Biotechnology Letters

, Volume 30, Issue 2, pp 243–252 | Cite as

Aptameric enzyme subunit for homogeneous DNA sensing

  • Kazunori IkebukuroEmail author
  • Wataru Yoshida
  • Koji Sode
Original Research Paper


We have developed an aptameric enzyme subunit (AES) which can detect the DNA in a homogeneous solution. The AES is an artificial enzyme subunit composed of an enzyme-inhibiting aptamer bearing a target-molecule binding site. We connected a probe DNA to a thrombin-inhibiting aptamer at its 5′ or 3′ end. The inhibitory activity of the thrombin-inhibiting aptamer bearing the probe DNA decreased compared to that of the original aptamer; however, it recovered upon hybridization with the target DNA. Using this AES, we were able to detect target DNAs by measuring the thrombin activity in a homogeneous solution.


Aptameric enzyme subunit Enzyme-inhibiting aptamer Homogeneous DNA sensing 



This work was supported by Industrial Technology Research Grant Program in 2006 from New Energy and Industrial Technology Development Organization (NEDO) of Japan.


  1. Bock LC, Griffin LC, Latham JA, Vermaas EH, Toole JJ (1992) Selection of single-stranded DNA molecules that bind and inhibit human thrombin. Nature 355:564–566PubMedCrossRefGoogle Scholar
  2. Dapic V, Abdomerovic V, Marrington R, Peberdy J, Rodger A, Trent JO, Bates PJ (2003) Biophysical and biological properties of quadruplex oligodeoxyribonucleotides. Nucleic Acids Res 31:2097–2107PubMedCrossRefGoogle Scholar
  3. Ellington AD, Szostak JW (1990) In vitro selection of RNA molecules that bind specific ligands. Nature 346:818–822PubMedCrossRefGoogle Scholar
  4. Fouchier RA, Bestebroer TM, Herfst S, Van Der Kemp L, Rimmelzwaan GF, Osterhaus AD (2000) Detection of influenza A viruses from different species by PCR amplification of conserved sequences in the matrix gene. J Clin Microbiol 38:4096–4101PubMedGoogle Scholar
  5. Galan JE, Curtiss R (1990) Expression of Salmonella typhimurium genes required for invasion is regulated by changes in DNA supercoiling. Infect Immun 58:1879–1885PubMedGoogle Scholar
  6. Ho HA, Boissinot M, Bergeron MG, Corbeil G, Dore K, Boudreau D, Leclerc M (2002) Angew. Chem., Int. Ed. Colorimetric and Fluorometric Detection of Nucleic Acids Using Cationic Polythiophene Derivatives. 114:1618–1621Google Scholar
  7. Ikebukuro K, Okumura Y, Sumikura K, Karube I (2005) A novel method of screening thrombin-inhibiting DNA aptamers using an evolution-mimicking algorithm. Nucleic Acids Res 33:e108PubMedCrossRefGoogle Scholar
  8. Isacsson J, Cao H, Ohlsson L, Nordgren S, Svanvik N, Westman G, Kubista M, Sjoback R, Sehlstedt U (2000) Rapid and specific detection of PCR products using light-up probes. Mol Cell Probes 14:321–328PubMedCrossRefGoogle Scholar
  9. Kuryavyi V, Majumdar A, Shallop A, Chernichenko N, Skripkin E, Jones R, Patel DJ (2001) A double chain reversal loop and two diagonal loops define the architecture of a unimolecular DNA quadruplex containing a pair of stacked G(syn)-G(syn)-G(anti)-G(anti) tetrads flanked by a G-(T-T) Triad and a T-T-T triple. J Mol Biol 310:181–194PubMedCrossRefGoogle Scholar
  10. Li Q, Luan G, Guo Q, Liang J (2002) A new class of homogeneous nucleic acid probes based on specific displacement hybridization. Nucleic Acids Res 30:E5PubMedCrossRefGoogle Scholar
  11. Macaya RF, Schultze P, Smith FW, Roe JA, Feigon J (1993) Thrombin-binding DNA aptamer forms a unimolecular quadruplex structure in solution. Proc Natl Acad Sci U. S. A. 90:3745–3749PubMedCrossRefGoogle Scholar
  12. Marra MA et al (2003) The Genome sequence of the SARS-associated coronavirus. Science 300:1399–1404PubMedCrossRefGoogle Scholar
  13. Masuko M, Ohtani H, Ebata K, Shimadzu A (1998) Optimization of excimer-forming two-probe nucleic acid hybridization method with pyrene as a fluorophore. Nucleic Acids Res 26:5409–5416PubMedCrossRefGoogle Scholar
  14. Pavlov V, Shlyahovsky B, Willner I (2005) Fluorescence detection of DNA by the catalytic activation of an aptamer/thrombin complex. J Am Chem Soc 127:6522–6523PubMedCrossRefGoogle Scholar
  15. Rota PA et al (2003) Characterization of a novel coronavirus associated with severe acute respiratory syndrome. Science 300:1394–1399PubMedCrossRefGoogle Scholar
  16. Saghatelian A, Guckian KM, Thayer DA, Ghadiri MR (2003) DNA detection and signal amplification via an engineered allosteric enzyme. J Am Chem Soc 125:344–345PubMedCrossRefGoogle Scholar
  17. Schultze P, Macaya RF, Feigon J (1994) Three-dimensional solution structure of the thrombin-binding DNA aptamer d(GGTTGGTGTGGTTGG). J Mol Biol 235:1532–1547PubMedCrossRefGoogle Scholar
  18. Smirnov I, Shafer RH (2000) Effect of loop sequence and size on DNA aptamer stability. Biochemistry 39:1462–1468PubMedCrossRefGoogle Scholar
  19. Stojanovic MN, de Prada P, Landry DW (2001) Catalytic molecular beacons. Chembiochem 2:411–415PubMedCrossRefGoogle Scholar
  20. Tian Y, He Y, Mao C (2006) Cascade signal amplification for DNA detection. Chembiochem 7:1862–1864PubMedCrossRefGoogle Scholar
  21. Tuerk C, Gold L (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249:505–510PubMedCrossRefGoogle Scholar
  22. Tyagi S, Kramer FR (1996) Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol 14:303–308PubMedCrossRefGoogle Scholar
  23. Wang DY, Sen D (2001) A novel mode of regulation of an RNA-cleaving DNAzyme by effectors that bind to both enzyme and substrate. J Mol Biol 310:723–734PubMedCrossRefGoogle Scholar
  24. Xiao Y, Pavlov V, Niazov T, Dishon A, Kotler M, Willner I (2004) Catalytic beacons for the detection of DNA and telomerase activity. J Am Chem Soc 126:7430–7431PubMedCrossRefGoogle Scholar
  25. Yoshida W, Sode K, Ikebukuro K (2006a) Aptameric enzyme subunit for biosensing based on enzymatic activity measurement. Anal Chem 78:3296–3303PubMedCrossRefGoogle Scholar
  26. Yoshida W, Sode K, Ikebukuro K (2006b) Homogeneous DNA sensing using enzyme-inhibiting DNA aptamers. Biochem Biophys Res Commun 348:245–252PubMedCrossRefGoogle Scholar
  27. Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  1. 1.Department of Biotechnology and Life ScienceTokyo University of Agriculture and TechnologyTokyoJapan

Personalised recommendations