Abstract
DNA- and MNAzymes are nucleic acid-based enzymes (NAzymes), which infiltrated the otherwise protein-rich field of enzymology three decades ago. The 10–23 core NAzymes are one of the most widely used and well-characterized NAzymes, but often require elevated working temperatures or additional complex modifications for implementation at standard room temperatures. Here, we present a generally applicable method, based on thermodynamic principles governing hybridization, to re-engineer the existing 10–23 core NAzymes for use at 23 °C. To establish this, we first assessed the activity of conventional NAzymes in the presence of cleavable and non-cleavable substrate at 23 °C as well as over a temperature gradient. These tests pointed towards a non-catalytic mechanism of signal generation at 23 °C, suggesting that conventional NAzymes are not suited for use at this temperature. Following this, several novel NAzyme-substrate complexes were re-engineered from the conventional ones and screened for their performance at 23 °C. The complex with substrate and substrate-binding arms of the NAzymes shortened by four nucleotides on each terminus demonstrated efficient catalytic activity at 23 °C. This has been further validated over a dilution of enzymes or enzyme components, revealing their superior performance at 23 °C compared to the conventional 10–23 core NAzymes at their standard operating temperature of 55 °C. Finally, the proposed approach was applied to successfully re-engineer three other new MNAzymes for activity at 23 °C. As such, these re-engineered NAzymes present a remarkable addition to the field by further widening the diverse repertoire of NAzyme applications.
Similar content being viewed by others
References
Berg J, Tymoczko J, Stryer L. Biochemistry, 7th edition. New York: W. H. Freeman. 2007. pp. 319–344.
Gesteland RF, Atkins JF. The RNA world: the nature of modern RNA suggests a prebiotic RNA world. New York: Cold Spring Harbor Laboratory Pr. 1993. p. 630
Wilson DS, Szostak JW. In vitro selection of functional nucleic acids. Annu Rev Biochem. 1999;68:611–47. https://doi.org/10.1146/annurev.biochem.68.1.611.
Walter NG, Engelke DR. Ribozymes: catalytic RNAs that cut things, make things, and do odd and useful jobs. Biologist. 2002;49:199–203.
Mitsuyasu RT, Merigan TC, Carr A, Zack JA, Winters MA, Workman C, et al. Phase 2 gene therapy trial of an anti-HIV ribozyme in autologous CD34+ cells. Nat Med. 2009;15:285–92. https://doi.org/10.1038/nm.1932.
Zhang Y, Wang J, Cheng H, Sun Y, Liu M, Wu Z, et al. Conditional control of suicide gene expression in tumor cells with theophylline-responsive ribozyme. Gene Ther. 2017;24:84–91. https://doi.org/10.1038/gt.2016.78.
Breaker RR, Joyce GF. The expanding view of RNA and DNA function. Chem Biol. 2014;21:1059–65. https://doi.org/10.3174/ajnr.A1256.Functional.
Silverman SK. Nucleic Acid Enzymes (Ribozymes and Deoxyribozymes): In Vitro Selection and Application. In: Begley TP, Wiley Encyclopedia of Chemical Biology. Hoboken, NJ: John Wiley & Sons, Inc. 2008. https://doi.org/10.1002/9780470048672.wecb406.
Kasprowicz A, Stokowa-Sołtys K, Jeżowska-Bojczuk M, Wrzesiński J, Ciesiołka J. Characterization of highly efficient RNA-cleaving DNAzymes that function at acidic pH with no divalent metal-ion cofactors. ChemistryOpen. 2017;6:46–56. https://doi.org/10.1002/open.201600141.
Nakano S, Horita M, Kobayashi M, Sugimoto N. Catalytic activities of ribozymes and DNAzymes in water and mixed aqueous media. Catalysts. 2017;7:355. https://doi.org/10.3390/catal7120355.
Nesbitt SM, Erlacher HA, Fedor MJ. The internal equilibrium of the hairpin ribozyme: temperature, ion and pH effects. J Mol Biol. 1999;286:1009–24. https://doi.org/10.1006/jmbi.1999.2543.
Perrotta AT, Been MD. HDV ribozyme activity in monovalent cations. Biochemistry. 2006;45:11357–65. https://doi.org/10.1021/bi061215+.
