Abstract
We have isolated RNA aptamers which are directed against the recombinant Syrian golden hamster prion protein rPrP23-231 (rPrPc) fused to glutathione S-transferase (GST). The aptamers did not recognize the fusion partner GST or the fusion protein GST::rPrP90-231 (rPrP27-30), which lacks 67 amino acids from the PrP N terminus. The aptamer-interacting region of PrPc was mapped to the N-terminal amino acids 23 to 52. Sequence analyses suggest that the RNA aptamers may fold into G-quartet-containing structural elements. Replacement of the G residues in the G quartet scaffold with uridine residues destroyed binding to PrP completely, strongly suggesting that the G quartet motif is essential for PrP recognition. Individual RNA aptamers interact specifically with prion protein in brain homogenates from wild-type mice (C57BL/6), hamsters (Syrian golden), and cattle as shown by supershifts obtained in the presence of anti-PrP antibodies. No interaction was observed with brain homogenates from PrP knockout mice (prn-p(0/0)). Specificity of the aptamer-PrP interaction was further confirmed by binding assays with antisense aptamer RNA or a mutant aptamer in which the guanosine residues in the G tetrad scaffold were replaced by uridine residues. The aptamers did not recognize PrP27-30 in brain homogenates from scrapie-infected mice. RNA aptamers may provide a first milestone in the development of a diagnostic assay for the detection of transmissible spongiform encephalopathies.
Full Text
The Full Text of this article is available as a PDF (1.6 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Alper T., Cramp W. A., Haig D. A., Clarke M. C. Does the agent of scrapie replicate without nucleic acid? Nature. 1967 May 20;214(5090):764–766. doi: 10.1038/214764a0. [DOI] [PubMed] [Google Scholar]
- Bolton D. C., Bendheim P. E., Marmorstein A. D., Potempska A. Isolation and structural studies of the intact scrapie agent protein. Arch Biochem Biophys. 1987 Nov 1;258(2):579–590. doi: 10.1016/0003-9861(87)90380-8. [DOI] [PubMed] [Google Scholar]
- Brandner S., Isenmann S., Raeber A., Fischer M., Sailer A., Kobayashi Y., Marino S., Weissmann C., Aguzzi A. Normal host prion protein necessary for scrapie-induced neurotoxicity. Nature. 1996 Jan 25;379(6563):339–343. doi: 10.1038/379339a0. [DOI] [PubMed] [Google Scholar]
- Büeler H., Aguzzi A., Sailer A., Greiner R. A., Autenried P., Aguet M., Weissmann C. Mice devoid of PrP are resistant to scrapie. Cell. 1993 Jul 2;73(7):1339–1347. doi: 10.1016/0092-8674(93)90360-3. [DOI] [PubMed] [Google Scholar]
- Caughey B., Raymond G. J., Ernst D., Race R. E. N-terminal truncation of the scrapie-associated form of PrP by lysosomal protease(s): implications regarding the site of conversion of PrP to the protease-resistant state. J Virol. 1991 Dec;65(12):6597–6603. doi: 10.1128/jvi.65.12.6597-6603.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Edenhofer F., Rieger R., Famulok M., Wendler W., Weiss S., Winnacker E. L. Prion protein PrPc interacts with molecular chaperones of the Hsp60 family. J Virol. 1996 Jul;70(7):4724–4728. doi: 10.1128/jvi.70.7.4724-4728.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Famulok M., Hüttenhofer A. In vitro selection analysis of neomycin binding RNAs with a mutagenized pool of variants of the 16S rRNA decoding region. Biochemistry. 1996 Apr 9;35(14):4265–4270. doi: 10.1021/bi952479r. [DOI] [PubMed] [Google Scholar]
- Geiger A., Burgstaller P., von der Eltz H., Roeder A., Famulok M. RNA aptamers that bind L-arginine with sub-micromolar dissociation constants and high enantioselectivity. Nucleic Acids Res. 1996 Mar 15;24(6):1029–1036. doi: 10.1093/nar/24.6.1029. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Griffith J. S. Self-replication and scrapie. Nature. 1967 Sep 2;215(5105):1043–1044. doi: 10.1038/2151043a0. [DOI] [PubMed] [Google Scholar]
- Groschup M. H., Langeveld J., Pfaff E. The major species specific epitope in prion proteins of ruminants. Arch Virol. 1994;136(3-4):423–431. doi: 10.1007/BF01321071. [DOI] [PubMed] [Google Scholar]
- 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]
- Hope J., Morton L. J., Farquhar C. F., Multhaup G., Beyreuther K., Kimberlin R. H. The major polypeptide of scrapie-associated fibrils (SAF) has the same size, charge distribution and N-terminal protein sequence as predicted for the normal brain protein (PrP). EMBO J. 1986 Oct;5(10):2591–2597. doi: 10.1002/j.1460-2075.1986.tb04539.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hope J., Multhaup G., Reekie L. J., Kimberlin R. H., Beyreuther K. Molecular pathology of scrapie-associated fibril protein (PrP) in mouse brain affected by the ME7 strain of scrapie. Eur J Biochem. 1988 Mar 1;172(2):271–277. doi: 10.1111/j.1432-1033.1988.tb13883.x. [DOI] [PubMed] [Google Scholar]
