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. 1996 Jun 15;24(12):2220–2227. doi: 10.1093/nar/24.12.2220

Characterisation of antibody-binding RNAs selected from structurally constrained libraries.

J Hamm 1
PMCID: PMC145926  PMID: 8710489

Abstract

Constrained RNA libraries of limited sequence complexity were constructed and used to select RNA molecules binding to the antigen binding site of an anti-ferritin antibody. The sequences required as primer-binding sites for the selection cycle were designed to form a predictable secondary structure, which greatly facilitated the characterisation of the secondary structures of the selected RNAs. RNA-antibody interactions were studied by real-time interaction analysis to study the dynamic aspects of binding and by circular dichroism spectroscopy to search for conformational changes upon binding. The selected RNAs were analysed with a binding site sequestering assay and were shown to compete with ferritin for binding to the antigen-binding site. The experiments described here indicate that the introduction of strong structural constraints does not have to interfere with the ability to select tightly and specifically binding RNA-molecules.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bock L. C., Griffin L. C., Latham J. A., Vermaas E. H., Toole J. J. Selection of single-stranded DNA molecules that bind and inhibit human thrombin. Nature. 1992 Feb 6;355(6360):564–566. doi: 10.1038/355564a0. [DOI] [PubMed] [Google Scholar]
  2. Burd C. G., Dreyfuss G. RNA binding specificity of hnRNP A1: significance of hnRNP A1 high-affinity binding sites in pre-mRNA splicing. EMBO J. 1994 Mar 1;13(5):1197–1204. doi: 10.1002/j.1460-2075.1994.tb06369.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Calnan B. J., Biancalana S., Hudson D., Frankel A. D. Analysis of arginine-rich peptides from the HIV Tat protein reveals unusual features of RNA-protein recognition. Genes Dev. 1991 Feb;5(2):201–210. doi: 10.1101/gad.5.2.201. [DOI] [PubMed] [Google Scholar]
  4. Daly T. J., Rusche J. R., Maione T. E., Frankel A. D. Circular dichroism studies of the HIV-1 Rev protein and its specific RNA binding site. Biochemistry. 1990 Oct 23;29(42):9791–9795. doi: 10.1021/bi00494a005. [DOI] [PubMed] [Google Scholar]
  5. Doudna J. A., Cech T. R., Sullenger B. A. Selection of an RNA molecule that mimics a major autoantigenic epitope of human insulin receptor. Proc Natl Acad Sci U S A. 1995 Mar 14;92(6):2355–2359. doi: 10.1073/pnas.92.6.2355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ellington A. D., Szostak J. W. In vitro selection of RNA molecules that bind specific ligands. Nature. 1990 Aug 30;346(6287):818–822. doi: 10.1038/346818a0. [DOI] [PubMed] [Google Scholar]
  7. Fedor M. J. A slippery grip. Nat Struct Biol. 1994 May;1(5):267–269. doi: 10.1038/nsb0594-267. [DOI] [PubMed] [Google Scholar]
  8. Gautheret D., Konings D., Gutell R. R. A major family of motifs involving G.A mismatches in ribosomal RNA. J Mol Biol. 1994 Sep 9;242(1):1–8. doi: 10.1006/jmbi.1994.1552. [DOI] [PubMed] [Google Scholar]
  9. Giver L., Bartel D., Zapp M., Pawul A., Green M., Ellington A. D. Selective optimization of the Rev-binding element of HIV-1. Nucleic Acids Res. 1993 Nov 25;21(23):5509–5516. doi: 10.1093/nar/21.23.5509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gold L., Polisky B., Uhlenbeck O., Yarus M. Diversity of oligonucleotide functions. Annu Rev Biochem. 1995;64:763–797. doi: 10.1146/annurev.bi.64.070195.003555. [DOI] [PubMed] [Google Scholar]
  11. Green L., Waugh S., Binkley J. P., Hostomska Z., Hostomsky Z., Tuerk C. Comprehensive chemical modification interference and nucleotide substitution analysis of an RNA pseudoknot inhibitor to HIV-1 reverse transcriptase. J Mol Biol. 1995 Mar 17;247(1):60–68. doi: 10.1006/jmbi.1994.0122. [DOI] [PubMed] [Google Scholar]
  12. Harada K., Frankel A. D. Identification of two novel arginine binding DNAs. EMBO J. 1995 Dec 1;14(23):5798–5811. doi: 10.1002/j.1460-2075.1995.tb00268.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Helmer-Citterich M., Rovida E., Luzzago A., Tramontano A. Modelling antibody-antigen interactions: ferritin as a case study. Mol Immunol. 1995 Sep;32(13):1001–1010. doi: 10.1016/0161-5890(95)00027-c. [DOI] [PubMed] [Google Scholar]
  14. Jaeger J. A., SantaLucia J., Jr, Tinoco I., Jr Determination of RNA structure and thermodynamics. Annu Rev Biochem. 1993;62:255–287. doi: 10.1146/annurev.bi.62.070193.001351. [DOI] [PubMed] [Google Scholar]
  15. Jenison R. D., Gill S. C., Pardi A., Polisky B. High-resolution molecular discrimination by RNA. Science. 1994 Mar 11;263(5152):1425–1429. doi: 10.1126/science.7510417. [DOI] [PubMed] [Google Scholar]
