Biologically inspired synthetic enzymes made from DNA.

In cells, DNA typically consists of two antiparallel strands arranged in a double-helical structure, which is central to its fundamental role in storing and transmitting genetic information. In laboratories, however, DNA can be readily synthesized as a single-stranded polymer that can adopt many other types of structures, including some that have been shown to catalyze chemical transformations. These catalytic DNA molecules are commonly referred to as DNAzymes, or deoxyribozymes. Thus far, DNAzymes have not been found in cells, but hundreds of structural and functional variations have been created in the laboratory. This alternative catalytic platform has piqued the curiosity of many researchers, including those who seek to exploit them in potential applications ranging from analytical tools to therapeutic agents. In this review, we explore the unconventional role of DNA as a biologically inspired synthetic enzyme.

[1]  Alavattam Sreedhara,et al.  Ligating DNA with DNA. , 2004, Journal of the American Chemical Society.

[2]  M. Famulok,et al.  Nucleic acid aptamers-from selection in vitro to applications in vivo. , 2000, Accounts of chemical research.

[3]  Chengde Mao,et al.  Putting a brake on an autonomous DNA nanomotor. , 2004, Journal of the American Chemical Society.

[4]  Yi Lu,et al.  Colorimetric Biosensors Based on DNAzyme-Assembled Gold Nanoparticles , 2004, Journal of Fluorescence.

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

[6]  L. Khachigian,et al.  New DNA enzyme targeting Egr-1 mRNA inhibits vascular smooth muscle proliferation and regrowth after injury , 1999, Nature Medicine.

[7]  S. Klußmann,et al.  Development of an automated in vitro selection protocol to obtain RNA-based aptamers: identification of a biostable substance P antagonist , 2005, Nucleic acids research.

[8]  Scott A Strobel,et al.  Catalytic strategies of self-cleaving ribozymes. , 2008, Accounts of chemical research.

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

[10]  G. C. Johns,et al.  The Promise and Peril of Continuous In Vitro Evolution , 2005, Journal of Molecular Evolution.

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

[12]  Yingfu Li,et al.  DNAzyme-mediated catalysis with only guanosine and cytidine nucleotides , 2008, Nucleic acids research.

[13]  L. Gold,et al.  Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. , 1990, Science.

[14]  S. Silverman,et al.  Site-selective depurination by a periodate-dependent deoxyribozyme. , 2007, Chemical communications.

[15]  D. Herschlag,et al.  Mechanistic aspects of enzymatic catalysis: lessons from comparison of RNA and protein enzymes. , 1997, Annual review of biochemistry.

[16]  Yingfu Li,et al.  Sequence diversity, metal specificity, and catalytic proficiency of metal-dependent phosphorylating DNA enzymes. , 2002, Chemistry & biology.

[17]  Burckhard Seelig,et al.  Selection and evolution of enzymes from a partially randomized non-catalytic scaffold , 2007, Nature.

[18]  M. Famulok,et al.  The Ca2+ Ion as a Cofactor for a Novel RNA-Cleaving Deoxyribozyme† , 1996 .

[19]  D. Chinnapen,et al.  A deoxyribozyme that harnesses light to repair thymine dimers in DNA , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[20]  M. Levy,et al.  ATP-dependent allosteric DNA enzymes. , 2002, Chemistry & biology.

[21]  G. F. Joyce,et al.  A ribozyme composed of only two different nucleotides , 2002, Nature.

[22]  Yingfu Li,et al.  DNA-enhanced peroxidase activity of a DNA-aptamer-hemin complex. , 1998, Chemistry & biology.

[23]  R. Cedergren,et al.  Mixed deoxyribo- and ribo-oligonucleotides with catalytic activity , 1990, Nature.

[24]  Yingfu Li,et al.  Tracing sequence diversity change of RNA-cleaving deoxyribozymes under increasing selection pressure during in vitro selection. , 2004, Biochemistry.

[25]  Yingfu Li,et al.  Diverse Evolutionary Trajectories Characterize a Community of RNA-Cleaving Deoxyribozymes: A Case Study into the Population Dynamics of In Vitro Selection , 2005, Journal of Molecular Evolution.

[26]  Yingfu Li,et al.  Efficient signaling platforms built from a small catalytic DNA and doubly labeled fluorogenic substrates , 2006, Nucleic acids research.

[27]  Gerald F. Joyce,et al.  A ribozyme that lacks cytidine , 1999, Nature.

