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Time-Lapse Single-Biomolecule Atomic Force Microscopy Investigation on Modified Graphite in Solution Evgeniy V. Dubrovin,*,†,‡,§ Marc Schac̈ htele,† Dmitry V. Klinov,‡ and Tilman E. Schaff̈ er† †

University of Tübingen, Institute of Applied Physics, Auf der Morgenstelle 10, 72076 Tübingen, Germany Federal Research and Clinical Center of Physical-Chemical Medicine, Malaya Pirogovskaya 1a, Moscow 119435, Russian Federation § Lomonosov Moscow State University, Leninskie gory 1-2, Moscow 119991, Russian Federation ‡

S Supporting Information *

ABSTRACT: Atomic force microscopy (AFM) of biomolecular processes at the single-molecule level can provide unique information for understanding molecular function. In AFM studies of biomolecular processes in solution, mica surfaces are predominantly used as substrates. However, owing to its high surface charge, mica may induce high local ionic strength in the vicinity of its surface, which may shift the equilibrium of studied biomolecular processes such as biopolymer adsorption or protein−DNA interaction. In the search for alternative substrates, we have investigated the behavior of adsorbed biomolecules, such as plasmid DNA and E. coli RNA polymerase σ70 subunit holoenzyme (RNAP), on highly oriented pyrolytic graphite (HOPG) surfaces modified with stearylamine and oligoglycine-hydrocarbon derivative (GM) monolayers using AFM in solution. We have demonstrated ionicstrength-dependent DNA mobility on GM HOPG and nativelike dimensions of RNAP molecules adsorbed on modified HOPG surfaces. We propose an approach to the real-time AFM investigation of transcription on stearylamine monolayers on graphite. We conclude that modified graphite allows us to study biomolecules and biomolecular processes on its surface at controlled ionic strength and may be used as a complement to mica in AFM investigations.



biomolecules should remain free enough to move or rotate.3,7 To meet both requirements, particular care should be paid to the ionic composition of the imaging solution, which modulates the interaction of DNA and protein with the substrate (usually mica). For example, it was proposed to use two different buffers for time-lapsed AFM studies of transcription: one for conducting transcription and the other one for AFM imaging.8−10 However, this significantly complicates and hampers such studies. Though different protocols for the AFM visualization of single-biomolecular processes on mica have been successfully established,1,11 they may be inconvenient for particular tasks. A mica surface is highly negatively charged in aqueous solution (∼2.1 e/nm2 without considering neutralization by the adsorbed counterions12). This negative charge promotes the formation of a counterion cloud, whose concentration at the surface may be several moles per liter even in relatively low ionic strength solution.13 In particular, the concentration of monovalent salt concentration at 1 nm distance from the mica surface is estimated to be larger than 300 mM, irrespective of the bulk monovalent salt concentration.14 The adsorbed DNA

INTRODUCTION Atomic force microscopy (AFM) has become a widespread tool for single-biomolecule studies in various fields of research including biophysics and molecular biology. Many insights into fundamental biological processes involving DNA, such as DNA transcription, replication, recombination, and repair, have been provided by AFM studies of the corresponding reaction products in air (reviewed in refs 1 and 2). One prerequisite of AFM analysis of DNA−protein complexes is the ability to transfer the complexes from solution to a two-dimensional surface without altering the spatial relationship between the protein and the DNA. This is usually achieved by an equilibrated deposition (i.e., by the deposition in a relaxed conformation) of DNA−protein complexes, whose interactions with the surface are dominated by DNA, which therefore determines the global conformation of the adsorbed complexes.3,4 In practice, equilibrated deposition is usually obtained by DNA−protein complex adsorption on mica surfaces in the presence of monovalent and divalent cations.5,6 The ability to operate in liquids makes AFM especially appealing for investigating biomolecular processes in an aqueous environment. For such investigations, there are two oppositional requirements: on the one hand, the adsorption of biomolecules on the substrate should be rather strong to make AFM imaging possible, but on the other hand these © XXXX American Chemical Society

