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/