Expanding targets of DNAzyme-based sensors through deactivation and activation of DNAzymes by single uracil removal: sensitive fluorescent assay of uracil-DNA glycosylase.

Although deoxyribozymes (DNAzymes) have been widely used as biosensors for the detection of their cofactors and the targets of related aptazymes, it is desirable to expand their range of analytes to take advantage of the DNAzyme-based signal amplification for more sensitive detections. In this study, the activity of uracil-DNA glycosylase (UNG) was successfully detected and quantified by deoxyuridine-modified DNAzymes that underwent UNG-dependent deactivation or activation. In one design, the indispensable thymidine T2.1 in the 8-17 DNAzyme was replaced with a deoxyuridine, resulting in minimal change of the DNAzyme's activity. Since UNG is capable of removing uracils from single- or double-stranded DNAs, the modified DNAzyme was deactivated when the uracil at the indispensable thymidine site was eliminated by UNG. In another design, introducing a deoxyuridine to the 3' position of the deoxycytidine C13 in the catalytic core of the same DNAzyme caused significant decrease of the activity. The removal of the interfering deoxyuridine by UNG, however, activated the DNAzyme. By monitoring the activity change of the DNAzymes through the fluorescence enhancement from the DNAzyme-catalyzed cleavage of DNA substrates labeled by a fluorophore and quencher pair, the UNG activity was measured based on UNG-dependent deactivation and activation of the DNAzymes. This method was found to be able to detect UNG activity as low as 0.0034 U/mL. Such a method can be applied to the detection of other nucleotide-modifying enzymes and expand the analyte range of DNAzyme-based biosensors.

[1]  Yi Lu,et al.  Label-free fluorescent functional DNA sensors using unmodified DNA: a vacant site approach. , 2010, Analytical chemistry.

[2]  Yi Lu,et al.  Abasic site-containing DNAzyme and aptamer for label-free fluorescent detection of Pb(2+) and adenosine with high sensitivity, selectivity, and tunable dynamic range. , 2009, Journal of the American Chemical Society.

[3]  Michael Famulok,et al.  Aptamers for allosteric regulation. , 2011, Nature chemical biology.

[4]  M. Saparbaev,et al.  Alternative nucleotide incision repair pathway for oxidative DNA damage , 2002, Nature.

[5]  Michael Famulok,et al.  Functional aptamers and aptazymes in biotechnology, diagnostics, and therapy. , 2007, Chemical reviews.

[6]  Yi Lu,et al.  A catalytic beacon sensor for uranium with parts-per-trillion sensitivity and millionfold selectivity , 2007, Proceedings of the National Academy of Sciences.

[7]  Juewen Liu,et al.  Functional DNA nanotechnology: emerging applications of DNAzymes and aptamers. , 2006, Current opinion in biotechnology.

[8]  S. Karp,et al.  Immunological lesions in human uracil DNA glycosylase: association with Bloom syndrome. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[9]  Yingfu Li,et al.  Probing the function of nucleotides in the catalytic cores of the 8-17 and 10-23 DNAzymes by abasic nucleotide and C3 spacer substitutions. , 2010, Biochemistry.

[10]  E. Kool,et al.  Direct fluorescence monitoring of DNA base excision repair. , 2012, Angewandte Chemie.

[11]  H. Krokan,et al.  Uracil DNa-glycosylase from HeLa cells: general properties, substrate specificity and effect of uracil analogs. , 1981, Nucleic acids research.

[12]  Yi Lu,et al.  A lead-dependent DNAzyme with a two-step mechanism. , 2003, Biochemistry.

[13]  Jing Li,et al.  A highly sensitive and selective catalytic DNA biosensor for lead ions [9] , 2000 .

[14]  Gerald F. Joyce,et al.  Selection in vitro of an RNA enzyme that specifically cleaves single-stranded DNA , 1990, Nature.

[15]  B. Liu,et al.  Real-time monitoring of uracil removal by uracil-DNA glycosylase using fluorescent resonance energy transfer probes. , 2007, Analytical biochemistry.

[16]  J. Storhoff,et al.  A DNA-based method for rationally assembling nanoparticles into macroscopic materials , 1996, Nature.

[17]  J. Stivers,et al.  2-Aminopurine fluorescence studies of base stacking interactions at abasic sites in DNA: metal-ion and base sequence effects. , 1998, Nucleic acids research.

[18]  G. Baldwin,et al.  A rapid reaction analysis of uracil DNA glycosylase indicates an active mechanism of base flipping , 2007, Nucleic acids research.

[19]  Juewen Liu,et al.  Biochemical Characterization of a Uranyl Ion‐Specific DNAzyme , 2009, Chembiochem : a European journal of chemical biology.

[20]  M. Chung,et al.  8-oxoguanine (8-hydroxyguanine) DNA glycosylase and its substrate specificity. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[21]  S. Markowitz,et al.  Removal of uracil by uracil DNA glycosylase limits pemetrexed cytotoxicity: overriding the limit with methoxyamine to inhibit base excision repair , 2012, Cell Death and Disease.

[22]  Itamar Willner,et al.  A DNAzyme cascade for the amplified detection of Pb(2+) ions or L-histidine. , 2008, Chemical communications.

[23]  G. F. Joyce,et al.  A general purpose RNA-cleaving DNA enzyme. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[24]  Yingfu Li,et al.  Dinucleotide junction cleavage versatility of 8-17 deoxyribozyme. , 2004, Chemistry & biology.

