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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 technologiesCell simulation
Cybercell: U of Alberta / U of Calgary
E-cell: Institute for Advanced Biosciences, Keio University
Long Term genomics technologiesSingle-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-hemolysinAqueous channel: 1.5 nm min. dia. 10 nm long
L. Z. Song et. al., Science 1996
Kasianowicz, Brandin, Branton, Deamer, Proc. Nat. Acad. Sci. 1996 J.Nakane, M. Akeson, A. Marziali , Electrophoresis, 2002
Long Term genomics technologiesSingle-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
polydA(50) @ 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
CGTTCGAACGCAAGCTTG
TTT 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
IL UL LL F S
IL
UL
LL
Biophysics Laboratory, Dept. of Chemistry & Biochemistry, U.C. Santa Cruz
Current blockage contains complex information on molecule geometry
0
0.5
1
1.5
2
2.5
3
0 5 10 15
Stem Length
Mea
n S
tem
Res
ista
nce
(Goh
ms) LL
UL
IL
May correspondto UL
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
avidin
biotin
Reporting
Pore
Position sensor
Sensing
RNA aptamers
TCA
DNA Structural
AssemblyBase pairing
Hairpins
~ 10 – 30 kT~ kT
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
Sensor Operation
avidin -
+ Probe capture
0
200 pA 200 mV
50 pA
R~ 1 G
R~ 4 G
A
I
R
V
Measured electrical characteristics
Sensor Operationavidin
-
+
+
-
Voltage reversal
0
-60 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
0
-60 mV
(with NO target bound )
Sensor Operationavidin
-
+
+
-0
R~ 10 G
R~ 1.5 G
Target dissociates and probe exits
pore
-60 mV
A successful analyte capture and release
-
+-
+
)/()/( bb ffkTEDoff eett
tD = relaxation time = (attempt rate)-1
Eb = free energy barrier height
f = applied force = zeV /l
fb = thermal force scale = kT / xbarrier
xbarrier = energy barrier width along the
reaction coordinate.
Arrhenius relationship
Find Eb, xbarrier
values for various molecules and applied potentials
Image: E.Evans
To first order, expect toff ~ e-V
Unbinding (and escape) probability accumulated over ~ 50 - 500 binding events: eg. 7c at –55mV
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 50 100 150 200ms
Pe
sc
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.01 0.1 1 10 100 1000 10000ms
itt
iiesc eatP /1)(
Targets
Perfect complement 3’ - GGTTTGGTTGGTGG – 5’
7c mismatch 3’ - GGTTTGCTTGGTGG – 5’
10c mismatch 3’ – GGTTTGGTTCGTGG – 5’
1A mismatch 3’ – AGTTTGGTTGGTGG – 5’
Probe BIOTIN – 5’ – (A51) 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
0.1
1
10
100
1000
10000
20 40 60 80 100mV
ms
7c14pc10c1a10c-27c-2Expon. (7c)Expon. (14pc)Expon. (10c)Expon. (1a)
Molecule Slope mV-1 Intercept at –10 mV
14 pc 0.16 16.7
1a 0.17 16.2 10c 0.10 10.2
7c 0.09 7.4
1a
10c
7c
14pc
)ln())27(
()ln( Db
off tkT
EV
mVl
xzt
+
- - - - -
4
6
8
10
12
14
16
18
20
20 25 30 35 40
Predicted binding energy in kT
Ln
(Tim
esc
ale
) in
terc
ep
t a
t +
10 m
V
Lifetime-force curve intercepts are consistent with predicted binding energies?
1a10c
7c
14pc8.11)(76.0int
Tk
EY
b
b
Acknowledgements
Jonathan NakaneMatthew WigginSibyl DrisslerDhruti Trivedi
This work is funded in part by NSERC
Tudor Costin Dr. Nick FameliDan GreenAviv Keshet
Prof. Steven PlotkinProf. Carl MichalDr. Mark Akeson (UCSC)
Nanosensor:
SCODA:
Joel Pel Prof. Lorne WhiteheadElliot HolthamDavid BroemelingRobin CoopeProf. Dan Bizzotto
This work is funded in part by NHGRI
http://www.physics.ubc.ca/~andre/