[28]  Levon M Khachigian,et al.  Transcription factor Egr-1 supports FGF-dependent angiogenesis during neovascularization and tumor growth , 2003, Nature Medicine.

[29]  A. Banerjea,et al.  Inhibition of HIV-1 gene expression by novel DNA enzymes targeted to cleave HIV-1 TAR RNA: potential effectiveness against all HIV-1 isolates. , 2003, Molecular therapy : the journal of the American Society of Gene Therapy.

[30]  G. F. Joyce,et al.  Mechanism and utility of an RNA-cleaving DNA enzyme. , 1998, Biochemistry.

[31]  R R Breaker,et al.  Capping DNA with DNA. , 2000, Biochemistry.

[32]  P. Yin,et al.  A DNAzyme that walks processively and autonomously along a one-dimensional track. , 2005, Angewandte Chemie.

[33]  J. Szostak,et al.  In vitro selection of RNA molecules that bind specific ligands , 1990, Nature.

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

[35]  C. Mao,et al.  DNAzyme amplification of molecular beacon signal. , 2005, Talanta.

[36]  Chunhai Fan,et al.  Construction of molecular logic gates with a DNA-cleaving deoxyribozyme. , 2006, Angewandte Chemie.

[37]  S. Silverman,et al.  Deoxyribozymes that synthesize branched and lariat RNA. , 2003, Journal of the American Chemical Society.

[38]  Yingfu Li,et al.  Characterization of long RNA-cleaving deoxyribozymes with short catalytic cores: the effect of excess sequence elements on the outcome of in vitro selection , 2006, Nucleic acids research.

[39]  T. Abe,et al.  A new modified DNA enzyme that targets influenza virus A mRNA inhibits viral infection in cultured cells , 2004, FEBS letters.

[40]  I. Willner,et al.  Amplified analysis of low-molecular-weight substrates or proteins by the self-assembly of DNAzyme-aptamer conjugates. , 2007, Journal of the American Chemical Society.

[41]  S. Silverman,et al.  A deoxyribozyme that synthesizes 2′,5′-branched RNA with any branch-site nucleotide , 2005, Nucleic acids research.

[42]  Eun Jeong Cho,et al.  Using a deoxyribozyme ligase and rolling circle amplification to detect a non-nucleic acid analyte, ATP. , 2005, Journal of the American Chemical Society.

[43]  Yingfu Li,et al.  Catalysis and Rational Engineering of trans‐Acting pH6DZ1, an RNA‐Cleaving and Fluorescence‐Signaling Deoxyribozyme with a Four‐Way Junction Structure , 2006, Chembiochem : a European journal of chemical biology.

[44]  Anastasia Khvorova,et al.  Fast cleavage kinetics of a natural hammerhead ribozyme. , 2004, Journal of the American Chemical Society.

[45]  Alessio Peracchi,et al.  Kinetic and thermodynamic characterization of the RNA-cleaving 8-17 deoxyribozyme. , 2004, Nucleic acids research.

[46]  Deoxyribozyme-catalyzed labeling of RNA. , 2007, Angewandte Chemie.

[47]  Levon M Khachigian,et al.  Inhibition of human breast carcinoma proliferation, migration, chemoinvasion and solid tumour growth by DNAzymes targeting the zinc finger transcription factor EGR-1. , 2004, Nucleic acids research.

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

[49]  Jie Liu,et al.  Inhibition of β-lactamase-mediated oxacillin resistance in Staphylococcus aureus by a deoxyribozyme , 2007, Acta Pharmacologica Sinica.

[50]  Darko Stefanovic,et al.  Deoxyribozyme-based three-input logic gates and construction of a molecular full adder. , 2006, Biochemistry.

[51]  Xiaoxing Luo,et al.  RESTORATION OF ANTIBIOTIC SUSCEPTIBILITY IN METHICILLIN‐RESISTANT STAPHYLOCOCCUS AUREUS BY TARGETING MECR1 WITH A PHOSPHOROTHIOATE DEOXYRIBOZYME , 2007, Clinical and experimental pharmacology & physiology.

[52]  J. Macdonald,et al.  Deoxyribozyme-based ligase logic gates and their initial circuits. , 2005, Journal of the American Chemical Society.

[53]  G. F. Joyce,et al.  Conversion of a ribozyme to a deoxyribozyme through in vitro evolution. , 2006, Chemistry & biology.

