We previously isolated nucleic acid-binding antibody fragments (Fab) from bacteriophage display libraries representing the immunoglobulin repertoire of autoimmune mice to expedite the analysis of antibody-DNA recognition. In the present study, the binding properties of one such anti-DNA Fab, high affinity single-stranded (ss) DNA-binding Fab (DNA-1), were defined using equilibrium gel filtration and fluorescence titration. Results demonstrated that DNA-1 had a marked preference for oligo(dT) (100 nM dissociation constant) and required oligo(dT) >5 nucleotides in length. A detailed analysis of the involvement of the individual heavy chain (H) complementarity-determining regions (CDR) ensued using previously constructed HCDR transplantation mutants between DNA-1 and low affinity ssDNA-binding The presence of circulating antibodies that recognize DNA or RNA is diagnostic of certain autoimmune diseases including systemic lupus erythematosus (1, 2). Anti-DNA antibodies are also produced in autoimmune mice and have facilitated the study of systemic lupus erythematosus and nucleic acid-protein interactions. The well developed hybridoma technology for monoclonal antibody production has made possible the production of numerous murine anti-DNA antibodies. Consequently, there is substantial amino acid sequence information on murine anti-DNA antibodies (3-5), although significantly less is known about the exact mechanism of binding and interactions between autoantibodies and DNA. Murine monoclonal anti-DNA antibodies exhibit a high frequency of basic and aromatic amino acid residues in the hypervariable or complementaritydetermining regions (CDR) 1 contained in the antigen binding fragment (Fab) (6, 7). Mutagenesis studies (8, 9) and threedimensional structure analyses of two Fab-oligo(dT) complexes (10, 11) have substantiated a direct role of these residues in nucleic acid interaction.In general, the heavy chain (H) is thought to contribute more than the light chain to antigen binding, especially through HCDR3 (12). HCDR transplantation studies of an anti-fluorescein single chain antibody (SCA) 4 -4-20 and an anti-ssDNA SCA BV04 -01 have analyzed the roles of individual HCDR in combining site formation (13). Data from the transplantation studies indicated that hybrids containing HCDR3 or HCDR1 of SCA BV04 -01 bound oligo(dT). No binding occurred, however, when both HCDR1 and -3 of SCA BV04 -01 were present in the context of HCDR2 of SCA 14). While studies have demonstrated the importance of HCDR3 in binding nucleic acid (15, 16), the roles of HCDR1 and -2 in this interaction are less clear.We previously isolated ssDNA-binding Fab from combinatorial bacteriophage display libraries derived from the immunoglobulin repertoire of an autoimmune MRL/lpr mouse (17). Immunological methods were used to characterize two of these Fab, in particular, a high affinity ssDNA-binding Fab (DNA-1) and a Fab (D5) that bound DNA poorly. DNA-1 and D5 were identical in genetic background and were very similar in amino acid sequence, suggesting t...
Protein recognition of RNA has been studied using Peptide Phage Display Libraries, but in the absence of RNA modifications. Peptides from two libraries, selected for binding the modified anticodon stem and loop (ASL) of human tRNA(LyS3) having 2-thiouridine (s(2)U34) and pseudouridine (psi39), bound the modified human ASL(Lys3)(s(2)U34;psi39) preferentially and had significant homology with RNA binding proteins. Selected peptides were narrowed to a manageable number using a less sensitive, but inexpensive assay before conducting intensive characterization. The affinity and specificity of the best binding peptide (with an N-terminal fluorescein) were characterized by fluorescence spectrophotometry. The peptide exhibited the highest binding affinity for ASL(LYS3)(s(2)U34; psi39), followed by the hypermodified ASL(Lys3) (mcm(5)s(2) U34; ms(2)t(6)A37) and the unmodified ASL(Lys3), but bound poorly to singly modified ASL(Lys3) constructs (psi39, ms(2)t(6)A37, s(2)34), ASL(Lys1,2) (t(6)A37) and Escherichia coli ASL(Glu) (s(2)U34). Thus, RNA modifications are potentially important recognition elements for proteins and can be targets for selective recognition by peptides.
