![Page 1: John Lund , Declan Ryan, Ranjana Mehta, Maryam Rahimi and Babak A. Parviz](https://reader031.vdocuments.us/reader031/viewer/2022013004/5681446e550346895db10059/html5/thumbnails/1.jpg)
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
![Page 2: John Lund , Declan Ryan, Ranjana Mehta, Maryam Rahimi and Babak A. Parviz](https://reader031.vdocuments.us/reader031/viewer/2022013004/5681446e550346895db10059/html5/thumbnails/2.jpg)
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
![Page 3: John Lund , Declan Ryan, Ranjana Mehta, Maryam Rahimi and Babak A. Parviz](https://reader031.vdocuments.us/reader031/viewer/2022013004/5681446e550346895db10059/html5/thumbnails/3.jpg)
BACKGROUND APPROACH STRETCHING SPECTROSCOPY
What do we need to detect?
DNA
4 possible basesHuman genome: ~ 3 billion bases long
![Page 4: John Lund , Declan Ryan, Ranjana Mehta, Maryam Rahimi and Babak A. Parviz](https://reader031.vdocuments.us/reader031/viewer/2022013004/5681446e550346895db10059/html5/thumbnails/4.jpg)
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
![Page 5: John Lund , Declan Ryan, Ranjana Mehta, Maryam Rahimi and Babak A. Parviz](https://reader031.vdocuments.us/reader031/viewer/2022013004/5681446e550346895db10059/html5/thumbnails/5.jpg)
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.
![Page 6: John Lund , Declan Ryan, Ranjana Mehta, Maryam Rahimi and Babak A. Parviz](https://reader031.vdocuments.us/reader031/viewer/2022013004/5681446e550346895db10059/html5/thumbnails/6.jpg)
BACKGROUND APPROACH STRETCHING SPECTROSCOPY
Inelastic Tunneling Spectroscopy
Science 1974
V
Electron Tunneling Current
![Page 7: John Lund , Declan Ryan, Ranjana Mehta, Maryam Rahimi and Babak A. Parviz](https://reader031.vdocuments.us/reader031/viewer/2022013004/5681446e550346895db10059/html5/thumbnails/7.jpg)
BACKGROUND APPROACH STRETCHING SPECTROSCOPY
Inelastic Tunneling Spectroscopy
Low V
Electron Tunneling Current
I
V
d2 I/d
V2
V
![Page 8: John Lund , Declan Ryan, Ranjana Mehta, Maryam Rahimi and Babak A. Parviz](https://reader031.vdocuments.us/reader031/viewer/2022013004/5681446e550346895db10059/html5/thumbnails/8.jpg)
BACKGROUND APPROACH STRETCHING SPECTROSCOPY
Inelastic Tunneling Spectroscopy
High V
Electron Tunneling Current
I
V
d2 I/d
V2
V
New Tunneling Pathway
![Page 9: John Lund , Declan Ryan, Ranjana Mehta, Maryam Rahimi and Babak A. Parviz](https://reader031.vdocuments.us/reader031/viewer/2022013004/5681446e550346895db10059/html5/thumbnails/9.jpg)
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
![Page 10: John Lund , Declan Ryan, Ranjana Mehta, Maryam Rahimi and Babak A. Parviz](https://reader031.vdocuments.us/reader031/viewer/2022013004/5681446e550346895db10059/html5/thumbnails/10.jpg)
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
![Page 11: John Lund , Declan Ryan, Ranjana Mehta, Maryam Rahimi and Babak A. Parviz](https://reader031.vdocuments.us/reader031/viewer/2022013004/5681446e550346895db10059/html5/thumbnails/11.jpg)
BACKGROUND APPROACH STRETCHING SPECTROSCOPY
Procedure
![Page 12: John Lund , Declan Ryan, Ranjana Mehta, Maryam Rahimi and Babak A. Parviz](https://reader031.vdocuments.us/reader031/viewer/2022013004/5681446e550346895db10059/html5/thumbnails/12.jpg)