Saran R, Liu J. A silver DNAzyme. Anal Chem. 2016;88:4014–20. https://doi.org/10.1021/acs.analchem.6b00327.
Nagraj N, Liu J, Sterling S, Wu J, Lu Y. DNAzyme catalytic beacon sensors that resist temperature-dependent variations. Chem Commun. 2009:4103. https://doi.org/10.1039/b903059j.
Kosman J, Juskowiak B. Peroxidase-mimicking DNAzymes for biosensing applications: a review. Anal Chim Acta. 2011;707:7–17.
Boersma AJ, Feringa BL, Roelfes G. Enantioselective Friedel–Crafts reactions in water using a DNA-based catalyst. Angew Chem Int Ed. 2009;48:3346–8. https://doi.org/10.1002/anie.200900371.
Li Y, Sen D. A catalytic DNA for porphyrin metallation. Nat Struct Biol. 1996;3:743–7. https://doi.org/10.1038/nsb0996-743.
Santoro SW, Joyce GF. A general purpose RNA-cleaving DNA enzyme. Proc Natl Acad Sci. 1997;94:4262–6. https://doi.org/10.1073/pnas.94.9.4262.
Zhou W, Saran R, Liu J. Metal sensing by DNA. Chem Rev. 2017;117:8272–325. https://doi.org/10.1021/acs.chemrev.7b00063.
Kim SU, Batule BS, Mun H, Shim W-B, Kim M-G. Ultrasensitive colorimetric detection of Salmonella enterica Typhimurium on lettuce leaves by HRPzyme-integrated polymerase chain reaction. Food Control. 2018;84:522–8. https://doi.org/10.1016/j.foodcont.2017.09.010.
Park Y, Lee CY, Kang S, Kim H, Park KS, Park HG. Universal, colorimetric microRNA detection strategy based on target-catalyzed toehold-mediated strand displacement reaction. Nanotechnology. 2018;29:085501. https://doi.org/10.1088/1361-6528/aaa3a3.
Chen F, Bai M, Cao K, Zhao Y, Cao X, Wei J, et al. Programming enzyme-initiated autonomous DNAzyme nanodevices in living cells. ACS Nano. 2017;11:11908–14. https://doi.org/10.1021/acsnano.7b06728.
Chen J, Zuehlke A, Deng B, Peng H, Hou X, Zhang H. A target-triggered DNAzyme motor enabling homogeneous, amplified detection of proteins. Anal Chem. 2017;89:12888–95. https://doi.org/10.1021/acs.analchem.7b03529.
Yang J, Tang M, Diao W, Cheng W, Zhang Y, Yan Y. Electrochemical strategy for ultrasensitive detection of microRNA based on MNAzyme-mediated rolling circle amplification on a gold electrode. Microchim Acta. 2016;183:3061–7. https://doi.org/10.1007/s00604-016-1958-5.
Tabrizi SN, Tan LY, Walker S, Poljak M, Twin J, Garland SM, et al. Multiplex assay for simultaneous detection of mycoplasma genitalium and macrolide resistance using plexzyme and plexprime technology. PLoS One. 2016;11:e0156740. https://doi.org/10.1136/sextrans-2015-052270.106.
Zhang P, He Z, Wang C, Chen J, Zhao J, Zhu X, et al. In situ amplification of intracellular microRNA with MNAzyme nanodevices for multiplexed imaging, logic operation, and controlled drug release. ACS Nano. 2015;9:789–98. https://doi.org/10.1021/nn506309d.
Li X, Cheng W, Li D, Wu J, Ding X, Cheng Q, et al. A novel surface plasmon resonance biosensor for enzyme-free and highly sensitive detection of microRNA based on multi component nucleic acid enzyme (MNAzyme)-mediated catalyzed hairpin assembly. Biosens Bioelectron. 2016;80:98–104. https://doi.org/10.1016/j.bios.2016.01.048.
Mokany E, Bone SM, Young PE, Doan TB, Todd AV. MNAzymes, a versatile new class of nucleic acid enzymes that can function as biosensors and molecular switches. J Am Chem Soc. 2010;132:1051–9. https://doi.org/10.1021/ja9076777.
Deborggraeve S, Dai JY, Xiao Y, Soh HT. Controlling the function of DNA nanostructures with specific trigger sequences. Chem Commun. 2013;49:397–9. https://doi.org/10.1039/C2CC36878A.