Received: June 28, 2017 Revised: August 29, 2017 Published: August 29, 2017 A

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(reviewed in refs 2,4,7, and 34−37), though only a few studies were conducted in real time in solution.8,9,38,39 However, some important aspects of AFM investigations of transcription have not been properly addressed, such as the characterization of the dimensions of the adsorbed free holoenzyme of E. coli RNAP, which should be considered when distinguishing DNA-RNAP complexes with different stoichiometry from each other or from other bloblike structures on DNA. Aiming to extend the applicability of modified graphite surfaces to real-time AFM studies of biomolecular processes in aqueous environments, we have characterized the real-time adsorption behavior and conformation of RNAP and plasmid DNA molecules on stearylamine and GM monolayers on HOPG. The GM HOPG surface was selected because of its known ability to absorb single DNA molecules in an extended conformation33,40 and to retain the nativelike dimensions of the adsorbed proteins.20,41 The potential suitability of stearylaminemodified HOPG as a substrate for the investigation of DNA− protein processes in solution is associated with the equilibrated adsorption of DNA on this surface, which was observed on a length scale of several hundred nanometers in AFM studies previously.31,32 Moreover, adsorbed DNA segments are considerably mobile on the stearylamine-modified HOPG surface and undergo out-of-plane movements,32 which is also favorable to the study of protein−DNA interactions. In this article, we show that these modified HOPG surfaces are suitable substrates for single-biomolecule AFM studies in solution. This allowed us to develop a procedure for timelapsed visualization of the transcription process on the stearylamine nanotemplate on HOPG in liquid. We conclude that modified graphite may be used instead of or as a complement to mica for AFM studies of biomolecules and biomolecular processes in aqueous solutions.

molecules are partially embedded in this counterion cloud because DNA must be located less than a Bjerrum length (∼0.7 nm) away from the mica surface to allow DNA adsorption.13,15 This may lead to locally uncontrolled conditions of the investigated biomolecular processes and, as a consequence, to an inappropriate interpretation of the obtained results. The intense dissociation of EcoRI-DNA complexes adsorbed on mica even in a solution with low ionic strength was explained by the ionic conditions in the vicinity of the mica surface, inducing a shift of the equilibrium of EcoRI-DNA complexes toward the equilibrium that prevails at high ionic strength.14 In another work, the local increase in the ionic strength by mica counterions was proposed as a reason for the observation that DNA molecules bound to avidin on mica cannot be cleaved by restriction endonucleases, irrespective of the ionic conditions.16 Moreover, in the last years a lot of evidence for the formation of potassium carbonate on a mica surface shortly after its cleavage in air has been accumulated. In this regard, there is an understanding that many studies of the structure of adsorbed water on mica surfaces may need to be revisited (reviewed in ref 17). Potassium and carbonate ions may also contribute to the ionic composition and ionic strength of the solution near the surface. The above-mentioned circumstances may complicate AFM studies (or their interpretation) of biomolecules including protein−DNA complexes on mica surfaces, especially at low ionic strengths. Therefore, the development of alternative procedures for reliable AFM studies of biomolecules, which are based on substrates other than mica, would allow us not only to extend the range of conditions for AFM investigations of biological processes but also, in some cases, to avoid possible artifacts connected with the above-mentioned peculiarities of mica. The characteristic features of highly oriented pyrolytic graphite (HOPG) (atomic flatness of its surface, charge neutrality, electric conductivity, and chemical inertness) have already made it a very popular support for AFM and other scanning probe microscopy techniques. However, the use of a bare HOPG surface as a substrate for AFM studies of DNA and proteins is limited owing to an impeded immobilization of single DNA molecules in an expanded conformation on that surface.18,19 Furthermore, HOPG induces severe denaturation of many proteins, which was shown both experimentally20−23 and in molecular dynamics simulations.24−26 Recent AFM studies have demonstrated the huge utilization potential of HOPG surfaces modified by organic monolayers as substrates for AFM studies of DNA and proteins. Self-assembled monolayers of amphiphiles such as dodecylamine, stearylamine, stearyl alcohol, and stearic acid on HOPG allow the adsorption of single, properly expanded DNA molecules on their surface.27−32 In AFM studies in air, the modification of HOPG with a monolayer of amphiphilic oligoglycine-hydrocarbon derivative (CH2)n(NCH2CO)m-NH2 (often referred to as a graphite modifier, or GM33) was shown to preserve the nativelike conformation of the adsorbed protein molecules and to provide even milder conditions for protein adsorption than mica.20 To our knowledge, amphiphilic monolayers on a HOPG surface have not been previously used as a support for AFM investigations of proteins and DNA−protein complexes in solution. Among fundamental molecular biological processes, transcription was probably the one studied most often with AFM



