Skip to content
Licensed Unlicensed Requires Authentication Published by De Gruyter September 30, 2020

Influence of monovalent metal ions on metal binding and catalytic activity of the 10–23 DNAzyme

Hannah Rosenbach, Jan Borggräfe, Julian Victor, Christine Wuebben, Olav Schiemann, Wolfgang Hoyer, Gerhard Steger, Manuel Etzkorn and Ingrid Span ORCID logo
From the journal Biological Chemistry

Abstract

Deoxyribozymes (DNAzymes) are single-stranded DNA molecules that catalyze a broad range of chemical reactions. The 10–23 DNAzyme catalyzes the cleavage of RNA strands and can be designed to cleave essentially any target RNA, which makes it particularly interesting for therapeutic and biosensing applications. The activity of this DNAzyme in vitro is considerably higher than in cells, which was suggested to be a result of the low intracellular concentration of bioavailable divalent cations. While the interaction of the 10–23 DNAzyme with divalent metal ions was studied extensively, the influence of monovalent metal ions on its activity remains poorly understood. Here, we characterize the influence of monovalent and divalent cations on the 10–23 DNAzyme utilizing functional and biophysical techniques. Our results show that Na+ and K+ affect the binding of divalent metal ions to the DNAzyme:RNA complex and considerably modulate the reaction rates of RNA cleavage. We observe an opposite effect of high levels of Na+ and K+ concentrations on Mg2+- and Mn2+-induced reactions, revealing a different interplay of these metals in catalysis. Based on these findings, we propose a model for the interaction of metal ions with the DNAzyme:RNA complex.


Corresponding authors: Manuel Etzkorn, Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Universitätsstr. 1, D-40225Düsseldorf, Germany; Institute for Biological Information Processing: Structural Biochemistry (IBI-7), Research Center Jülich, Wilhelm-Johnen-Str., D-52428Jülich, Germany; and Ingrid Span, Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Universitätsstr. 1, D-40225Düsseldorf, Germany, E-mail: (M. Etzkorn), (I. Span)

Funding source: Chemical Industry Fund

Award Identifier / Grant number: 196/05

Award Identifier / Grant number: 700080

Funding source: German Academic Scholarship Foundation

Funding source: German Research Foundation

Award Identifier / Grant number: 103/2

Award Identifier / Grant number: 103/4

Acknowledgments

We gratefully thank D. Riesner for fruitful discussions and Matthias R. Steger for help with python programming.

  1. Author contribution: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

  2. Research funding: This work was supported by the Chemical Industry Fund (Li 196/05 to I.S. and Hoe 700080 to H.R.); the German Academic Scholarship Foundation (to H.R.); and the German Research Foundation (ET 103/2, ET 103/4 to M.E.).

  3. Conflict of interest statement: The authors declare that they have no conflict of interest regarding the contents of this article.

References

Bobbin, M.L. and Rossi, J.J. (2016). RNA interference (RNAi)-based therapeutics: delivering on the promise? Annu. Rev. Pharmacol. Toxicol. 56: 103–122, https://doi.org/10.1146/annurev-pharmtox-010715-103633.10.1146/annurev-pharmtox-010715-103633Search in Google Scholar

Boots, J.L., Canny, M.D., Azimi, E., and Pardi, A. (2008). Metal ion specificities for folding and cleavage activity in the Schistosoma hammerhead ribozyme. RNA 14: 2212–2222, https://doi.org/10.1261/rna.1010808.10.1261/rna.1010808Search in Google Scholar

Breaker, R.R. and Joyce, G.F. (1994). A DNA enzyme that cleaves RNA. Chem. Biol. 1: 223–229, https://doi.org/10.1016/1074-5521(94)90014-0.10.1016/1074-5521(94)90014-0Search in Google Scholar

Brown, A.K., Li, J., Pavot, C.M.B., and Lu, Y. (2003). A lead-dependent DNAzyme with a two-step mechanism. Biochemistry 42: 7152–7161, https://doi.org/10.1021/bi027332w.10.1021/bi027332wSearch in Google Scholar

Carmi, N., Balkhi, S.R., and Breaker, R.R. (1998). Cleaving DNA with DNA. Proc. Natl. Acad. Sci. USA 95: 2233–2237, https://doi.org/10.1073/pnas.95.5.2233.10.1073/pnas.95.5.2233Search in Google Scholar

