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 – 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 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 nm in diameter Dwell time of 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 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 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

Results 24

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 Web. 13 June "Towards the 15-minute Genome." The Economist. The Economist Newspaper, 12 Mar Web. 17 June Uddin A, Yemenicioglu S, Chen C-H, Corigliano E, Milaninia K and Theogarajan L. Integration of solid-state nanopores in a 0.5Â um CMOS foundry process. Nanotechnology. IOPScience, 31 October Web. 2 July 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),

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), Niederweis, M. (2003). "Mycobacterial Porins - new channel proteins in unique outer membranes." Molecular Microbiology, 49(5), 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 and EEC ] 36

Questions? 37