A Synthetic Electronic Nanopore for DNA

Sequencing

Mr. Aaron Choi, Computer Science, Sophomore

Mr. Davis Sneider, Biomedical Engineering, Sophomore

Mr. Saifuddin Aijaz, Chemical Engineering, Pre-Junior

Mentors:

Dr. David Wendell, Assistant Professor, Environmental Engineering

Dr. Vasile Nistor, Assistant Professor, Biomedical Engineering

Ms. Elizabeth Wurtzler, Graduate Student, Environmental Engineering

1

Introduction

• Background• Goals & Tasks• Time Schedule

– What we’ve done• Inserting DNA

– What we’re looking for, what we’ve found• Findings• Conclusion

2

DNA Sequencing Methods

• 454 pyrosequencing– DNA is amplified inside water droplets (emulsion PCR) with

each drop containing a single DNA template attached to a single primer-coated bead that forms a clonal colony.

– 700 bp read length– 1 million reads per run– ≈ $2500per run

• Ion Torrent– dNTP is incorporated and is used by determining if a

hydrogen ion is released from the dNTP forming a bond– Up to 400 bp read length– Up to 80 million reads per run– ≈ $750 per run.

3

• Illumina Dye sequencing– DNA and primers are put on

a slide, amplified with polymerase so DNA colonies can form. Then nucleotides are added and a camera takes images of the nucleotides

– 50-300 bp per read– Up to 3 billion reads per run– ≈ $2000 per run

4

DNA Sequencing Methods

Current Problem

• DNA sequencing can cost several thousand dollars and take about a week

• Nanopore technology can save a lot of money and reduce the time to one day

5

Nanopores: What are they?

• They are extremely small holes.• They have potential applications for

many kinds of developing technology

Oxford Nanopore Technologies

6

• Hydrophilic inner channel, hydrophobic outer protein membrane

• 1.4-2.8 nm in diameter

• Dwell time of 8-10ms/ bp

7

Mycobacterium Smegmatis Porin A (MspA)

Nature.com

8

Alpha Hemolysin (αHL)

• Hydrophilic inner channel, hydrophobic outer protein membrane

• 1.4-4.6 nm in diameter• Dwell time of 0.0151

ms/bp

Nature.com

9

Oxford Nanopore Technology

• “Oxford Nanopore Technologies® is developing a new generation of nanopore-based electronic systems for analysis of single molecules…”

• Use α-hemolysin nanopore

10

• Commercialize GridION™ systems• Chip containing thousands

of microwells with individual charges and a single nanopore

Oxford Nanopore Technology

Hydraphile Nanopore

• A synthetic nanopore, created by Dr. George Gokel at University of Missouri, St. Louis

• Lariat Ethers– Excellent cation selectivity– Excellent binding and release kinetics

Royal Society of Chemistry http://pubs.rsc.org/en/content/articlehtml/2000/cc/a903825f

11

Why use it?

Synthetic–More functional over broader

range–Easier to store

12

Applications

• We could detect cancer earlier and much more efficiently

• DNA sequencing allows us to find many genetic disorders

• Ability to detect viruses

13

Our Goals

• To determine which buffer works best• To test the hydraphile’s sequencing

ability

14

Tasks

• Use Clampfit to analyze data from four buffers

• Run items through nanopore:– DNA– Ion Solutions

15

Time Schedule

16

Conclusion from buffers tests

• Out of the four solutions used, it was determined that KCl is the best choice to use for nanopore sequencing as it gives a more stable membrane.

17

Potassium Buffer

• 1M KCl Buffer, with 5mM HEPES

• pH 7.8• Able to get data

with ease• Analyzing Data

– Clampex• 100< data points

Glogster.com

18

Nanopore Insertion

19

Pore Diameter Estimation

20

Use event data to:• Find conductance of individual

events• Estimate pore diameter by

comparing conductance to that of other pores

Detecting DNA Current Change

• Inserting DNA causes resistances in the current across the membrane– Negative charge across membrane

www.ks.uiuc.edu

21

DNA Passing

22

What We Measured

• 2 major measurements– Blockage %– Dwell Time (ms)

• DNA length– 250 bp– 500 bp– 1,000 bp– 2,500 bp– 5,000 bp

23

Results24

Results

25

26

Results

27

Results

28

Results

29

Results

30

Results

31

Results

• Found: • blockage % for multiple lengths

of DNA• dwell times for multiple lengths

of DNA• Proved that DNA can pass

through the hydraphile nanopore

What Does It Mean & What Is It Useful For?

• Blockage %– Tells us how much of the nanopore has been

blocked– Helps us identify approximate width of DNA/RNA

strand

• Event Duration– Tells us how long it took the DNA

segment to pass through the nanopore– Helps us identify approximate length

of the DNA/RNA strand

32

Conclusions

• Hydraphile:– Can pass DNA– Long dwell times are good for sequencing

• Requires more research– More conformations suggested by

conductance data

33

References

• Gokel, George. Hydraphiles: Design, Synthesis and Analysis of a Family of Synthetic, Cation-conducting Channels. Tech. Royal Society of Chemistry, 24 Dec. 1999. Web. 13 June 2014.

• "Towards the 15-minute Genome." The Economist. The Economist Newspaper, 12 Mar. 2011. Web. 17 June 2014.

• Uddin A, Yemenicioglu S, Chen C-H, Corigliano E, Milaninia K and Theogarajan L. Integration of solid-state nanopores in a 0.5 um CMOS foundry process. Nanotechnology. IOPScience, 31 October 2013. Web. 2 July 2014.

• Wendell, D., Jing, P., Geng, J., Subramaniam, V., Lee, T. J., Montemagno, C., and Guo, P. (2009). "Translocation of double-stranded DNA through membrane-adapted phi29 motor protein nanopores." Nat Nano, 4(11), 765-772.

34

References (cont.)

• Butler, T. Z., Pavlenok, M., and Derrington, I. M. (2008). "Single-molecule DNA detection with an engineered MspA protein nanopore." Proc. Natl. Acad. Sciences, 105(52), 20647-20652.

• Niederweis, M. (2003). "Mycobacterial Porins - new channel proteins in unique outer membranes." Molecular Microbiology, 49(5), 1167-1177.

• Shoseyov, O., and Levy, I. (2008). NanoBioTechnology Bio Inspired Devices and Materials of the Future, Humana Press, New Jersey.

 

35

Thank You!

• We would like to thank NSF for funding our research [Grant ID No.: DUE-0756921 and EEC-1004623]

36

Questions?

37