Design of antisense oligonucleotides stabilized by locked nucleic acids

Kurreck, Jens
Wyszko, Eliza
Gillen, Clemens
Erdmann, Volker A.

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
³Grünenthal GmbH, Molekulare Pharmakologie, Zieglerstrasse 6, 52078 Aachen, Germany

Received January 31, 2002; Revised and Accepted March 5, 2002

^*To whom correspondence should be addressed. Tel: +49 30 83 85 6002; Fax: +49 30 83 85 6413; Email: [email protected]

The authors wish to be known that, in their opinion, the first two authors should be regarded as joint First Authors

Nucleic Acids Research 30(9):p 1911-1918, May 1, 2002.

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 T_m by only <1°C per modification and the T_m 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 t_1/2=∼1.5 h to t_1/2=∼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.