EXPERIMENTAL SECTION

HOPG Modification. For HOPG modification with stearylamine, 40 μL of a 100 μg/mL stearylamine (Sigma-Aldrich, USA) solution in propanol-2 (p.a.) was spin-coated onto a freshly cleaved HOPG surface (ZYB Quality, mosaic spread 0.8−1.2°, NT-MDT, Russia) at 150 rotations per second at ∼22 °C. For HOPG modification with GM, 10 μL of a 0.01 mg/mL water solution of GM (Nanotuning, Chernogolovka, Russia) was deposited onto a freshly cleaved HOPG surface and incubated for about 1 min. Then, the droplet was removed from the surface by a flow of nitrogen. Sample Preparation. For time-lapse AFM studies of adsorbed plasmid DNA, a 20−40 μL portion of pSF-OXB19 DNA (3845 base pairs, Oxford Genetics, U.K.) diluted to a concentration of 0.2 μg/mL in either Milli-Q water or NaCl (p.a.) solution was deposited on a modified HOPG surface prior to AFM imaging. For AFM studies of RNA polymerase (RNAP), a 10 μL portion of a 50-fold water-diluted E. coli RNA polymerase σ70 subunit holoenzyme (New England Biolabs, USA; final protein concentration ∼20 μg/mL) was deposited on a modified HOPG surface prior to AFM imaging. Stalled elongation complexes were prepared by the incubation of plasmid DNA templates (5−10 nM) with RNAP (30−50 nM) in E. coli RNA polymerase reaction buffer (New England Biolabs, USA; 40 mM Tris-HCl, 150 mM KCl, 10 mM MgCl2, 1 mM dithiothreitol, 0.01% Triton X-100, pH 7.5) for 5 min at 37 °C, followed by the simultaneous addition of the incomplete set of ribonucleoside triphosphates (ATP, UTP, CTP, i.e. lacking GTP) to yield 200 μM (each). The total reaction volume was 5 μL. Upon incubation for 10 min at 37 °C, the reaction was stopped by cooling at 5 °C. For time-lapsed AFM studies of transcription, 15−20 μL of freshly made stalled elongation complexes, 30−50-fold diluted in 50 mM NaCl, was deposited onto stearylamine-modified HOPG and scanned continuously in the same droplet. After an appropriate surface region B

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Figure 1. Time-lapse tapping-mode AFM height images of plasmid DNA adsorbed on a GM HOPG surface (a) in water, (b) in 5 mM NaCl, and (c) in 100 mM NaCl solution. Arrows in (b) indicate lateral DNA segmental movement. The time of each AFM image is provided relative to the first AFM image in the sequence (in min). The sizes of AFM images are (a) 570 × 720 nm2, (b) 300 × 300 nm2, and (c) 500 × 500 nm2. Analysis of AFM Images. AFM images were processed in NanoScope Analysis (Bruker, USA) and Femtoscan software (Advanced Technologies Center, Russia). The height analysis of the protein globules was manually determined in Femtoscan. AFM movies were made in ImageJ42 or in NanoScope Analysis. The estimation of the mean square distance ⟨R2⟩ between two internal points on a DNA plasmid was done in Scilab 5.3.343 as described previously.31,32 In brief, the contours of the DNA molecules were digitized and the distances between all pairs of points on a DNA strand separated by a length s along the contour (with a step length equal to the current length separation s) were averaged for each s ranging from 2 nm to the maximal traced DNA length with 2 nm steps. The scaling exponent ν was determined from the linear regression of the experimental dependence ln(⟨R2⟩) = f(ln(s)). The error value for v corresponds to the standard deviation of the regression coefficient.

