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. 2019 Nov 4;9(61):35820-35824.
doi: 10.1039/c9ra06797c. eCollection 2019 Oct 31.

Bulky cations greatly increase the turnover of a native hammerhead ribozyme

Affiliations

Bulky cations greatly increase the turnover of a native hammerhead ribozyme

Shu-Ichi Nakano et al. RSC Adv. .

Abstract

Methods to facilitate the catalytic turnover of ribozymes are required for advancing oligonucleotide-based technologies. This study examined tetraalkylammonium ions for their ability to increase the efficiency of catalytic turnover of a native hammerhead ribozyme. Kinetic analysis showed that large tetraalkylammonium ions significantly increased the turnover rate of the ribozyme and was much more effective than poly(ethylene glycol) (PEG) and urea. The magnitude of the rate increase depended on the concentrations of Mg2+ and tetrapentylammonium ions, and the rate was enhanced by more than 180-fold at the optimal concentrations of these salts. The results provide physical insights into interactions of ribozymes with large cationic molecules through electrostatic forces and steric hindrance.

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Conflict of interest statement

There are no conflicts to declare.

Figures

Fig. 1
Fig. 1. (A) Secondary structure of the complex of the HH10 ribozyme (red) and the RNA substrate (blue) investigated in this study. The site of cleavage is indicated by an arrow. (B) The kinetic trace of the ribozyme-catalyzed substrate cleavage under the multiple-turnover condition (5.0 nM ribozyme and 100 nM substrate) conducted with 10 mM MgCl2 at 37 °C in the absence of additives. (C–E) Increases in the turnover rate by the addition of different amounts (weight per volume) of PEG200 (C), PEG8000 (D), or urea (E) to the reaction solution.
Fig. 2
Fig. 2. (A) Kinetic traces for the substrate cleavage under the multiple-turnover condition in the presence of 100 mM TBA (blue triangles) or 100 mM TPeA (red closed squares). In one case, 100 mM TPeA was added 3 h after the addition of MgCl2 (indicated by an arrow) to initiate substrate cleavage, represented by open squares. (B) Relative rate increases (relative to control) of multiple-turnover cleavage (black) and single-turnover cleavage (gray) by the addition of 20% PEGs, 2 M urea, 100 mM NH4+, or 100 mM tetraalkylammonium ions.
Fig. 3
Fig. 3. Temperature dependence of the turnover rate in the absence (black circles) and presence of TBA (blue triangles) or TPeA (red squares) at 100 mM. Some error bars are within the size of the symbols.
Fig. 4
Fig. 4. (A) Effects of the TPeA concentration on the turnover rate in the presence of 10 mM (circles), 5.0 mM (triangles), or 2.5 mM MgCl2 (squares). (B) Effects of MgCl2 concentration on the turnover rate in the presence of 100 mM TPeA. Data in the absence of TPeA are shown in the inset. (C) The turnover rates at different concentrations of TPeA and MgCl2, represented by the numbers and gray shades.

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References

    1. Bourdeau V. Ferbeyre G. Pageau M. Paquin B. Cedergren R. Nucleic Acids Res. 1999;27:4457–4467. doi: 10.1093/nar/27.22.4457. - DOI - PMC - PubMed
    1. Barciszewska M. Z. Szymanski M. Wyszko E. Pas J. Rychlewski L. Barciszewski J. Mutat. Res. 2005;589:103–110. - PubMed
    1. Salehi-Ashtiani K. Luptak A. Litovchick A. Szostak J. W. Science. 2006;313:1788–1792. doi: 10.1126/science.1129308. - DOI - PubMed
    1. Martick M. Horan L. H. Noller H. F. Scott W. G. Nature. 2008;454:899–902. doi: 10.1038/nature07117. - DOI - PMC - PubMed
    1. de la Peña M. Garcia-Robles I. EMBO Rep. 2010;11:711–716. doi: 10.1038/embor.2010.100. - DOI - PMC - PubMed