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

Sylvain Geny, Pedro M D Moreno, Tomasz Krzywkowski, Olof Gissberg, Nicolai K Andersen, Abdirisaq J Isse, Amro M El-Madani, Chenguang Lou, Y Vladimir Pabon, Brooke A Anderson, Eman M Zaghloul, Rula Zain, Patrick J Hrdlicka, Per T Jørgensen, Mats Nilsson, Karin E Lundin, Erik B Pedersen, Jesper Wengel, C I Edvard Smith

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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.

Original languageEnglish
JournalNucleic Acids Research
Volume44
Issue number5
Pages (from-to)2007-2019
ISSN0305-1048
DOIs
Publication statusPublished - 18. Mar 2016

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Geny, S., Moreno, P. M. D., Krzywkowski, T., Gissberg, O., Andersen, N. K., Isse, A. J., ... Smith, C. I. E. (2016). Next-generation bis-locked nucleic acids with stacking linker and 2'-glycylamino-LNA show enhanced DNA invasion into supercoiled duplexes. Nucleic Acids Research, 44(5), 2007-2019. https://doi.org/10.1093/nar/gkw021
Geny, Sylvain ; Moreno, Pedro M D ; Krzywkowski, Tomasz ; Gissberg, Olof ; Andersen, Nicolai K ; Isse, Abdirisaq J ; El-Madani, Amro M ; Lou, Chenguang ; Pabon, Y Vladimir ; Anderson, Brooke A ; Zaghloul, Eman M ; Zain, Rula ; Hrdlicka, Patrick J ; Jørgensen, Per T ; Nilsson, Mats ; Lundin, Karin E ; Pedersen, Erik B ; Wengel, Jesper ; Smith, C I Edvard. / Next-generation bis-locked nucleic acids with stacking linker and 2'-glycylamino-LNA show enhanced DNA invasion into supercoiled duplexes. In: Nucleic Acids Research. 2016 ; Vol. 44, No. 5. pp. 2007-2019.
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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.",
author = "Sylvain Geny and Moreno, {Pedro M D} and Tomasz Krzywkowski and Olof Gissberg and Andersen, {Nicolai K} and Isse, {Abdirisaq J} and El-Madani, {Amro M} and Chenguang Lou and Pabon, {Y Vladimir} and Anderson, {Brooke A} and Zaghloul, {Eman M} and Rula Zain and Hrdlicka, {Patrick J} and J{\o}rgensen, {Per T} and Mats Nilsson and Lundin, {Karin E} and Pedersen, {Erik B} and Jesper Wengel and Smith, {C I Edvard}",
note = "{\circledC} The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.",
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Geny, S, Moreno, PMD, Krzywkowski, T, Gissberg, O, Andersen, NK, Isse, AJ, El-Madani, AM, Lou, C, Pabon, YV, Anderson, BA, Zaghloul, EM, Zain, R, Hrdlicka, PJ, Jørgensen, PT, Nilsson, M, Lundin, KE, Pedersen, EB, Wengel, J & Smith, CIE 2016, 'Next-generation bis-locked nucleic acids with stacking linker and 2'-glycylamino-LNA show enhanced DNA invasion into supercoiled duplexes', Nucleic Acids Research, vol. 44, no. 5, pp. 2007-2019. https://doi.org/10.1093/nar/gkw021

Next-generation bis-locked nucleic acids with stacking linker and 2'-glycylamino-LNA show enhanced DNA invasion into supercoiled duplexes. / Geny, Sylvain; Moreno, Pedro M D; Krzywkowski, Tomasz; Gissberg, Olof; Andersen, Nicolai K; Isse, Abdirisaq J; El-Madani, Amro M; Lou, Chenguang; Pabon, Y Vladimir; Anderson, Brooke A; Zaghloul, Eman M; Zain, Rula; Hrdlicka, Patrick J; Jørgensen, Per T; Nilsson, Mats; Lundin, Karin E; Pedersen, Erik B; Wengel, Jesper; Smith, C I Edvard.

In: Nucleic Acids Research, Vol. 44, No. 5, 18.03.2016, p. 2007-2019.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Next-generation bis-locked nucleic acids with stacking linker and 2'-glycylamino-LNA show enhanced DNA invasion into supercoiled duplexes

AU - Geny, Sylvain

AU - Moreno, Pedro M D

AU - Krzywkowski, Tomasz

AU - Gissberg, Olof

AU - Andersen, Nicolai K

AU - Isse, Abdirisaq J

AU - El-Madani, Amro M

AU - Lou, Chenguang

AU - Pabon, Y Vladimir

AU - Anderson, Brooke A

AU - Zaghloul, Eman M

AU - Zain, Rula

AU - Hrdlicka, Patrick J

AU - Jørgensen, Per T

AU - Nilsson, Mats

AU - Lundin, Karin E

AU - Pedersen, Erik B

AU - Wengel, Jesper

AU - Smith, C I Edvard

N1 - © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

PY - 2016/3/18

Y1 - 2016/3/18

N2 - 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.

AB - 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.

U2 - 10.1093/nar/gkw021

DO - 10.1093/nar/gkw021

M3 - Journal article

C2 - 26857548

VL - 44

SP - 2007

EP - 2019

JO - Nucleic Acids Research

JF - Nucleic Acids Research

SN - 0305-1048

IS - 5

ER -