BACKGROUND APPROACH STRETCHING SPECTROSCOPY
Procedure
![Page 13: John Lund , Declan Ryan, Ranjana Mehta, Maryam Rahimi and Babak A. Parviz](https://reader031.vdocuments.us/reader031/viewer/2022013004/5681446e550346895db10059/html5/thumbnails/13.jpg)
BACKGROUND APPROACH STRETCHING SPECTROSCOPY
Procedure
![Page 14: John Lund , Declan Ryan, Ranjana Mehta, Maryam Rahimi and Babak A. Parviz](https://reader031.vdocuments.us/reader031/viewer/2022013004/5681446e550346895db10059/html5/thumbnails/14.jpg)
BACKGROUND APPROACH STRETCHING SPECTROSCOPY
Procedure
![Page 15: John Lund , Declan Ryan, Ranjana Mehta, Maryam Rahimi and Babak A. Parviz](https://reader031.vdocuments.us/reader031/viewer/2022013004/5681446e550346895db10059/html5/thumbnails/15.jpg)
BACKGROUND APPROACH STRETCHING SPECTROSCOPY
3D AFM image of bare HOPG before combing DNA
Results
![Page 16: John Lund , Declan Ryan, Ranjana Mehta, Maryam Rahimi and Babak A. Parviz](https://reader031.vdocuments.us/reader031/viewer/2022013004/5681446e550346895db10059/html5/thumbnails/16.jpg)
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.
![Page 17: John Lund , Declan Ryan, Ranjana Mehta, Maryam Rahimi and Babak A. Parviz](https://reader031.vdocuments.us/reader031/viewer/2022013004/5681446e550346895db10059/html5/thumbnails/17.jpg)
BACKGROUND APPROACH STRETCHING SPECTROSCOPY
3D AFM image of coiled phage ss-DNA deposited on HOPG prior
to molecular combing.
![Page 18: John Lund , Declan Ryan, Ranjana Mehta, Maryam Rahimi and Babak A. Parviz](https://reader031.vdocuments.us/reader031/viewer/2022013004/5681446e550346895db10059/html5/thumbnails/18.jpg)
BACKGROUND APPROACH STRETCHING SPECTROSCOPY
3D AFM image of phage ss-DNA completely elongated on HOPG after the completion of the molecular combing procedure.
![Page 19: John Lund , Declan Ryan, Ranjana Mehta, Maryam Rahimi and Babak A. Parviz](https://reader031.vdocuments.us/reader031/viewer/2022013004/5681446e550346895db10059/html5/thumbnails/19.jpg)
BACKGROUND APPROACH STRETCHING SPECTROSCOPY
STM results
![Page 20: John Lund , Declan Ryan, Ranjana Mehta, Maryam Rahimi and Babak A. Parviz](https://reader031.vdocuments.us/reader031/viewer/2022013004/5681446e550346895db10059/html5/thumbnails/20.jpg)
BACKGROUND APPROACH STRETCHING SPECTROSCOPY
![Page 21: John Lund , Declan Ryan, Ranjana Mehta, Maryam Rahimi and Babak A. Parviz](https://reader031.vdocuments.us/reader031/viewer/2022013004/5681446e550346895db10059/html5/thumbnails/21.jpg)
BACKGROUND APPROACH STRETCHING SPECTROSCOPY
Gold substrate
Pt/Ir STM tip
Tunnelingcurrent
A’s
Tunneling spectroscopy on gold
![Page 22: John Lund , Declan Ryan, Ranjana Mehta, Maryam Rahimi and Babak A. Parviz](https://reader031.vdocuments.us/reader031/viewer/2022013004/5681446e550346895db10059/html5/thumbnails/22.jpg)
BACKGROUND APPROACH STRETCHING SPECTROSCOPY
Spectroscopy on poly A’s
![Page 23: John Lund , Declan Ryan, Ranjana Mehta, Maryam Rahimi and Babak A. Parviz](https://reader031.vdocuments.us/reader031/viewer/2022013004/5681446e550346895db10059/html5/thumbnails/23.jpg)
BACKGROUND APPROACH STRETCHING SPECTROSCOPY
Spectroscopy on poly C’s
