An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

1. Generation of reporter mRNA

1. Modify a DNA transcription template that encodes a bulk-assay “untagged” reporter mRNA by inserting an N-terminus epitope tag-encoding sequence to generate a DNA transcription template for a “tagged” reporter mRNA (Figure 2A).
   NOTE: 3xFLAG/anti-FLAG interaction is recommended for this assay due to its superior sensitivity and the short 3xFLAG tag length. However, the assay is compatible with other epitope/antibody pairs.
2. Synthesize untagged and tagged RNAs using linearized DNA templates and an in vitro transcription kit (see Table of Materials). Typically, 10–20 pmol of in vitro transcribed RNA is sufficient for subsequent RNA processing to allow a systematic single-molecule study.
3. Cap the RNA 5′ end (Figure 2B) with an m⁷G capping kit (see Table of Materials) following the manufacturer’s recommendation.
4. Biotinylate the RNA 3′ end (Figure 2B) using a biotinylation kit (see Table of Materials) as per the protocol provided with the kit. Purify RNAs by phenol-chloroform extraction followed by purification with a spin column. Elute RNA in 10–20 μL of RNase-free water. Approximately 40–50% of the uncapped input RNA is recovered.
5. Assess RNA integrity and concentration by denaturing acrylamide gel electrophoresis and RNA staining, as described previously²⁹.
6. Perform bulk cell-free translation³⁰ with the 3′-end-biotinylated tagged reporter mRNA to confirm that the modified mRNA preserves the translation properties that are of interest. As an example, cap-dependent translation can be confirmed by measuring and comparing reporter activities in bulk cell-free translation reactions with uncapped and capped reporter mRNAs (see Representative Results).

2. Flow chamber preparation

1. Select an appropriate coverslip thickness, as specified by the microscope objective spec sheet. Select a glass or quartz slide for objective- or prism-type total internal reflection fluorescence (TIRF) imaging systems³¹, respectively.
2. Perform cleaning, salinization, PEGylation, and chamber assembly of coverslip and slide following the protocol described by Chandradoss et al.³² with the following modifications.
   1. Wash slides containing drilled holes in 10% alkaline liquid detergent (v/v) for 20 min with sonication. Combine the slides and coverslips for all remaining cleaning steps.
   2. Skip the acetone wash step. Extend the Piranha etching incubation time from 20 min to 1 h. Incubate the first-round PEGylation for 3 h. Incubate the second-round PEGylation with MS(PEG)₄ for 3 h.

3. Equipment preparation

1. At least 3 h before performing a single-molecule experiment, turn on the microscope incubator and reduce the airflow to a low rate to avoid excessive acoustic disturbance of experiments. Set the incubator temperature to the optimal temperature for the cell-free translation system.
   NOTE: Translation is extremely sensitive to temperature. The optimal reaction temperature is specific for each cell extract system. Reaction temperature characterization is a vital step in the development of a bulk cell-free translation system. This single-molecule assay should be conducted under the same optimal temperature as the corresponding bulk translation condition.
2. At least 1 h before performing a single-molecule experiment turn the laser on by pressing the laser power button behind the laser box, turn the laser safety key, and press the start button; turn on the microscope and tap on the touch screen control panel to select the imaging mode, objective, and filter set.
3. Turn on the EMCCD camera, the Prior stage controller, and the syringe pump by pressing their power buttons. Turn on the computer and open software Andor Solis (software 1) to control the EMCCD camera, TirfCtrl (software 2) to control the laser, PriorTest (software 3) to control the motorized stage, and Coolterm to control the syringe pump. Allow the camera to cool and stabilize to its designated working temperature before data acquisition begins.
4. In software 2, confirm that the correct parameters are set for laser wavelength, objective type, and refractive index of glass and sample. Click the Connect button. To set the laser intensity, click the Control button next to the laser wavelength, then in the laser control pop up window, drag the intensity slide bar. Set the laser to the desired angle by dragging the fiber position slide bar or inputting the corresponding penetration depth. Ensure that the Shutter box is checked to maintain the laser in the Off mode when not imaging.
   NOTE: Open and close the laser shutter by checking the Shutter box. When initially establishing the assay, different laser intensities and incident angles should be tested for optimal single-molecule detection. The optimal laser intensity balances bright single-molecule fluorescence and fast photobleaching of the fluorophores. The incident angle should be increased beyond the critical angle to reduce the high fluorescent background from the diffusing labeled antibodies until mRNA-bound antibodies are clearly resolved. As a reference, a 532 nm laser was set to 10 μW at the objective with the laser incident angle set to 71.5° in a study¹⁵ using Cy3-labeled anti-FLAG with a labeling ratio of 2–7 dyes per antibody.
5. In software 1, choose Kinetic under the Acquisition Mode, input the parameters for exposure time, kinetic series length, and EM gain value. Choose the directory and assign filenames for saving the data image file.
6. Set the syringe pump to a modest to fast flow rate for the subsequent tubing washing step.
   Pipette an aliquot of 70% ethanol into a microfuge tube, insert the syringe pump tubing end into the ethanol solution, and use Coolterm to toggle the syringe pump between withdraw and dispense modes three times to wash the tubing. Repeat using RNase-free water.
   NOTE: The tubing length must be long enough to allow the translation mix to be dispensed at step 5 to only contact the tubing, not the syringe. The rinse volume here should be greater than the volume of dispensed translation mix to ensure that the translation mix at step 5 remains in contact with sanitized tubing.
7. After the final water rinse has been dispensed, remove residual water from the inside of the tubing by dabbing the tubing end on a clean tissue wipe. Maintain the tubing end dry by setting it into a clean microfuge tube.