  16. Joyce G. F. In vitro evolution of nucleic acids. Curr Opin Struct Biol. 1994;4:331–336. doi: 10.1016/s0959-440x(94)90100-7. [DOI] [PubMed] [Google Scholar]
  17. Kenan D. J., Tsai D. E., Keene J. D. Exploring molecular diversity with combinatorial shape libraries. Trends Biochem Sci. 1994 Feb;19(2):57–64. doi: 10.1016/0968-0004(94)90033-7. [DOI] [PubMed] [Google Scholar]
  18. Lauhon C. T., Szostak J. W. RNA aptamers that bind flavin and nicotinamide redox cofactors. J Am Chem Soc. 1995 Feb 1;117(4):1246–1257. doi: 10.1021/ja00109a008. [DOI] [PubMed] [Google Scholar]
  19. Lehman N., Joyce G. F. Evolution in vitro of an RNA enzyme with altered metal dependence. Nature. 1993 Jan 14;361(6408):182–185. doi: 10.1038/361182a0. [DOI] [PubMed] [Google Scholar]
  20. Lorsch J. R., Szostak J. W. In vitro evolution of new ribozymes with polynucleotide kinase activity. Nature. 1994 Sep 1;371(6492):31–36. doi: 10.1038/371031a0. [DOI] [PubMed] [Google Scholar]
  21. Luzzago A., Arosio P., Iacobello C., Ruggeri G., Capucci L., Brocchi E., De Simone F., Gamba D., Gabri E., Levi S. Immunochemical characterization of human liver and heart ferritins with monoclonal antibodies. Biochim Biophys Acta. 1986 Jul 25;872(1-2):61–71. doi: 10.1016/0167-4838(86)90147-0. [DOI] [PubMed] [Google Scholar]
  22. Luzzago A., Cesareni G. Isolation of point mutations that affect the folding of the H chain of human ferritin in E.coli. EMBO J. 1989 Feb;8(2):569–576. doi: 10.1002/j.1460-2075.1989.tb03411.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Luzzago A., Felici F., Tramontano A., Pessi A., Cortese R. Mimicking of discontinuous epitopes by phage-displayed peptides, I. Epitope mapping of human H ferritin using a phage library of constrained peptides. Gene. 1993 Jun 15;128(1):51–57. doi: 10.1016/0378-1119(93)90152-s. [DOI] [PubMed] [Google Scholar]
  24. Mattaj I. W., De Robertis E. M. Nuclear segregation of U2 snRNA requires binding of specific snRNP proteins. Cell. 1985 Jan;40(1):111–118. doi: 10.1016/0092-8674(85)90314-9. [DOI] [PubMed] [Google Scholar]
  25. Morton T. A., Myszka D. G., Chaiken I. M. Interpreting complex binding kinetics from optical biosensors: a comparison of analysis by linearization, the integrated rate equation, and numerical integration. Anal Biochem. 1995 May 1;227(1):176–185. doi: 10.1006/abio.1995.1268. [DOI] [PubMed] [Google Scholar]
  26. Pizzi E., Cortese R., Tramontano A. Mapping epitopes on protein surfaces. Biopolymers. 1995 Nov;36(5):675–680. doi: 10.1002/bip.360360513. [DOI] [PubMed] [Google Scholar]
  27. Pley H. W., Flaherty K. M., McKay D. B. Three-dimensional structure of a hammerhead ribozyme. Nature. 1994 Nov 3;372(6501):68–74. doi: 10.1038/372068a0. [DOI] [PubMed] [Google Scholar]
  28. Puglisi J. D., Tan R., Calnan B. J., Frankel A. D., Williamson J. R. Conformation of the TAR RNA-arginine complex by NMR spectroscopy. Science. 1992 Jul 3;257(5066):76–80. doi: 10.1126/science.1621097. [DOI] [PubMed] [Google Scholar]
  29. Robertson D. L., Joyce G. F. Selection in vitro of an RNA enzyme that specifically cleaves single-stranded DNA. Nature. 1990 Mar 29;344(6265):467–468. doi: 10.1038/344467a0. [DOI] [PubMed] [Google Scholar]
  30. Tsai D. E., Harper D. S., Keene J. D. U1-snRNP-A protein selects a ten nucleotide consensus sequence from a degenerate RNA pool presented in various structural contexts. Nucleic Acids Res. 1991 Sep 25;19(18):4931–4936. doi: 10.1093/nar/19.18.4931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Tsai D. E., Kenan D. J., Keene J. D. In vitro selection of an RNA epitope immunologically cross-reactive with a peptide. Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):8864–8868. doi: 10.1073/pnas.89.19.8864. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Tuerk C., Gold L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science. 1990 Aug 3;249(4968):505–510. doi: 10.1126/science.2200121. [DOI] [PubMed] [Google Scholar]
  33. Tuerk C., MacDougal S., Gold L. RNA pseudoknots that inhibit human immunodeficiency virus type 1 reverse transcriptase. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):6988–6992. doi: 10.1073/pnas.89.15.6988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Tuschl T., Gohlke C., Jovin T. M., Westhof E., Eckstein F. A three-dimensional model for the hammerhead ribozyme based on fluorescence measurements. Science. 1994 Nov 4;266(5186):785–789. doi: 10.1126/science.7973630. [DOI] [PubMed] [Google Scholar]
  35. Wilson C., Szostak J. W. In vitro evolution of a self-alkylating ribozyme. Nature. 1995 Apr 27;374(6525):777–782. doi: 10.1038/374777a0. [DOI] [PubMed] [Google Scholar]

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