Measuring and Modeling the Kinetics of Individual DNA–DNA Polymerase Complexes on a Nanopore

The assembly of a DNA–DNA polymerase binary complex is the precursory step in genome replication, in which the enzyme binds to the 3′ junction created when a primer binds to its complementary substrate. In this study, we use an active control method for observing the binding interaction between Klenow fragment (exo-) (KF) in the bulk-phase chamber above an α-hemolysin (α-HL) nanopore and a single DNA molecule tethered noncovalently in the nanopore. Specifically, the control method regulates the temporal availability of the primer-template DNA to KF binding and unbinding above the nanopore, on millisecond-to-second time scales. Our nanopore measurements support a model that incorporates two mutually exclusive binding states of KF to DNA at the primer-template junction site, termed “weakly bound” and “strongly bound” states. The composite binding affinity constant, the equilibrium constant between the weak and strong states, and the unbound-to-strong association rate are quantified from the data using derived modeling analysis. The results support that the strong state is in the nucleotide incorporation pathway, consistent with other nanopore assays. Surprisingly, the measured unbound-to-strong association process does not fit a model that admits binding of only free (unbound) KF to the tethered DNA but does fit an association rate that is proportional to the total (unbound and DNA-bound) KF concentration in the chamber above the nanopore. Our method provides a tool for measuring pre-equilibrium kinetics one molecule at a time, serially and for tens of thousands of single-molecule events, and can be used for other polynucleotide-binding enzymes

Kinetic Mechanism at the Branchpoint between the DNA Synthesis and Editing Pathways in Individual DNA Polymerase Complexes

Exonucleolytic editing of incorrectly incorporated nucleotides by replicative DNA polymerases (DNAPs) plays an essential role in the fidelity of DNA replication. Editing requires that the primer strand of the DNA substrate be transferred between the DNAP polymerase and exonuclease sites, separated by a distance that is typically on the order of ∼30 Å. Dynamic transitions between functional states can be quantified with single-nucleotide spatial precision and submillisecond temporal resolution from ionic current time traces recorded when individual DNAP complexes are held atop a nanoscale pore in an electric field. In this study, we have exploited this capability to determine the kinetic relationship between the translocation step and primer strand transfer between the polymerase and exonuclease sites in complexes formed between the replicative DNAP from bacteriophage Φ29 and DNA. We demonstrate that the pathway for primer strand transfer from the polymerase to exonuclease site initiates prior to the translocation step, while complexes are in the pre-translocation state. We developed a mathematical method to determine simultaneously the forward and reverse translocation rates and the rates of primer strand transfer in both directions between the polymerase and the exonuclease sites, and we have applied it to determine these rates for Φ29 DNAP complexes formed with a DNA substrate bearing a fully complementary primer–template duplex. This work provides a framework that will be extended to determine the kinetic mechanisms by which incorporation of noncomplementary nucleotides promotes primer strand transfer from the polymerase site to the exonuclease site

Table1_Population pharmacokinetic modeling of ilaprazole in healthy subjects and patients with duodenal ulcer in China.DOCX

Aims: This study aimed to develop a population pharmacokinetic (PopPK) model of ilaprazole in healthy subjects and patients with duodenal ulcer in Chinese and investigate the effect of potential covariates on pharmacokinetic (PK) parameters.Methods: Pharmacokinetic data from 4 phase I clinical trials and 1 phase IIa clinical trial of ilaprazole were included in PopPK analysis. Phoenix NLME 8.3 was used to establish a PopPK model and quantify the effects of covariate, such as demographic data, biochemical indicators and disease state on the PK parameters of ilaprazole. The final model was evaluated by goodness-of-fit plots, bootstrap analysis, and visual predictive check.Results: A two-compartment model with first-order elimination successfully described the pharmacokinetic properties of ilaprazole. In the final PopPK model, body weight and sex were identified as statistically significant covariates for volume of peripheral compartment (Vp) and clearance of central compartment (CL), respectively, and disease status was also screened as a significant covariate affecting both CL and Vp. The validation results demonstrated the good predictability of the model, which was accurate and reliable.Conclusion: This is the first population pharmacokinetics study of ilaprazole in the Chinese, and the PopPK model developed in this study is expected to be helpful in providing relevant PK parameters and covariates information for further studies of ilaprazole.</p

Predicting the Pharmacokinetics of Orally Administered Drugs across BCS Classes 1–4 by Virtual Bioequivalence Model

To evaluate the influence of solubility and permeability on the pharmacokinetic prediction performance of orally administered drugs using avirtual bioequivalence (VBE) model, a total of 23 orally administered drugs covering Biopharmaceutics Classification System (BCS) classes 1–4 were selected. A VBE model (i.e., a physiologically based pharmacokinetic model integrated with dissolution data) based on a B2O simulator was applied for pharmacokinetic (PK) prediction in a virtual population. Parameter sensitivity analysis was used for input parameter selection. The predictive performances of PK parameters (i.e., AUC0–t, Cmax, and Tmax), PK profiles, and bioequivalence (BE) results were evaluated using the twofold error, average fold error (AFE), absolute average fold error (AAFE), and BE reassessment metrics. All models successfully simulated the mean PK profiles, with AAFE < 2 and AFE ranging from 0.58 to 1.66. As for the PK parameters, except for the time of peak concentration, Tmax, of isosorbide mononitrate, other simulated PK parameters were all within a twofold error. The simulated PK behaviors were comparable to the observed ones, both for test (T) and reference (R) products, and the simulated T/R arithmetic mean ratios were all within 0.88–1.16 of the observed values. These four evaluation metrics were distributed equally among BCS class 1–4 drugs. The VBE model showed powerful performance to predict the PK behavior of orally administered drugs with various combinations of solubility and permeability, irrespective of the BCS category

