Design of antisense oligonucleotides stabilized by locked nucleic acids

Nucleic Acids Res. 2002 May 1;30(9):1911-8. doi: 10.1093/nar/30.9.1911.

Abstract

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 degrees C per LNA depending on the positions of the modified residues. 2'-O-methyl nucleotides increase the T(m) by only <1 degree 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) = approximately 1.5 h to t(1/2) = approximately 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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Drug Design
  • Drug Stability
  • Half-Life
  • Humans
  • Kinetics
  • Nucleic Acid Denaturation
  • Oligodeoxyribonucleotides, Antisense / metabolism
  • Oligonucleotides, Antisense / blood
  • Oligonucleotides, Antisense / chemistry*
  • Oligonucleotides, Antisense / metabolism*
  • RNA, Messenger / metabolism
  • Receptors, Drug / genetics
  • Ribonuclease H / chemistry
  • Ribonucleotides / chemistry
  • TRPV Cation Channels
  • Temperature
  • Thionucleotides / metabolism

Substances

  • Oligodeoxyribonucleotides, Antisense
  • Oligonucleotides, Antisense
  • RNA, Messenger
  • Receptors, Drug
  • Ribonucleotides
  • TRPV Cation Channels
  • TRPV1 receptor
  • Thionucleotides
  • Ribonuclease H