with adsorbed stalled elongation complexes was found and several consequent AFM images were recorded, 1 μL of the ribonucleotide solution set (New England Biolabs, USA) containing 10 mM of all four ribonucleotides (ATP, GTP, UTP, and CTP) was added to the droplet on the surface. AFM images were continuously recorded after the addition of ribonucleotides. Atomic Force Microscopy. AFM experiments were performed on a MultiMode 8 atomic force microscope with a Nanoscope V controller (Bruker, USA) in tapping and Peakforce tapping mode at 22 °C. For imaging in liquid, we have used a tapping-mode fluid cell equipped with a fluorosilicone O-ring to minimize evaporation. The time interval between initial sample deposition and the start of AFM scanning in liquid was about 5 min. For tapping and Peakforce tapping mode in liquid, we used the F cantilevers of the MLCT series (Bruker, USA), which have a spring constant of ∼0.6 N/m and a resonance frequency in water of ∼30 kHz. For tapping mode in air, PPP-NCHR cantilevers (NanoWorld, Switzerland) with a spring constant of ∼42 N/m and a resonance frequency of ∼330 kHz were used. To minimize the effective force applied by a cantilever, we have maximized the set-point amplitude in tapping mode and minimized the set-point value in Peakforce tapping mode. In the latter case, it was set below 0.05 V, which corresponded to peak forces below 100 pN. The line scan rate was typically 2.1 Hz for tapping mode and 0.5−1 Hz for Peakforce tapping mode, with 1024 × 1024 or 512 × 512 pixels per image.



RESULTS AND DISCUSSION Adsorption of Plasmid DNA on Modified HOPG. First, we have studied the adsorption behavior of plasmid DNA molecules onto GM HOPG in water by recording sequential AFM images (Figure 1a; Movie 1 in the Supporting Information contains the complete image sequence). The time (in minutes) of each AFM image is provided relative to the first AFM image in the sequence. The presented AFM data

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limit allows us to neglect the effect of circularity, which leads to a symmetrical shape of the dependence of ⟨R2⟩ on s relative to L/2 (for pSF-OXB19 DNA, L ≈ 1.3 μm).45 For small s values, the linear regression gives ν ≈ 1 (dashed line in Figure 2), indicating a rodlike regime, which is expected for length scales less than the persistence length of a polymer.46 The linear regression for larger s (s > 55 nm, solid line in Figure 2) gives ν = 0.59 ± 0.01. This value together with the presence of selfcrossings of a DNA contour corresponds to the case of a projection of its three-dimensional conformation onto the surface,44 which also supports the notion of kinetic trapping of DNA by the GM HOPG surface in water. The observed change in the scaling is consistent with previous investigations of the conformation of adsorbed DNA.45,46 To elucidate the effect of the ionic strength on the mobility of DNA molecules adsorbed onto GM HOPG, we have performed similar AFM experiments in 5 mM (Figure 1b) and 100 mM (Figure 1c) NaCl solutions. (Movies 2 and 3 in the Supporting Information contain the complete image sequences.) DNA thermal motion on GM HOPG surfaces can be clearly observed in both solutions but to different extents. In the AFM images obtained in 5 mM NaCl, the motion of particular segments can be observed on a time scale of several minutes (Figure 1b, arrows), whereas the majority of the DNA molecules remained immobile. (In Figure 1b, the shape of the DNA did not significantly change during 54 min of observation.) The movement of DNA molecules in 100 mM NaCl is much more pronounced: entire DNA contours change their positions after several minutes of observation, and the shape of the adsorbed DNA molecules changes completely in about half an hour (Figure 1c). So we can conclude that the lateral mobility of DNA molecules adsorbed on GM HOPG increases when the ionic strength of the solution increases. The

