Analytical and Bioanalytical Chemistry

, Volume 411, Issue 1, pp 205–215 | Cite as

Re-engineering 10–23 core DNA- and MNAzymes for applications at standard room temperature

  • Karen Ven
  • Saba Safdar
  • Annelies Dillen
  • Jeroen Lammertyn
  • Dragana Spasic
Research Paper


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.


DNAzyme MNAzyme Room temperature 10–23 core 


Funding information

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).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2018_1429_MOESM1_ESM.pdf (216 kb)
ESM 1 (PDF 180 kb)


  1. 1.
    Berg J, Tymoczko J, Stryer L. Biochemistry, 7th edition. New York: W. H. Freeman. 2007. pp. 319–344. Google Scholar
  2. 2.
    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. 630Google Scholar
  3. 3.
    Wilson DS, Szostak JW. In vitro selection of functional nucleic acids. Annu Rev Biochem. 1999;68:611–47. Scholar
  4. 4.
    Walter NG, Engelke DR. Ribozymes: catalytic RNAs that cut things, make things, and do odd and useful jobs. Biologist. 2002;49:199–203.PubMedGoogle Scholar
  5. 5.
    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. Scholar
  6. 6.
    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. Scholar
  7. 7.
    Breaker RR, Joyce GF. The expanding view of RNA and DNA function. Chem Biol. 2014;21:1059–65. Scholar
  8. 8.
    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.
  9. 9.
    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. Scholar
  10. 10.
    Nakano S, Horita M, Kobayashi M, Sugimoto N. Catalytic activities of ribozymes and DNAzymes in water and mixed aqueous media. Catalysts. 2017;7:355. Scholar
  11. 11.
    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. Scholar
  12. 12.
    Perrotta AT, Been MD. HDV ribozyme activity in monovalent cations. Biochemistry. 2006;45:11357–65. Scholar
  13. 13.
    Saran R, Liu J. A silver DNAzyme. Anal Chem. 2016;88:4014–20. Scholar
  14. 14.
    Nagraj N, Liu J, Sterling S, Wu J, Lu Y. DNAzyme catalytic beacon sensors that resist temperature-dependent variations. Chem Commun. 2009:4103.
  15. 15.
    Kosman J, Juskowiak B. Peroxidase-mimicking DNAzymes for biosensing applications: a review. Anal Chim Acta. 2011;707:7–17.CrossRefGoogle Scholar
  16. 16.
    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. Scholar
  17. 17.
    Li Y, Sen D. A catalytic DNA for porphyrin metallation. Nat Struct Biol. 1996;3:743–7. Scholar
  18. 18.
    Santoro SW, Joyce GF. A general purpose RNA-cleaving DNA enzyme. Proc Natl Acad Sci. 1997;94:4262–6. Scholar
  19. 19.
    Zhou W, Saran R, Liu J. Metal sensing by DNA. Chem Rev. 2017;117:8272–325. Scholar
  20. 20.
    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. Scholar
  21. 21.
    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. Scholar
  22. 22.
    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. Scholar
  23. 23.
    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. Scholar
  24. 24.
    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. Scholar
  25. 25.
    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. Scholar
  26. 26.
    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. Scholar
  27. 27.
    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. Scholar
  28. 28.
    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. Scholar
  29. 29.
    Deborggraeve S, Dai JY, Xiao Y, Soh HT. Controlling the function of DNA nanostructures with specific trigger sequences. Chem Commun. 2013;49:397–9. Scholar
  30. 30.
    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. Scholar
  31. 31.
    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. Scholar
  32. 32.
    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.
  33. 33.
    Saran R, Liu J. A comparison of two classic Pb2+−dependent RNA-cleaving DNAzymes. Inorg Chem Front. 2016;3:494–501. Scholar
  34. 34.
    Li J, Lu Y. A highly sensitive and selective catalytic DNA biosensor for lead ions. J Am Chem Soc. 2000;122:10466–7. Scholar
  35. 35.
    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. Scholar
  36. 36.
    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. Scholar
  37. 37.
    Gao J, Shimada N, Maruyama A. Enhancement of deoxyribozyme activity by cationic copolymers. Biomater Sci. 2015;3:308–16. Scholar
  38. 38.
    Gao J, Shimada N, Maruyama A. MNAzyme-catalyzed nucleic acid detection enhanced by a cationic copolymer. Biomater Sci. 2015;3:716–20. Scholar
  39. 39.
    Levy M, Ellington AD. Exponential growth by cross-catalytic cleavage of deoxyribozymogens. Proc Natl Acad Sci U S A. 2003;100:6416–21. Scholar
  40. 40.
    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. Scholar
  41. 41.
    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. Scholar
  42. 42.
    So PTC, Dong CY. Fluorescence spectrophotometry. Chichester: John Wiley & Sons, Ltd. 2001. pp. 426–469.
  43. 43.
    Tomin VI. Effect of temperature on the dynamic quenching of the dual fluorescence of molecules. Opt Spectrosc. 2008;104:838–45. Scholar
  44. 44.
    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. Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Karen Ven
    • 1
  • Saba Safdar
    • 1
  • Annelies Dillen
    • 1
  • Jeroen Lammertyn
    • 1
  • Dragana Spasic
    • 1
  1. 1.Department of Biosystems, Biosensors GroupKU Leuven - University of LeuvenLeuvenBelgium

Personalised recommendations