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Nucleic Acids Research, 2002, Vol. 30, No. 9 1911-1918
© 2002 Oxford University Press

Design of antisense oligonucleotides stabilized by locked nucleic acids

Jens Kurreck1, Eliza Wyszko1,2, Clemens Gillen3 and Volker A. Erdmann1,*

1Freie Universität Berlin, Institut für Chemie/Biochemie, Thielallee 63, 14195 Berlin, Germany, 2Institute of Bioorganic Chemistry of the Polish Academy of Sciences, Noskowsiego 12, 61794 Poznan, Poland and 3Grünenthal GmbH, Molekulare Pharmakologie, Zieglerstrasse 6, 52078 Aachen, Germany

The design of antisense oligonucleotides containing locked nucleic acids (LNA) was optimized and compared to intensively studied DNA oligonucleotides, phosphorothioates and 2'-O-methyl gapmers. In contradiction to the literature, a stretch of seven or eight DNA monomers in the center of a chimeric DNA/LNA oligonucleotide is necessary for full activation of RNase H to cleave the target RNA. For 2'-O-methyl gapmers a stretch of six DNA monomers is sufficient to recruit RNase H. Compared to the 18mer DNA the oligonucleotides containing LNA have an increased melting temperature of 1.5–4°C per LNA depending on the positions of the modified residues. 2'-O-methyl nucleotides increase the Tm by only <1°C per modification and the Tm of the phosphorothioate is reduced. The efficiency of an oligonucleotide in supporting RNase H cleavage correlates with its affinity for the target RNA, i.e. LNA > 2'-O-methyl > DNA > phosphorothioate. Three LNAs at each end of the oligonucleotide are sufficient to stabilize the oligonucleotide in human serum 10-fold compared to an unmodified oligodeoxynucleotide (from t1/2 = ~1.5 h to t1/2 = ~15 h). These chimeric LNA/DNA oligonucleotides are more stable than isosequential phosphorothioates and 2'-O-methyl gapmers, which have half-lives of 10 and 12 h, respectively.

* To whom correspondence should be addressed. Tel: +49 30 83 85 6002; Fax: +49 30 83 85 6413; Email: erdmann{at}chemie.fu-berlin.deThe authors wish to be known that, in their opinion, the first two authors should be regarded as joint First Authors


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