4. Immobilization of 3′-end biotinylated mRNA

1. Maintain the cell extract and translation mix frozen on dry ice until just before their use. Thaw the remaining frozen reagents and maintain them on ice.
2. Place the flow chamber into the microscope sample holder with the coverslip side facing down and secure it in place with tape. Use tape to cover the inlets and outlets of flow channels that are not in use.
3. Pipette a 1.5x volume (relative to channel volume; also applies to other steps in step 4 and step 5) of 0.2 mg/mL streptavidin into the flow channel and incubate for 10 min at room temperature.
4. To remove unbound streptavidin, wash the channel three times by pipetting in a 3x volume of T50 wash buffer (20 mM Tris HCl, pH 7.0, 50 mM NaCl, 0.2 U/μL RNase inhibition reagent) per wash.
   NOTE: It is important to prevent liquid waste from flowing back into the channel. If the outlet of the flow channel is not connected to a waste collection tube, place a folded tissue paper near the outlet to absorb the liquid that flows out of the channel.
5. Transfer the translation extract and mix from dry ice to ice and allow them to thaw.
6. Put a drop of immersion oil on the objective. Place the microscope sample holder with the flow chamber into the microscope stage. Bring the objective close to the coverslip until immersion oil on the objective contacts the coverslip. Move the sample stage so that the position of the flow channel is directly above the objective lens.
7. In software 2, open the laser shutter and adjust the laser intensity level.
8. On the microscope control touch screen, select the Focus tab and click the Focus Search and Start button. A beep is heard when a focus plane is successfully achieved.
9. Image the flow channel surface in Live mode in software 1. Optimize the focus for a sharper image by adjusting the objective position with the fine step control on the touch screen.
10. In software 3, move the stage to evaluate the level of fluorescence background at various areas of the flow channel detection surface. If the fluorescence is low, continue with the experiment. If the fluorescence background is excessively high, consider discarding the flow channel.
11. In software 2, close the laser shutter.
12. Pipette out the T50 wash buffer from the flow channel and immediately pipette in a 2x volume of biotinylated mRNA solution to the flow channel. Incubate for 15 min.
    NOTE: For each batch of mRNA preparation, mRNA concentrations and incubation times should be tested to achieve the mRNA surface density necessary for single-molecule detection. Appropriate mRNA surface density mediates antibody binding that shows fluorescent spots with no more than ~5% overlapping (see Representative Results). As a starting point, biotinylated mRNA at a concentration of 2–20 nM is recommended.
13. Wash the channel three times by pipetting a 3x volume of translation compatible buffer per wash.