[54]  L. Khachigian,et al.  DNAzymes targeting the transcription factor Egr‐1 reduce myocardial infarct size following ischemia–reperfusion in rats , 2006, Journal of thrombosis and haemostasis : JTH.

[55]  S. Silverman Artificial Functional Nucleic Acids: Aptamers, Ribozymes, and Deoxyribozymes Identified by In Vitro Selection , 2009 .

[56]  M. Cairns,et al.  Brothers in arms: DNA enzymes, short interfering RNA, and the emerging wave of small-molecule nucleic acid-based gene-silencing strategies. , 2007, The American journal of pathology.

[57]  S. Stass,et al.  Angiogenic inhibition mediated by a DNAzyme that targets vascular endothelial growth factor receptor 2. , 2002, Cancer research.

[58]  R R Breaker,et al.  Phosphorylating DNA with DNA. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[59]  H. Suenaga,et al.  Novel Approach to Quantitative Detection of Specific rRNA in a Microbial Community, Using Catalytic DNA , 2005, Applied and Environmental Microbiology.

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

[61]  Whitney E. Purtha,et al.  General deoxyribozyme-catalyzed synthesis of native 3'-5' RNA linkages. , 2005, Journal of the American Chemical Society.

[62]  S. Silverman,et al.  DNA and RNA can be equally efficient catalysts for carbon-carbon bond formation. , 2008, Journal of the American Chemical Society.

[63]  D. Crothers,et al.  RNase H cleavage for processing of in vitro transcribed RNA for NMR studies and RNA ligation. , 1996, RNA.

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

[65]  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.

[66]  R. Collins,et al.  Exceptionally fast self-cleavage by a Neurospora Varkud satellite ribozyme. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

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

[68]  Juewen Liu,et al.  Miniaturized lead sensor based on lead-specific DNAzyme in a nanocapillary interconnected microfluidic device. , 2005, Environmental science & technology.

[69]  R. Momparler,et al.  Cleavage of intracellular hepatitis C RNA in the virus core protein coding region by deoxyribozymes , 2006, Journal of viral hepatitis.

[70]  A. Peracchi Preferential Activation of the 8–17 Deoxyribozyme by Ca 2+ Ions , 2000, The Journal of Biological Chemistry.

[71]  T. Schlick,et al.  In vitro RNA random pools are not structurally diverse: a computational analysis. , 2005, RNA.

[72]  C. Geyer,et al.  Use of intrinsic binding energy for catalysis by a cofactor-independent DNA enzyme. , 2000, Journal of molecular biology.

[73]  A. J. Bennet,et al.  A ribozyme and a catalytic DNA with peroxidase activity: active sites versus cofactor-binding sites. , 1999, Chemistry & biology.

[74]  L. Khachigian,et al.  c-Jun Regulates Vascular Smooth Muscle Cell Growth and Neointima Formation after Arterial Injury , 2002, The Journal of Biological Chemistry.

[75]  Sequence elements outside the hammerhead ribozyme catalytic core enable intracellular activity , 2003, Nature Structural Biology.

[76]  R. Cedergren,et al.  The conformation of single-stranded nucleic acids tDNA versus tRNA. , 1990, European journal of biochemistry.

[77]  D. Bartel,et al.  Accessing rare activities from random RNA sequences: the importance of the length of molecules in the starting pool. , 1997, Chemistry & biology.

[78]  M. Levy,et al.  Direct selection of trans-acting ligase ribozymes by in vitro compartmentalization. , 2005, RNA.

[79]  Yingfu Li,et al.  In vitro selection of small RNA-cleaving deoxyribozymes that cleave pyrimidine–pyrimidine junctions , 2008, Nucleic acids research.

[80]  Gerald F. Joyce,et al.  Crystal structure of an 82-nucleotide RNA–DNA complex formed by the 10-23 DNA enzyme , 1999, Nature Structural Biology.

[81]  M. Levy,et al.  In Vitro Selection of a Deoxyribozyme That Can Utilize Multiple Substrates , 2002, Journal of Molecular Evolution.

[82]  Timothy P. Mui,et al.  Convergent and general one-step DNA-catalyzed synthesis of multiply branched DNA. , 2008, Organic letters.

[83]  Eric A. Althoff,et al.  Kemp elimination catalysts by computational enzyme design , 2008, Nature.

