Design of antisense oligonucleotides stabilized by locked nucleic acids

• Published in: Nucleic acids research 2002-05, Vol.30 (9), p.1911-1918
• Main Authors: Kurreck, Jens, Wyszko, Eliza, Gillen, Clemens, Erdmann, Volker A.
• Format: Article
• Language: English
• Subjects: Drug Design; Drug Stability; Half-Life; Humans; Kinetics; Nucleic Acid Denaturation; Oligodeoxyribonucleotides, Antisense - metabolism; Oligonucleotides, Antisense - blood; Oligonucleotides, Antisense - chemistry; Oligonucleotides, Antisense - metabolism; phosphorothioate; Receptors, Drug - genetics; ribonuclease H; Ribonuclease H - chemistry; Ribonucleotides - chemistry; RNA, Messenger - metabolism; Temperature; Thionucleotides - metabolism; TRPV Cation Channels
• ISSN: 0305-1048; 1362-4962; 1362-4962
• DOI: 10.1093/nar/30.9.1911

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 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.