hydrophobic interactions retard proteins upon translocation through silicon nitride nanopores

1
1074-Pos Board B829 Quantifying Short-Lived Events in Multi-State Ionic Channel Measurements Arvind Balijepalli 1,2 , Canute Vaz 1 , Jessica Ettedgui 1 , Andrew T. Cornio 1 , Joseph W.F. Robertson 1 , Kin P. Cheung 1 , Richard W. Pastor 2 , John J. Kasianowicz 1 . 1 NIST, Gaithersburg, MD, USA, 2 NIH, Rockville, MD, USA. We present a new technique that considerably improves the resolution and accuracy of single molecule measurements with nanopores. Molecular inter- actions with nanopores are characterized by electrical measurements of discrete changes in the channel conductance. By representing physical com- ponents of the system with electrical equivalents, we used circuit theory to model response of the system to a stimulus (e.g., a molecule entering the channel). This allowed us to characterize short-lived events where the ionic current does not reach a steady state value, and were previously not analyzed. Applying this technique to measurements of poly(ethylene glycol) (PEG) molecules with the a-hemolysin (aHL) nanopore resulted in remarkable im- provements in accuracy and the number of detected events. When measuring polydisperse PEG (mean molecular weights of 400 g/mol and 600 g/mol), the new method recovered z 18-fold more events per unit time, compared with existing techniques, and discriminated molecules with as few as 8 monomers (PEG8). We validated the measurement of PEG with an aHL nanopore using results from a recently published study (Balijepalli et. al, J Am Chem Soc, 135: 7064, 2013) that refined a previous analytical theory with molecular dy- namics simulations. Fitting this model to the newly obtained experimental data resulted in excellent agreement of both the blockade depth (the ratio of the ionic current when a molecule occupies the pore to the open channel current) and the residence times of the molecule in the channel, over the entire measurement range (PEG8 to PEG19). Finally, we applied the new analysis technique to recover the sequence of a known DNA strand with 26 bases, from a published ionic current trace (Manrao et. al, Nat. Biotechnol. 30: 349, 2012). The technique detected systematic fluctuations in the ionic current that were as small as 0.9 5 0.04 pA. 1075-Pos Board B830 Fast, Label-Free Force Spectroscopy of Histone-DNA Interactions in Individual Nucleosomes using Nanopores Andrey Ivankin 1 , Spencer Carson 1 , Shannon R.M. Kinney 2 , Meni Wanunu 1 . 1 Physics, Northeastern University, Boston, MA, USA, 2 Pharmaceutical and Administrative Sciences, Western New England University, Springfield, MA, USA. Eukaryotic DNA is packaged into nucleosomes, each comprised of ~147 base pairs of DNA wrapped 1.7 turns around histone octamers. Nucleosome organi- zation inherently limits the accessibility of regulatory proteins to genes, which serves as a sophisticated mechanism to control transcription, replication, and repair processes in a cell. While it is known that dynamic modulation of nucle- osomal structures is achieved via epigenetic modifications of histone proteins and DNA and by ATP-dependent remodelers, the mechanisms by which these enzyme-assisted modifications affect intranucleosomal interactions remain elusive. Forster resonance energy transfer (FRET), atomic force microscopy, and optical tweezers, all of these methods, though have provided valuable insight, require time-consuming sample labeling and/or surface immobilization. Herein we report a novel approach for fast, label-free probing of DNA-histone interactions in individual nucleosomes. We use small 3 nm diameter solid- state nanopores to unravel individual DNA/histone complexes for the first time. Three force regimes can be distinguished: (1) at low voltages no nucleosome-related events are detected; (2) at moderate voltages nucleosome collisions with the pore are observed, although force is insufficient for nucleosome unraveling; (3) at voltages above the critical nucleosomes are captured and unraveled by the pore. We also find that the unraveling time depends on the applied electrophoretic force, and our results are in line with pre- vious studies that employ optical tweezers. Our approach for studying nucleo- somal interactions can greatly accelerate the understanding of fundamental mechanisms by which transcription, replication, and repair processes in a cell are modulated through DNA-histone interactions, as well as in diagnosis of dis- eases with abnormal patterns of DNA and histone modifications. 