Surface plasmon field-enhanced fluorescence spectroscopy studies of primer extension reactions

Abstract

Surface plasmon field-enhanced fluorescence spectroscopy (SPFS) utilizes the evanescent electromagnetic field of a surface plasmon to excite chromophors in close proximity to the surface. While conventional surface plasmon resonance spectroscopy allows the observation of surface reactions by means of refractive index changes, SPFS additionally provides a channel for the read-out of fluorescence changes. Thus, the detection limit for low mass compounds, whose adsorption is only accompanied by small refractive index changes, can be substantially improved by fluorescent labeling. In this study, we present the first example that utilizes SPFS to follow the dynamics of an enzymatic reaction. The elongation of surface-tethered DNA has been observed by the incorporation of Cy5-labeled nucleotides into the nascent strand by the action of DNA polymerase I (Klenow fragment). The technique offers a rapid way to determine the binding constant and the catalytic activity of a DNA processing enzyme, here exemplified by the Klenow fragment. Furthermore, the effect of mispaired bases in the primer/template duplex and the influence of different label densities have been studied. The resulting sensitivity for nucleotide incorporation, being in the femtomolar regime, combined with the specificity of the enzyme for fully complementary DNA duplexes suggest the application of this assay as a powerful tool for DNA detection.

Figure 1

Scheme of the SPFS set-up.

Figure 2

Schematic representation of the surface architecture.

Figure 3

Example for a primer extension assay monitored by SPFS. First, KF (7.6 nM) was added together with Cy5–dCTP (1 μM). The extension process was not initiated at this point, since the first base on the template strand required the incorporation of dATP. The reflectivity increase after KF injection corresponds to the formation of a binary DNA–KF complex (solid line). Injection of the complete dNTP mixture (1 μM each) caused the release of KF and a rapid fluorescence increase (open circles) demonstrating the incorporation of Cy5-labeled nucleotides into surface-attached DNA. The refractive index change owing to the primer extension was below the sensitivity limit of SPR which is why the reflectivity returned to the initial value after the removal of the enzyme.

Figure 4

(a) SPFS scan curves recorded after the elongation of P45/Te50G1close using different mole fractions of Cy5–dCTP, x_Label. The total dNTP concentration was 1 μM and KF was bound to DNA from a 7.6 nM solution before the addition of dNTP. One exemplary reflectivity curve is shown as a dotted line; the fluorescence curves are given as straight lines and marked with the corresponding mole fractions. (b) Plot of the final fluorescence values versus x_Label according to the raw data shown on top. The plot is not linear revealing the non-stochastic nature of the label incorporation. The inset shows the SPFS scan curve monitored after primer extension at a mole fraction x_Label = 0.001. The yielded fluorescence intensity corresponds to a label density of 6 Cy5-molecules per μm².

Figure 5

Influence of the amount of bound KF on the velocity of DNA replication and KF release. Shown are five sets of experiments that were carried out with varying bulk concentrations of KF (between 1.5 and 30 nM). DNA synthesis was initiated by the addition of identical amounts of dNTPs (10 μM each with x_Cy5 = 0.025). (a) Time evolution of the reflectivity (solid dots). The exponential decay of the curves after dNTP addition was fitted using Equation 2 (solid lines). Owing to different time courses, curves corresponding to very low KF concentrations are shown in a separate panel (top). (b) Fluorescence curves monitored simultaneously with the reflectivity curves shown in (a). The time of dNTP injection is offset to zero and the curves are shifted relative to each other for a clearer presentation. The inset explains the temporal connection between the fluorescence and the reflectivity data. (c) Affinity of the KF to recessed DNA duplexes. Plotting the immobilized amount of KF according to (a) versus the corresponding solution concentration (closed squares) yields K_A = 1.5 × 10⁸ M⁻¹ using a 1:1 binding model (solid line). (d) Plot of the initial slopes of the fluorescence curves as displayed in (b) versus surface concentration of KF (closed circles). The primer extension rate was found to depend linearly on the amount of bound KF. Also plot of Δt, which is the time that elapses between the start of the fluorescence rise and that of the reflectivity decay, versus KF concentration (open squares). (e) Change of binding affinity after complete primer extension. Plotting τ⁻¹ versus [KF] produced a linear curve that determines the affinity constant to be K_A = 3.7 × 10⁷ M⁻¹.

Figure 6

Replication rates as a function of total dNTP concentration. x_Label was 0.5 and identical surface concentration of KF were applied (80 ng/cm²). (a) Time courses of the reflectivity starting from the time of dNTP injection. (b) The fluorescence slopes were proportional to the dNTP substrate concentration. (c) Lineweaver–Burk plot yielding K_M = 3.4 μM.

Figure 7

Influence of single base mismatches in the primer/template region on the primer extension and the KF binding. While the internal mismatch 3 nt upstream the 3′-terminus of the primer decelerated the elongation process (blue), a terminal mismatch inhibited the reaction entirely (red). The extension of the fully matched primer carried out at identical conditions is shown in black.