Nanoscale pores have potential to be used as biosensors and are an established tool for analysing the structure and composition of single DNA or RNA molecules 1-3 . Recently, nanopores have been used to measure the binding of enzymes to their DNA substrates 4,5 . In this technique, a polynucleotide bound to an enzyme is drawn into the nanopore by an applied voltage. The force exerted on the charged backbone of the polynucleotide by the electric field is used to examine the enzyme-polynucleotide interactions. Here we show that a nanopore sensor can accurately identify DNA templates bound in the catalytic site of individual DNA polymerase molecules. Discrimination among unbound DNA, binary DNA/polymerase complexes, and ternary DNA/polymerase/ deoxynucleotide triphosphate complexes was achieved in real time using finite state machine logic. This technique is applicable to numerous enzymes that bind or modify DNA or RNA including exonucleases, kinases and other polymerases.We describe a nanopore device that monitors ionic current through a single protein pore inserted in a lipid bilayer (Fig. 1a). The limiting aperture of the pore is just sufficient to accommodate single-stranded DNA (ssDNA) 6,7 , and the adjacent pore vestibule can accommodate doublestranded (duplex) DNA (dsDNA) 7-9 . In the absence of DNA, the open channel current (I o ) through the α-haemolysin pore is 60 pA at 180 mV applied potential in 0.3 M KCl. DNA capture in the nanopore results in a decrease in the current (I). The DNA resides in the pore for a time (t D ) until it leaves, moving to the trans compartment (Fig. 1b). These two parameters, I and t D , together with current noise, are typically used to report results from nanopore experiments 6,10-19 .We used a nanopore instrument to probe the interaction of the Klenow fragment (KF) of Escherichia coli DNA polymerase I with its DNA substrate. This substrate is a duplex DNA formed by base-pairing of a short ssDNA primer with a longer template DNA. The KF catalyses DNA replication by the sequential addition of nucleotides to the primer strand, dictated by Watson-Crick complementarity to the template strand 20 . In contrast with earlier studies examining Exonuclease I/DNA complexes 4 and EcoRI/DNA complexes 5 , our nanopore Capture and translocation of a model DNA template (14 bp hairpin with a 36-nucleotide 5′ overhang and 2′-3′ dideoxycytidine terminus) resulted in a cluster of events with a median duration of 1 ms and an average blockade amplitude I = 20 pA (Fig. 2a). When the KF (2 μM) was subsequently added to the cis compartment under conditions where catalytic activity had been demonstrated in separate experiments (see Supplementary Information, Fig. S1), a second population of events emerged with a 3-ms median dwell time and a higher blockade current (I = 23 pA, Fig. 2b). This class of events is enzyme-concentration-dependent (see Supplementary Information, Fig. S2 and Table S1), consistent with nanopore capture of a DNA/ KF binary complex.Addition of a deoxynucleotide triphosphat...
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 α-hemolysin 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 sub-millisecond 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 purpose of this session is to present to the CDC audience a set of molecular-level biological research venues in which dynamics and control is, or can be, of use. With the large and growing number of people within the control community interested in biology, experiment driven research at the interface of biology and control is enticing. In our session, four different research venues are presented: 1) feedback control of individual DNA molecules in a nanopore, with a goal of DNA sequencing; 2) DNA secondary structure kinetics, with the goal of improving design and synthesis of DNA logic circuits that may be used, for example, for in vivo feedback control of biological processes; 3) an artificial DNA nanomotor, for which feedback compensation improves robustness and performance; and, 4) cooperative protein interactions in muscle force generation, in which identification of feedback mechanisms can ultimately improve therapeutic methods for post-cardiac arrest rehabilitation.
This paper demonstrates feedback voltage control of individual DNA hairpin molecules captured in a nanopore. A finite state machine is used to program voltage control logic, executed on a field-programmable gate array, for rapid detection and regulation of hundreds of DNA hairpins, one at a time. Prompt voltage reduction is used for extension of the dwell time of DNA hairpins in the nanopore. Then, voltage reversal after a preset dwell time is used for automated expulsion of molecules prior to hairpin unzipping. The demonstrated control authority of single molecular complexes captured in the nanopore device is an integral part of our ongoing research for direct monitoring and control of enzyme-bound biopolymers.
This paper demonstrates feedback voltage control of a single DNA molecule tethered in a biological nanopore. The nanopore device monitors ionic current through a single protein pore inserted in a lipid bilayer. The limiting aperture of the pore is just sufficient (1.5 nm diameter) to accommodate single-stranded DNA. The tethered DNA is double stranded on each end, with a single stranded segment that traverses the pore. Voltage control is used to regulate the motion of the tethered DNA, for repeated capture and subsequent voltage-promoted dissociation of DNA-binding enzymes above the nanopore. In initial experiments using the Klenow fragment of Escherichia coli DNA polymerase I, control of 8 independent tethered DNA molecules yielded 337 dissociation events in a period of 380 seconds. The resulting distribution of DNA-protein dissociation times can be used to model the free energy profile of dissociation. Moreover, the approach is applicable to numerous enzymes that bind or modify DNA or RNA including exonucleases, kinases, and other polymerases.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.