Cepeda-Plaza, M. and Peracchi, A. (2020). Insights into DNA catalysis from structural and functional studies of the 8–17 DNAzyme. Org. Biomol. Chem. 18: 1697–1709, https://doi.org/10.1039/c9ob02453k.10.1039/C9OB02453KSearch in Google Scholar

Chandra, M., Sachdeva, A., and Silverman, S.K. (2009). DNA-catalyzed sequence-specific hydrolysis of DNA. Nat. Chem. Biol. 5: 718–720, https://doi.org/10.1038/nchembio.201.10.1038/nchembio.201Search in Google Scholar

Cheng, X., Liu, X., Bing, T., Cao, Z., and Shangguan, D. (2009). General peroxidase activity of G-quadruplex–hemin complexes and its application in ligand screening. Biochemistry 48: 7817–7823, https://doi.org/10.1021/bi9006786.10.1021/bi9006786Search in Google Scholar

Chinnapen, D.J.F. and Sen, D. (2004). A deoxyribozyme that harnesses light to repair thymine dimers in DNA. Proc. Natl. Acad. Sci. USA 101: 65–69, https://doi.org/10.1073/pnas.0305943101.10.1073/pnas.0305943101Search in Google Scholar

Cho, E.A., Moloney, F.J., Cai, H., Au-Yeung, A., China, C., Scolyer, R.A., Yosufi, B., Raftery, M.J., Deng, J.Z., Morton, S.W., et al. (2013). Safety and tolerability of an intratumorally injected DNAzyme, Dz13, in patients with nodular basal-cell carcinoma: a phase 1 first-in-human trial (DISCOVER). Lancet 381: 1835–1843, https://doi.org/10.1016/s0140-6736(12)62166-7.10.1016/S0140-6736(12)62166-7Search in Google Scholar

Cuenoud, B. and Szostak, J.W. (1995). A DNA metalloenzyme with DNA ligase activity. Nature 375: 611–614, https://doi.org/10.1038/375611a0.10.1038/375611a0Search in Google Scholar

Dahm, S.A.C., Derrick, W.B., and Uhlenbeck, O.C. (1993). Evidence for the role of solvated metal hydroxide in the hammerhead cleavage mechanism. Biochemistry 32: 13040–13045, https://doi.org/10.1021/bi00211a013.10.1021/bi00211a013Search in Google Scholar

Eaton, G.R., Eaton, S.S., Barr, D.P., and Weber, R.T. (2010). Quantitative EPR: a practitioners guide. Vienna: Springer.10.1007/978-3-211-92948-3Search in Google Scholar

Flynn-Charlebois, A., Wang, Y., Prior, T.K., Rashid, I., Hoadley, K.A., Coppins, R.L., Wolf, A.C., and Silverman, S.K. (2003). Deoxyribozymes with 2′-5′ RNA ligase activity. J. Am. Chem. Soc. 125: 2444–2454, https://doi.org/10.1021/ja028774y.10.1021/ja028774ySearch in Google Scholar

Fokina, A.A., Stetsenko, D.A., and François, J.C. (2015). DNA enzymes as potential therapeutics: towards clinical application of 10–23 DNAzymes. Expet Opin. Biol. Ther. 15: 689–711, https://doi.org/10.1517/14712598.2015.1025048.10.1517/14712598.2015.1025048Search in Google Scholar

Freisinger, E. and Sigel, R.K.O. (2007). From nucleotides to ribozymes-A comparison of their metal ion binding properties. Coord. Chem. Rev. 251: 1834–1851, https://doi.org/10.1016/j.ccr.2007.03.008.10.1016/j.ccr.2007.03.008Search in Google Scholar

Graphics Layout Engine, URL http://glx.sourceforge.net/ (Accessed 5 8 20).Search in Google Scholar

Gu, H., Furukawa, K., Weinberg, Z., Berenson, D.F., and Breaker, R.R. (2013). Small, highly active DNAs that hydrolyze DNA. J. Am. Chem. Soc. 135: 9121–9129, https://doi.org/10.1021/ja403585e.10.1021/ja403585eSearch in Google Scholar

He, Q.C., Zhou, J.M., Zhou, D.M., Nakamatsu, Y., Baba, T., and Taira, K. (2002). Comparison of metal-ion-dependent cleavages of RNA by a DNA enzyme and a hammerhead ribozyme. Biomacromolecules 3: 69–83, https://doi.org/10.1021/bm010095c.10.1021/bm010095cSearch in Google Scholar

