Journal of the American Chemical Society, Jan 14, 2011
The distance between electrodes in a tunnel junction cannot be determined from the external movem... more The distance between electrodes in a tunnel junction cannot be determined from the external movement applied to the electrodes because of interfacial forces that distort the electrode geometry at the nanoscale. These distortions become particularly complex when molecules are present in the junction, as demonstrated here by measurements of the AC response of a molecular junction over a range of conductivities from microsiemens to picosiemens. Specific chemical interactions within the junction lead to distinct features in break-junction data, and these have been used to determine the electrode separation in a junction functionalized with 4(5)-(2-mercaptoethyl)-1H-imidazole-2-carboxamide, a reagent developed for reading DNA sequences.
4(5)-(2-mercaptoethyl)-1H-imidazole-2-carboxamide is a molecule that has multiple hydrogen bondin... more 4(5)-(2-mercaptoethyl)-1H-imidazole-2-carboxamide is a molecule that has multiple hydrogen bonding sites and a short flexible linker. When tethered to a pair of electrodes, it traps target molecules in a tunnel junction. Surprisingly large recognition-tunneling signals are generated for all naturally occurring DNA bases A, C, G, T and 5-methyl-cytosine. Tunnel current spikes are stochastic and broadly distributed, but characteristic enough so that individual bases can be identified as a tunneling probe is scanned over DNA oligomers. Each base yields a recognizable burst of signal, the duration of which is controlled entirely by the probe speed, down to speeds of 1 nm s -1, implying a maximum off-rate of 3 s -1 for the recognition complex. The same measurements yield a lower bound on the on-rate of 1 M -1 s -1. Despite the stochastic nature of the signals, an optimized multiparameter fit allows base calling from a single signal peak with an accuracy that can exceed 80% when a single ...
... Brett Gyarfas , Bryan Wiggins and KW Hipps*. Materials Science Program and Department of Ch... more ... Brett Gyarfas , Bryan Wiggins and KW Hipps*. Materials Science Program and Department of Chemistry, Washington State University, Pullman, Washington 99164-4630. J. Phys. Chem. ... 1. Chen, F. ; He, J. ; Nuckolls, C. ; Roberts, T. ; Klare, J. ; Lindsay, S. Nano Lett. ...
Gold has been the metal of choice for research on molecular tunneling junctions, but it is incomp... more Gold has been the metal of choice for research on molecular tunneling junctions, but it is incompatible with complementary metal-oxide-semiconductor fabrication because it forms deep level traps in silicon. Palladium electrodes do not contaminate silicon, and also give higher tunnel current signals in the molecular tunnel junctions that we have studied. The result is cleaner signals in a recognition-tunneling junction that recognizes the four natural DNA bases as well as 5-methyl cytosine, with no spurious background signals. More than 75% of all the recorded signal peaks indicate the base correctly.
DNA polymerases catalyze template-dependent genome replication. The assembly of a high affinity t... more DNA polymerases catalyze template-dependent genome replication. The assembly of a high affinity ternary complex between these enzymes, the double strand-single strand junction of their DNA substrate, and the deoxynucleoside triphosphate (dNTP) complementary to the first template base in the polymerase active site is essential to this process. We present a single molecule method for iterative measurements of DNA-polymerase complex assembly with high temporal resolution, using active voltage control of individual DNA substrate molecules tethered noncovalently in an αhemolysin nanopore. DNA binding states of the Klenow fragment of Escherichia coli DNA polymerase I (KF) were diagnosed based upon their ionic current signature, and reacted to with submillisecond precision to execute voltage changes that controlled exposure of the DNA substrate to KF and dNTP. Precise control of exposure times allowed measurements of DNA-KF complex assembly on a time scale that superimposed with the rate of KF binding. Hundreds of measurements were made with a single tethered DNA molecule within seconds, and dozens of molecules can be tethered within a single experiment. This approach allows statistically robust analysis of the assembly of complexes between DNA and RNA processing enzymes and their substrates at the single molecule level.
The human proteome has millions of protein variants due to alternative RNA splicing and posttrans... more The human proteome has millions of protein variants due to alternative RNA splicing and posttranslational modifications, and variants that are related to diseases are frequently present in minute concentrations. For DNA and RNA, low concentrations can be amplified using the polymerase chain reaction, but there is no such reaction for proteins. Therefore, the development of single molecule protein sequencing is a critical step in the search for protein biomarkers. Here we show that single amino acids can be identified by trapping the molecules between two electrodes that are coated with a layer of recognition molecules and measuring the electron tunneling current across the junction. A given molecule can bind in more than one way in the junction, and we therefore use a machine-learning algorithm to distinguish between the sets of electronic 'fingerprints' associated with each binding motif. With this recognition tunneling technique, we are able to identify D, L enantiomers, a methylated amino acid, isobaric isomers, and short peptides. The results suggest that direct electronic sequencing of single proteins could be possible by sequentially measuring the products of processive exopeptidase digestion, or by using a molecular motor to pull proteins through a tunnel junction integrated with a nanopore.