1076-Pos Board B831 Controlling the Mechanism of DNA transport through Synthetic Nanopores Meni Wanunu. Physics, Northeastern University, Boston, MA, USA. Solid-state nanopores have emerged as a useful tool for studying biopolymers. The ability to map regions of interest along stretched biopolymers, as well as to detect subtle changes in conformation, makes nanopores attractive single- molecule sensors. Using solid-state nanopores with diameters in the range of 3-10 nm, DNA transport has been too fast but seems to be more regulated. In contrast, transport through smaller pores in the 3-5 nm range is noticeably slower, yet sticking and other interactions complicate the transport dynamics. Therefore, a full understanding of the translocation process requires exploring regimes where polymer linearization is coupled to regulated transport. In this study, we report on an interesting nanopore geometry regime that allows for regulated DNA transport process, while slowing down DNA in order to achieve true detection from individual base pairs. We will show that transport matches theoretical prediction in this geometric regime, and that these results allow us to obtain quantitative information on polymer length with unprece- dented detail. 1077-Pos Board B832 Hydrophobic Interactions Retard Proteins upon Translocation through Silicon Nitride Nanopores Ruoshan Wei, Ulrich Rant. Walter Schottky Institut, Technische Universitaet Munich, Garching, Germany. Over the last years, translocation experiments with proteins through solid-state nanopores have consistently produced anomalously long residence times that are far beyond a ballistic passage process. It has been suggested that attractive interactions to the pore wall might cause a retardation of the protein, but the origin of these interactions has remained elusive up to now. Here we present experimental evidence that this interaction is of hydrophobic nature: We quantitatively compared the translocation times of a variety of different proteins from our own experiments as well as from other authors and analyzed the hydrophobicity of the respective protein surfaces from available atomic structure data. We found that the occurrence of very long translocation times correlates with the existence of large hydrophobic patches on the protein surface. This strongly suggests that hydrophobic interactions are a dominant factor in determining the passage time of proteins through artificial nanopores, and that it is necessary to engineer the hydrophobicity of artificial nanopores in order to control the passage of proteins through the pore. 1078-Pos Board B833 Direct and Simultaneous Force and Current Measurements of Single- Stranded DNA in Synthetic Nanopores Edward M. Nelson, Hui Li, Gregory Timp. Department of Electrical Engineering, University of Notre Dame, Notre Dame, IN, USA. We report direct and simultaneous measurements of the forces and currents associated with the translocation of a single-stranded DNA molecule tethered to an AFM cantilever (Figure a) through synthetic pores 1.2 to 3.5 nm in diameter in 852 nm thick silicon nitride membranes. These measurements were performed to determine the force required and the electrical signal available for sequencing a single molecule of DNA in a pore small enough to affect the configuration of the molecule. The measurements revealed that ssDNA either translocated the nanopore in a ‘‘stick-slip’’ motion character- ized by multiple stretching and rupture events or slid with a relatively constant net force between 10 and 60 pN (Figure b). While the tip moved at a constant velocity, minute <1 pN and <20 pA fluctuations in the force and current, respectively, were observed every 0.35-0.60 nm (Figure c) in homo/heteropolymers of ssDNA, which were attributed to individual nucleotides translating through the nanopore in a turnstile-like motion. These results indicate that synthetic nanopores <2 nm in diameter may offer the resolution to sequence individual bases of DNA, provided the molecule slides through the pore. 1079-Pos Board B834 Osmotically-Driven Transport through Carbon Nanotube Pores Kyunghoon Kim 1,2 , Jia Geng 2,3 , Ramya Tunuguntla 4 , Caroline Ajo-Franklin 2,5 , Costas P. Grigoropoulos 1 , Aleksandr Noy 3,4 . 1 Mechanical Engineering, Univ. of California, Berkeley, Berkeley, CA, USA, 2 The Molecular Foundry, Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA, 3 School of Natural Sciences, Univ. of California, Merced, Merced, CA, USA, 4 Biology and Biotechnology Division, Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, Livermore, CA, USA, 5 Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. Sunday, February 16, 2014 213a

