Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.
The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.
Single molecule (SM) techniques have greatly developed over the past thirty years to respond to the need of overcoming some of the limitations of traditional, bulk solution measurements 1-3. The manipulation of single biological molecules has created the opportunity to measure mechanical properties of biopolymers4 and control the mechanical parameters of protein-protein5 and protein-DNA interactions6,7. SM fluorescence detection, on the other hand, represents an incredibly versatile tool for studying protein activity in vitro and in vivo, leading to the possibility of localizing and tracking single molecules with nanometer precision. Through fitting of the instrument point-spread-function to the SM image, in fact, one can accomplish localization with a precision depending mainly on signal-to-noise ratio (SNR) and reaching a limit of about one nanometer8,9. These methodologies find powerful applications in the study of the dynamics of motor proteins, as well as of the diffusion processes underlying target search in DNA-binding proteins. The capability of determining diffusion constants as a function of the DNA sequence, residence time on the target and accurately measuring the DNA length explored during one-dimensional diffusion events, represent a powerful tool for the study of protein-DNA interaction dynamics and for the investigation of the mechanisms of specific target search.
Recently, the combination of these two techniques has produced a new generation of experimental setups10-14 enabling the simultaneous manipulation of a biological substrate (for example an actin filament or a DNA molecule) and detection/localization of an interacting partner enzyme (for example myosin or a DNA-binding protein). The advantages of these techniques mainly rest on the possibility of exerting mechanical control over the trapped polymer, thus enabling the study of interaction dynamics versus forces or torques. Also, the methodology allows measuring biochemical reactions far from the surface, avoiding one of the main limitations of classic SM methods, i.e., the need for immobilization of the molecules under study on a surface (glass slide or microspheres).
The combination of two single molecules techniques requires overcoming several technical difficulties, mainly arising from the requirements of mechanical stability and adequate SNR (especially when requiring localization with nm precision)15. Particularly, when coupling SM fluorescence detection with optical tweezers, the reduction of noise and photobleaching from the trapping infrared lasers16 and the control of biochemical buffers for assembly of the biological complexes and performance of the experimental measurements11 are of paramount importance. Here, we describe the methods for performing successful measurements in a dual trapping/SM Fluorescence localization setup. The methodology is illustrated with the example of lactose repressor protein (LacI) fluorescently labeled (with Atto532) and detected as it binds to a DNA molecule (trapped between two optical tweezers) containing specific LacI binding sequences (i.e., operators). We demonstrate the effectiveness of the method in detecting binding of LacI to DNA and diffusion along its contour in the target search process. The method is applicable to any combination of DNA sequence and DNA-binding protein, as well as to other systems (microtubules or actin filaments and the motor proteins interacting with them).
1. Optical Tweezers Setup with Nanometer Stability
The experimental setup must provide two optical tweezers with pointing stability at the nanometer level and intensity fluctuations of the trapping laser below 1%. Combination of these conditions will assure nanometer stability of the dumbbell under typical tension (1 pN – few tens of pN), trap stiffness (0.1 pN/nm) and measurement bandwidth (image acquisition rate 20 sec-1). A scheme of the experimental setup is depicted in Figure 1.
2. Single Molecule Localization with Nanometer Accuracy
3. Microfluidics
To achieve a precise control of buffer exchange and 'dumbbell' assembly, i.e., the anchoring of a single DNA molecule between two optically trapped microspheres, a custom-built laminar multichannel flow system must be developed. This is composed by a flow chamber, a pressure-reservoir and a pressure control system (Figure 2).
4. DNA Tailing with Biotinylated Deoxycytidine Triphosphate (biotin-dCTP)
The DNA molecule should be longer than 1 μm to facilitate its extension under flow and anchoring to the trapped beads. Moreover, a long DNA will prevent the IR trapping light from illuminating the DNA binding protein. This is of special relevance because absorption of IR light from an excited chromophore results in increased photobleaching. NOTE: In the present study, the DNA molecule was 3.7 μm long. The molecule contained three copies of the primary operator O1 and one copy of each of the two auxiliary operators, O2 and O3. These sequences have been inserted in the middle of the molecule, thus resulting approximately equidistant from the trapped beads and IR laser beams.
5. Labeling Protein Thiol Groups with ATTO532
Labeling through modification of cysteine residues must not alter the protein activity. To overcome this problem, we used a LacI mutant, LacIQ231C, which carries just one cysteine per monomer at position 231. This mutant preserves the same characteristics of the wild type and chemical modifications at position 231 have been shown to not interfere with protein stability 22. NOTE: We labeled the protein with ATTO532 maleimide.