![Page 24: John Lund , Declan Ryan, Ranjana Mehta, Maryam Rahimi and Babak A. Parviz](https://reader031.vdocuments.us/reader031/viewer/2022013004/5681446e550346895db10059/html5/thumbnails/24.jpg)
BACKGROUND APPROACH STRETCHING SPECTROSCOPY
Spectroscopy on poly G’s
![Page 25: John Lund , Declan Ryan, Ranjana Mehta, Maryam Rahimi and Babak A. Parviz](https://reader031.vdocuments.us/reader031/viewer/2022013004/5681446e550346895db10059/html5/thumbnails/25.jpg)
BACKGROUND APPROACH STRETCHING SPECTROSCOPY
Spectroscopy on poly T’s
![Page 26: John Lund , Declan Ryan, Ranjana Mehta, Maryam Rahimi and Babak A. Parviz](https://reader031.vdocuments.us/reader031/viewer/2022013004/5681446e550346895db10059/html5/thumbnails/26.jpg)
BACKGROUND APPROACH STRETCHING SPECTROSCOPY
Deviation from blank gold
![Page 27: John Lund , Declan Ryan, Ranjana Mehta, Maryam Rahimi and Babak A. Parviz](https://reader031.vdocuments.us/reader031/viewer/2022013004/5681446e550346895db10059/html5/thumbnails/27.jpg)
BACKGROUND APPROACH STRETCHING SPECTROSCOPY
Confirmation of IETS
![Page 28: John Lund , Declan Ryan, Ranjana Mehta, Maryam Rahimi and Babak A. Parviz](https://reader031.vdocuments.us/reader031/viewer/2022013004/5681446e550346895db10059/html5/thumbnails/28.jpg)
BACKGROUND APPROACH STRETCHING SPECTROSCOPY
Measurement on stretched dsDNA
20 nm
![Page 29: John Lund , Declan Ryan, Ranjana Mehta, Maryam Rahimi and Babak A. Parviz](https://reader031.vdocuments.us/reader031/viewer/2022013004/5681446e550346895db10059/html5/thumbnails/29.jpg)
BACKGROUND APPROACH STRETCHING SPECTROSCOPY
Tip steering approach
![Page 30: John Lund , Declan Ryan, Ranjana Mehta, Maryam Rahimi and Babak A. Parviz](https://reader031.vdocuments.us/reader031/viewer/2022013004/5681446e550346895db10059/html5/thumbnails/30.jpg)
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
![Page 31: John Lund , Declan Ryan, Ranjana Mehta, Maryam Rahimi and Babak A. Parviz](https://reader031.vdocuments.us/reader031/viewer/2022013004/5681446e550346895db10059/html5/thumbnails/31.jpg)
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)
![Page 32: John Lund , Declan Ryan, Ranjana Mehta, Maryam Rahimi and Babak A. Parviz](https://reader031.vdocuments.us/reader031/viewer/2022013004/5681446e550346895db10059/html5/thumbnails/32.jpg)
Undigested phage ds-DNA on HOPG
AFM images
![Page 33: John Lund , Declan Ryan, Ranjana Mehta, Maryam Rahimi and Babak A. Parviz](https://reader031.vdocuments.us/reader031/viewer/2022013004/5681446e550346895db10059/html5/thumbnails/33.jpg)
ds-DNA Hind III digest on HOPG with 10 mM MgCl2
AFM images
![Page 34: John Lund , Declan Ryan, Ranjana Mehta, Maryam Rahimi and Babak A. Parviz](https://reader031.vdocuments.us/reader031/viewer/2022013004/5681446e550346895db10059/html5/thumbnails/34.jpg)
phage ss-DNA Hind III digest on HOPG with 10 mM MgCl2
AFM images
![Page 35: John Lund , Declan Ryan, Ranjana Mehta, Maryam Rahimi and Babak A. Parviz](https://reader031.vdocuments.us/reader031/viewer/2022013004/5681446e550346895db10059/html5/thumbnails/35.jpg)
STM imaging of ssDNA on HOPG
![Page 36: John Lund , Declan Ryan, Ranjana Mehta, Maryam Rahimi and Babak A. Parviz](https://reader031.vdocuments.us/reader031/viewer/2022013004/5681446e550346895db10059/html5/thumbnails/36.jpg)