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

Teunissen, S.W.M.; Stassen, M.; Pruijn, Ger J. M.; Venrooij, W.J. van; Hoet, R.M.A.

doi:10.1017/s1355838298980633

Cited by 10 publications

(7 citation statements)

References 25 publications

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“…Thus, as a crystallization chaperone, Fab2 plays an important role in crystal packing via interactions with both protein and RNA. (14,44) and rheumatoid arthritis (15). The successful isolation of synthetic Fabs that bind tightly to ⌬C209 P4-P6 and to a diverse panel of other structured RNAs (J.-D.Y., Y. Koldobskaya, J. Min, and J.A.P., unpublished work) suggests that the paucity of available anti-RNA antibodies probably reflects nucleolytic instability of RNA during in vivo immunization, rather than some fundamental limitation in the ability of natural antibody repertoires to bind RNA.…”

Section: Statistical Comparison Of Fab2-⌬c209 P4-p6 Binding Interfacementioning

confidence: 99%

Synthetic antibodies for specific recognition and crystallization of structured RNA

Tereshko

Frederiksen

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

123

157

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Antibodies that bind protein antigens are indispensable in biochemical research and modern medicine. However, knowledge of RNA-binding antibodies and their application in the ever-growing RNA field is lacking. Here we have developed a robust approach using a synthetic phage-display library to select specific antigenbinding fragments (Fabs) targeting a large functional RNA. We have solved the crystal structure of the first Fab-RNA complex at 1.95 Å. Capability in phasing and crystal contact formation suggests that the Fab provides a potentially valuable crystal chaperone for RNA. The crystal structure reveals that the Fab achieves specific RNA binding on a shallow surface with complementaritydetermining region (CDR) sequence diversity, length variability, and main-chain conformational plasticity. The Fab-RNA interface also differs significantly from Fab-protein interfaces in amino acid composition and light-chain participation. These findings yield valuable insights for engineering of Fabs as RNA-binding modules and facilitate further development of Fabs as possible therapeutic drugs and biochemical tools to explore RNA biology.antigen-binding fragments ͉ x-ray crystallography A ntibodies are integral components of the immune system and represent a rapidly growing sector of the biotechnology industry (1, 2). Clinically, antibodies serve as diagnostic markers for disease antigens and play increasingly important roles as therapeutic agents for a wide range of diseases (3). Antibodies also provide invaluable biomedical research tools, serving to define the components and functions of macromolecular complexes, to establish cellular distributions of proteins, and to facilitate structural analysis as chaperones for crystallization of membrane proteins (4-6). Hybridoma and other technologies have yielded antibodies against a vast array of specific antigens (2). An enormous body of literature documents the molecular details of antibody interactions with a variety of antigens, including proteins (7), polysaccharides (8), and small haptens (9). However, much less information (and, in particular, no structural information) exists for antibody-RNA interactions.The relative absence of antibodies that bind RNA from the immunologic repository is striking, especially considering that recent genome-wide analyses of the metazoan transcriptome have revealed the presence of vast numbers of noncoding RNAs, including silencing RNAs, riboswitches, catalytic RNAs, and a multitude of other functional RNA moleucles (10,11). A large number of these RNAs adopt complex three-dimensional architectures that frequently act in complex with proteins to mediate their biological function (12, 13). Nevertheless, with the exception of a handful of examples, mostly isolated from the sera of autoimmune patients (14-17), we know little about anti-RNA antibodies and their recognition of nucleic acids. This dearth of information reflects our inability to elicit antibodies against RNA by using traditional approaches. RNA appears to lack immunogenic potency...

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Section: Statistical Comparison Of Fab2-⌬c209 P4-p6 Binding Interfacementioning

confidence: 99%

Synthetic antibodies for specific recognition and crystallization of structured RNA

Tereshko

Frederiksen

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

123

157

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“…29 The anti-U1-stemloop II scFv antibody immunoprecipitated the U1 snRNA as well as its cleavage products ( Figure 6A, upper panel). In contrast, the anti-TMG cap antibody precipitated full-length U1 snRNA but not its cleavage products, indicating that the TMG cap was absent from the latter ( Figure 6A, lower panel).…”

Section: Apoptotically Cleaved U1 Snrna Lacks a 227-trimethylguanosmentioning

confidence: 99%

“…Since the anti-VSV-G-tag mAb was of the IgG1 isotype, the beads were precoated with rabbit anti-mouse IgG (Dako) for 2 h at 48C. The periplasmic fractions of tagged scFv anti-U1C (K36) 23 and scFv anti-U1-stemloop II (Z5scFv3) 29 were coupled to protein A-agarose via the anti-VSV-Gtag antibodies for 2 h at 48C. Anti-2,2,7-trimethylguanosine (TMG) cap antibodies (20 mg of H20, Euro-Diagnostica BV) were coupled to 20 ml protein A-agarose.…”

Section: Immunoprecipitationmentioning

confidence: 99%

The fate of U1 snRNP during anti-Fas induced apoptosis: specific cleavage of the U1 snRNA molecule

et al. 2000

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During apoptosis, the U1-70K protein, a component of the spliceosomal U1 snRNP complex, is specifically cleaved by the enzyme caspase-3, converting it into a C-terminally truncated 40-kDa fragment. In this study, we show that the 40-kDa U1-70K fragment is still associated with the complete U1 snRNP complex, and that no obvious modifications occur with the U1 snRNP associated proteins U1A, U1C and Sm-B/B'. Furthermore, it is described for the first time that the U1 snRNA molecule, which is the backbone of the U1 snRNP complex, is modified during apoptosis by the specific removal of the first 5 ± 6 nucleotides including the 2,2,7-trimethylguanosine (TMG) cap. The observations that U1 snRNA cleavage is very specific (no such modifications were detected for the other U snRNAs tested) and that U1 snRNA cleavage is markedly inhibited in the presence of caspase inhibitors, indicate that an apoptotically activated ribonuclease is responsible for the specific modification of U1 snRNA during apoptosis. Cell Death and Differentiation (2000) 7, 70 ± 79.

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“…Beyond generating antibodies against a wide variety of proteins (Dong et al, 2003;Smith et al, 2003;Chang et al, 2006;Marcus et al, 2006), it has also been used to produce antibodies specific for unconventional antigens including small peptides (Rodriguez-Diaz et al, 2004), DNA photoproducts (Zavala et al, 2000), snRNAs (Teunissen et al, 1998) and lipids (Takkinen et al, 1996). However, this technology has not been efficient in producing antibodies that target subunits of macromolecular complexes (Rubinstein et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

A fast and efficient procedure to produce scFvs specific for large macromolecular complexes

Doudna

2007

Journal of Immunological Methods

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We have expanded the application of antibody phage display to a new type of antigen: ribonucleoprotein (RNP) complexes. We describe a simple and efficient method for screening antibodies specific for large intact RNPs and individual components. We also describe a fast and easy method to overcome the abundance of amber stop codons in the positive phage clones. The resulting antibodies have been used in ELISA and Western blot analysis.

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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

Abstract: ABSTRACT

Cited by 10 publications

References 25 publications

Synthetic antibodies for specific recognition and crystallization of structured RNA

Synthetic antibodies for specific recognition and crystallization of structured RNA

The fate of U1 snRNP during anti-Fas induced apoptosis: specific cleavage of the U1 snRNA molecule

A fast and efficient procedure to produce scFvs specific for large macromolecular complexes

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