[84]  R R Breaker,et al.  An amino acid as a cofactor for a catalytic polynucleotide. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[85]  K. Entian,et al.  Analysis of 2'-O-methylated nucleosides and pseudouridines in ribosomal RNAs using DNAzymes. , 2007, Analytical biochemistry.

[86]  R R Breaker,et al.  Structural diversity of self-cleaving ribozymes. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[87]  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.

[88]  R. Cedergren,et al.  Minimum ribonucleotide requirement for catalysis by the RNA hammerhead domain. , 1992, Biochemistry.

[89]  Dipankar Sen,et al.  A catalytic DNA for porphyrin metallation , 1996, Nature Structural Biology.

[90]  R R Breaker,et al.  Characterization of a DNA-cleaving deoxyribozyme. , 2001, Bioorganic & medicinal chemistry.

[91]  Selection of deoxyribozyme ligases that catalyze the formation of an unnatural internucleotide linkage. , 2001, Bioorganic & medicinal chemistry.

[92]  S. Silverman,et al.  Deoxyribozymes: useful DNA catalysts in vitro and in vivo , 2008, Cellular and Molecular Life Sciences.

[93]  S. Silverman,et al.  DNA-catalyzed formation of nucleopeptide linkages. , 2008, Angewandte Chemie.

[94]  W. Chiuman,et al.  DNAzymes: from creation in vitro to application in vivo. , 2004, Current pharmaceutical biotechnology.

[95]  J. Knowles,et al.  Evolution of enzyme function and the development of catalytic efficiency. , 1976, Biochemistry.

[96]  S. Silverman,et al.  In vitro evolution of an RNA-cleaving DNA enzyme into an RNA ligase switches the selectivity from 3'-5' to 2'-5'. , 2003, Journal of the American Chemical Society.

[97]  R R Breaker,et al.  Cleaving DNA with DNA. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[98]  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.

[99]  S. Silverman In vitro selection, characterization, and application of deoxyribozymes that cleave RNA , 2005, Nucleic acids research.

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

[101]  S. Silverman,et al.  Deoxyribozymes with 2'-5' RNA ligase activity. , 2003, Journal of the American Chemical Society.

[102]  D. Stefanovic,et al.  Deoxyribozyme-based half-adder. , 2003, Journal of the American Chemical Society.

[103]  Robert M. Smith† and,et al.  The pH-Rate Profile for the Hydrolysis of a Peptide Bond , 1998 .

[104]  Ronald R. Breaker,et al.  In vitro selection of self-cleaving DNAs. , 1996, Chemistry & biology.

[105]  Yi Lu,et al.  Dissecting metal ion-dependent folding and catalysis of a single DNAzyme. , 2007, Nature chemical biology.

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

[107]  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.

[108]  Y. Aoyama,et al.  Amplified nucleic acid sensing using programmed self-cleaving DNAzyme. , 2003, Journal of the American Chemical Society.

[109]  Li Yingfu,et al.  Functional nucleic acids for analytical applications , 2009 .

[110]  S. Silverman,et al.  Catalytic DNA (deoxyribozymes) for synthetic applications-current abilities and future prospects. , 2008, Chemical communications.

[111]  P. Bevilacqua,et al.  Nucleobase catalysis in ribozyme mechanism. , 2006, Current opinion in chemical biology.

[112]  S. Silverman,et al.  Ty1 reverse transcriptase does not read through the proposed 2',5'-branched retrotransposition intermediate in vitro. , 2007, RNA.

[113]  S. Silverman,et al.  Efficient one-step synthesis of biologically related lariat RNAs by a deoxyribozyme. , 2005, Angewandte Chemie.

[114]  S. Silverman,et al.  Directing the outcome of deoxyribozyme selections to favor native 3'-5' RNA ligation. , 2005, Biochemistry.

[115]  G. F. Joyce,et al.  A DNA enzyme with N-glycosylase activity. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[116]  J. Szostak,et al.  A DNA metalloenzyme with DNA ligase activity , 1995, Nature.

[117]  Peter F M Choong,et al.  DNAzyme technology and cancer therapy: cleave and let die , 2008, Molecular Cancer Therapeutics.

[118]  M. Stojanović,et al.  Homogeneous assays based on deoxyribozyme catalysis. , 2000, Nucleic acids research.

[119]  Yi Lu,et al.  Improving fluorescent DNAzyme biosensors by combining inter- and intramolecular quenchers. , 2003, Analytical chemistry.

