Next Article in Journal
Theaflavin-3,3′-Digallate Suppresses Human Ovarian Carcinoma OVCAR-3 Cells by Regulating the Checkpoint Kinase 2 and p27 kip1 Pathways
Next Article in Special Issue
Site-Specific Cleavage of RNAs Derived from the PIM1 3′-UTR by a Metal-Free Artificial Ribonuclease
Previous Article in Journal
Increased Phenolic Content and Enhanced Antioxidant Activity in Fermented Glutinous Rice Supplemented with Fu Brick Tea
Previous Article in Special Issue
Synthesis of Glycosidic (β-1′′→6, 3′ and 4′) Site Isomers of Neomycin B and Their Effect on RNA and DNA Triplex Stability
Article Menu
Issue 4 (February-2) cover image

Export Article

Open AccessArticle
Molecules 2019, 24(4), 672; https://doi.org/10.3390/molecules24040672

Further Probing of Cu2+-Dependent PNAzymes Acting as Artificial RNA Restriction Enzymes

1
Department of Biosciences and Nutrition, Karolinska Institutet, Neo, 141 83 Huddinge, Stockholm, Sweden
2
Department of Chemistry, University of Turku, FI-20014 Turku, Finland
3
Institut für Pharmazeutische Wissenschaften (IPW), Eidgenössische Technische Hochschule Vladimir-Prelog-Weg 1-5/10, 8093 Zürich, Switzerland
*
Author to whom correspondence should be addressed.
Academic Editor: Derek J. McPhee
Received: 30 January 2019 / Revised: 11 February 2019 / Accepted: 13 February 2019 / Published: 14 February 2019
Full-Text   |   PDF [1345 KB, uploaded 14 February 2019]   |  

Abstract

Peptide nucleic acid (PNA)-neocuproine conjugates have been shown to efficiently catalyse the cleavage of RNA target sequences in the presence of Cu2+ ions in a site-specific manner. These artificial enzymes are designed to force the formation of a bulge in the RNA target, the sequence of which has been shown to be key to the catalytic activity. Here, we present a further investigation into the action of Cu2+-dependent PNAzymes with respect to the dependence on bulge composition in 3- and 4-nucleotide bulge systems. Cu2+-dependent PNAzymes were shown to have a clear preference for 4-nucleotide bulges, as the cleavage of 3-nucleotide bulge-forming RNA sequences was significantly slower, which is illustrated by a shift in the half-lives from approximately 30 min to 24 h. Nonetheless, the nucleotide preferences at different positions in the bulge displayed similar trends in both systems. Moreover, the cleavage site was probed by introducing critical chemical modifications to one of the cleavage site nucleotides of the fastest cleaved 4-nucleotide RNA bulge. Namely, the exclusion of the exocyclic amine of the central adenine and the replacement of the 2′-hydroxyl nucleophile with 2′-H or 2′-OMe substituents in the RNA severely diminished the rate of RNA cleavage by the Cu2+-dependent PNAzyme, giving insight into the mechanism of cleavage. Moreover, the shorter recognition arm of the RNA/PNAzyme complex was modified by extending the PNAzyme by two additional nucleobases. The new PNAzyme was able to efficiently promote the cleavage of RNA when fully hybridised to a longer RNA target and even outperform the previous fastest PNAzyme. The improvement was demonstrated in cleavage studies with stoichiometric amounts of either PNAzyme present, and the extended PNAzyme was also shown to give turnover with a 10-fold excess of the RNA target. View Full-Text
Keywords: artificial ribonuclease; peptide nucleic acid; RNA cleavage; catalysis artificial ribonuclease; peptide nucleic acid; RNA cleavage; catalysis
Figures

Graphical abstract

This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).

Supplementary material

SciFeed

Share & Cite This Article

MDPI and ACS Style

Luige, O.; Murtola, M.; Ghidini, A.; Strömberg, R. Further Probing of Cu2+-Dependent PNAzymes Acting as Artificial RNA Restriction Enzymes. Molecules 2019, 24, 672.

Show more citation formats Show less citations formats

Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.

Related Articles

Article Metrics

Article Access Statistics

1

Comments

[Return to top]
Molecules EISSN 1420-3049 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert
Back to Top