Research ArticleBIOPHYSICS

Label-free optical detection of single enzyme-reactant reactions and associated conformational changes

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Science Advances  29 Mar 2017:
Vol. 3, no. 3, e1603044
DOI: 10.1126/sciadv.1603044
  • Fig. 1 Methods for the detection of sm-DNA/Pol interactions.

    (A) Schematic of the prism-based microcavity sensor setup. The inset shows an image of NR scatterers bound to the equatorial plane of a microsphere. (B) Typical transmission spectra showing a WGM (Lorentzian dip) before (blue) and during (red) a DNA/polymerase interaction. (C) Conceptual representation of the two different approaches used for monitoring DNA/polymerase interactions (immo-DNA and immo-Pol scheme) and (D) the corresponding resonance traces, exhibiting spike signals caused by the respective DNA/polymerase interactions.

  • Fig. 2 Near field–based transduction mechanism.

    (A to C) Spatial distributions of the near field’s intensity and the parameters associated with the respective movement of the polymerase volume. (A) Distance between the NR and the polymerase. (B) Changing angle between both arms with fixed base. (C) Changing angle between both arms with fixed left arm. a.u., arbitrary units. (D and E) Dependence of the volume-integrated intensity I, normalized to Vm, on the parameters associated with (A) to (C).

  • Fig. 3 sm-DNA/Pol interaction signals using immo-DNA scheme.

    (A) Example resonance traces exhibiting spike patterns caused by Taq (top; blue) and KF (bottom; maroon) polymerase/DNA interactions and the different noise levels found for ptDNA-functionalized NRs (maroon), unfunctionalized NRs (green), and in the absence of KF (light blue). (B) Distributions of the average spike amplitudes Embedded Image and (C) durations Δτ obtained for Taq (blue) and KF (red) interacting with ptDNA in the presence of dNTP. The concentrations of Taq, KF, and dNTP were kept to ≈200 nM, 200 nM, and 50 μM, respectively.

  • Fig. 4 sm-DNA/Pol interaction signals using the immo-Pol scheme.

    (A) Average spike amplitude Embedded Image distributions obtained for Pfu/ptDNA/dNTP interactions showing the evolution of overall signal amplitude with increasing temperature and enzyme activity. Peak center positions Embedded Image extracted via lognormal fits (solid lines) are indicated by dashed lines. (B) Embedded Image for different DNA polymerase species (Taq, KF, and Pfu) and temperatures. (C) Distributions of spike durations Δτ obtained for Pfu/ptDNA/dNTP interactions at two different temperatures. The concentrations of ptDNA and dNTPs in the solution were kept to 1 and 50 μM, respectively.

  • Fig. 5 Different conformational transitions in the presence and absence of dNTPs.

    (A) Comparison between average spike amplitude Embedded Image distributions obtained for Pfu/DNA interactions in the absence (top) and presence (bottom) of dNTPs (50 μM) at 296 and 315 K. Arrhenius plots displaying the temperature dependence of (B) Embedded Image and off-rates koff (D) found for Pfu in the presence and absence of dNTPs. (C) Change of the spike duration distributions at two different temperatures in the absence of dNTPs. (A) and (C) were obtained with the same Pfu/NR-modified microsphere, whereas (B) and (D) show data obtained with six different sensors.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/3/e1603044/DC1

    section S1. Real-time monitoring of the NR immobilization procedure

    section S2. Single-molecule analysis

    section S3. Control experiments

    section S4. PCR experiments

    section S5. KF interactions with ssDNA and ptDNA/dNTP

    section S6. Signal comparisons between Pfu-immobilized NRS and bare NRS interacting with a WGM cavity

    section S7. Pfu polymerase interactions with two different lengths of ptDNA

    section S8. Additional information

    section S9. Discussion on the detection of freely diffusing polymerase molecules

    fig. S1. Time-traced WGM resonance positions (Δ) and linewidth broadening (ΔΓ) with step-like changes induced by binding of individual NRs (dimensions, 25 nm × 49 nm; λlaser = 642 nm).