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Page 1: Hydrophobic Interactions Retard Proteins upon Translocation through Silicon Nitride Nanopores

Sunday, February 16, 2014 213a

1074-Pos Board B829Quantifying Short-Lived Events in Multi-State Ionic ChannelMeasurementsArvind Balijepalli1,2, Canute Vaz1, Jessica Ettedgui1, Andrew T. Cornio1,Joseph W.F. Robertson1, Kin P. Cheung1, Richard W. Pastor2,John J. Kasianowicz1.1NIST, Gaithersburg, MD, USA, 2NIH, Rockville, MD, USA.We present a new technique that considerably improves the resolution andaccuracy of single molecule measurements with nanopores. Molecular inter-actions with nanopores are characterized by electrical measurements ofdiscrete changes in the channel conductance. By representing physical com-ponents of the system with electrical equivalents, we used circuit theory tomodel response of the system to a stimulus (e.g., a molecule entering thechannel). This allowed us to characterize short-lived events where the ioniccurrent does not reach a steady state value, and were previously not analyzed.Applying this technique to measurements of poly(ethylene glycol) (PEG)molecules with the a-hemolysin (aHL) nanopore resulted in remarkable im-provements in accuracy and the number of detected events. When measuringpolydisperse PEG (mean molecular weights of 400 g/mol and 600 g/mol), thenew method recovered z 18-fold more events per unit time, compared withexisting techniques, and discriminated molecules with as few as 8 monomers(PEG8). We validated the measurement of PEG with an aHL nanopore usingresults from a recently published study (Balijepalli et. al, J Am Chem Soc,135: 7064, 2013) that refined a previous analytical theory with molecular dy-namics simulations. Fitting this model to the newly obtained experimentaldata resulted in excellent agreement of both the blockade depth (the ratioof the ionic current when a molecule occupies the pore to the open channelcurrent) and the residence times of the molecule in the channel, over theentire measurement range (PEG8 to PEG19). Finally, we applied the newanalysis technique to recover the sequence of a known DNA strand with 26bases, from a published ionic current trace (Manrao et. al, Nat. Biotechnol.30: 349, 2012). The technique detected systematic fluctuations in the ioniccurrent that were as small as 0.9 5 0.04 pA.

1075-Pos Board B830Fast, Label-Free Force Spectroscopy of Histone-DNA Interactions inIndividual Nucleosomes using NanoporesAndrey Ivankin1, Spencer Carson1, Shannon R.M. Kinney2, Meni Wanunu1.1Physics, Northeastern University, Boston, MA, USA, 2Pharmaceutical andAdministrative Sciences, Western New England University, Springfield, MA,USA.Eukaryotic DNA is packaged into nucleosomes, each comprised of ~147 basepairs of DNA wrapped 1.7 turns around histone octamers. Nucleosome organi-zation inherently limits the accessibility of regulatory proteins to genes, whichserves as a sophisticated mechanism to control transcription, replication, andrepair processes in a cell. While it is known that dynamic modulation of nucle-osomal structures is achieved via epigenetic modifications of histone proteinsand DNA and by ATP-dependent remodelers, the mechanisms by which theseenzyme-assisted modifications affect intranucleosomal interactions remainelusive. Forster resonance energy transfer (FRET), atomic force microscopy,and optical tweezers, all of these methods, though have provided valuableinsight, require time-consuming sample labeling and/or surface immobilization.Herein we report a novel approach for fast, label-free probing of DNA-histoneinteractions in individual nucleosomes. We use small 3 nm diameter solid-state nanopores to unravel individual DNA/histone complexes for thefirst time. Three force regimes can be distinguished: (1) at low voltages nonucleosome-related events are detected; (2) at moderate voltages nucleosomecollisions with the pore are observed, although force is insufficient fornucleosome unraveling; (3) at voltages above the critical nucleosomes arecaptured and unraveled by the pore. We also find that the unraveling timedepends on the applied electrophoretic force, and our results are in line with pre-vious studies that employ optical tweezers. Our approach for studying nucleo-somal interactions can greatly accelerate the understanding of fundamentalmechanisms by which transcription, replication, and repair processes in a cellare modulated through DNA-histone interactions, as well as in diagnosis of dis-eases with abnormal patterns of DNA and histone modifications.