5. Translation mix assembly and delivery to a flow channel for single-molecule detection

1. On ice, assemble 3x volumes of the translation mix³⁰ in a microcentrifuge tube following the bulk translation protocol from step 1.7, except omit mRNA and supplement with an optimized concentration of fluorescently labeled antibodies. Briefly spin the microcentrifuge tube to collect the translation mix at the bottom of the tube.
   NOTE: When establishing the assay, different antibody concentrations are tested. The optimal concentration shows low amounts of nonspecific antibody binding to the detection surface and fast antibody binding to the nascent peptide (see steps 7.1. and 7.2., respectively, for assay calibration details).
2. Transfer the translation mix to the inside of a 0.7 mL microfuge tube cap. Set the syringe pump to the withdraw mode. Insert the pump tubing end into the translation mix. Start the syringe withdrawal to collect the translation mix into the pump tubing. Reverse the syringe pump setting to the dispensing mode and set the flow to an optimal rate for translation mix delivery.
   NOTE: Avoid generating bubbles in the translation mix when transferring it into the pump tubing. Avoid withdrawing the translation mix too deep into the part of the tubing that was not sanitized and rinsed in step 3.6. When establishing the assay, different delivery flow rates are tested to determine the optimal rate (see step 6.2. for details on assay calibration).
3. If there is a gap between the tubing end and translation mixture, start the syringe pump and allow the translation mixture to reach the tubing end, then stop the syringe pump.
4. Insert the pump tubing end into the inlet of the flow channel. Avoid introducing air bubbles into the translation mix when performing the connection. Stabilize the connection by screwing the custom-made metal bracket onto the microscope sample holder plate. Assess the microscope focus and imaging area fluorescence.
   NOTE: The tubing can be clamped to the flow chamber with a custom part as previously described³³. Other types of fluidics systems can also be used for this assay³⁴. The field of view should appear similar before and after connecting the tubing. If more fluorescent spots appear after the connection, the translation mix is likely leaking from the delivery tubing. Depending on the application, the channel with leaked translation mix may need to be discarded.
5. Confirm that the movie acquisition parameters are correct and that the appropriate filename and directory have been entered for saving files.
6. Move the stage to image an area in the center of the flow channel detection surface. On the microscope control touch screen, select the Continuous AF tab and click the Focus Search and Start button to maintain the focal position during the experiment. A beep is heard when a focus plane is successfully achieved.
7. In software 2, open the laser shutter. In software 1, click the Take Signal button to start recording. After approximately 5 s of recording, enter the Run command in Coolterm to deliver the translation mix into the flow channel. Record for up to 2 h with a time resolution of 0.5–2 s/frame.
   NOTE: The maximum span of data acquisition depends on how fast the translation extract loses activity over time, which can be conveniently characterized by performing bulk cell-free translation with luciferase reporter mRNA and measuring luciferase activity in the translation reactions at different time points³⁵.
8. When data acquisition is complete, turn off the laser, turn off the Continuous AF on the microscope control touch screen, and disassemble the tubing from the flow chamber.
9. Pipette out the translation mixture from the flow channel inlet, transfer it to a microfuge tube, and snap-freeze the solution by placing the tube in liquid nitrogen. Later store at -80 °C.
10. Wash the syringe pump tubing three times with RNase-free water, then three times with 70% ethanol. Withdraw 70% ethanol into the syringe pump tubing for storage.
11. Turn off the microscope and all accessories. Clean up.

6. Data analysis

NOTE: Data analysis for this assay requires common methods in single-molecule biophysical studies. All computationally intensive steps can be achieved using existing algorithms and software (references are included below at their corresponding steps).

