A solid-state nanopore sensor is an effective method to obtain information about single biomolecules.^[1–4] The idea of nanopore sensing comes from the Coulter-counter method to evaluate particles in solution.^[2–4] In a container full of electrolyte, the container is divided into two chambers by an insulating chip with a small pore. When a molecule is translocating through the pore under an electric field, the conductance of the system will decrease, and the decrease is related to the geometry and charge of the molecule. A nanopore sensor works by analyzing the change of the current caused by molecule translocation across the nanopore. In previous research, a solid-state nanopore sensor has been shown to be a reliable and accurate method for detecting nucleic acids,^[5–12] protein,^[13–16] and synthetic structures.^[17–22]

Proteins are crucial to most biological processes, such as enzymes, and they are dynamic during the catalytic process. The dynamics of protein are reflected in its conformational change, which is caused by local and collective thermal motion of atoms and chains. Many studies have shown that the conformational change of a protein highly influences its function.^[23–28] Many techniques have been used to probe protein flexibility, such as Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD),^[28] x-ray crystallography,^[29] and nuclear magnetic resonance (NMR).^[30] Nevertheless, in those techniques, chemical modification or crystallization of protein is needed, which may influence the structure of the protein and lead to inaccurate structural information being obtained. Compared to other methods, a solid-state nanopore can gauge the label-free protein in solvent, similar to intracellular environment, and it can also analyze protein in real time. Recently, Waduge et al. demonstrated that the breadth of current blockades increases with more flexible proteins by measuring a set of proteins.^[31,32] A protein with larger flexibility can have more possible configurations when it passes through the nanopore, thus it will provide wider distributions of current blockages, resulting in wider full-width at half-maximum (FWHM) values from Gaussian fits to the current blockade histograms. In this method, FWHM values obtained from Gaussian fits to the current blockade histograms could be used an indicator of protein flexibility. A larger FWHM value suggests higher flexibility of the protein.

T7 RNA polymerase (RNAP) is one of the ideal models for studying the interaction between DNA promoter and polymerase because it can easily form a tight binary complex with DNA promoter.^[33–36] T7 RNA polymerase is a DNA-dependent RNA polymerase which consists of a single polypeptide chain and has a molecular weight of 99 kDa. As Fig. 1(a) shows, T7 RNAP is constituted by N-terminal domain (8–325), thumb sub-domain (326–411), palm sub-domain (421–449, 528–553 and 785–879), palm insertion module (450–527), fingers sub-domain (554–739 and 759–784), specificity loop (740–769), and extended foot module (838–879).^[37] The N-terminal domain is flexible in solution, which interacts with upstream regions of promoter and RNA to begin the catalysis.^[38] The thumb sub-domain is a poorly ordered part of RNAP, and affects the affinity of template DNA towards T7 RNAP.^[35,39] The palm sub-domain is disordered in electron density maps, and directly participates in catalysis.^[40,41] The fingers sub-domain is comparatively ordered, and supports the specificity loop, which is involved in recognizing promoter.^[42] The palm insertion module is a 77 residues insertion in the palm sub-domain, working as a switch to shut down the channel of binding nucleic acid with its conformation changing in this process.^[43] The extended foot module interacts with incoming rNTPs and promoter to continue the transcription, and its flexibility in solution changes after forming complex.^[40,44]

Fig. 1.

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Fig. 1. (color online) (a) PDB-based cartoon shows the structure of the T7 RNAP and complex with DNA promoter, which are colored by domain and module, with the N-terminal domain (red), the thumb (green), the palm (blue), the palm insertion module (yellow), the fingers (cyan), the specificity loop (orange). (b) Schematic of the nanopore sensor used in this work. A SiN membrane separates the electrolyte-filled space into two chambers which are connected by a nanoscale pore. An applied voltage between the electrodes generates an electrical field to drive the protein crossing the pore. (c) TEM image of a 10 nm nanopore used in this work, and a size comparison with T7 RNAP.