Inhibitory effects of components from root exudates of Welsh onion against root knot nematodes

<div><p>Root-knot nematodes (RKNs; <i>Meloidogyne</i> spp.) are obligate endoparasites that infect many crops and cause severe yield losses. In this research, we studied the effect of Welsh onion, grown as a companion plant, on the resistance of cucumber plants to RKN infection and analyzed the most abundant components of Welsh onion root exudates. The results showed that, when grown with Welsh onion as a companion plant, cucumber roots had 77.0% fewer root knots and egg masses than the control cucumber roots. Welsh onion root exudates were collected and extracted with chloroform, ethyl ether, <i>n</i>-butanol and ethyl acetate. High concentrations of the extracts from the Welsh onion root exudates decreased the hatchability of RKN eggs. In particular, the inhibitory effect of the <i>n</i>-butanol extract was significant and the hatchability of RKN eggs did not exceed 10%. Gas chromatographic–mass spectrometric analysis revealed that the most abundant component in the <i>n</i>-butanol extract was 4-hydroxy-benzeneethanol. Treatment with 1.2 mM 4-hydroxy-benzeneethanol decreased egg hatchability to 40%, whereas treatment with 9.6 mM or a higher concentration of 4-hydroxy-benzeneethanol decreased egg hatchability to less than 10%. In addition, 1.2 mM or a higher concentration of 4-hydroxy-benzeneethanol decreased the activity of the second-stage juvenile (J2). Higher 4-hydroxy-benzeneethanol concentrations (9.8 and 19.2 mM) were lethal to RKNs to some extent, with death rates greater than 50% at 48 h of treatment. The present results suggest that cultivation with Welsh onion as a companion plant may represent an alternative to the application of synthetic nematicides, with fewer side effects. We confirmed that 4-hydroxy-benzeneethanol is a natural effective nematicide.</p></div

Dynamics of the Translocation Step Measured in Individual DNA Polymerase Complexes

Complexes formed between the bacteriophage phi29 DNA polymerase (DNAP) and DNA fluctuate between the pre-translocation and post-translocation states on the millisecond time scale. These fluctuations can be directly observed with single-nucleotide precision in real-time ionic current traces when individual complexes are captured atop the α-hemolysin nanopore in an applied electric field. We recently quantified the equilibrium across the translocation step as a function of applied force (voltage), active-site proximal DNA sequences, and the binding of complementary dNTP. To gain insight into the mechanism of this step in the DNAP catalytic cycle, in this study, we have examined the stochastic dynamics of the translocation step. The survival probability of complexes in each of the two states decayed at a single exponential rate, indicating that the observed fluctuations are between two discrete states. We used a robust mathematical formulation based on the autocorrelation function to extract the forward and reverse rates of the transitions between the pre-translocation state and the post-translocation state from ionic current traces of captured phi29 DNAP–DNA binary complexes. We evaluated each transition rate as a function of applied voltage to examine the energy landscape of the phi29 DNAP translocation step. The analysis reveals that active-site proximal DNA sequences influence the depth of the pre-translocation and post-translocation state energy wells and affect the location of the transition state along the direction of the translocation

Kinetic Mechanisms Governing Stable Ribonucleotide Incorporation in Individual DNA Polymerase Complexes

Ribonucleoside triphosphates (rNTPs) are frequently incorporated during DNA synthesis by replicative DNA polymerases (DNAPs), and once incorporated are not efficiently edited by the DNAP exonucleolytic function. We examined the kinetic mechanisms that govern selection of complementary deoxyribonucleoside triphosphates (dNTPs) over complementary rNTPs and that govern the probability of a complementary ribonucleotide at the primer terminus escaping exonucleolytic editing and becoming stably incorporated. We studied the quantitative responses of individual Φ29 DNAP complexes to ribonucleotides using a kinetic framework, based on our prior work, in which transfer of the primer strand from the polymerase to exonuclease site occurs prior to translocation, and translocation precedes dNTP binding. We determined transition rates between the pre-translocation and post-translocation states, between the polymerase and exonuclease sites, and for dNTP or rNTP binding, with single-nucleotide spatial precision and submillisecond temporal resolution, from ionic current time traces recorded when individual DNAP complexes are held atop a nanopore in an electric field. The predominant response to the presence of a ribonucleotide in Φ29 DNAP complexes before and after covalent incorporation is significant destabilization, relative to the presence of a deoxyribonucleotide. This destabilization is manifested in the post-translocation state prior to incorporation as a substantially higher rNTP dissociation rate and manifested in the pre-translocation state after incorporation as rate increases for both primer strand transfer to the exonuclease site and the forward translocation, with the probability of editing not directly increased. In the post-translocation state, the primer terminal 2′-OH group also destabilizes dNTP binding

The main compositions of Welsh onion root exudates extracted with <i>n</i>-butanol.

<p>The main compositions of Welsh onion root exudates extracted with <i>n</i>-butanol.</p

Effect of different concentrations of 4-hydroxy-benzeneethanol on RKN growth and survival.

<p>Each point is the average of three individual experiments. Error bars represent the standard deviation.</p

Effects of Welsh onion as a companion plant on root-knot nematode (RKN) infections of cucumber plants.

<p>(A) Cucumber plants cultivated in monoculture (CK) or with Welsh onion (T). (B) Roots of CK cucumber plants. (C) Roots of T cucumber plants. (D) Cucumber production during the preproduction and total production stages. (E) Root fresh weight and the number of RKNs, eggs, and adult female RKNs in cucumber roots. Each point is the average of three individual experiments. Error bars represent the standard deviation. * indicates a significant difference (P ≤ 0.05).</p