Antisense oligonucleotides selected by hybridisation to scanning arrays are effective reagents <em>in vivo</em>

M Sohail

H Hochegger

A Klotzbücher

R Guellec

T Hunt

E Southern

Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK, ^{Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD, UK,}^{KTB GmbH, Institut für Molekulare Onkologie, Breisacher Strasse 117, D-79106 Freiburg, Germany and}^{Unité de Biologie et Genetique du Development, CNRS UPR 41, Universite Rennes I, Avenue du General Leclerc, 35042 Rennes, France}

^{To whom correspondence should be addressed. Tel: +44 1865 275224; Fax: +44 1865 275259; Email:}ku.ca.xo.hcoib@liahosm The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors

Received 2001 Feb 8; Revised 2001 Mar 27; Accepted 2001 Mar 27.

Abstract

Transcripts representing mRNAs of three Xenopus cyclins, B1, B4 and B5, were hybridised to arrays of oligonucleotides scanning the first 120 nt of the coding region to assess the ability of the immobilised oligonucleotides to form heteroduplexes with their targets. Oligonucleotides that produced high heteroduplex yield and others that showed little annealing were assayed for their effect on translation of endogenous cyclin mRNAs in Xenopus egg extracts and their ability to promote cleavage of cyclin mRNAs in oocytes by RNase H. Excellent correlation was found between antisense potency and affinity of oligonucleotides for the cyclin transcripts as measured by the array, despite the complexity of the cellular environment.

Abstract

^{Arbitrary units.}

^{Both these oligonucleotides inhibited only one of the two bands (see text for details).}

^{Oligonucleotides used in microinjection.}

ACKNOWLEDGEMENTS

The authors would like to thank Lydie Baudouin, Stephan Geley and Hiro Mahbubani for their help and advice and the ICRF Biological Resource Unit for providing rabbit polyclonal antisera. M.S. was funded by the Medical Research Council, UK and H.H. by the Boehringer Ingelheim Fonds.