Horton, T.E., Clardy, D.R., and DeRose, V.J. (1998). Electron paramagnetic resonance spectroscopic measurement of Mn2+ binding affinities to the hammerhead ribozyme and correlation with cleavage activity. Biochemistry 37: 18094–18101, https://doi.org/10.1021/bi981425p.10.1021/bi981425pSearch in Google Scholar

Hunsicker, L.M. and DeRose, V.J. (2000). Activities and relative affinities of divalent metals in unmodified and phosphorothioate-substituted hammerhead ribozymes. J. Inorg. Biochem. 80: 271–281, https://doi.org/10.1016/s0162-0134(00)00079-9.10.1016/S0162-0134(00)00079-9Search in Google Scholar

Hunsicker-Wang, L., Vogt, M., and Derose, V.J. (2009). EPR methods to study specific metal-ion binding sites in RNA. Methods Enzymol. 468: 335–367, https://doi.org/10.1016/s0076-6879(09)68016-2.10.1016/S0076-6879(09)68016-2Search in Google Scholar

Kisseleva, N., Khvorova, A., Westhof, E., and Schiemann, O. (2005). Binding of manganese(II) to a tertiary stabilized hammerhead ribozyme as studied by electron paramagnetic resonance spectroscopy. RNA 11: 1–6, https://doi.org/10.1261/rna.7127105.10.1261/rna.7127105Search in Google Scholar

Krug, N., Hohlfeld, J.M., Kirsten, A.M., Kornmann, O., Beeh, K.M., Kappeler, D., Korn, S., Ignatenko, S., Timmer, W., Rogon, C., et al. (2015). Allergen-induced asthmatic responses modified by a GATA3-specific DNAzyme. N. Engl. J. Med. 372: 1987–1995, https://doi.org/10.1056/nejmoa1411776.10.1056/NEJMoa1411776Search in Google Scholar

Ladbury, J.E. and Chowdhry, B.Z. (1996). Sensing the heat: the application of isothermal titration calorimetry to thermodynamic studies of biomolecular interactions. Chem. Biol. 3: 791–801, https://doi.org/10.1016/s1074-5521(96)90063-0.10.1016/S1074-5521(96)90063-0Search in Google Scholar

Li, Y. and Breaker, R.R. (1999). Phosphorylating DNA with DNA. Proc. Natl. Acad. Sci. USA 96: 2746–2751, https://doi.org/10.1073/pnas.96.6.2746.10.1073/pnas.96.6.2746Search in Google Scholar

Li, Y. and Sen, D. (1996). A catalytic DNA for porphyrin metalation. Nat. Struct. Biol. 3: 743–747, https://doi.org/10.1038/nsb0996-743.10.1038/nsb0996-743Search in Google Scholar

Li, Y., Liu, Y., and Breaker, R.R. (2000). Capping DNA with DNA. Biochemistry 39: 3106–3114, https://doi.org/10.1021/bi992710r.10.1021/bi992710rSearch in Google Scholar

Liu, H., Yu, X., Chen, Y., Zhang, J., Wu, B., Zheng, L., Haruehanroengra, P., Wang, R., Li, S., Lin, J.,et al. (2017). Crystal structure of an RNA-cleaving DNAzyme. Nat. Commun. 8: 2006, https://doi.org/10.1038/s41467-017-02203-x.10.1038/s41467-017-02203-xSearch in Google Scholar

McGhee, C.E., Loh, K.Y., and Lu, Y. (2017). DNAzyme sensors for detection of metal ions in the environment and imaging them in living cells. Curr. Opin. Biotechnol. 45: 191–201, https://doi.org/10.1016/j.copbio.2017.03.002.10.1016/j.copbio.2017.03.002Search in Google Scholar

Murray, J.B., Seyhan, A.A., Walter, N.G., Burke, J.M., and Scott, W.G. (1998). The hammerhead, hairpin and VS ribozymes are catalytically proficient in monovalent cations alone. Chem. Biol. 5: 587–595, https://doi.org/10.1016/s1074-5521(98)90116-8.10.1016/S1074-5521(98)90116-8Search in Google Scholar

Newville, M., Otten, R., Nelson, A., Ingargiola, A., Stensitzki, T., Allan, D., Fox, A., Carter, F., Michał Pustakhod, D., Ram, Y., Glenn Deil, C., Stuermer Beelen, A., Frost, O., Zobrist, N., Pasquevich, G., Hansen, A.L.R., Spillane, T., Caldwell, S., Polloreno, A., andrewhannum Zimmermann, J., Borreguero, J., Fraine, J., deep-42-thought Maier, B.F., Gamari, B., Almarza, A. (2019). https://lmfit/lmfit-py 1.0.0.Search in Google Scholar