Journal of the American Chemical Society, Jan 14, 2011
The distance between electrodes in a tunnel junction cannot be determined from the external movem... more The distance between electrodes in a tunnel junction cannot be determined from the external movement applied to the electrodes because of interfacial forces that distort the electrode geometry at the nanoscale. These distortions become particularly complex when molecules are present in the junction, as demonstrated here by measurements of the AC response of a molecular junction over a range of conductivities from microsiemens to picosiemens. Specific chemical interactions within the junction lead to distinct features in break-junction data, and these have been used to determine the electrode separation in a junction functionalized with 4(5)-(2-mercaptoethyl)-1H-imidazole-2-carboxamide, a reagent developed for reading DNA sequences.
4(5)-(2-mercaptoethyl)-1H-imidazole-2-carboxamide is a molecule that has multiple hydrogen bondin... more 4(5)-(2-mercaptoethyl)-1H-imidazole-2-carboxamide is a molecule that has multiple hydrogen bonding sites and a short flexible linker. When tethered to a pair of electrodes, it traps target molecules in a tunnel junction. Surprisingly large recognition-tunneling signals are generated for all naturally occurring DNA bases A, C, G, T and 5-methyl-cytosine. Tunnel current spikes are stochastic and broadly distributed, but characteristic enough so that individual bases can be identified as a tunneling probe is scanned over DNA oligomers. Each base yields a recognizable burst of signal, the duration of which is controlled entirely by the probe speed, down to speeds of 1 nm s -1, implying a maximum off-rate of 3 s -1 for the recognition complex. The same measurements yield a lower bound on the on-rate of 1 M -1 s -1. Despite the stochastic nature of the signals, an optimized multiparameter fit allows base calling from a single signal peak with an accuracy that can exceed 80% when a single ...
... Brett Gyarfas , Bryan Wiggins and KW Hipps*. Materials Science Program and Department of Ch... more ... Brett Gyarfas , Bryan Wiggins and KW Hipps*. Materials Science Program and Department of Chemistry, Washington State University, Pullman, Washington 99164-4630. J. Phys. Chem. ... 1. Chen, F. ; He, J. ; Nuckolls, C. ; Roberts, T. ; Klare, J. ; Lindsay, S. Nano Lett. ...
Gold has been the metal of choice for research on molecular tunneling junctions, but it is incomp... more Gold has been the metal of choice for research on molecular tunneling junctions, but it is incompatible with complementary metal-oxide-semiconductor fabrication because it forms deep level traps in silicon. Palladium electrodes do not contaminate silicon, and also give higher tunnel current signals in the molecular tunnel junctions that we have studied. The result is cleaner signals in a recognition-tunneling junction that recognizes the four natural DNA bases as well as 5-methyl cytosine, with no spurious background signals. More than 75% of all the recorded signal peaks indicate the base correctly.
DNA polymerases catalyze template-dependent genome replication. The assembly of a high affinity t... more DNA polymerases catalyze template-dependent genome replication. The assembly of a high affinity ternary complex between these enzymes, the double strand-single strand junction of their DNA substrate, and the deoxynucleoside triphosphate (dNTP) complementary to the first template base in the polymerase active site is essential to this process. We present a single molecule method for iterative measurements of DNA-polymerase complex assembly with high temporal resolution, using active voltage control of individual DNA substrate molecules tethered noncovalently in an αhemolysin nanopore. DNA binding states of the Klenow fragment of Escherichia coli DNA polymerase I (KF) were diagnosed based upon their ionic current signature, and reacted to with submillisecond precision to execute voltage changes that controlled exposure of the DNA substrate to KF and dNTP. Precise control of exposure times allowed measurements of DNA-KF complex assembly on a time scale that superimposed with the rate of KF binding. Hundreds of measurements were made with a single tethered DNA molecule within seconds, and dozens of molecules can be tethered within a single experiment. This approach allows statistically robust analysis of the assembly of complexes between DNA and RNA processing enzymes and their substrates at the single molecule level.
The human proteome has millions of protein variants due to alternative RNA splicing and posttrans... more The human proteome has millions of protein variants due to alternative RNA splicing and posttranslational modifications, and variants that are related to diseases are frequently present in minute concentrations. For DNA and RNA, low concentrations can be amplified using the polymerase chain reaction, but there is no such reaction for proteins. Therefore, the development of single molecule protein sequencing is a critical step in the search for protein biomarkers. Here we show that single amino acids can be identified by trapping the molecules between two electrodes that are coated with a layer of recognition molecules and measuring the electron tunneling current across the junction. A given molecule can bind in more than one way in the junction, and we therefore use a machine-learning algorithm to distinguish between the sets of electronic 'fingerprints' associated with each binding motif. With this recognition tunneling technique, we are able to identify D, L enantiomers, a methylated amino acid, isobaric isomers, and short peptides. The results suggest that direct electronic sequencing of single proteins could be possible by sequentially measuring the products of processive exopeptidase digestion, or by using a molecular motor to pull proteins through a tunnel junction integrated with a nanopore.
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