In the binding process, promoter sequence-specific recognition is accomplished by the specificity loop. Meanwhile, at position −17 to −13, AT-rich promoter sequence is recognized by a loop in the N-terminal domain, which proceeds the transcription. DNA strands are then melted by the fingers sub-domain and remain as single-stranded until the position +1, and the complex is prepared during the transcription at the active site of the palm sub-domain.^[33,37,45] The crystal structure of the polymerase shows the diameter of about 8 nm for T7 RNA polymerase. In a solution of pH = 8.0, T7 RNAP will be negatively charged. Studying the flexibility of T7 RNA and its complex may reveal a more detailed understanding of the interaction between RNA polymerase and its promoter.

The static structure of T7 RNAP and its complex with DNA promoter has been already studied by using x-ray crystallography,^[35,36] but the conformational change of T7 RNAP complex in solvent remains unclear. In this article, we take advantage of solid-state nanopore to probe T7 RNAP and its complex in solution. The translocation events of T7 RNAP and its complex are analyzed, and FWHM values from Gaussian fits of blockages are calculated, respectively. We find that compared to T7 RNAP, the FWHM value is larger in its complex, suggesting a larger flexibility of T7 RNAP complex, which means that the flexibility of T7 RNAP increases after binding DNA promoter. This increase may relate to the function changes of T7 RNAP after initiation. Our results further support that the flexibility change is due to a breath-like 70 nm³ volume change.

To study the conformational change of T7 RNAP binding DNA promoter, three series of nanopore experiments were carried out where T7 RNAP sample only, DNA promoter only, and T7 RNAP complex with DNA promoter were used as three sets of target molecules, respectively. A schematic of the experimental setup is shown in Fig. 1(b), as the negatively charged sample molecules are translocated through the drilled SiN nanopore under an electric field. The typical TEM-drilled SiN nanopore ( ) is shown in Fig. 1(c), which is only a little bit larger than the T7 RNAP molecule.

The current baseline through the nanopore is rather smooth (Fig. 2(a)) with low noise (Fig. S2 of the supporting information). The DNA promoter is not detected through nanopore experiments due to its small size and limited temporal resolution (Fig. 2(b)). Once the T7 RNAP sample is added into the nanopore chamber, a large number of translocation events are observed as conductance dropping, as shown in Fig. 2(c). From previous reports, one can obtain the volume of the detected molecules through nanopore experiments by using the following equation:^[46,47]

Fig. 2.

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	Fig. 2. (color online) Current traces captured by Axon 200B for electrolyte (a), DNA promoter (b), T7 RNAP (c), and complex (d) obtained by a 10 nm nanopore in a condition of 0.15 M NaCl, pH = 8, and applied voltage of 100 mV. (e) Scatter plot of ΔG vs log dwell-times (td) for T7 RNAP with a 10 nm nanopore and applied voltage 100 mV.

The bias voltage is the primary parameter that one can alter in nanopore experiment and offers the electric field force which drives the protein through the nanopore. Obviously, a larger bias can lead to a higher translocation speed of the protein across the pore, which will be reflected by shorter time durations of translocation events. Meanwhile, a larger external electric field can also cause a higher capture rate of translocating molecules.^[48] Figures 3(a)–3(c) show three time duration histograms of T7 RNAP blockage events under three different voltage biases (60 mV, 100 mV, 140 mV). One can see that the average time duration becomes shorter under larger voltage bias, indicating that the blockage events are caused by smooth translocation of T7 RNAP molecules. The capture rate shows a linear increase as a function of voltage when the voltage is smaller than 100 mV (Fig. 3(d)). However, the capture rate decreases when the voltage bias is larger than 100 mV, because the time duration of translocation event is too short to be identified under larger voltage due to limited temporal resolution ( ) in normal nanopore experimental setup. The bandwidth of the amplifier circuit limits the resolution of duration, and shorter event under larger voltage ( in this case) cannot be detected due to the limited temporal resolution. For voltage bias lower than 100 mV, the event duration is sufficiently long for current blockage analysis, which can provide a good capture rate for data analysis.

Fig. 3.

