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Single molecule technologies for genomics

Single molecule technologies for genomics. Andre Marziali Department of Physics and Astronomy University of British Columbia Vancouver, Canada. Long term needs of genomics:. Selected technology challenges. Sequencing and genotyping technologies to reduce costs ..

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Single molecule technologies for genomics

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  1. Single molecule technologies for genomics Andre Marziali Department of Physics and Astronomy University of British Columbia Vancouver, Canada

  2. Long term needs of genomics: Selected technology challenges • Sequencing and genotyping technologies toreduce costs.. • In vivo, real-time monitoring of gene expression .. F. Collins et al , Nature, 2003

  3. Genomics = Electronics ? H. McAdams – Science 1995 M. Elowitz, S.Leibler, Nature, 2000

  4. Genomics tools Electronics tools

  5. Genomics needs SPICE…. First principles (solid state physics) --- device behavior --- circuit behavior First principles (chemistry / biophysics) --- macromolecule behavior --- cell behavior Protein folding, molecular modifications, molecule structures… Networks, interactions, pathways etc..

  6. Long Term genomics technologies Cell simulation Cybercell: U of Alberta / U of Calgary E-cell: Institute for Advanced Biosciences, Keio University

  7. Long Term genomics technologies Single-molecule technologies: the $1000 genome Single molecule, long read DNA sequencing M. J. Levene,1 J. Korlach,1,2 S. W. Turner,1* M. Foquet,1 H. G. Craighead,1 W. W. Webb1† Science, 2003

  8. Long Term genomics technologies Single-molecule technologies: nanopore based detection Alpha-hemolysin Aqueous channel: 1.5 nm min. dia. 10 nm long Engleman, et. al. Science 1996 L. Z. Song et. al., Science 1996 • A cytolytic toxin produced by S. aureus, spontaneously forms heptameric membrane pores • Aqueous channel is permeable to ssDNA but not dsDNA. Kasianowicz, Brandin, Branton, Deamer, Proc. Nat. Acad. Sci. 1996 J.Nakane, M. Akeson, A. Marziali , Electrophoresis,2002

  9. Engineered pore-polymer assemblies can be used as single-molecule sensors • PEG molecules tethered inside nanopores can act as single molecule protein detectors. L. Movileanu et. al., Nature 2001

  10. Single molecule DNA detection with nanopores 120 pA A 15 pA ~ 2 ms 1M KCl Alpha-HL DNA Lipid bilayer Decrease in KCl mediated current can be used to detect pore blockage by a single DNA molecule Drawing courtesy of M. Akeson - UCSC Kasianowicz, Brandin, Branton, Deamer, PNAS 1996 Applications Single molecule sensor DNA sequencing

  11. Nanopore-based DNA concentration sensor • DNA sequencing in this manner is made difficult by the short residence time of DNA in the pore polydA(50) @ 240 mV Measured current through pore vs. time Event rate

  12. Hairpins trapped in pore allow long integration times 8 bp dsDNA GC 4 T loop T CGTTCGAAC GCAAGCTTG T T T Vercoutere et. al. NAR, 2003 Biophysics Laboratory, Dept. of Chemistry & Biochemistry, U.C. Santa Cruz

  13. Terminal base pair analysis Current blockage signature is a reliable indicator of terminal base pair identity. Vercoutere et. al. NAR, 2003 Biophysics Laboratory, Dept. of Chemistry & Biochemistry, U.C. Santa Cruz

  14. Current blockage contains complex information on molecule geometry IL UL LL F S UL IL LL Vercoutere et. al., Nucleic Acids Research, 2003 Biophysics Laboratory, Dept. of Chemistry & Biochemistry, U.C. Santa Cruz

  15. Electrical pore impedance as an indicator of molecule position • Impedance measurement of blocked pore yields Angstrom resolution at room temperature! 3.2 A Biophysics Laboratory, Dept. of Chemistry & Biochemistry, U.C. Santa Cruz

  16. Single Molecule Nano-sensor

  17. A trans-membrane single-molecule nanosensor • Long-term goals of our nanosensor project: • Real-time measurements on single cells. • Synthetic nanosensors for genotyping applications

  18. Sensor Components: Position sensor RNA aptamers avidin Pore A C Hairpins Reporting T Sensing Structural DNA avidin Base pairing Assembly biotin ~ 10 – 30 kT ~ kT

  19. The world’s smallest fishing rod: A trans-membrane, sequence-specific sensor probe sequence: biotin-5’-(A)51CCAAACCAACCACC-3’ Manuscript submitted: Jonathan Nakane, Matthew Wiggin, Andre Marziali

  20. Sensor Operation - avidin I A + R V Measured electrical characteristics 200 mV 200 pA Probe capture R~ 1 GW 50 pA R~ 4 GW 0

  21. Sensor Operation avidin - + - + Voltage reversal R~ 4 GW Probe exits pore 0 -60 mV R~ 1.5 GW

  22. Sensor Operation - avidin + Probe capture R~ 1 GW R~ 4 GW 0

  23. Sensor Operation - avidin + - + (with NO target bound ) Reverse pore impedance is greater for the trapped molecule 0 -60 mV R~ 10 GW

  24. Sensor Operation avidin - + - + Target dissociates and probe exits pore R~ 10 GW 0 -60 mV R~ 1.5 GW

  25. A successful analyte capture and release - + + -

  26. To first order, expect toff ~ e-aV Arrhenius relationship Image: E.Evans tD = relaxation time = (attempt rate)-1 Eb = free energy barrier height f = applied force = zeV /Dl fb = thermal force scale = kT / Dxbarrier Dxbarrier= energy barrier width along the reaction coordinate. Find Eb, Dxbarrier values for various molecules and applied potentials

  27. Unbinding (and escape) probability accumulated over ~ 50 - 500 binding events: eg. 7c at –55mV

  28. Four 14-mer oligonucleotides differing by a single base were used to test the sensor. 1 14 Probe BIOTIN – 5’ – (A51) CCAAACCAACCACC - 3’ Targets Perfect complement 3’ - GGTTTGGTTGGTGG – 5’ 7c mismatch 3’ - GGTTTGCTTGGTGG – 5’ 10c mismatch 3’ – GGTTTGGTTCGTGG – 5’ 1A mismatch 3’ – AGTTTGGTTGGTGG – 5’

  29. Lifetime-force curves for 14-mer DNA molecules with single nucleotide mutations 1a 10c 7c 14pc + - - - - -

  30. Lifetime-force curve intercepts are consistent with predicted binding energies? 14pc 1a 10c 7c

  31. Acknowledgements Nanosensor: Tudor Costin Dr. Nick Fameli Dan Green Aviv Keshet Jonathan Nakane Matthew Wiggin Sibyl Drissler Dhruti Trivedi Prof. Steven Plotkin Prof. Carl Michal Dr. Mark Akeson (UCSC) This work is funded in part by NSERC SCODA: Joel Pel Prof. Lorne Whitehead Elliot Holtham David Broemeling Robin Coope Prof. Dan Bizzotto This work is funded in part by NHGRI http://www.physics.ubc.ca/~andre/

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