A Synthetic Electronic Nanopore for DNA Sequencing

Submitted To

The 2014 Summer NSF CEAS REU Program Part of

NSF Type 1 STEP Grant

&

NSF REU Site Sustainable Urban Environments

Sponsored ByThe National Science Foundation

Grant ID No.: DUE-0756921 and EEC-1004623

College of Engineering and Applied Science University of Cincinnati

Cincinnati, Ohio

Prepared By

Aaron Choi, Computer Science, University of CincinnatiDavis Sneider, Biomedical Engineering, The George Washington University

Saifuddin Aijaz, Chemical Engineering, University of Cincinnati

Report Reviewed By:

David Wendell, PhD, PE

Faculty MentorAssistant Professor

Department of Biomedical, Chemical and Environmental EngineeringUniversity of Cincinnati

July 29, 2014

A Synthetic Electronic Nanopore for DNA Sequencing

Submitted toThe 2014 Summer NSF REU Program

Sponsored ByThe National Science Foundation

Grant ID No.: DUE-0756921

College of Engineering and Applied ScienceUniversity of Cincinnati

Cincinnati, Ohio

Prepared By:Mr. Aaron Choi, Computer Science, University of Cincinnati

Mr. Davis Sneider, Biomedical Engineering, The George Washington University

Mr. Saifuddin Aijaz, Chemical Engineering, University of Cincinnati

Report Review By:

Margaret J. Kupferle, PhD, PEREU Faculty Co-Mentor

Department of Biomedical, Chemical, and Environmental EngineeringUniversity of Cincinnati

July 18, 2014

Abstract _____________________________________________________________________________________

DNA sequencing currently impacts numerous aspects of daily life. With sequencing, it is possible to: identify genes for genetic disorders such as cancer or cystic fibrosis, identify suspects in crimes, differentiate different pathogens and be used in the field of genetic engineering. It is expected to advance the fields of medicine and biotechnology. Sequencing will be helpful in combatting genetic disease and stopping the spread of pathogens. However, current sequencing methods are expensive and time consuming, prohibiting widespread use. Nanopore sequencing, could become an inexpensive alternative to current methods, but studies have been limited to only a few nanopores. This study examines the stability and selectivity of the hydraphile nanopore during DNA passage events. The hydraphile nanopore has been identified as a candidate for future sequencing work due to its demonstrated stability and selectivity. Sequencing methods blah, blah, blah...However, current sequencing methods are expensive and time consuming, prohibiting widespread use. Nanopore sequencing could become an inexpensive alternative to current methods, but studies have been limited to only a few nanopores. This study examines the stability and selectivity of the hydraphile nanopore during DNA passage events. The hydraphile nanopore has been identified as candidate for future sequencing work due to its demonstrated stability and selectivity. .

Introduction________________________________________________________________________________

DNA sequencing is essential to understanding the code of life, allowing identification of any DNA abnormalities. These abnormalities are the causes of many serious mutations and diseases such as cancer, cystic fibrosis, and sickle cell. (National Human Genome Research Institute, 2011) . Many sequencing techniques are currently available including the shotgun method, the Illumina dye sequencing, Sanger sequencing and 454 sequencing.1 (National Human Genome Research Institute, 2011) Current generation sequencing methods such as Illumina are capable of generating up to 300 million unique reads per run. However, these methods can cost $10,000a few thousand dollars and take up to a week to completerequire a substantial amount of money and time,month to amplify, analyze, and get the results from a sample. (University of Wisconsin Biotechnology Center, 2014) as well as amplification of the sample. This required amplification step can introduce bias, preventing a complete analysis. They require amplification of DNA as well as other expensive materials.Other issues involve individual processes such as tag counting which leads to greater error as well as bias in the ratios. (Shendure, 2008) DNA sequencing can cost $10,000 and take up to a week  with our current technology. Next generation technologies aim to reduce costs to $1,000 and increase efficiency. (National Institutes of Health, 2013)y.1

With the present expense and time of current sequencing technologies, scientists have discovered a cheaper more viable alternative in nanoporesNanopores have been examined as an alternative to current sequencing techniques. During nanopore

sequencing, DNA translocates through the pore, resulting in varying electrical signals depending on the nitrogen base present in the pore. Advantages to nanopore sequencing methods include,Nanopores have the ability to read long base pair chains with great speed, (Timp, 2010)d.2 and the potential for single molecule sequencing. A single molecule sequencing method serves to reduce the cost and time required in current methods and removes any bias that could be introduced by amplification and tag counting methods. Additionally, nanopores remove the cost of amplification procedures with their ability of single molecule sensitivity.  Despite the clear positives of such sequencing, drawbacks still are present. These drawbacks are evident in pore structure and molecular configuration. (Timp, 2010).2 The configuration is vital to which ions are able to pass through, making the proper configuration of the nanopore a crucial factor.

