Inhibition of the in vitro replication of DNA by an aptamer-protein complex in an autonomous DNA machine.

DNA replication plays a central role in living organisms. Unregulated or uncontrollable DNA replication is well known to result in many pathological states, such as cancer, autoimmune diseases, and viral/bacterial infections. We report that an aptamer-protein complex could indirectly inhibit in vitro replication of DNA. An isothermal DNA machine based on the strand-displacement amplification is employed to support our assumption. An antithrombin aptamer sequence is rationally encoded into the DNA replication template. Once thrombin binds to the template, the as-formed aptamer-protein complexes can, in turn, become a barrier to the polymerase and inhibit the DNA replication activities in both static and dynamic modes. The inhibition is successfully confirmed by both fluorescence and gel-electrophoresis experiments. Considering the availability of a broad library of aptamers and the existence of various DNA/protein interactions, our results imply the possibility for the rational regulation of DNA replication in vivo.

[1]  S. Linder,et al.  Mechanisms of action of DNA-damaging anticancer drugs in treatment of carcinomas: is acute apoptosis an "off-target" effect? , 2007, Mini reviews in medicinal chemistry.

[2]  Nobuko Hamaguchi,et al.  Aptamer-based biosensor arrays for detection and quantification of biological macromolecules. , 2003, Analytical biochemistry.

[3]  L. Hurley,et al.  A DNA polymerase stop assay for G-quadruplex-interactive compounds. , 1999, Nucleic acids research.

[4]  D. Bunka,et al.  Aptamers come of age – at last , 2006, Nature Reviews Microbiology.

[5]  John J McGuire,et al.  Anticancer antifolates: current status and future directions. , 2003, Current pharmaceutical design.

[6]  E. Vermaas,et al.  Selection of single-stranded DNA molecules that bind and inhibit human thrombin , 1992, Nature.

[7]  R. Breaker.  DNA aptamers and DNA enzymes. , 1997, Current opinion in chemical biology.

[8]  L. Hurley,et al.  DNA as a target for drug action. , 1988, Trends in pharmacological sciences.

[9]  Itamar Willner,et al.  Spotlighting of cocaine by an autonomous aptamer-based machine. , 2007, Journal of the American Chemical Society.

[10]  N. Janjić,et al.  Novel approach to specific growth factor inhibition in vivo: antagonism of platelet-derived growth factor in glomerulonephritis by aptamers. , 1999, The American journal of pathology.

[11]  D. Patel,et al.  Guanine residues in d(T2AG3) and d(T2G4) form parallel-stranded potassium cation stabilized G-quadruplexes with anti glycosidic torsion angles in solution. , 1992, Biochemistry.

[12]  Laurence H. Hurley,et al.  DNA and its associated processes as targets for cancer therapy , 2002, Nature Reviews Cancer.

[13]  Robert M. Dirks,et al.  Triggered amplification by hybridization chain reaction. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[14]  Chunhai Fan,et al.  Aptamer-based biosensors , 2008 .

[15]  L. Loeb,et al.  DNA polymerases and human disease , 2008, Nature Reviews Genetics.

[16]  Andrew D Ellington,et al.  Aptamer therapeutics advance. , 2006, Current opinion in chemical biology.

[17]  Andreas Reuter,et al.  Eine DNA‐basierte Maschine, die Thrombin abwechselnd binden und wieder freigeben kann , 2004 .

[18]  S. Mccurdy,et al.  The single-stranded DNA aptamer-binding site of human thrombin. , 1993, The Journal of biological chemistry.

[19]  A. Berdis.  DNA polymerases as therapeutic targets. , 2008, Biochemistry.

[20]  K. Woodford,et al.  A novel K(+)-dependent DNA synthesis arrest site in a commonly occurring sequence motif in eukaryotes. , 1994, The Journal of biological chemistry.

[21]  Itamar Willner,et al.  A virus spotlighted by an autonomous DNA machine. , 2006, Angewandte Chemie.

[22]  Friedrich C Simmel,et al.  A DNA-based machine that can cyclically bind and release thrombin. , 2004, Angewandte Chemie.

[23]  S. Klußmann,et al.  The aptamer handbook : functional oligonucleotides and their applications , 2006 .

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

[25]  Itamar Willner,et al.  Electronic aptamer-based sensors. , 2007, Angewandte Chemie.

[26]  Itamar Willner,et al.  DNA-based machines. , 2006, Organic & biomolecular chemistry.

[27]  Sara Tombelli,et al.  New trends in affinity sensing: aptamers for ligand binding , 2003 .

[28]  Itamar Willner,et al.  Optical analysis of Hg2+ ions by oligonucleotide-gold-nanoparticle hybrids and DNA-based machines. , 2008, Angewandte Chemie.

[29]  R. Parwaresch,et al.  Human DNA-Topoisomerases – Diagnostic and Therapeutic Implications for Cancer , 2000, Oncology Research and Treatment.

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

[31]  Sanjay Tyagi,et al.  Molecular Beacons: Probes that Fluoresce upon Hybridization , 1996, Nature Biotechnology.

[32]  D. Patel,et al.  Adaptive recognition by nucleic acid aptamers. , 2000, Science.

[33]  D. Shank,et al.  Isothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase system. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[34]  G. F. Joyce,et al.  Directed evolution of nucleic acid enzymes. , 2003, Annual review of biochemistry.

[35]  P. Bellecave,et al.  Selection of DNA aptamers that bind the RNA-dependent RNA polymerase of hepatitis C virus and inhibit viral RNA synthesis in vitro. , 2003, Oligonucleotides.

[36]  Michael Famulok,et al.  9.2 N&V 665 NEW MH , 2006 .

[37]  G. Walker.  Empirical aspects of strand displacement amplification. , 1993, PCR methods and applications.

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

[39]  I. Roy,et al.  Therapeutic applications of aptamers , 2008, Expert opinion on investigational drugs.

[40]  L. Hurley,et al.  G-quadruplex DNA: a potential target for anti-cancer drug design. , 2000, Trends in pharmacological sciences.

[41]  G. Walker,et al.  Strand displacement amplification--an isothermal, in vitro DNA amplification technique. , 1992, Nucleic acids research.

[42]  S. Chou,et al.  DNA aptamers as potential anti-HIV agents. , 2005, Trends in biochemical sciences.