Direct Electronic Identification of Oligonucleotides with Inelastic

Electron Tunneling Spectroscopy

John Lund, Declan Ryan, Ranjana Mehta, Maryam Rahimi and Babak A. Parviz

Center of Excellence in Genomic Sciences

Microscale Life Sciences Center

University of Washington

USA

BACKGROUND APPROACH STRETCHING SPECTROSCOPY

Sequencing the Human Genome

When ~2001Present (2007)

Our Goal (2014)

Cost$3-5

Billion$10

Million$1000

Time 5-7 Years 6 Months 1 Week

Uses the entire sequencing capacity of a large center (~4 in the USA)

Enables personalized medicine

BACKGROUND APPROACH STRETCHING SPECTROSCOPY

What do we need to detect?

DNA

4 possible basesHuman genome: ~ 3 billion bases long

BACKGROUND APPROACH STRETCHING SPECTROSCOPY

3D AFM image of phage ss-DNA completely elongated on

HOPG with molecular combing

STM Tip

Conductive Substrate

All-Electronic SequencingSequencing technique:a. Stretch ss-DNA on a conductive surface

(e.g. graphite, atomically flat gold, etc)b. Perform a rough scan with a scanning tunneling

microscope to locate the molecule on the surfacec. Follow the molecule on the surface with computer

controlled STM tip and decipher the bases

Attributes of the technique:a. Single molecule (no PCR necessary)b. No labels; no chemical modification/manipulation of

the DNAc. Can be performed in principle on very long strands

(thousands to millions of bases)d. Can be parallelized by using a multiple probe

systeme. Can be very fast depending on the STM system

and algorithms used.

DNA

BACKGROUND APPROACH STRETCHING SPECTROSCOPY

How Fast Can STMs Work?

Carbon atoms on the surface of HOPG

imaged at tip speed of 40000 nm/s

This is equivalent to reading a whole bacterial genome in 10 seconds.

BACKGROUND APPROACH STRETCHING SPECTROSCOPY

Inelastic Tunneling Spectroscopy

Science 1974

V

Electron Tunneling Current

BACKGROUND APPROACH STRETCHING SPECTROSCOPY

Inelastic Tunneling Spectroscopy

Low V

Electron Tunneling Current

I

V

d2 I/d

V2

V

BACKGROUND APPROACH STRETCHING SPECTROSCOPY

Inelastic Tunneling Spectroscopy

High V

Electron Tunneling Current

I

V

d2 I/d

V2

V

New Tunneling Pathway

BACKGROUND APPROACH STRETCHING SPECTROSCOPY

Molecular Extension

•Our stretching approach employs molecular combing to orient DNA molecules on atomically flat surfaces

•The interaction of the DNA and surface is tuned using coordinating ions or self-assembled monolayers

•DNA molecules are stretched by a receding meniscus between a substrate

Substrate

DNA Molecule

Droplet Meniscus

BACKGROUND APPROACH STRETCHING SPECTROSCOPY

Experimental Details

•We verified our technique with two phage genome systems

•The Hind III digest of phage DNA, which yields 8 fragments with effective size range of 125 bp to 23 kb

•Virion X174 DNA is ss, covalently closed, circular, and 5,386 bases in length

•The ds- phage DNA was disrupted to ss-DNA by heating for 5 minutes and immediately cooling on ice

•MgCl2 is used to mediate adhesion between the DNA and freshly-cleaved graphite surface

BACKGROUND APPROACH STRETCHING SPECTROSCOPY

Procedure

BACKGROUND APPROACH STRETCHING SPECTROSCOPY

Procedure

BACKGROUND APPROACH STRETCHING SPECTROSCOPY

Procedure

BACKGROUND APPROACH STRETCHING SPECTROSCOPY

Procedure

BACKGROUND APPROACH STRETCHING SPECTROSCOPY

3D AFM image of bare HOPG before combing DNA

Results

BACKGROUND APPROACH STRETCHING SPECTROSCOPY

3D AFM image of phage ds-DNA completely elongated on HOPG with molecular combing. The DNA goes over multiple domains on the graphite surface.

BACKGROUND APPROACH STRETCHING SPECTROSCOPY

3D AFM image of coiled phage ss-DNA deposited on HOPG prior

to molecular combing.

BACKGROUND APPROACH STRETCHING SPECTROSCOPY

3D AFM image of phage ss-DNA completely elongated on HOPG after the completion of the molecular combing procedure.

BACKGROUND APPROACH STRETCHING SPECTROSCOPY

STM results

BACKGROUND APPROACH STRETCHING SPECTROSCOPY

BACKGROUND APPROACH STRETCHING SPECTROSCOPY

Gold substrate

Pt/Ir STM tip

Tunnelingcurrent

A’s

Tunneling spectroscopy on gold

BACKGROUND APPROACH STRETCHING SPECTROSCOPY

Spectroscopy on poly A’s

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Spectroscopy on poly C’s

BACKGROUND APPROACH STRETCHING SPECTROSCOPY

Spectroscopy on poly G’s

BACKGROUND APPROACH STRETCHING SPECTROSCOPY

Spectroscopy on poly T’s

BACKGROUND APPROACH STRETCHING SPECTROSCOPY

Deviation from blank gold

BACKGROUND APPROACH STRETCHING SPECTROSCOPY

Confirmation of IETS

BACKGROUND APPROACH STRETCHING SPECTROSCOPY

Measurement on stretched dsDNA

20 nm

BACKGROUND APPROACH STRETCHING SPECTROSCOPY

Tip steering approach

Conclusions

• All-electronic genome sequencing requires cost-effective and reproducible methods for extension of DNA on atomically flat surfaces

• Molecular combing offers a simple and cost-effective method for stretching DNA on surfaces

• IETS is a promising method for identifying DNA bases on conductive substrates using STM

• We have measured IETS spectra on 5-mer DNA bases on gold and will apply our approach to sequencing strands of DNA in the future

AcknowledgmentsOur Research Group Members•Postdoctoral Research Fellows

–Declan Ryan–Maryam Rahimi–Ranjana Mehta–Xiaorong Xiang (now at Intel)

•Graduate Students–Jianchun Dong–Harvey Ho–Sam Kim (with D. Meldrum)–John Lund–Coretta Maremma–Chris Morris–Ehsan Saeedi–Angela Shum–Andrei Afanasiev–Jean Wang (with Lih Lin)

•Undergraduate Students–Lisa Oh–James Etzkorn

Funding for our group:National Institutes of Health (NIH)Gordon and Betty Moore FoundationNational Science Foundation (NSF)National Academies Keck Future Initiative (NAKFI)Defense Advanced Research Project Agency (DARPA)Office of Naval Research (ONR)University Initiative Fund (UIF) at UWUW Technology Gap Innovation Fund (TGIF)

Undigested phage ds-DNA on HOPG

AFM images

ds-DNA Hind III digest on HOPG with 10 mM MgCl2

AFM images

phage ss-DNA Hind III digest on HOPG with 10 mM MgCl2

AFM images

STM imaging of ssDNA on HOPG