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Interaction of DNA with engineered pores How to sequence individual ssDNA molecules

Interaction of DNA with engineered pores How to sequence individual ssDNA molecules -I identify nucleotides the tool: ion flux through a protein nanopore affect the ion flux by non-covalent interactions between pore and bases -II control how nucleotides are fed to the recognition site

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Interaction of DNA with engineered pores How to sequence individual ssDNA molecules

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  1. Interaction of DNA with engineered pores How to sequence individual ssDNA molecules -I identify nucleotides the tool: ion flux through a protein nanopore affect the ion flux by non-covalent interactions between pore and bases -II control how nucleotides are fed to the recognition site Enzymes not covalently attached attached covalently or fused

  2. affect the ion flux by non-covalent interactions between pore and bases

  3. Triple mutant in KCl C10A10C10A10C10A10 WT in KCl C10A10C10A10C10A10 Dwell time 0.23 ms

  4. Triple mutant in KI 1M A25C25 Triple mutant in CaCl2 1M A25C25

  5. Triple mutant in CaCl2 1M A25C25

  6. Triple mutant in BaCl2 1M A25C25

  7. Triple mutant in BaCl2 1M C10A10C10A10C10A10

  8. WT in BaCl2 1M C10A10C10A10C10A10

  9. Position of the mutation RR1 mutant KCl 1M A25C25

  10. Next experiments for base identification Hold the adapter into place covalently attach the adapter polymerise a ring inside the pore Customise the adapter

  11. II control the threading speed Enzymes seem to be the only option Helicase Exonuclease P4 for RNA

  12. http://www.rcsb.org/pdb/explore/images.do?structureId=1AVQ Lambda Exonuclease MgCl2 dependent Salt sensitive pH 9.5 Work out how much MgCl2 could be tolerated (use of ml spectrometry)

  13. Single-Molecule Kinetics of Exonuclease Reveal Base Dependence and Dynamic DisorderAntoine M. van Oijen,1 Paul C. Blainey,1 Donald J. Crampton,2 Charles C. Richardson,2 Tom Ellenberger,2 X. Sunney Xie1*

  14. RNA work

  15. 127R C10A10C10A10C10A10 CaCl2 0.5M P4 +ATP

  16. Summary: With graphted single strand in the vestibule: create a recognition site Same can be done with hairpins With a PEG lead: thread ssDNA through adapters attach adapters inside the pore engineer subunits so that a ring forms when protein assembles Investigate working conditions for lambda exonuclease in high salt Identify enzymes that could be fused with the protein pore P4 studies: produce larger ssRNA

  17. Beginning

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