indicate that DNA molecules adsorbed from water stay almost completely immobile (Figure 1a, Movie 1) on GM HOPG. This observation supports the idea that DNA molecules are kinetically trapped by the surface and cannot equilibrate on it. To characterize the polymer conformation, the scaling exponent ν may be used, which relates the mean square endto-end distance ⟨Rends2⟩ to the contour length L of the polymer by the formula: ⟨Rends2⟩ = const × L2ν.44 For an estimation of the scaling exponent of a circular polymer (DNA plasmid), we have used the mean square distance ⟨R2⟩ between two internal points of a polymer molecule (internal end-to-end distance) and the length s along the DNA contour between these points.45 The dependence of ln(⟨R2⟩) on ln(s) obtained after digitization of the DNA contours from the AFM images was analyzed for s ≤ 250 nm (Figure 2). We assume that the chosen

Figure 2. ln(⟨R2⟩) as a function of ln(s) for plasmid DNA adsorbed on a GM HOPG surface in water. The dashed and solid lines are fits for the estimation of scaling exponent ν on different length scales.

Figure 3. AFM height images of the E. coli RNA polymerase σ70 subunit holoenzyme adsorbed on (a) GM HOPG and (b) the stearylamine monolayer on HOPG obtained in Peakforce tapping mode in water. (c, d) Corresponding height distributions of RNAPs. (The Gaussian fit is shown by a solid line.) The number of measured proteins was N = 910 and 183 for (c) and (d), respectively. The size of the AFM images is 1 × 1 μm2. D

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Figure 4. Time-lapse Peakforce tapping-mode AFM images of stalled elongation complexes adsorbed on stearylamine-modified HOPG (a, b) before and (c, d) after the addition of ribonucleotides. The arrow denotes an RNAP molecule. The time of each AFM image is provided relative to the first AFM image in the sequence (in min). The size of the AFM images is 500 × 500 nm2.

stearylamine-modified HOPG obtained in water (Figure 3a,b) were highly reproducible in repetitive AFM scans and did not reveal any movements or protein desorption events. RNAP molecules show a distinct globular shape without any signs of denaturation such as protein spreading on the surface.20,49 Their height distributions and corresponding Gaussian fits (solid lines) are presented in the histograms (Figure 3c,d). The mean height of RNAP obtained on GM HOPG (8.6 ± 1.1 nm) is very close to that on stearylamine-modified HOPG (8.8 ± 1.0 nm; the error is the standard deviation). The diameter of the RNAP globule estimated from its molecular mass (449 kDa50) with the assumption of a spherical shape is ∼11 nm.51 However, the available crystal structure of the RNAP holoenzyme demonstrates deviations from the spherical shape: RNAP has a complex elongated form with a maximum diameter of about 14 nm and a minimum diameter of about 10 nm.52 This may result in varying heights of adsorbed RNAP molecules. The height of RNAP adsorbed on the modified HOPG surfaces obtained in Peakforce tapping mode in water is very close to the minimum diameter (10 nm) from the crystal structure. (The trend toward smaller values may be a consequence of the local probe-sample geometry53 or of a deformation of the protein molecules that is induced by the imaging force.) Therefore, we assume that the RNAP does not experience a significant structural reorganization upon adsorption on modified HOPG surfaces and may remain fully functional on these surfaces. Approach for AFM Studies of Transcription on Stearylamine-Modified HOPG. For time-lapse investigation of transcription, we have used stearylamine-modified HOPG, for which we showed that adsorbed DNA molecules adopt a two-dimensional equilibrated conformation31,32 and RNAP molecules stably adsorb in a nativelike conformation. We recorded time-lapsed AFM images of stalled elongation complexes on stearylamine-modified HOPG in Peakforce tapping mode (Figure 4a,b; Movie 5 in the Supporting Information contains the complete image sequence). A globule of ∼8 nm in height (RNA polymerase molecule, see Figure 3b,d) is located on the circular DNA plasmid (Figure 4, arrow). Though we observe the typical nanotemplate-directed motion of DNA on stearylamine nanotemplates,32 the stalled elongation complex remains immobile on the surface (Figure 4a,b). At a certain point in time, the transcription was resumed by the addition of all four NTPs to the imaging solution (between Figure 4b and c). Subsequent dissociation of RNAP from the plasmid DNA can clearly be observed (Figure 4c). This is consistent with the native behavior of RNAP, which

