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

Abstract

Monitoring the kinetics and conformational dynamics of single enzymes is crucial to better understand their biological functions because these motions and structural dynamics are usually unsynchronized among the molecules. However, detecting the enzyme-reactant interactions and associated conformational changes of the enzyme on a single-molecule basis remains as a challenge to established optical techniques because of the commonly required labeling of the reactants or the enzyme itself. The labeling process is usually nontrivial, and the labels themselves might skew the physical properties of the enzyme. We demonstrate an optical, label-free method capable of observing enzymatic interactions and associated conformational changes on a single-molecule level. We monitor polymerase/DNA interactions via the strong near-field enhancement provided by plasmonic nanorods resonantly coupled to whispering gallery modes in microcavities. Specifically, we use two different recognition schemes: one in which the kinetics of polymerase/DNA interactions are probed in the vicinity of DNA-functionalized nanorods, and the other in which these interactions are probed via the magnitude of conformational changes in the polymerase molecules immobilized on nanorods. In both approaches, we find that low and high polymerase activities can be clearly discerned through their characteristic signal amplitude and signal length distributions. Furthermore, the thermodynamic study of the monitored interactions suggests the occurrence of DNA polymerization. This work constitutes a proof-of-concept study of enzymatic activities using plasmonically enhanced microcavities and establishes an alternative and label-free method capable of investigating structural changes in single molecules.

Keywords: conformational change; optical microcavity; polymerase; protein-DNA interactions; single enzyme dynamics; single molecule reaction; whispering Gallery mode.

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 V_m, 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 $\overline{\mathrm{\Delta }\mathrm{\lambda }}$ 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 $\overline{\mathrm{\Delta }\mathrm{\lambda }}$ distributions obtained for Pfu/ptDNA/dNTP interactions showing the evolution of overall signal amplitude with increasing temperature and enzyme activity. Peak center positions ${\overline{\mathrm{\Delta }\mathrm{\lambda }}}_{\mathrm{c}}$ extracted via lognormal fits (solid lines) are indicated by dashed lines. (B) ${\overline{\mathrm{\Delta }\mathrm{\lambda }}}_{\mathrm{c}}$ 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 $\overline{\mathrm{\Delta }\mathrm{\lambda }}$ 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) ${\overline{\mathrm{\Delta }\mathrm{\lambda }}}_{\mathrm{c}}$ and off-rates k_off (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.