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	Fig. 3. (color online) Histogram of log dwell-time (td) for T7 RNAP in certain applied voltages of (a) 60 mV, (b) 100 mV, and (c) 140 mV. (d) The capture rate as a function of applied voltage for T7 RNAP. The blue straight line is obtained by binomial fitting of first 3 points and coordinate zero.

After analyzing the translocation events of T7 RNAP molecules, we did nanopore experiments with T7 RNAP complex. Figure 2(d) shows the current trace of translocation events of complex with T7 RNAP and DNA promoter. Figures 4(a) and 4(b) show the contour plots of T7 RNAP and complex translocation events under bias 100 mV, respectively. In both contour diagrams, the fraction blockage is gathered at 0.4, indicating that binding DNA has little impact on size change of T7 RNAP. This character is also found in T7 RNAP and complex structures from x-ray crystallography.^[35,36] In the complex contour diagram (Fig. 4(b)), the fraction blockage distribution of translocation events is more dispersed than that in T7 RNAP (Fig. 4(a). In particular, more events appear in the area with a smaller blockage that we consider T7 RNAP as a shrink state. Meanwhile, time duration is centered at in both cases.

Fig. 4.

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	Fig. 4. (color online) Color contour plots of vs log dwell-time (td) for T7 1142 events (a) and complex 1214 events (b) with a 10 nm nanopore and applied voltage of 100 mV. Histograms of for T7 (c) and complex (d) events. The histograms are fitted with Gaussian distributions (blue curves).

To probe the flexibility of these two samples, we calculate the FWHM values from Gaussian fits to the fraction blockage of translocation events of T7 RNAP and complex (Figs. 4(c) and 4(d)). From the histogram, it is evident that the FWHM of complex is larger than that of T7 RNAP. The T7 RNAP sample shows FWHM of 0.179, and the complex sample shows FWHM of 0.207. From previous study,^[31,32] larger FWHM suggests a higher flexibiliy of translcoating molecules. The larger FWHM of the complex sample suggests a greater flexibility in T7 RNAP binding DNA promoter complex. This difference is contributed by events with fraction blockage in the range of 0.3 to 0.35. Compared to the most probable range of 0.4–0.45 (Figs. 4(c) and 4(d)), there is a quarter diminution of blockage. If we regard all of those events as a sphere across the pore, by using Eq. (1), we could easily calculate the volume change in complex to be about one fourth, which is 70 nm³. We did the same experiment using another nanopore ( ). Very similar results were found that FWHM increases from Gaussian fits to the fraction blockage of translocation events in T7 RNAP and DNA complex samples (Fig. S4 of the supporting information).

From the increase of FWHM (Figs. 4(c) and 4(d)), we can obtain that T7 RNAP becomes more flexible after binding DNA promoter to form complex. This increase comes from extension of distribution of events blockage, which suggests that the complex is much easier to reach a shrink state. In the binding process, DNA promoter activates the T7 RNAP to open this state. Therefore, the flexibility of the complex performs like breathing, which has two states alternating with time. This breath takes 70 nm³ volume change of the T7 RNAP, which is too large if only module is involved. Consequently, domain and sub-domain participate in this conformational change, while thumb and fingers sub-domains may impact the promoter in the whole binding process.

In summary, we used SiN nanopore sensor to probe T7 RNAP and its complex with DNA promoter. Duration histograms of T7 RNAP under different bias voltages indicate smooth translocation of detected molecules. Furthermore, the FWHM values of Gaussian fits to the blockade histograms of T7 RNAP and complex translocation events indicate that the complex is more flexible in our experiment. Our analysis suggests that T7 RNAPʼs flexibility changes after binding DNA promoter and this dynamic change activates a state that decreases the volume of T7 RNAP. Moreover, we calculated the decreased volume of T7 RNAP and have supposed that this decrease is caused by major component of T7 RNAP, such as thumb and fingers sub-domains. Our findings may be helpful to reveal the interaction of T7 RNAP and its promoter, and it gives an insight into the protein activity variations caused by conformational change.