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

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

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

Genomics tools Electronics tools

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..

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

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

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

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

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

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

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

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

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

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

Single Molecule Nano-sensor

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

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

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

Sensor Operation avidin - + Probe capture pA 200 mV 50 pA R~ 1 G  R~ 4 G  A I R V Measured electrical characteristics

Sensor Operation avidin Voltage reversal mV Probe exits pore R~ 4 G  R~ 1.5 G 

Sensor Operation avidin - + Probe capture 0 R~ 1 G  R~ 4 G 

Sensor Operation avidin Reverse pore impedance is greater for the trapped molecule R~ 10 G  mV (with NO target bound )

Sensor Operation avidin R~ 10 G  R~ 1.5 G  Target dissociates and probe exits pore -60 mV

A successful analyte capture and release

t D = relaxation time = (attempt rate) -1 E b = free energy barrier height f = applied force = zeV /  l f b = thermal force scale = kT /  x barrier  x barrier = energy barrier width along the reaction coordinate. Arrhenius relationship Find E b,  x barrier values for various molecules and applied potentials Image: E.Evans To first order, expect t off ~ e -  V

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

Targets Perfect complement3’ - GGTTTGGTTGGTGG – 5’ 7c mismatch3’ - GGTTTGCTTGGTGG – 5’ 10c mismatch3’ – GGTTTGGTTCGTGG – 5’ 1A mismatch3’ – AGTTTGGTTGGTGG – 5’ Probe BIOTIN – 5’ – (A 51 ) CCAAACCAACCACC - 3’ 1 14 Four 14-mer oligonucleotides differing by a single base were used to test the sensor.

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

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

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