Electronic control of DNA polymerase binding and unbinding to single DNA molecules.

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 alpha-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 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.

Feedback control of a DNA molecule tethered in a nanopore to repeatedly probe DNA-binding enzymes.

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.

Sequence-specific detection of individual DNA polymerase complexes in real time using a nanopore.

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. Recently, nanopores have been used to measure the binding of enzymes to their DNA substrates. 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.