[25]  Ronald R. Breaker,et al.  Natural and engineered nucleic acids as tools to explore biology , 2004, Nature.

[26]  Itamar Willner,et al.  Amplified detection of DNA through an autocatalytic and catabolic DNAzyme-mediated process. , 2011, Angewandte Chemie.

[27]  Chengde Mao,et al.  An autonomous DNA nanomotor powered by a DNA enzyme. , 2004, Angewandte Chemie.

[28]  G. Shen,et al.  Unimolecular catalytic DNA biosensor for amplified detection of L-histidine via an enzymatic recycling cleavage strategy. , 2011, Analytical chemistry.

[29]  Bin Liu,et al.  A rapid fluorescence assay for hSMUG1 activity based on modified molecular beacon. , 2011, Molecular and cellular probes.

[30]  I. Willner,et al.  Functional nucleic acid nanostructures and DNA machines. , 2010, Current opinion in biotechnology.

[31]  Yi Lu,et al.  Using personal glucose meters and functional DNA sensors to quantify a variety of analytical targets. , 2011, Nature chemistry.

[32]  E. Winer,et al.  Poly(ADP-Ribose) Polymerase Inhibition: “Targeted” Therapy for Triple-Negative Breast Cancer , 2010, Clinical Cancer Research.

[33]  Jian-hui Jiang,et al.  A target-activated autocatalytic DNAzyme amplification strategy for the assay of base excision repair enzyme activity. , 2012, Chemical communications.

[34]  Paul C J Kamer,et al.  Bioinspired catalyst design and artificial metalloenzymes. , 2011, Chemistry.

[35]  Yi Lu,et al.  Catalytic and molecular beacons for amplified detection of metal ions and organic molecules with high sensitivity. , 2010, Analytical chemistry.

[36]  Yi Lu,et al.  A DNAzyme catalytic beacon sensor for paramagnetic Cu2+ ions in aqueous solution with high sensitivity and selectivity. , 2007, Journal of the American Chemical Society.

[37]  Juewen Liu,et al.  Functional nucleic acid sensors. , 2009, Chemical reviews.

[38]  Yingfu Li,et al.  Biologically inspired synthetic enzymes made from DNA. , 2009, Chemistry & biology.

[39]  Marcel Hollenstein,et al.  A highly selective DNAzyme sensor for mercuric ions. , 2008, Angewandte Chemie.

[40]  Shizuka Nakayama,et al.  Colorimetric split G-quadruplex probes for nucleic acid sensing: improving reconstituted DNAzyme's catalytic efficiency via probe remodeling. , 2009, Journal of the American Chemical Society.

[41]  Juewen Liu,et al.  Colorimetric Cu2+ detection with a ligation DNAzyme and nanoparticles. , 2007, Chemical communications.

[42]  Yi Lu,et al.  Rational design of "turn-on" allosteric DNAzyme catalytic beacons for aqueous mercury ions with ultrahigh sensitivity and selectivity. , 2007, Angewandte Chemie.

[43]  Yingfu Li,et al.  An efficient RNA-cleaving DNA enzyme that synchronizes catalysis with fluorescence signaling. , 2003, Journal of the American Chemical Society.

[44]  S. Aguirre,et al.  Fluorogenic DNAzyme probes as bacterial indicators. , 2011, Angewandte Chemie.

[45]  Yi Lu,et al.  A colorimetric lead biosensor using DNAzyme-directed assembly of gold nanoparticles. , 2003, Journal of the American Chemical Society.

[46]  Kevin W Plaxco,et al.  Electrochemical detection of parts-per-billion lead via an electrode-bound DNAzyme assembly. , 2007, Journal of the American Chemical Society.

[47]  J Li,et al.  In vitro selection and characterization of a highly efficient Zn(II)-dependent RNA-cleaving deoxyribozyme. , 2000, Nucleic acids research.

[48]  J. Tainer,et al.  Uracil-DNA glycosylase-DNA substrate and product structures: conformational strain promotes catalytic efficiency by coupled stereoelectronic effects. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[49]  Tao Li,et al.  A lead(II)-driven DNA molecular device for turn-on fluorescence detection of lead(II) ion with high selectivity and sensitivity. , 2010, Journal of the American Chemical Society.

[50]  Alessio Peracchi,et al.  Prospects for antiviral ribozymes and deoxyribozymes , 2004, Reviews in medical virology.

[51]  Yi Lu.  New transition-metal-dependent DNAzymes as efficient endonucleases and as selective metal biosensors. , 2002, Chemistry.

[52]  A. Pyle,et al.  Using DNAzymes to cut, process, and map RNA molecules for structural studies or modification. , 2000, Methods in enzymology.

[53]  J. Stivers,et al.  A mechanistic perspective on the chemistry of DNA repair glycosylases. , 2003, Chemical reviews.

[54]  Itamar Willner,et al.  DNAzymes for sensing, nanobiotechnology and logic gate applications. , 2008, Chemical Society reviews.

[55]  R R Breaker,et al.  A DNA enzyme that cleaves RNA. , 1994, Chemistry & biology.

[56]  Yingfu Li,et al.  Nucleic acid aptamers and enzymes as sensors. , 2006, Current opinion in chemical biology.