    fig. S2. Single-molecule statistics.

    fig. S3. Control experiments.

    fig. S4. Gel of the PCR with citrate NRs and Taq/Pfu polymerase.

    fig. S5. Comparison of KF interactions associated with ssDNA and ptDNA/dNTP.

    fig. S6. Comparison of transient changes in WGM response position and WGM linewidth as induced by NRs (before and after Pfu-surface functionalization) entering the WGM’s evanescent field.

    fig. S7. Pfu-polymerase interactions with two different lengths of ptDNA strands.

    fig. S8. Sketch of the model used to perform Monte Carlo simulations.

    fig. S9. Probabilities of a polymerase molecule to be still located inside the detection volume after a certain duration of time as obtained from Monte Carlo simulations using different initial molecule positions.

    fig. S10. Probabilities of detecting resonance shifts with amplitudes of 15 (black) and 30 (red) fm of perturbations with a certain duration as shifts above 3σ (solid lines) or σ (dashed lines) during one wavelength sweep of the laser (20 ms).

    fig. S11. Zoom in showing probabilities Pdet and Ploc plotted on the interval and for the cases where they overlap with nonzero values.

    fig. S12. Zoom in showing Pres.

    table S1. Numerical values used in the fitting process of PPoisson to Pmeas in fig. S2, alongside the respective coefficients of determination R2.

    table S2. Numerical values of the results shown in the Arrhenius plots of Fig. 5.

    References (2527)

  • Supplementary Materials

    This PDF file includes:

    • section S1. Real-time monitoring of the NR immobilization procedure
    • section S2. Single-molecule analysis
    • section S3. Control experiments
    • section S4. PCR experiments
    • section S5. KF interactions with ssDNA and ptDNA/dNTP
    • section S6. Signal comparisons between Pfu-immobilized NRS and bare NRS interacting with a WGM cavity
    • section S7. Pfu polymerase interactions with two different lengths of ptDNA
    • section S8. Additional information
    • section S9. Discussion on the detection of freely diffusing polymerase molecules
    • fig. S1. Time-traced WGM resonance positions (Δ) and linewidth broadening (ΔΓ) with step-like changes induced by binding of individual NRs (dimensions, 25 nm× 49 nm; λlaser = 642 nm).
    • fig. S2. Single-molecule statistics.
    • fig. S3. Control experiments.
    • fig. S4. Gel of the PCR with citrate NRs and Taq/Pfu polymerase.
    • fig. S5. Comparison of KF interactions associated with ssDNA and ptDNA/dNTP.
    • fig. S6. Comparison of transient changes in WGM response position and WGM
      linewidth as induced by NRs (before and after Pfu-surface functionalization) entering the WGM’s evanescent field.
    • fig. S7. Pfu-polymerase interactions with two different lengths of ptDNA strands.
    • fig. S8. Sketch of the model used to perform Monte Carlo simulations.
    • fig. S9. Probabilities of a polymerase molecule to be still located inside the detection volume after a certain duration of time as obtained from Monte Carlo simulations using different initial molecule positions.
    • fig. S10. Probabilities of detecting resonance shifts with amplitudes of 15 (black) and 30 (red) fm of perturbations with a certain duration as shifts above 3σ (solid lines) or σ (dashed lines) during one wavelength sweep of the laser (20 ms).
    • fig. S11. Zoom in showing probabilities Pdet and Ploc plotted on the interval and for the cases where they overlap with nonzero values.
    • fig. S12. Zoom in showing Pres.
    • table S1. Numerical values used in the fitting process of PPoisson to Pmeas in fig. S2,
      alongside the respective coefficients of determination R2.
    • table S2. Numerical values of the results shown in the Arrhenius plots of Fig. 5.
    • References (25–27)

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