1076-Pos Board B831Controlling the Mechanism of DNA transport through SyntheticNanoporesMeni Wanunu.Physics, Northeastern University, Boston, MA, USA.Solid-state nanopores have emerged as a useful tool for studying biopolymers.The ability to map regions of interest along stretched biopolymers, as well asto detect subtle changes in conformation, makes nanopores attractive single-

molecule sensors. Using solid-state nanopores with diameters in the range of3-10 nm, DNA transport has been too fast but seems to be more regulated. Incontrast, transport through smaller pores in the 3-5 nm range is noticeablyslower, yet sticking and other interactions complicate the transport dynamics.Therefore, a full understanding of the translocation process requires exploringregimes where polymer linearization is coupled to regulated transport. In thisstudy, we report on an interesting nanopore geometry regime that allows forregulated DNA transport process, while slowing down DNA in order toachieve true detection from individual base pairs. We will show that transportmatches theoretical prediction in this geometric regime, and that these resultsallow us to obtain quantitative information on polymer length with unprece-dented detail.

1077-Pos Board B832Hydrophobic Interactions Retard Proteins upon Translocation throughSilicon Nitride NanoporesRuoshan Wei, Ulrich Rant.Walter Schottky Institut, Technische Universitaet Munich, Garching,Germany.Over the last years, translocation experiments with proteins through solid-statenanopores have consistently produced anomalously long residence times thatare far beyond a ballistic passage process. It has been suggested that attractiveinteractions to the pore wall might cause a retardation of the protein, but theorigin of these interactions has remained elusive up to now.Here we present experimental evidence that this interaction is of hydrophobicnature: We quantitatively compared the translocation times of a variety ofdifferent proteins from our own experiments as well as from other authorsand analyzed the hydrophobicity of the respective protein surfaces fromavailable atomic structure data. We found that the occurrence of very longtranslocation times correlates with the existence of large hydrophobic patcheson the protein surface. This strongly suggests that hydrophobic interactions area dominant factor in determining the passage time of proteins through artificialnanopores, and that it is necessary to engineer the hydrophobicity of artificialnanopores in order to control the passage of proteins through the pore.

1078-Pos Board B833Direct and Simultaneous Force and Current Measurements of Single-Stranded DNA in Synthetic NanoporesEdward M. Nelson, Hui Li, Gregory Timp.Department of Electrical Engineering, University of Notre Dame,Notre Dame, IN, USA.We report direct and simultaneous measurements of the forces and currentsassociated with the translocation of a single-stranded DNA molecule tetheredto an AFM cantilever (Figure a) through synthetic pores 1.2 to 3.5 nm indiameter in 852 nm thick silicon nitride membranes. These measurementswere performed to determine the force required and the electrical signalavailable for sequencing a single molecule of DNA in a pore small enoughto affect the configuration of the molecule. The measurements revealed thatssDNA either translocated the nanopore in a ‘‘stick-slip’’ motion character-ized by multiple stretching and rupture events or slid with a relativelyconstant net force between 10 and 60 pN (Figure b). While the tip movedat a constant velocity, minute <1 pN and <20 pA fluctuations in the forceand current, respectively, were observed every 0.35-0.60 nm (Figure c)in homo/heteropolymers of ssDNA, which were attributed to individual

nucleotides translatingthrough the nanopore ina turnstile-like motion.These results indicate thatsynthetic nanopores <2nm in diameter may offerthe resolution to sequenceindividual bases of DNA,provided the moleculeslides through the pore.

1079-Pos Board B834Osmotically-Driven Transport through Carbon Nanotube PoresKyunghoon Kim1,2, Jia Geng2,3, Ramya Tunuguntla4,Caroline Ajo-Franklin2,5, Costas P. Grigoropoulos1, Aleksandr Noy3,4.1Mechanical Engineering, Univ. of California, Berkeley, Berkeley, CA,USA, 2The Molecular Foundry, Materials Sciences Division, LawrenceBerkeley National Laboratory, Berkeley, CA, USA, 3School of NaturalSciences, Univ. of California, Merced, Merced, CA, USA, 4Biology andBiotechnology Division, Physical and Life Sciences Directorate, LawrenceLivermore National Laboratory, Livermore, CA, USA, 5Physical BiosciencesDivision, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.