Two major nanopores have been introduced and widely studied for their sequencing ability, Alpha Hemolysin ( HL) and Mycobacterium smegmatis porin A α(MspA). HL isα a heptamer, composed of a hydrophilic inner channel and a hydrophobic outer layer. (Song 1996) This protein structure has a diameter of approximately 14 Å-46 Å, allowing it to only pass single stranded DNA. (Song 1996) The MspA pore is a tetramer, also being composed of a hydrophobic outer protein layer (Neiderweis 2003) and a diameter of 21.4 ±7Å. (Shoseyov and Levy 2008)Nanopore sequencing is possible by applying an electric current across the molecular configuration which in turn is given a specific resistance when DNA translocates through the nanopore. As each individual base passes through, different electrical signals are received, allowing the base-pair order to be determined. In order to thread the DNA however, the pore must first be large enough for DNA to fit through. Double-stranded DNA has a diameter around 2.6-2.9 nm. Single-stranded DNA in comparison to dsDNA has about half of the diameter which calls for a pore of around 1-1.5 nm in diameter for ssDNA to fit through.

Nanopores are typically made up of proteins, however the nanopore used in this experiment isIn order to minimize the drawbacks associated with other nanopores, this study examines a synthetic nanopore composed of hydraphiles. The hydraphiles are composed of Lariat ethers and carbon chains ,(Fig.ure 1) highlighted in the figure below.

Fig. 1

The organic compound encircled in Fig. 1the figure is one of the lariat ethers, each of which have a diameter of about 1 nm.which compose is tthe central structure of the hydraphile. The hydraphile is 40 Åangstroms long in total, with each of the carbon chains being about 15 Åangstroms long.  This hydraphile nanopore has been used in experiments before, one of which tested its usagepreviously tested as a possible cure for cancer by upsetting the ion balance in the cancer cells and an experiment which discovered this conformation of the hydraphile with information on the fluorescent properties of the pore. (Negin et al., 2008), (Gokel 2000)3,4 While studies have been performed to further examine the properties of the hydraphile and investigate its toxicity, DNA passage characteristics have yet to be examined. Other experimentation included testing of other molecules to use as the head groups, how short the optimal length of the hydraphile should be, and the hydraphile’s toxicity to cells (Negin et al., 2008), (Gokel 2000) . 3,4 This study investigates the performance of the hydraphile nanopore under various buffer conditions and demonstrates DNA translocation through the nanopore.

The hydraphile nanopore was created by Dr. George Gokel and its ability to sequence and sense DNA isn’t fully tapped. The ability of this nanopore will be tested by running multiple base pair lengths of DNA through the pore, collecting data on the resistances caused by the passage as well as the dwell time of the specific lengths. In addition to sequencing, sensing of molecules such as the norovirus will aid in the detection of diseases and viruses, helping to treat and eliminate them from the human body. The hydraphile nanopore is expected to be able to run DNA through its core.

Materials & Methods______________________________________________________________________

All chemicals were purchased from Sigma Aldrich unless otherwise stated.  The hydraphile nanopore was a generous gift from George Gokel.  Hydraphile vesicles were prepared with 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPHPC, Avanti Polar Lipids, Alabaster, AL) at a concentration of 10mg/mL in a film rehydration

Fig. 1: Hydraphile Diagram

process.  After rehydration, vesicles were extruded at 200nm and stored at -80oC until use.  Three buffers of 5mM HEPES and either 1M KCl, MgCl2, or NaCl at pH 7.8 and a seawater solution (National Center for Marine Algae and Biota, East Boothbay, ME) were used for initial conductance measurements.  A minimum of 100 data points were taken per solution.  For DNA passage data, 25ng of Fermentas NoLimits DNA fragments (250, 500, 1000, and 2500 bp) were used (Thermo Scientific, Pittsburgh, PA).  A minimum of 200 data points were taken per length.  