[120]  S. Silverman,et al.  A deoxyribozyme that forms a three-helix-junction complex with its RNA substrates and has general RNA branch-forming activity. , 2005, Journal of the American Chemical Society.

[121]  Yingfu Li,et al.  Sequence-function relationships provide new insight into the cleavage site selectivity of the 8–17 RNA-cleaving deoxyribozyme , 2008, Nucleic acids research.

[122]  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.

[123]  C. Geyer,et al.  Evidence for the metal-cofactor independence of an RNA phosphodiester-cleaving DNA enzyme. , 1997, Chemistry & biology.

[124]  T. Cech,et al.  The Ribosome Is a Ribozyme , 2000, Science.

[125]  R. Breaker Engineered allosteric ribozymes as biosensor components. , 2002, Current opinion in biotechnology.

[126]  A. Ellington,et al.  Cofactor‐Assisted Self‐Cleavage in DNA Libraries with a 3′–5′‐Phosphoramidate Bond , 1997 .

[127]  Darko Stefanovic,et al.  Deoxyribozyme-based logic gates. , 2002, Journal of the American Chemical Society.

[128]  R. Raines,et al.  Structural determinants of enzymatic processivity. , 1994, Biochemistry.

[129]  Juewen Liu,et al.  Metal-dependent global folding and activity of the 8-17 DNAzyme studied by fluorescence resonance energy transfer. , 2007, Journal of the American Chemical Society.

[130]  L. Khachigian,et al.  Effect of deoxyribozymes targeting c-Jun on solid tumor growth and angiogenesis in rodents. , 2004, Journal of the National Cancer Institute.

[131]  Eric A. Althoff,et al.  De Novo Computational Design of Retro-Aldol Enzymes , 2008, Science.

[132]  T. Applegate,et al.  DzyNA-PCR: use of DNAzymes to detect and quantify nucleic acid sequences in a real-time fluorescent format. , 2000, Clinical chemistry.

[133]  T. Steitz,et al.  The structural basis of ribosome activity in peptide bond synthesis. , 2000, Science.

[134]  David R. Liu,et al.  Nucleic acid evolution and minimization by nonhomologous random recombination , 2002, Nature Biotechnology.

[135]  R. Breaker,et al.  A common speed limit for RNA-cleaving ribozymes and deoxyribozymes. , 2003, RNA.

[136]  Nicodemus Tedla,et al.  Suppression of vascular permeability and inflammation by targeting of the transcription factor c-Jun , 2006, Nature Biotechnology.

[137]  Peter G. Schultz,et al.  Antibody-catalyzed porphyrin metallation. , 1990, Science.

[138]  M. Cairns,et al.  Nucleic acid mutation analysis using catalytic DNA. , 2000, Nucleic acids research.

[139]  Jennifer A. Doudna,et al.  The chemical repertoire of natural ribozymes , 2002, Nature.

[140]  Yi Lu,et al.  Adenosine-dependent assembly of aptazyme-functionalized gold nanoparticles and its application as a colorimetric biosensor. , 2004, Analytical chemistry.

[141]  Nucleic acid sequence analysis using DNAzymes. , 2004, Methods in molecular biology.

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

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

[144]  D. H. Burke,et al.  Extraordinary rates of transition metal ion-mediated ribozyme catalysis. , 2006, RNA.

[145]  M. Helm,et al.  Use of DNAzymes for site-specific analysis of ribonucleotide modifications. , 2007, RNA.

[146]  Darko Stefanovic,et al.  A deoxyribozyme-based molecular automaton , 2003, Nature Biotechnology.

[147]  A. Ferré-D’Amaré,et al.  Use of cis- and trans-ribozymes to remove 5' and 3' heterogeneities from milligrams of in vitro transcribed RNA. , 1996, Nucleic acids research.

[148]  Ronald R. Breaker,et al.  Kinetics of RNA Degradation by Specific Base Catalysis of Transesterification Involving the 2‘-Hydroxyl Group , 1999 .

[149]  Juewen Liu,et al.  FRET study of a trifluorophore-labeled DNAzyme. , 2002, Journal of the American Chemical Society.

[150]  Multiple occurrences of an efficient self-phosphorylating deoxyribozyme motif. , 2007, Biochemistry.

[151]  S. Silverman,et al.  Experimental tests of two proofreading mechanisms for 5'-splice site selection. , 2006, ACS chemical biology.