A Synthetic Electronic Nanopore for DNA Sequencing

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 By

The 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 Cincinnati

Davis Sneider, Biomedical Engineering, The George Washington University

Saifuddin Aijaz, Chemical Engineering, University of Cincinnati

Report Reviewed By:

David Wendell, PhD, PE

Faculty Mentor

Assistant Professor

Department of Biomedical, Chemical and Environmental Engineering

University of Cincinnati

July 29, 2014

A Synthetic Electronic Nanopore for DNA Sequencing

Submitted to

The 2014 Summer NSF REU Program

Sponsored By

The National Science Foundation

Grant ID No.: DUE-0756921

College of Engineering and Applied Science

University 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, PE

REU Faculty Co-Mentor

Department of Biomedical, Chemical, and Environmental Engineering

University 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 (αHL) and Mycobacterium smegmatis porin A (MspA). αHL is 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 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 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)10

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 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.