Peptides that bind either U1 small nuclear RNA (U1 snRNA) or the anticodon stem and loop of yeast tRNA(Phe) (tRNA(ACPhe)) were selected from a random-sequence, 15-amino acid bacteriophage display library. An experimental system, including an affinity selection method, was designed to identify primary RNA-binding peptide sequences without bias to known amino acid sequences and without incorporating nonspecific binding of the anionic RNA backbone. Nitrocellulose binding assays were used to evaluate the binding of RNA by peptide-displaying bacteriophage. Amino acid sequences of RNA-binding bacteriophage were determined from the foreign insert DNA sequences, and peptides corresponding to the RNA-binding bacteriophage inserts were chemically synthesized. Peptide affinities for the RNAs (Kd approximately 0.1-5.0 microM) were analyzed successfully using fluorescence and circular dichroism spectroscopies. These methodologies demonstrate the feasibility of rapidly identifying, isolating, and initiating the analyses of small peptides that bind to RNAs in an effort to define better the chemistry, structure, and function of protein-RNA complexes.
The heavy chain complementarity-determining region 3 (HCDR3) of the anti-oligo(dT) recombinant antibody fragment, DNA-1, contributes significantly to antigen binding (Komissarov, A. A., Calcutt, M. J., Marchbank, M. T., Peletskaya, E. N., and Deutscher, S. L. (1996) J. Biol. Chem. 271, 12241-12246). In the present study, the role of separate HCDR3 residues of DNA-1 in interaction with oligo(dT) was elucidated. Based on a molecular model of the combining site, residues at the base (Arg 98 and Asp 108 ) and in the middle (Tyr 101-ArgPro-Tyr-Tyr 105 ) of HCDR3 were predicted to support the loop conformation and directly contact the ligand, respectively. Twenty-five site-specific mutants were produced as hexahistidine-tagged proteins, purified, and examined for binding to (dT) 15 Antibodies comprise a valuable class of protein produced in mammals and other species in response to antigen. The importance of antibodies is highlighted by their vital roles in immune protection. Mammalian immune systems are capable of generating millions of different antibody specificities in response to foreign antigens. For unknown reasons, a breakdown in immune tolerance can occur resulting in activation of self-reactive B and T cells. This abnormal cascade of events leads to autoimmune disease. A hallmark of the autoimmune disorders systemic lupus erythematosus (SLE) 1 and mixed connective tissue disease is the presence of serum antibodies that recognize nucleic acids (1-3). The presence of high levels of circulating antibodies that bind double-stranded (ds) DNA is diagnostic for SLE, and certain anti-DNA immune complexes contribute to disease pathology (4, 5). It is uncertain what are the distinguishing features of pathogenic versus non-pathogenic antibodies, however. Studies of the binding properties of anti-DNA antibodies have been facilitated by methods to select for DNA-binding antibody fragments (Fab) from bacteriophage display libraries and the ability to produce large quantities of the Fab in Escherichia coli. We previously isolated an anti-single-stranded (ss) DNA-binding Fab, DNA-1, from a bacteriophage display library derived from the immunoglobulin repertoire of an autoimmune MRL/MpJ-lpr/lpr (MRL/lpr) mouse (6). The MRL/lpr murine library was enriched in anti-DNA Fab since these mice spontaneously produce anti-ssDNA and anti-dsDNA antibodies and develop an SLE-like syndrome. DNA-1 was shown to preferentially bind to oligo(dT) of 15 nucleotides or greater in length, with an equilibrium dissociation constant (K d ) of 150 -200 nM (7, 8). The regions responsible for DNA binding are known to reside in the three heavy (H) and light (L) chain complementarity-determining regions (CDRs) of the Fab. It has been shown that the H chain contributes more to the interaction with DNA than the L chain (9). Comparison of HCDR transplantation mutants between DNA-1 and a Fab that bound poorly to DNA demonstrated that HCDR3 of DNA-1 was critical for oligo(dT) binding (7,8). The importance of HCDR3 in binding to other DNA molecules includin...
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