this recombinant monoclonal anti-U1 snRNA scFv might be very useful in studies on U1 snRNPs and its involvement in cellular processes like splicing.
تدمد: 1355-8382
DOI: 10.1017/s1355838298980633
URL الوصول: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::9bb2eace7cc26d1009257ed7ba826871
https://doi.org/10.1017/s1355838298980633
Rights: OPEN
رقم الانضمام: edsair.doi.dedup.....9bb2eace7cc26d1009257ed7ba826871
قاعدة البيانات: OpenAIRE
الوصف
تدمد:13558382
DOI:10.1017/s1355838298980633