6. Combined Optical Trapping and Single-molecule Fluorescence Imaging Experiments
In a successful experiment, one (or more) labeled proteins undergo binding/unbinding and/or monodimensional diffusion along the DNA molecule (Figure 3A). Localization of proteins along the DNA molecule allows the quantification of kinetic parameters as a function of the DNA sequence. When buffer conditions causing 1D diffusion are applied, it is possible to follow protein trajectories, and determine, for example, the diffusion coefficient D1D.
The precise position of a single spot, in each frame, can be determined by fitting the Point Spread Function (PSF) with a bidimensional Gaussian function or, alternatively, when handling a large data set, with a more rapid, recently published algorithm (Radial Symmetry Center, RSC)26,27. The latter method determines the point of maximal radial symmetry of the image, attaining a localization precision that is typically comparable with Gaussian fitting. In both cases, tracking can be achieved through MATLAB routines that can be accessed freely27.
Figure 3A shows a kymogram of a typical experiment in which a single LacI molecule diffuses along non-specific DNA sequences while another LacI molecule is specifically bound to one operator, located in the center of the DNA molecule. Figure 3B shows the position of the two LacI molecules (obtained using RSC) along the direction connecting the centers of the two traps (x), as a function of time. From the position of the molecule diffusing along the DNA molecule, we calculated the Mean Square Displacement (MSD) at different time intervals according to the following equation:
(1)
where N is the total number of positions measured and n is the measurement index going from 1 to N. In case of pure unidimensional Brownian diffusion, the MSD is directly proportional to nΔt (Δt is the time interval between two consecutive frames, 100 msec in our experiments), with a slope equal to twice the diffusion coefficient (MSD(n,N) = 2D1nΔt). Figure 4 shows the MSD vs nΔt plot for the molecule diffusing along DNA. The diffusion coefficient of the protein can be readily obtained from a linear fit of the MSD vs nΔt plot. Note that as n increases, the number of points for calculating MSD from Eq. 1 consequently decreases. The error in the evaluation of the MSD thus increases with n. For this reason, we chose to limit our analysis to n=N/4. This is anyway a quite large value compared to the N/10 used by many labs.
Figure 1. The experimental setup consists of: halogen lamp (H), condenser (C), sample (S), piezo translators (x-y and z), objective (O), a low-magnification camera (CCD 200X) and a high-magnification camera (CCD 2,000X) used for the nm-stabilization feedback (BS is a 50:50 beam splitter cube). Double optical tweezers are inserted and extracted from the optical axis of the microscope through dichroic mirrors (D2 and D3) and comprise: Nd:YAG laser (1,064 nm), optical isolator (OI), λ/2 waveplates, polarizing beam splitter cubes (PBS), acousto-optic deflectors (AOD), 1,064 nm interferential filters (F1 and F2), quadrant detector photodiodes (QDP). Signals from QDPs were acquired through an analog-to-digital converter (ADC) and elaborated with a FPGA board. Two custom-built direct digital synthesizers (DDS) drove the AODs to control traps’ position. A digital input-output board (DIO) controlled the aperture of two solenoid valves (V1 and V2) connected to a compressor (+0.5 atm) and ambient pressure (+0 atm), respectively. A Labview software monitored the pressure inside the pressure reservoir (C1) through a pressure meter (PM) and automatically opened one of the two valves to reach the desired pressure level. Fluorescence excitation was provided by a duplicated Nd:YAG laser (532 nm) and the image projected on an electron multiplied camera (EMCCD). M is a movable mirror, F3 an emission filter. Please click here to view a larger version of this figure.
Figure 2. (A) Flow system diagram. The pressure-control system is composed by a pressure meter (PM) and two solenoid valves V1 and V2 connected to a compressor at +0.5 atm (PRS) and to ambient pressure (ATM), respectively. X1 and X2 represent 3-way connectors. The aperture of the valves is finely adjusted to reach the desired pressure in the pressure reservoir C1 with 4 buffer containers. Each inlet tube includes an independent on/off valve at the entrance of the multichannel flow chamber. The outlet tubing is connected to the waste container C2 placed at ambient pressure. Drawings are not to scale. (B) Schematic representation of the multichannel flow chamber. Four laminar flows separately translocate functionalized polystyrene beads, DNA molecules, imaging buffer and labeled proteins. Two beads are caught with dual optical tweezers and moved to the DNA channel to allow DNA anchoring in between them. A DNA force-extension analysis is performed in the buffer channel to verify the presence of a single DNA molecule and, just afterwards, the DNA is incubated in the protein channel. The inset shows a magnification of the final molecular construct, ready for single-molecule imaging in the buffer channel. Drawings are not to scale. Please click here to view a larger version of this figure.