- Huizenga D. E., Szostak J. W. A DNA aptamer that binds adenosine and ATP. Biochemistry. 1995 Jan 17;34(2):656–665. doi: 10.1021/bi00002a033. [DOI] [PubMed] [Google Scholar]
- 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]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Lasmézas C. I., Deslys J. P., Robain O., Jaegly A., Beringue V., Peyrin J. M., Fournier J. G., Hauw J. J., Rossier J., Dormont D. Transmission of the BSE agent to mice in the absence of detectable abnormal prion protein. Science. 1997 Jan 17;275(5298):402–405. doi: 10.1126/science.275.5298.402. [DOI] [PubMed] [Google Scholar]
- 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]
- Macaya R. F., Schultze P., Smith F. W., Roe J. A., Feigon J. Thrombin-binding DNA aptamer forms a unimolecular quadruplex structure in solution. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3745–3749. doi: 10.1073/pnas.90.8.3745. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Osborne Scott E., Ellington Andrew D. Nucleic Acid Selection and the Challenge of Combinatorial Chemistry. Chem Rev. 1997 Apr 1;97(2):349–370. doi: 10.1021/cr960009c. [DOI] [PubMed] [Google Scholar]
- Perini F., Vidal R., Ghetti B., Tagliavini F., Frangione B., Prelli F. PrP27-30 is a normal soluble prion protein fragment released by human platelets. Biochem Biophys Res Commun. 1996 Jun 25;223(3):572–577. doi: 10.1006/bbrc.1996.0936. [DOI] [PubMed] [Google Scholar]
- Prusiner S. B., Groth D. F., Bolton D. C., Kent S. B., Hood L. E. Purification and structural studies of a major scrapie prion protein. Cell. 1984 Aug;38(1):127–134. doi: 10.1016/0092-8674(84)90533-6. [DOI] [PubMed] [Google Scholar]
- Prusiner S. B., McKinley M. P., Bowman K. A., Bolton D. C., Bendheim P. E., Groth D. F., Glenner G. G. Scrapie prions aggregate to form amyloid-like birefringent rods. Cell. 1983 Dec;35(2 Pt 1):349–358. doi: 10.1016/0092-8674(83)90168-x. [DOI] [PubMed] [Google Scholar]
- Prusiner S. B., McKinley M. P., Groth D. F., Bowman K. A., Mock N. I., Cochran S. P., Masiarz F. R. Scrapie agent contains a hydrophobic protein. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6675–6679. doi: 10.1073/pnas.78.11.6675. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Prusiner S. B. Novel proteinaceous infectious particles cause scrapie. Science. 1982 Apr 9;216(4542):136–144. doi: 10.1126/science.6801762. [DOI] [PubMed] [Google Scholar]
- Schätzl H. M., Da Costa M., Taylor L., Cohen F. E., Prusiner S. B. Prion protein gene variation among primates. J Mol Biol. 1995 Jan 27;245(4):362–374. doi: 10.1006/jmbi.1994.0030. [DOI] [PubMed] [Google Scholar]
- Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wang K. Y., Krawczyk S. H., Bischofberger N., Swaminathan S., Bolton P. H. The tertiary structure of a DNA aptamer which binds to and inhibits thrombin determines activity. Biochemistry. 1993 Oct 26;32(42):11285–11292. doi: 10.1021/bi00093a004. [DOI] [PubMed] [Google Scholar]
- Wang K. Y., McCurdy S., Shea R. G., Swaminathan S., Bolton P. H. A DNA aptamer which binds to and inhibits thrombin exhibits a new structural motif for DNA. Biochemistry. 1993 Mar 2;32(8):1899–1904. doi: 10.1021/bi00059a003. [DOI] [PubMed] [Google Scholar]
- Weiss S., Famulok M., Edenhofer F., Wang Y. H., Jones I. M., Groschup M., Winnacker E. L. Overexpression of active Syrian golden hamster prion protein PrPc as a glutathione S-transferase fusion in heterologous systems. J Virol. 1995 Aug;69(8):4776–4783. doi: 10.1128/jvi.69.8.4776-4783.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weiss S., Häusl G., Famulok M., König B. The multimerization state of retroviral RNA is modulated by ammonium ions and affects HIV-1 full-length cDNA synthesis in vitro. Nucleic Acids Res. 1993 Oct 25;21(21):4879–4885. doi: 10.1093/nar/21.21.4879. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weiss S., König B., Müller H. J., Seidel H., Goody R. S. Synthetic human tRNA(UUULys3) and natural bovine tRNA(UUULys3) interact with HIV-1 reverse transcriptase and serve as specific primers for retroviral cDNA synthesis. Gene. 1992 Feb 15;111(2):183–197. doi: 10.1016/0378-1119(92)90686-j. [DOI] [PubMed] [Google Scholar]
- Weiss S., Rieger R., Edenhofer F., Fisch E., Winnacker E. L. Recombinant prion protein rPrP27-30 from Syrian golden hamster reveals proteinase K sensitivity. Biochem Biophys Res Commun. 1996 Feb 6;219(1):173–179. doi: 10.1006/bbrc.1996.0201. [DOI] [PubMed] [Google Scholar]
- Wiegand T. W., Williams P. B., Dreskin S. C., Jouvin M. H., Kinet J. P., Tasset D. High-affinity oligonucleotide ligands to human IgE inhibit binding to Fc epsilon receptor I. J Immunol. 1996 Jul 1;157(1):221–230. [PubMed] [Google Scholar]
- Williamson J. R. G-quartets in biology: reprise. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3124–3124. doi: 10.1073/pnas.90.8.3124. [DOI] [PMC free article] [PubMed] [Google Scholar]