Bilayer lipid membrane (BLM) measurements were taken using the BCH-1 chamber (Eastern Scientific LLC, Rockville, MD).  Electrical measurements were taken using the Axon Axopatch 200B and Axopatch Digidata (Molecular Devices, Carlsbad, CA) and data collection and analysis were performed using Clampex and Clampfit software.  Voltages ranged from 50-100mV and membranes were painted using a DPHPC-hexane solution.  

Results and Discussion___________________________________________________________________

Conductance of the hydraphile nanopore was examined using seawater and buffers containing NaCl, MgCl2, and KCl. Of the four buffers tested, the hydraphile nanopore showed conductance in the presence of seawater, NaCl, and KCl, while no conductance was observed in the presence of MgCl2... The KCl buffer has an average conductance of 1.76 nS, the Seawater buffer has an average resistance of 1.06 nS, and the NaCl buffer has an average resistance of 1.54 nS (Fig. 2). The hydraphile showed the least variability in the Seawater buffer; however, the KCl buffer was chosen for continued DNA passage work due to improved pore stability and the ability to take large amounts of data as the Seawater buffer resulted in blocked pores and the NaCl buffer caused rapid membrane collapse.

Conductance of the hydraphile nanopore was examined using seawater and buffers containing NaCl, MgCl2, and KCl. Out ofOf the four buffers which were tested, only three were successful, giving data showing that a current of ions was indeed able to flow through the hydraphile nanoporethe hydraphile nanopore showed conductance in the presence of seawater, NaCl, and KCl, while no conductance was observed in the presence of MgCl2. Out of the three

buffers that were successful in allowing the ion current to pass through, there was a varying effect of the buffer on the current. After analyzing the data in Clampfit, the average jump for each nanopore opening was determined. The KCl buffer has an

Fig. 2: Buffer and average conductance.Fig. 2 2: Buffer and average conductance.

average resistance of 1.76nS nano siemens and the NaCl buffer has an average resistance of 1.54 nano siemensnS. The hydraphile showed the least variability in

the NaCl buffer; however, the KCl buffer was chosen for continued DNA passage work due to improved pore stability.

The buffer that is used for DNA sequencing was determined by comparing their resistances. Whichever buffer has the least standard deviation should be used due to

lower variance in measurements because the lower deviation allows the researchers have a more reliable buffer which in turn allows them to have more certainty in disturbances being blockages by DNA rather than instability in the membrane due to the buffer. However, the KCl buffer was used due to greater

membrane stability during testing, which is vital to the passage of DNA through the nanopore.

Fig. 2: Relationship between pore diameter and conductance for three nanopores. [uses data from 5]Fig. 2 [uses data from 5]

The estimated conductance per pore obtained from the KCL KCl buffer solution data is approximately 2.8 nano SiemensnS with a standard deviation of nearly 1.3 nano Siemens S ([Fig. 23)]. Using a correlation between conductance and nanopore size it is estimated that the hydraphile nanopore is within a range of 2.1 to 3.1 nmanometers in diameter, large enough to pass double stranded DNA (diameter ≈ =2nm). making it large enough to pass double stranded DNA, which is approximately 2 nanometers wide. In comparison, the protein nanopore GP10 is about 3.6 nanometers in diameter and can be used to pass double stranded DNA as well. (Wendell, 2009)1 0

Fig. 3: Relationship between pore diameter and conductance for three nanopores. [Uses data from 11]

Fig. 3

Fig. 4

Other results as shown in the charts above obtained over the duration of experimentation were dwell time ranges for various DNA strand lengths of . 250, 500, 1000, 2500, and 5000 basepairs (bp)p base pair lengths were run through the nanopore causing negative resistancesused to determine the DNA passage characteristics of the hydraphile nanopore. The 250 bp strands had The charts above show that the a majority of DNA passage events occur in between two to four