Figure 3. Localization of single LacI molecules interacting with a stretched DNA molecule. (A) The vertical axis of the kymogram represents the x coordinate, parallel to the DNA molecule, while the horizontal axis is time (100 msec acquisition time). (B) X position of the LacI molecule vs time. The position of the molecules was determined by RSC. Images were acquired at 100 msec exposure time and 1.25 x 10-2 W cm-2 excitation laser intensity. Please click here to view a larger version of this figure.
Figure 4. Plot of the MSD vs time for a single LacI molecule diffusing along a stretched DNA molecule. The MSD is calculated from the position record represented in Figure 3B (red). The diffusion coefficient D1D, obtained from linear regression of the data depicted in the figure, is 0.030 ± 0.002 μm2 sec-1.
In the last decade, single molecule manipulation and imaging techniques have seen great progress in terms of spatial and temporal resolution. The combination of manipulation and imaging techniques is at the base of powerful instruments that now allow the control of the mechanical conditions of a single biological polymer, such as DNA, RNA or cytoskeletal filaments, and the simultaneous localization of single proteins interacting with the same polymer. Controlling the mechanical conditions of the trapped polymer is of particular interest. In fact, in living cells, nucleic acids are continuously undergoing mechanical stress caused by interacting enzymes and proteins; similarly, a complex network of interacting proteins and molecular motors modulates tension on cytoskeletal filaments. On the other hand, the possibility to detect and precisely localize proteins interacting with nucleic acids, allows the measurement of molecular interactions as a function of their position along the DNA or RNA sequence.
Combining single-molecule manipulation and imaging techniques requires careful design of the experimental setup and the biological constructs. Optical traps must provide a stable support, ensuring nm-level stability of the suspended polymer. Such stability can be reached through careful isolation of the experimental apparatus from the numerous sources of mechanical noise and by minimizing laser pointing and amplitude fluctuations. Precise (sub-nm) movements of the optical traps are achieved using AODs driven by DDSs. Here, we illustrated a step-by-step protocol to optimize and check optical tweezers’ stability at the nm-level.
Simple wide-field epi-fluorescence microscopy coupled to an EMCCD camera is used to localize single chromophores interacting with the suspended polymer. Our experimental assay is limited to long DNA or RNA molecules (≥6 kbp) to ensure a spatial separation between the trapping laser beam and the fluorescent probe. In fact, superposition of the near-infrared trapping laser with the visible excitation laser would lead to enhanced photobleaching of the chromophores due to absorption of the near-infrared light while the chromophore is in the excited state16. Dumbbell assembly is another tricky procedure that is better obtained using a multichannel flow-cell, for which we provided a detailed description. We indicated how to optimize the buffer flow and how to avoid air bubbles, which can otherwise seriously compromise the experiments. We described protocols for labeling the DNA extremities with biotin, which is required for dumbbell assembly using streptavidin-coated beads, and protocols for protein labeling. Finally, we illustrated step-by-step operations to perform experiments in which the binding and diffusion of a single lactose repressor molecule on a stretched DNA molecule is quantified.
The authors have nothing to disclose.
We thank Gijs Wuite, Erwin J.G. Peterman, and Peter Gross for help with the microfluidics and Alessia Tempestini for help with sample preparation. This research was funded by the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement n° 284464 and from the Italian Ministry for Education, University and Research FIRB 2011 RBAP11X42L006, Futuro in Ricerca 2013 RBFR13V4M2, and in the framework of the Flagship Project NANOMAX.
