Next-generation bis-locked nucleic acids with stacking linker and 2'-glycylamino-LNA show enhanced DNA invasion into supercoiled duplexes

Nucleic Acids Res. 2016 Mar 18;44(5):2007-19. doi: 10.1093/nar/gkw021. Epub 2016 Feb 8.


Targeting and invading double-stranded DNA with synthetic oligonucleotides under physiological conditions remain a challenge. Bis-locked nucleic acids (bisLNAs) are clamp-forming oligonucleotides able to invade into supercoiled DNA via combined Hoogsteen and Watson-Crick binding. To improve the bisLNA design, we investigated its mechanism of binding. Our results suggest that bisLNAs bind via Hoogsteen-arm first, followed by Watson-Crick arm invasion, initiated at the tail. Based on this proposed hybridization mechanism, we designed next-generation bisLNAs with a novel linker able to stack to adjacent nucleobases, a new strategy previously not applied for any type of clamp-constructs. Although the Hoogsteen-arm limits the invasion, upon incorporation of the stacking linker, bisLNA invasion is significantly more efficient than for non-clamp, or nucleotide-linker containing LNA-constructs. Further improvements were obtained by substituting LNA with 2'-glycylamino-LNA, contributing a positive charge. For regular bisLNAs a 14-nt tail significantly enhances invasion. However, when two stacking linkers were incorporated, tail-less bisLNAs were able to efficiently invade. Finally, successful targeting of plasmids inside bacteria clearly demonstrates that strand invasion can take place in a biologically relevant context.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Binding Sites
  • DNA, Bacterial / antagonists & inhibitors
  • DNA, Bacterial / chemistry
  • DNA, Bacterial / metabolism*
  • DNA, Superhelical / chemistry
  • DNA, Superhelical / metabolism*
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Glycine / analogs & derivatives*
  • Models, Molecular
  • Molecular Sequence Data
  • Nucleic Acid Hybridization
  • Oligonucleotides / chemical synthesis
  • Oligonucleotides / metabolism*
  • Oligonucleotides, Antisense / chemical synthesis
  • Oligonucleotides, Antisense / metabolism*
  • Plasmids / chemistry
  • Plasmids / metabolism
  • Solid-Phase Synthesis Techniques
  • Static Electricity
  • Structure-Activity Relationship


  • DNA, Bacterial
  • DNA, Superhelical
  • Oligonucleotides
  • Oligonucleotides, Antisense
  • locked nucleic acid
  • Glycine