observed effect may be explained by the Debye screening in electrolyte solutions, which weakens the electrostatic attraction between oppositely charged DNA molecules and amine groups on the GM HOPG surface. The decreased attraction between DNA and the surface leads, in turn, to the increase in DNA mobility. Because the Debye length is inversely proportional to the square root of the ionic strength of the solution, we observe a different extent of DNA motion at different NaCl concentrations. For stearylamine nanotemplates on HOPG (Movie 4 in the Supporting Information), the dynamic behavior of the adsorbed pSF plasmid is in agreement with previously reported data for phage T7 DNA.32 In particular, the motion of DNA segments along the stearylamine lamellas is observed. The adsorption of DNA on GM HOPG proceeds in a different way: we do not observe preferred directions of adsorbed DNA molecules or their motion (Figure 1b,c; Movies 2 and 3 in the Supporting Information). Moreover, DNA molecules are kinetically trapped on GM HOPG, but they are not traped on stearylamine nanotemplates even at low salt concentration, where they remain considerably mobile and adopt a twodimensional compact globule conformation (ν ≈ 0.5).32 In other words, the interaction of DNA is much stronger for GM HOPG than for stearylamine nanotemplates. This may be related to the higher surface density of positively charged amine groups on GM HOPG as compared to stearylamine nanotemplates. Adsorption of RNAP on Modified HOPG. Upon adsorption on a substrate, protein molecules may experience conformational changes including partial or complete denaturation.20,47 The use of a substrate that retains the native conformation of RNAP upon its adsorption is essential for AFM investigations of transcription in real time. The morphology and height of an adsorbed protein molecule obtained by AFM may indicate the protein’s conformation on the surface.20 Using AFM, we have characterized the height of the E. coli RNA polymerase σ70 holoenzyme subunit adsorbed on modified HOPG surfaces. Minimization of the forces exerted by an AFM tip on a surface is a cornerstone for obtaining unperturbed vertical dimensions of a soft protein globule. To achieve accurate height measurements, we have used Peakforce tapping mode, which exerts very low forces on the sample.48 [For comparison, tapping-mode AFM images and respective height analysis of adsorbed protein globules are shown in the Supporting Information (Section 2 and Figures S1 and S2)]. Peakforce AFM images of RNAPs adsorbed on GM HOPG and E

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dissociates from a DNA template after finishing the transcription of a gene. After the dissociation, the free DNA molecule moved away from the RNAP molecule, which stayed adsorbed in the same location on the surface (Figure 4d). The unchanged position of the RNAP molecule on the surface shows that DNA moves relative to the adsorbed enzyme molecule during transcription. This conclusion is in agreement with the above presented observations of immobile RNAPs and mobile DNA on stearylamine-modified HOPG. In some experiments, RNAP desorbed from the surface after dissociation from the DNA (Figure S3 in the Supporting Information). In this work, the RNA transcripts were not registered in the AFM images, possibly because they did not adsorb to the surface. Furthermore, the scan rate used in our experiments (3−10 min per image) has allowed us to observe only the starting and final stages of the transcription. In future studies, increasing the scan rate (e.g., by decreasing the pixel resolution of the AFM images) and decreasing the rate of transcription (e.g., by decreasing the concentration of NTPs) could make it possible to observe the movement of RNA polymerase along the DNA template.8,9 The developed approach may be also used for the investigation of transcription with high-speed AFM, which is capable of obtaining a subsecond time resolution.38,39,54 The obtained results for DNA and RNA polymerase adsorption on modified HOPG in aqueous environments have allowed us to develop a new approach for AFM studies of transcription in solution. Owing to the beneficial surface properties of the stearylamine-modified HOPG, this approach allows us to conduct and visualize transcription in the same buffer under the controlled conditions. Modified HOPG surfaces may also be used as a substrate for AFM studies of other single-molecule biological processes that involve DNA−protein interactions: DNA replication, recombination, repair, etc. The application of modified graphite allows us to avoiding high local ionic concentrations, which may form on mica surfaces that are often used as a substrate for biomolecular AFM imaging in solution.

Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.7b02220. Description of the supplementary movies. Tapping-mode AFM of RNAP on modified HOPG. Supplementary figures. (PDF) Sequence of tapping-mode AFM height images of plasmid DNA adsorbed onto GM HOPG in water acquired with a frequency of 1 image in ∼8 min. (The size of the frames is 2 × 2 μm2.) (AVI) Sequence of tapping-mode AFM height images of plasmid DNA adsorbed onto GM HOPG in 5 mM NaCl solution acquired with a frequency of 1 image in ∼8 min. (The size of the frames is 350 × 360 nm2.) (AVI) Sequence of tapping-mode AFM height images of plasmid DNA adsorbed onto GM HOPG in 100 mM NaCl solution acquired with a frequency of 1 image in ∼7 min. (The size of the frames is 2 × 2 μm2.) (AVI) Sequence of tapping-mode AFM phase images of plasmid DNA adsorbed on a stearylamine monolayer on HOPG in 5 mM NaCl solution acquired with a frequency of 1 image in ∼4 min. (The size of the frames is 2 × 2 μm2.) (AVI) Sequence of Peakforce tapping-mode AFM height images of stalled elongation complexes on a stearylamine monolayer on HOPG before and after the addition of NTPs. The AFM images were acquired with a frequency of 1 image in ∼10 min. (The size of the frames is 440 × 550 nm2.) (AVI)



AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID



Evgeniy V. Dubrovin: 0000-0001-8883-5966

CONCLUSIONS Using AFM, we have investigated and compared the behavior of adsorbed DNA and RNAP molecules on modified HOPG surfaces (GM HOPG and stearylamine-modified HOPG) in aqueous solutions. At low ionic strength (below 5 mM), GM HOPG kinetically traps DNA molecules on its surface. At higher ionic strength (100 mM), adsorbed DNA molecules become mobile on GM HOPG as a result of the partial screening of the DNA−surface electrostatic interaction. DNA molecules adsorbed on stearylamine-modified HOPG are considerably mobile even at low ionic strength and can equilibrate on this surface. Height analysis performed in Peakforce tapping mode shows that RNAP retains its nativelike conformation and probably its functional activity when adsorbed on modified HOPG surfaces. We have directly demonstrated that stearylamine-modified HOPG is a suitable substrate for real-time AFM studies of transcription in solution. This substrate may be used as a complement to a mica substrate, which may induce high local ionic strength in the vicinity of its surface. This substrate may be used not only for the study of transcription but also for the investigation of other DNA−protein interactions at a single-molecule level.

Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS We thank Dr. Olga Koroleva and Dr. Valery Drutsa for valuable consultations concerning biochemical procedures used in this work and Johanna Hutterer for valuable advice. E.V.D. acknowledges the support by the Russian Science Foundation (14-25-00013).



ABBREVIATIONS HOPG, highly oriented pyrolytic graphite; RNAP, RNA polymerase; AFM, atomic force microscopy; GM, graphite surface modifier (CH2)n(NCH2CO)m-NH2



REFERENCES

(1) Lyubchenko, Y. L.; Shlyakhtenko, L. S. Imaging of DNA and Protein-DNA Complexes with Atomic Force Microscopy. Crit. Rev. Eukaryotic Gene Expression 2016, 26 (1), 63−96. (2) Hansma, H. G.; Golan, R.; Hsieh, W.; Daubendiek, S. L.; Kool, E. T. Polymerase Activities and RNA Structures in the Atomic Force Microscope. J. Struct. Biol. 1999, 127 (3), 240−247.

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DOI: 10.1021/acs.langmuir.7b02220 Langmuir XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.langmuir.7b02220 Langmuir XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.langmuir.7b02220 Langmuir XXXX, XXX, XXX−XXX