Nowakowski, J., Shim, P.J., Prasad, G.S., Stout, C.D., and Joyce, G.F. (1999). Crystal structure of an 82-nucleotide RNA–DNA complex formed by the 10–23 DNA enzyme. Nat. Struct. Biol. 6: 151–156, https://doi.org/10.1038/5839.10.1038/5839Search in Google Scholar

Okumoto, Y. and Sugimoto, N. (2000). Effects of metal ions and catalytic loop sequences on the complex formation of a deoxyribozyme and its RNA substrate. J. Inorg. Biochem. 82: 189–195, https://doi.org/10.1016/s0162-0134(00)00159-8.10.1016/S0162-0134(00)00159-8Search in Google Scholar

Pechlaner, M. and Sigel, R.K.O. (2012). Characterization of metal ion–nucleic acid interactions insolution. In: Sigel, A., Sigel, H., and Sigel, R. K O. (Eds.), Interplay between metal ions and nucleic acids: metal ions in life science. Dordrecht: Springer, pp. 1–42.10.1007/978-94-007-2172-2_1Search in Google Scholar

Perrotta, A.T. and Been, M.D. (2006). HDV ribozyme activity in monovalent cations. Biochemistry 45: 11357–11365, https://doi.org/10.1021/bi061215+.10.1021/bi061215+Search in Google Scholar

Poland, D. (1978). Cooperative equilibria in physical biochemistry. Oxford: Clarendon Press. ISBN-10:019854622X; ISBN-13: 978-0198546221.Search in Google Scholar

Python Software Foundation, URL https://www.python.org/ (Accessed 5 August 20).Search in Google Scholar

Record, M.T., Lohman, T.M., and de Haseth, P. (1976). Ion effects on ligand–nucleic acid interactions. J. Mol. Biol. 107: 145–158, https://doi.org/10.1016/s0022-2836(76)80023-x.10.1016/S0022-2836(76)80023-XSearch in Google Scholar

Rosenbach, H., Victor, J., Etzkorn, M., Steger, G., Riesner, D., and Span, I. (2020). Molecular features and metal ions that influence 10–23 DNAzyme activity. Molecules 25: 3100, https://doi.org/10.3390/molecules25133100.10.3390/molecules25133100Search in Google Scholar

Santiago, F.S., Atkins, D.G., and Khachigian, L.M. (1999). Vascular smooth muscle cell proliferation and regrowth after mechanical injury in vitro are Egr-1/NGFI-A-dependent. Am. J. Pathol. 155: 897–905, https://doi.org/10.1016/s0002-9440(10)65189-9.10.1016/S0002-9440(10)65189-9Search in Google Scholar

Santoro, S.W. and Joyce, G.F. (1998). Mechanism and utility of an RNA-cleaving DNA enzyme. Biochemistry 37: 13330–13342, https://doi.org/10.1021/bi9812221.10.1021/bi9812221Search in Google Scholar PubMed

Santoro, S.W. and Joyce, G.F. (1997). A general purpose RNA-cleaving DNA enzyme. Proc. Natl. Acad. Sci. USA 94: 4262–4266, https://doi.org/10.1073/pnas.94.9.4262.10.1073/pnas.94.9.4262Search in Google Scholar PubMed PubMed Central

Sawata, S., Shimayama, T., Komiyama, M., Kumar, P.K.R., Nishikawa, S., and Taira, K. (1993). Enhancement of the cleavage rates of DNA-armed hammerhead ribozymes by various divalent metal ions. Nucleic Acids Res. 21: 5656–5660, https://doi.org/10.1093/nar/21.24.5656.10.1093/nar/21.24.5656Search in Google Scholar PubMed PubMed Central

Schiemann, O., Fritscher, J., Kisseleva, N., Sigurdsson, S.T., and Prisner, T.F. (2003). Structural investigation of a high-affinity MnII binding site in the hammerhead ribozyme by EPR spectroscopy and DFT calculations. Effects of neomycin B on metal–ion binding. Chembiochem 4: 1057–1065, https://doi.org/10.1002/cbic.200300653.10.1002/cbic.200300653Search in Google Scholar

Schnabl, J. and Sigel, R.K.O. (2010). Controlling ribozyme activity by metal ions. Curr. Opin. Chem. Biol. 14: 269–275, https://doi.org/10.1016/j.cbpa.2009.11.024.10.1016/j.cbpa.2009.11.024Search in Google Scholar