Name of Material/ Equipment | Company | Catalog Number | Comments/Description | Web Address |
elastomeric isolators | Newport | Newdamp | Choose the appropriate Newdamp elastomer depending on the microscope weight and resonance frequencies | http://www.newport.com |
optical isolator | Optics for Research | IO-3-YAG-VHP | http://www.ofr.com | |
Nd:YAG laser, 1064 nm wavelength | Spectra-Physics | Millennia IR | http://www.newport.com/ | |
acousto-optic deflectors (AODs) | A&A optoelectronic | DTS-XY 250 | http://www.aaoptoelectronic.com/ | |
Direct Digital Synthesizers | Analog Devices | http://www.analog.com/ | ||
quadrant detector photodiodes | OSI optoelectronics | SPOT-15-YAG | http://www.osioptoelectronics.com | |
DIO and FPGA board | National Instruments | NI-PCI-7830R | http://www.ni.com | |
Halogen lamp | Schott | KL 1500 LCD | http://www.schott.com | |
Condenser | Olympus | U-AAC 1.4NA Aplanat Apchromat | http://www.olympus-global.com/en/ | |
Objective | Nikon | CFI Plan Apochromat 60x 1.2NA water immersion | http://www.nikoninstruments.com | |
532 nm laser | Coherent | Sapphire | http://www.coherent.com | |
CCD 200X and 2000X | Hamamatsu | XC-ST70 CE | http://www.hamamatsu.com | |
electron-multiplied CCD | Hamamatsu | C9100-13 | http://www.hamamatsu.com/ | |
piezo stage with nm-accuracy | Physik Instrumente | P-527.2CL | http://www.physikinstrumente.com/ | |
Emission Filter | Chroma Technologies | 600/100m | http://www.chroma.com | |
silica beads (1.54 mm) | Bangs Laboratories | SS04N/5303 | http://www.bangslabs.com/ | |
Albumin from bovine serum (BSA) | Sigma Aldrich | B4287 | http://www.sigmaaldrich.com/ | |
pentyl acetate | Sigma Aldrich | 46022 | Flammable liquid and vapour (No 1272/2008) | http://www.sigmaaldrich.com/ |
nitrocellulose | Sigma Aldrich | N8267-5EA | Flammable solid (No 1272/2008) | http://www.sigmaaldrich.com/ |
heat block | MPM Instruments Srl | M502-HBD | with 2 removable blocks; preheated at 120° C | http://www.mpminstruments.com |
NanoPort assemblies | Upchurch Scientific Inc. | N-333 | http://www.upchurch.com/ | |
polyetheretherketone tubing | Upchurch Scientific Inc. | 1535 | http://www.upchurch.com/ | |
home-made metallic holder for the assembly of the flow-chamber pressure reservoir made of Plexiglass | ||||
luer lock-tip syringes 2.5 mL | Terumo | SS 02LZ1 | http://www.terumomedical.com | |
shut-off valves | Upchurch Scientific, Inc. | P-732 | http://www.upchurch.com/ | |
flangeless fittings | Upchurch Scientific, Inc. | LT-111 | http://www.upchurch.com/ | |
fluorinated ethylene propylene tubing | Upchurch Scientific, Inc. | 1549 | http://www.upchurch.com/ | |
two computer-controlled solenoid valves | Clippard, Cincinnati, USA | ET-2-H-M5 | http://www.clippard.com | |
pressure transducer | Druck LTD | PTX 1400 | ||
biotin-14-dCTP | Life Technologies | 19518-018 | http://www.lifetechnologies.com/ | |
Terminal deoxynucleotidyl Transferase (TdT) | Thermoscientific | EP0161 | http://www.thermoscientificbio.com/ | |
ATTO532 maleimide | Sigma Aldrich | 68499 | http://www.sigmaaldrich.com/ | |
N,N-dimethylformamide (DMF) | Sigma Aldrich | 227056 | Combustible Liquid, Harmful by skin absorption., Irritant, Teratogen. H226; H303; H312; H316; H319; H331; H360; P201; P261; P280;P305; P351; P338; P311 | http://www.sigmaaldrich.com/ |
Tris-(2-carboxyethyl)phosphine hydrochloride (TCEP) | Sigma Aldrich | C4706 | http://www.sigmaaldrich.com/ | |
L-Glutathione reduced (GSH) | Sigma Aldrich | G4251 | Acute toxicity, Oral (Category 5), H303 | http://www.sigmaaldrich.com/ |
Amicon Ultra-15, PLQK Ultracel-PL Membrane, 10 kDa cutoff spin concentrators | Merck Millipore | UFC901024 | http://www.merckmillipore.it/ | |
streptavidin-coated polystyrene beads 1,87 µm | Spherotech, Inc. | SVP-15-5 | http://www.spherotech.com/ |