title: Next-generation bis-locked nucleic acids with stacking linker and 2'-glycylamino-LNA show enhanced DNA invasion into supercoiled duplexes.
abstract: 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.
authors: Department of Laboratory Medicine, Karolinska Institutet and Clinical Research Center, Karolinska University Hospital Huddinge, SE-141 86 Stockholm, Sweden.
authors: Sylvain
authors: S
authors: Geny
authors: Department of Laboratory Medicine, Karolinska Institutet and Clinical Research Center, Karolinska University Hospital Huddinge, SE-141 86 Stockholm, Sweden INEB-Instituto de Engenharia Biomedica, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal.
authors: Pedro M D
authors: PM
authors: Moreno
authors: Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Stockholm, SE-171 21, Sweden.
authors: Tomasz
authors: T
authors: Krzywkowski
authors: Department of Laboratory Medicine, Karolinska Institutet and Clinical Research Center, Karolinska University Hospital Huddinge, SE-141 86 Stockholm, Sweden.
authors: Olof
authors: O
authors: Gissberg
authors: Nucleic Acid Center, Department of Physics, Chemistry and Pharmacy, Nucleic Acid Centre, University of Southern Denmark, 5230 Odense, Denmark.
authors: Nicolai K
authors: NK
authors: Andersen
authors: Nucleic Acid Center, Department of Physics, Chemistry and Pharmacy, Nucleic Acid Centre, University of Southern Denmark, 5230 Odense, Denmark.
authors: Abdirisaq J
authors: AJ
authors: Isse
authors: Nucleic Acid Center, Department of Physics, Chemistry and Pharmacy, Nucleic Acid Centre, University of Southern Denmark, 5230 Odense, Denmark.
authors: Amro M
authors: AM
authors: El-Madani
authors: Nucleic Acid Center, Department of Physics, Chemistry and Pharmacy, Nucleic Acid Centre, University of Southern Denmark, 5230 Odense, Denmark.
authors: Chenguang
authors: C
authors: Lou
authors: Department of Laboratory Medicine, Karolinska Institutet and Clinical Research Center, Karolinska University Hospital Huddinge, SE-141 86 Stockholm, Sweden.
authors: Y Vladimir
authors: YV
authors: Pabon
authors: Department of Chemistry, University of Idaho, Moscow, ID 83844, USA.
authors: Brooke A
authors: BA
authors: Anderson
authors: Department of Laboratory Medicine, Karolinska Institutet and Clinical Research Center, Karolinska University Hospital Huddinge, SE-141 86 Stockholm, Sweden.
authors: Eman M
authors: EM
authors: Zaghloul
authors: Department of Laboratory Medicine, Karolinska Institutet and Clinical Research Center, Karolinska University Hospital Huddinge, SE-141 86 Stockholm, Sweden Centre for Rare Diseases, Department of Clinical Genetics, Karolinska University Hospital, SE-171 76 Stockholm, Sweden.
authors: Rula
authors: R
authors: Zain
authors: Department of Chemistry, University of Idaho, Moscow, ID 83844, USA.
authors: Patrick J
authors: PJ
authors: Hrdlicka
authors: Nucleic Acid Center, Department of Physics, Chemistry and Pharmacy, Nucleic Acid Centre, University of Southern Denmark, 5230 Odense, Denmark.
authors: Per T
authors: PT
authors: Jørgensen
authors: Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Stockholm, SE-171 21, Sweden.
authors: Mats
authors: M
authors: Nilsson
authors: Department of Laboratory Medicine, Karolinska Institutet and Clinical Research Center, Karolinska University Hospital Huddinge, SE-141 86 Stockholm, Sweden.
authors: Karin E
authors: KE
authors: Lundin
authors: Nucleic Acid Center, Department of Physics, Chemistry and Pharmacy, Nucleic Acid Centre, University of Southern Denmark, 5230 Odense, Denmark.
authors: Erik B
authors: EB
authors: Pedersen
authors: Nucleic Acid Center, Department of Physics, Chemistry and Pharmacy, Nucleic Acid Centre, University of Southern Denmark, 5230 Odense, Denmark.
authors: Jesper
authors: J
authors: Wengel
authors: Department of Laboratory Medicine, Karolinska Institutet and Clinical Research Center, Karolinska University Hospital Huddinge, SE-141 86 Stockholm, Sweden edvard.smith@ki.se.
authors: C I Edvard
authors: CI
authors: Smith
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D004269:DNA, Bacterial / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D004278:DNA, Superhelical / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005998:Glycine / Q000031:analogs & derivatives*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000378:metabolism*; D016376:Oligonucleotides, Antisense / Q000138:chemical synthesis / Q000378:metabolism*; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D060327:Solid-Phase Synthesis Techniques; D055672:Static Electricity; D013329:Structure-Activity Relationship
chemical_list: D004269:DNA, Bacterial; D004278:DNA, Superhelical; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; C477371:locked nucleic acid; D005998:Glycine