Fig. 34: Graph of the blockage percentages compared

to the dwelltime in ms in the KCl buffer

Fig. 45: Graph of the blockage percentage versus the

dwell time in ms in the Kcl buffer

milli -seconds [Fig. 35], with typical blockages of and cause the pore to be 40%- to 80% blocked [Fig. 4] for DNA which is 250 base pairs long. The data obtained will be used to help predict average dwell times for various lengths of DNA and RNA was used compare the resistance caused by single stranded DNA to the resistance caused by double stranded DNA. The 250 bp strands showed 46 XXX instances ( 7.97YYY%) of blockage events over 100%. Also note that there are some events which appear to have a blockage percent much larger than 100 percent. Since these values have been calculated using an average conductance value with aGiven the large pore size estimate, it islis likely that a range of pore sizes and configurations are present, each with varying conductancesconductance. standard deviation these large valuesThe blockage events over 100% can could be representative of small pores which would have a greater blockage percentage for the same length of DNA.

Fig. 67: Graph of the blockage percentage versus the dwell time in ms in the KCl buffer for 500 bp.

Fig. 767: Graph of the blockage percentage versus the dwell time in ms in the KCl buffer for 1000 bp.

T Including those points, the average blockage percent of the 250 bp [Fig. 5] in KCL bufferase pair sample was 75% with an average dwell time per bpase pair of 0.01.19 *10^-05 msS, a relatively long amount of time for a single base pair passage. This compares well to -α alpha – hemolysin, which has a dwell time of 0.0151 ms per bp This showsing that the hydraphile nanopore is more ssensitive which is promising enough for nanopore sequencing technology as long dwell times and high sensitivity high sensibility are key. DNA segments of 500 bp [Fig. 6], 1000 bp [Fig. 7], and 2500 bp [Fig. 8] in KCl buffer had average dwell times per bp of 0.0085 ms, 0.0020 ms, and 0.0010 ms, respectively. When analyzing data for 250 bp [Fig. 10]Base Pair DNA segments in the NaCl buffer the average dwell time per bp was 0.012 ms, just larger than the dwell time observed in KCl. The 5000 bp [Fig. 9] DNA segments in NaCl buffer had an average dwell time per bp of 0.00087 ms.

The shorter dwell times per base pair found on increasingly longer DNA segments can be attributed to higher charges which are applied by larger molecules.

Fig. 5: Graph of the blockage percentage versus the dwell time in mS in the KCl buffer.Fig. 5

Something to note is that events found for 500 bp [Fig. 65.] and and 2500 bp [Fig. 8] base pairs there were no events with a blockage percent greater than 100 percent% while the 250 bp [Fig. 5] had 46 events and 1000 bp [Fig. 7] had 28 events greater than 100% blockage respectively. This may be due to the source of the DNA fragments, as Tthe DNA used for these two tests were both from a different batch from than the DNA used for 250 bp and 1000 bp.base pairs, which may explain this pattern.

Fig. 11: DNA in electrophoresis gel

DNA passage through hydraphile nanopore is confirmed by gel electrophoresis. The red box in Fig. 11 highlights the tested DNA that is present in the gel.

The average blockage percent of the 250 base pair sample was 75% with an average dwell time per base pair of 1.19*10^-05 mS, a relatively long amount of time for a single base pair passage. This shows that the hydraphile nanopore is more sensitive which is promising for nanopore sequencing technology as long dwell times and high sensibility are key.

Some generalizations about the performance of the nanopore hereBased upon the longer dwell times in some instances in and the large blockage percentage, the hydraphile can be identified as a good sensor. It has potential to be a good nanopore option for sequencing due to it’s sensibility however further studies must be conducted.. From here, more tests with the different base pair lengths would need to be run due to the broad range of resultslarge variations seen in the results. This would either confirm that the hydraphile simply produces a broad range of results or that the odd values were outliers. Also, some greater understanding of the configuration of the lariat ethers would perhaps disprove the blockage percentages whichpercentages that are over 100%, leading to a more accurate average blockage percentage. However due to time constraints, an understanding and study of this complex concept wasn’t reached.

Acknowledgements ________________________________________________________________________

This material is based upon work supported by the National Science Foundation under Grant Nos. DUE 0756921 and EEC 1004623. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.

We would like to thank Dr. David Wendell and Ms. Elizabeth Wurtzler for their lab equipment and endless help in our research. We finally would like to thank the NSF for our funding through grant no: DUE - 075692.

References __________________________________________________________________________________

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