Silverman, S.K. (2015). Pursuing DNA catalysts for protein modification. Acc. Chem. Res. 48: 1369–1379, https://doi.org/10.1021/acs.accounts.5b00090.10.1021/acs.accounts.5b00090Search in Google Scholar

Silverman, S.K. (2016). Catalytic DNA: scope, applications, and biochemistry of deoxyribozymes. Trends Biochem. Sci. 41: 595–609, https://doi.org/10.1016/j.tibs.2016.04.010.10.1016/j.tibs.2016.04.010Search in Google Scholar

Sugimoto, N., Okumoto, Y., and Ohmichi, T. (1999). Effect of metal ions and sequence of deoxyribozymes on their RNA cleavage activity. J. Chem. Soc. Perkin Trans. 2: 1381–1386, https://doi.org/10.1039/a901461f.10.1039/a901461fSearch in Google Scholar

Torabi, S.F., Wu, P., McGhee, C.E., Chen, L., Hwang, K., Zheng, N., Cheng, J., and Lu, Y. (2015). In vitro selection of a sodium-specific DNAzyme and its application in intracellular sensing. Proc. Natl. Acad. Sci. USA 112: 5903–5908, https://doi.org/10.1073/pnas.1420361112.10.1073/pnas.1420361112Search in Google Scholar

Travascio, P., Li, Y., and Sen, D. (1998). DNA-enhanced peroxidase activity of a DNA aptamer–hemin complex. Chem. Biol. 5: 505–517, https://doi.org/10.1016/s1074-5521(98)90006-0.10.1016/S1074-5521(98)90006-0Search in Google Scholar

Victor, J., Steger, G., and Riesner, D. (2017). Inability of DNAzymes to cleave RNA in vivo is due to limited Mg2+ concentration in cells. Eur. Biophys. J. 47: 1–11, https://doi.org/10.1007/s00249-017-1270-2.10.1007/s00249-017-1270-2Search in Google Scholar PubMed

Virtanen, P., Gommers, R., Oliphant, T. E., Haberland, M., Reddy, T., Cournapeau, D., Burovski, E., Peterson, P., Weckesser, W., Bright, J., et al. (2020). SciPy 1.0: fundamental algorithms for scientific computing in Python. Nat. Methods 17: 261–272, https://doi.org/10.1038/s41592-019-0686-2.10.1038/s41592-019-0686-2Search in Google Scholar PubMed PubMed Central

Wang, Y., Liu, E., Lam, C.H., and Perrin, D.M. (2018). A densely modified M2+-independent DNAzyme that cleaves RNA efficiently with multiple catalytic turnover. Chem. Sci. 9: 1813–1821, https://doi.org/10.1039/c7sc04491g.10.1039/C7SC04491GSearch in Google Scholar PubMed PubMed Central

Wang, M., Zhang, H., Zhang, W., Zhao, Y., Yasmeen, A., Zhou, L., Yu, X., and Tang, Z. (2014). In vitro selection of DNA-cleaving deoxyribozyme with site-specific thymidine excision activity. Nucleic Acids Res. 42: 9262–9269, https://doi.org/10.1093/nar/gku592.10.1093/nar/gku592Search in Google Scholar PubMed PubMed Central

Wu, Y., Yu, L., McMahon, R., Rossi, J.J., Forman, S.J., and Snyder, D.S. (1999). Inhibition of bcr-abl oncogene expression by novel deoxyribozymes (DNAzymes). Hum. Gene Ther. 10: 2847–2857, https://doi.org/10.1089/10430349950016573.10.1089/10430349950016573Search in Google Scholar PubMed

Young, D.D., Lively, M.O., and Deiters, A. (2010). Activation and deactivation of DNAzyme and antisense function with light for the photochemical regulation of gene expression in mammalian cells. J. Am. Chem. Soc. 132: 6183–6193, https://doi.org/10.1021/ja100710j.10.1021/ja100710jSearch in Google Scholar PubMed PubMed Central


Supplementary Material

Supplementary Figures S1–S13 and Tables S1–4 are available online.

The online version of this article offers supplementary material (https://doi.org/10.1515/hsz-2020-0207).


Received: 2020-06-03
Accepted: 2020-09-13
Published Online: 2020-09-30
Published in Print: 2020-11-18

© 2020 Walter de Gruyter GmbH, Berlin/Boston