ACKNOWLEDGEMENTS

References

1. Walder R.Y. and Walder,J.A. (1988) Role of RNase H in hybrid-arrested translation by antisense oligonucleotides. Proc. Natl Acad. Sci. USA, 85, 5011–5015.
2. Minshull J. and Hunt,T. (1986) The use of single-stranded DNA and RNase H to promote quantitative ‘hybrid arrest of translation’ of mRNA/DNA hybrids in reticulocyte lysate cell-free translations. Nucleic Acids Res., 14, 6433–6451.
3. Zamecnik P.C., Goodchild,J., Taguchi,Y. and Sarin,P.S. (1986) Inhibition of replication and expression of human T-cell lymphotropic virus type III in cultured cells by exogenous synthetic oligonucleotides complementary to viral RNA. Proc. Natl Acad. Sci. USA, 83, 4143–4146.
4. Branch A.D(1998) Antisense drug discovery: can cell-free screens speed the process? Antisense Nucleic Acid Drug Dev., 8, 249–254. [[PubMed][Google Scholar]
5. Woolf T.M., Melton,D.A. and Jennings,C.G. (1992) Specificity of antisense oligonucleotides in vivo. Proc. Natl Acad. Sci. USA, 89, 7305–7309.
6. Bramlage B., Luzi,E. and Eckstein,F. (1998) Designing ribozymes for the inhibition of gene expression. Trends Biotechnol., 16, 434–438. [[PubMed]
7. Ho S.P., Bao,Y., Lesher,T., Malhotra,R., Ma,L.Y., Fluharty,S.Jand Sakai,R.R. (1998) Mapping of RNA accessible sites for antisense experiments with oligonucleotide libraries. Nat. Biotechnol., 16, 59–63. [[PubMed][Google Scholar]
8. Milner N., Mir,K.U. and Southern,E.M. (1997) Selecting effective antisense reagents on combinatorial oligonucleotide arrays. Nat. Biotechnol., 15, 537–541. [[PubMed]
9. Patzel V. and Sczakiel,G. (1998) Theoretical design of antisense RNA structures substantially improves annealing kinetics and efficacy in human cells. Nat. Biotechnol., 16, 64–68. [[PubMed]
10. Wagner R.W., Matteucci,M.D., Grant,D., Huang,T. and Froehler,B.C. (1996) Potent and selective inhibition of gene expression by an antisense heptanucleotide. Nat. Biotechnol., 14, 840–844. [[PubMed]
11. Southern E.M., Case-Green,S.C., Elder,J.K., Johnson,M., Mir,K.U., Wang,Land Williams,J.C. (1994) Arrays of complementary oligonucleotides for analysing the hybridisation behaviour of nucleic acids. Nucleic Acids Res., 22, 1368–1373. [Google Scholar]
12. Minshull J. and Hunt,T. (1992) Antisense ablation of mRNA in frog and rabbit cell-free systems. In Murray,J.A.H. (ed.) Antisense RNA and DNA. Wiley-Liss, New York, NY, pp. 195–212.
13. Sohail M. and Southern,E.M. (2001) Hybridization of antisense reagents to RNA. Curr. Opin. Mol. Ther., 2, 264–271. [[PubMed]
14. Sohail M. and Southern,E.M. (2000) Antisense arrays. Mol. Cell Biol. Res. Commun., 3, 67–72. [[PubMed]
15. Matson R.S., Rampal,J.B. and Coassin,P.J. (1994) Biopolymer synthesis on polypropylene supports. I. Oligonucleotides. Anal. Biochem., 217, 306–310. [Erratum, Anal. Biochem. (1994) 220, 225] [[PubMed]
16. Sohail M. and Southern,E.M. (2001) Using oligonucleotide scanning arrays to find effective antisense reagents. In Pampal,J.B. (ed.) Methods in Molecular Biology—Oligonucleotide Array Methods and Protocols. Humana Press, Totowa, NJ, in press. [[PubMed]
17. Murray A.W. (1991) Cell cycle extracts. In Kay,B.K. and Peng,H.B. (eds) Xenopus laevis: Practical Uses in Cell and Molecular Biology. Academic Press, San Diego, CA, Vol. 36, pp. 573–597.
18. Eppig J.J. and Dumont,J.N. (1976) Defined nutrient medium for the in vitro maintenance of Xenopus laevis oocytes. In Vitro, 12, 418–427. [[PubMed]
19. Heasman J., Holwill,S. and Wylie,C.C. (1991) Fertilization of cultured Xenopus oocytes and use in studies of maternally inherited molecules. In Kay,B.K. and Peng,H.B. (eds) Xenopus laevis: Practical Uses in Cell and Molecular Biology. Academic Press, San Diego, CA, Vol. 36, pp. 213–230. [[PubMed]
20. Sambrook J., Fritsch,E.F. and Maniatis,T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
21. Thibier C., De Smedt,V., Poulhe,R., Huchon,D., Jessus,C. and Ozon,R. (1997) In vivo regulation of cytostatic activity in Xenopus metaphase II-arrested oocytes. Dev. Biol., 185, 55–66. [[PubMed]
22. Kobayashi H., Stewart,E., Poon,R.Y. and Hunt,T. (1994) Cyclin A and cyclin B dissociate from p34cdc2 with half-times of 4 and 15 h, respectively, regardless of the phase of the cell cycle. J. Biol. Chem., 269, 29153–29160. [[PubMed]
23. Sagata N., Oskarsson,M., Copeland,T., Brumbaugh,J. and Vande Woude,G.F. (1988) Function of c-mos proto-oncogene product in meiotic maturation in Xenopus oocytes. Nature, 335, 519–525. [[PubMed]
24. Sheets M.D., Wu,M. and Wickens,M. (1995) Polyadenylation of c-mos mRNA as a control point in Xenopus meiotic maturation. Nature, 374, 511–516. [[PubMed]
25. Donis-Keller H(1979) Site specific enzymatic cleavage of RNA. Nucleic Acids Res., 7, 179–192. [Google Scholar]
26. Minshull J., Murray,A., Colman,A. and Hunt,T. (1991) Xenopus oocyte maturation does not require new cyclin synthesis. J. Cell Biol., 114, 767–772.
27. Kozak M(1991) Structural features in eukaryotic mRNAs that modulate the initiation of translation. J. Biol. Chem., 266, 19867–19870. [[PubMed][Google Scholar]
28. Tu G.C., Cao,Q.N., Zhou,F. and Israel,Y. (1998) Tetranucleotide GGGA motif in primary RNA transcripts. Novel target site for antisense design. J. Biol. Chem., 273, 25125–25131. [[PubMed]
29. Agrawal S(1999) Importance of nucleotide sequence and chemical modifications of antisense oligonucleotides. Biochim. Biophys. Acta, 1489, 53–68. [[PubMed][Google Scholar]
30. Smetsers T.F., Boezeman,J.B. and Mensink,E.J. (1996) Bias in nucleotide composition of antisense oligonucleotides. Antisense Nucleic Acid Drug Dev., 6, 63–67. [[PubMed]
31. Matveeva O.V., Tsodikov,A.D., Giddings,M., Freier,S.M., Wyatt,J.R., Spiridonov,A.N., Shabalina,S.A., Gesteland,R.F. and Atkins,J.F. (2000) Identification of sequence motifs in oligonucleotides whose presence is correlated with antisense activity. Nucleic Acids Res., 28, 2862–2865.
32. Lehmann M.J., Patzel,V. and Sczakiel,G. (2000) Theoretical design of antisense genes with statistically increased efficacy. Nucleic Acids Res., 28, 2597–2604.
33. Vickers T.A., Wyatt,J.R. and Freier,S.M. (2000) Effects of RNA secondary structure on cellular antisense activity. Nucleic Acids Res., 28, 1340–1347.
34. Patzel V., Steidl,U., Kronenwett,R., Haas,R. and Sczakiel,G. (1999) A theoretical approach to select effective antisense oligodeoxyribonucleotides at high statistical probability. Nucleic Acids Res., 27, 4328–4334.
35. Mir K.U. and Southern,E.M. (1999) Determining the influence of structure on hybridization using oligonucleotide arrays. Nat. Biotechnol., 17, 788–792. [[PubMed]