LNA nucleotides improve cleavage efficiency of singular and binary hammerhead ribozymes

Janne K Christiansen, Sune Lobedanz, Khalil Arar, Jesper Wengel, Birte Vester

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

Variants of trans-acting hammerhead ribozymes were modified with Locked Nucleic Acid (LNA) nucleotides to reduce their size, to improve access to their RNA target and to explore combinational properties of binary constructs. Using low Mg(2+) concentrations and low substrate and ribozyme concentrations, it was found that insertion of LNA monomers into the substrate binding arms allowed these to be shortened and results in a very active enzyme under both single and multiple turnover conditions. Incorporation of a mix of LNA and DNA residues further increased the multiple turnover cleavage activity. At high Mg(2+) concentrations or high substrate and ribozyme concentrations, the enhancing effect of LNA incorporation was even more prominent. Using LNA in the stem of Helix II diminished cleavage activity, but allowed deletion of the tetra-loop and thus separating the ribozyme into two molecules with each half binding to the substrate. Efficient, binary hammerhead ribozymes were pursued in a combinatorial approach using a 6-times 5 library, which was analysed concerning the best combinations, buffer conditions and fragment ratios.
Original languageEnglish
JournalBioorganic & Medicinal Chemistry
Volume15
Issue number18
Pages (from-to)6135-6143
Number of pages8
ISSN0968-0896
DOIs
Publication statusPublished - 15. Sep 2007

Fingerprint

Nucleotides
Catalytic RNA
Substrates
Libraries
Buffers
Monomers
hammerhead ribozyme
locked nucleic acid
RNA
Molecules
DNA
Enzymes

Keywords

  • Magnesium
  • Nucleic Acid Conformation
  • Oligonucleotides
  • Oligonucleotides, Antisense
  • RNA
  • RNA, Catalytic
  • Substrate Specificity

Cite this

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title = "LNA nucleotides improve cleavage efficiency of singular and binary hammerhead ribozymes",
abstract = "Variants of trans-acting hammerhead ribozymes were modified with Locked Nucleic Acid (LNA) nucleotides to reduce their size, to improve access to their RNA target and to explore combinational properties of binary constructs. Using low Mg(2+) concentrations and low substrate and ribozyme concentrations, it was found that insertion of LNA monomers into the substrate binding arms allowed these to be shortened and results in a very active enzyme under both single and multiple turnover conditions. Incorporation of a mix of LNA and DNA residues further increased the multiple turnover cleavage activity. At high Mg(2+) concentrations or high substrate and ribozyme concentrations, the enhancing effect of LNA incorporation was even more prominent. Using LNA in the stem of Helix II diminished cleavage activity, but allowed deletion of the tetra-loop and thus separating the ribozyme into two molecules with each half binding to the substrate. Efficient, binary hammerhead ribozymes were pursued in a combinatorial approach using a 6-times 5 library, which was analysed concerning the best combinations, buffer conditions and fragment ratios.",
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LNA nucleotides improve cleavage efficiency of singular and binary hammerhead ribozymes. / Christiansen, Janne K; Lobedanz, Sune; Arar, Khalil; Wengel, Jesper; Vester, Birte.

In: Bioorganic & Medicinal Chemistry, Vol. 15, No. 18, 15.09.2007, p. 6135-6143.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - LNA nucleotides improve cleavage efficiency of singular and binary hammerhead ribozymes

AU - Christiansen, Janne K

AU - Lobedanz, Sune

AU - Arar, Khalil

AU - Wengel, Jesper

AU - Vester, Birte

PY - 2007/9/15

Y1 - 2007/9/15

N2 - Variants of trans-acting hammerhead ribozymes were modified with Locked Nucleic Acid (LNA) nucleotides to reduce their size, to improve access to their RNA target and to explore combinational properties of binary constructs. Using low Mg(2+) concentrations and low substrate and ribozyme concentrations, it was found that insertion of LNA monomers into the substrate binding arms allowed these to be shortened and results in a very active enzyme under both single and multiple turnover conditions. Incorporation of a mix of LNA and DNA residues further increased the multiple turnover cleavage activity. At high Mg(2+) concentrations or high substrate and ribozyme concentrations, the enhancing effect of LNA incorporation was even more prominent. Using LNA in the stem of Helix II diminished cleavage activity, but allowed deletion of the tetra-loop and thus separating the ribozyme into two molecules with each half binding to the substrate. Efficient, binary hammerhead ribozymes were pursued in a combinatorial approach using a 6-times 5 library, which was analysed concerning the best combinations, buffer conditions and fragment ratios.

AB - Variants of trans-acting hammerhead ribozymes were modified with Locked Nucleic Acid (LNA) nucleotides to reduce their size, to improve access to their RNA target and to explore combinational properties of binary constructs. Using low Mg(2+) concentrations and low substrate and ribozyme concentrations, it was found that insertion of LNA monomers into the substrate binding arms allowed these to be shortened and results in a very active enzyme under both single and multiple turnover conditions. Incorporation of a mix of LNA and DNA residues further increased the multiple turnover cleavage activity. At high Mg(2+) concentrations or high substrate and ribozyme concentrations, the enhancing effect of LNA incorporation was even more prominent. Using LNA in the stem of Helix II diminished cleavage activity, but allowed deletion of the tetra-loop and thus separating the ribozyme into two molecules with each half binding to the substrate. Efficient, binary hammerhead ribozymes were pursued in a combinatorial approach using a 6-times 5 library, which was analysed concerning the best combinations, buffer conditions and fragment ratios.

KW - Magnesium

KW - Nucleic Acid Conformation

KW - Oligonucleotides

KW - Oligonucleotides, Antisense

KW - RNA

KW - RNA, Catalytic

KW - Substrate Specificity

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DO - 10.1016/j.bmc.2007.06.045

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VL - 15

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SN - 0968-0896

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