Torabi S-F, Wu P, McGhee CE, Chen L, Hwang K, Zheng N, et al. In vitro selection of a sodium-specific DNAzyme and its application in intracellular sensing. Proc Natl Acad Sci. 2015;112:5903–8. https://doi.org/10.1073/pnas.1420361112.
Mazumdar D, Nagraj N, Kim HK, Meng X, Brown AK, Sun Q, et al. Activity, folding and Z-DNA formation of the 8-17 DNAzyme in the presence of monovalent ions. J Am Chem Soc. 2009;131:5506–15. https://doi.org/10.1021/ja8082939.
Zhou W, Zhang Y, Ding J, Liu J. In vitro selection in serum: RNA-cleaving DNAzymes for measuring Ca2+ and Mg2+. ACS Sens. 2016;1:600–6. https://doi.org/10.1021/acssensors.5b00306.
Saran R, Liu J. A comparison of two classic Pb2+−dependent RNA-cleaving DNAzymes. Inorg Chem Front. 2016;3:494–501. https://doi.org/10.1039/C5QI00125K.
Li J, Lu Y. A highly sensitive and selective catalytic DNA biosensor for lead ions. J Am Chem Soc. 2000;122:10466–7. https://doi.org/10.1021/ja0021316.
Ren K, Wu J, Ju H, Yan F. Target-driven triple-binder assembly of MNAzyme for amplified electrochemical immunosensing of protein biomarker. Anal Chem. 2015;87:1694–700. https://doi.org/10.1021/ac504277z.
Schubert S, Gül DC, Grunert HP, Zeichhardt H, Erdmann VA, Kurreck J. RNA cleaving “10-23” DNAzymes with enhanced stability and activity. Nucleic Acids Res. 2003;31:5982–92. https://doi.org/10.1093/nar/gkg791.
Gao J, Shimada N, Maruyama A. Enhancement of deoxyribozyme activity by cationic copolymers. Biomater Sci. 2015;3:308–16. https://doi.org/10.1039/C4BM00256C.
Gao J, Shimada N, Maruyama A. MNAzyme-catalyzed nucleic acid detection enhanced by a cationic copolymer. Biomater Sci. 2015;3:716–20. https://doi.org/10.1039/C4BM00449C.
Levy M, Ellington AD. Exponential growth by cross-catalytic cleavage of deoxyribozymogens. Proc Natl Acad Sci U S A. 2003;100:6416–21. https://doi.org/10.1073/pnas.1130145100.
Gerasimova YV, Cornett EM, Edwards E, Su X, Rohde KH, Kolpashchikov DM. Deoxyribozyme cascade for visual detection of bacterial RNA. Chembiochem. 2013;14:2087–90. https://doi.org/10.1002/cbic.201300471.
Bone SM, Hasick NJ, Lima NE, Erskine SM, Mokany E, Todd AV. DNA-only cascade: a universal tool for signal amplification, enhancing the detection of target analytes. Anal Chem. 2014;86:9106–13. https://doi.org/10.1021/ac501811r.
So PTC, Dong CY. Fluorescence spectrophotometry. Chichester: John Wiley & Sons, Ltd. 2001. pp. 426–469. https://doi.org/10.1038/npg.els.0002978.
Tomin VI. Effect of temperature on the dynamic quenching of the dual fluorescence of molecules. Opt Spectrosc. 2008;104:838–45. https://doi.org/10.1134/S0030400X08060064.
Gao Z, Hou L, Xu M, Tang D. Enhanced colorimetric immunoassay accompanying with enzyme cascade amplification strategy for ultrasensitive detection of low-abundance protein. Sci Rep. 2015;4:1–8. https://doi.org/10.1038/srep03966.
Funding
This work has received funding from Research Foundation−Flanders (FWO SB/1S30116N, FWO G086114, FWO G084818N) and the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement no. 675412 (H2020-MSCA-ITN-ND4ID).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Electronic supplementary material
ESM 1
(PDF 180 kb)
Rights and permissions
About this article
Cite this article
Ven, K., Safdar, S., Dillen, A. et al. Re-engineering 10–23 core DNA- and MNAzymes for applications at standard room temperature. Anal Bioanal Chem 411, 205–215 (2019). https://doi.org/10.1007/s00216-018-1429-4
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00216-018-1429-4