1. Optional: If applicable, convert the acquired image series file to a format that is compatible with the chosen image processing software or programming language.
2. Determine the translation reaction starting point and reagent exchange time.
   NOTE: Due to the diffusing fluorescent antibodies in the translation mix, the background fluorescence intensity of an imaged area will increase during the in-channel reagent exchange from translation compatible buffer to translation mix. The translation reaction starting point is set as the time point when the reagent exchange is complete. This completion point is found by measuring the background fluorescence intensity during the translation mix delivery and identifying the time point when the increasing fluorescence intensity plateaus¹⁵, as described below.
   1. Calculate the total fluorescence counts per image frame and plot the corresponding time profile. Identify the sudden increase in total fluorescence in the time profile plot.
      NOTE: The total fluorescence increase is due to the increasing concentration of fluorescently labeled antibody during the reagent exchange.
   2. Identify the end point of the total fluorescence increase phase and set this time point as the starting point (i.e., time 0) of the translation reaction. Identify both the start and end points of the total fluorescence increase phase and set the time difference as the reagent exchange time.
      NOTE: The total time-span of reagent exchange depends on the flow channel dimensions and the selected flow rate. It is possible that some translation factors can start binding to mRNA before the reagent exchange completes, which could reduce the resolution of the determined first-round initiation time. It is therefore recommended to test different flow rates when setting up the assay and select flow rates that allow reagent exchange completion within 3–4 s for data acquisition speeds of 0.5–2 s/frame.
3. Optional: Perform movie drift correction.
   NOTE: The positions of bound antibodies in the data image might change over time due to drift of microscope parts and accessories. Drift that is visually noticeable in a movie requires correction before proceeding further with data analysis. Several spatial drift correction algorithms and scripts are available^36,37,38.
   1. In each image, find the local maxima in pixel counts to determine the positions of bound antibodies in each frame with pixel-level accuracy. Set a threshold for the pixel count such that only spots that are clearly above the background noise are selected.
   2. Calculate the changes of individual antibody positions in each direction between two consecutive image frames.
   3. Plot the histogram of antibody positional changes between two consecutive frames and do so separately for each direction. Gaussian fit each histogram and set the center position of peaks as the directional drift between two consecutive frames.
   4. To counteract the observed drift, shift each image frame in the opposite direction by the calculated drift amount.
4. Construct single-molecule trajectories.
   NOTE: Detection/tracking software is available to identify bright spots and extract their intensities from data image series^39,40.
   1. Identify antibody positions as described in step 6.3.1.
      NOTE: Visual inspection is recommended to ensure that the chosen threshold does not give rise to excessive over- or under-picking of particles. Visual inspection can be achieved by overlaying the identified centroid positions over each image frame and visually assessing their agreement (see Representative Results).
   2. Combine the picked particles from all frames and remove the redundant particle positions.
   3. Visually inspect the images of bound antibodies and select a square area enclosing the pixels with counts that are clearly above background and representative of individually bound antibody.
      NOTE: The point spread function (i.e., the size of the square for a single molecule) is determined by the optical setup and the detector pixel size.
   4. For each particle position, construct a single-molecule trajectory by calculating the total intensity of all pixels in the square area around the particle position. Trajectories are constructed for each position, frame by frame, and for the whole data movie.
5. Optional: Apply a non-linear forward-backward filter⁴¹ to reduce background noise in single-molecule trajectories.
6. Optional: Digitize trajectories with existing step-detection algorithms^42,43,44,45.
   NOTE: The choice of step-detection algorithm depends on the complexity of the single-molecule dynamics. For the simplest scenario of only isolated ribosome translation (i.e. no polysome formation) and good signal to noise ratio, a threshold application to the fluorescence counts is sufficient for identifying the timing of antibody binding and dissociation events in the single-molecule trajectories. Visual inspection of at least 100 trajectories for each condition is recommended to ensure that trajectory steps have been appropriately selected by a step-detection strategy.
7. Determine the time of the first antibody binding event for each trajectory (i.e., first arrival time, t₁), either using digitized trajectories or applying a threshold to undigitized trajectories.
   NOTE: The median value of the first arrival time distribution can be compared between different translation conditions. Alternatively, the first arrival time distribution can be fitted to a shifted (3-parameter) log-normal function to determine the mean value¹⁵ <t₁>. Because the first arrival time distribution is typically skewed and has a long tail, do not calculate the mean by directly averaging over the observed first arrival times.
8. Optional: Dwell time analysis and calculation of average peptide synthesis rate.
   1. Use digitized trajectories to identify monosome (single ribosome) translation events in each trajectory and calculate the single binding event dwell times as the time difference between corresponding antibody binding and dissociation events.
      NOTE: Step-detection algorithms for digitizing trajectories are generally less reliable when multiple molecular events overlap in time. Excluding polysome events from dwell time analysis is therefore recommended. For highly active translation systems that are enriched with polysome events and infrequently display monosome events, a mixture of labeled and unlabeled antibodies can be used to increase the chance of observing isolated translation events in single-molecule trajectories.
   2. Fit the distribution to a log-normal function and use the fitted function to calculate the average dwell time.
      NOTE: Calculating the mean directly by averaging the observed dwell times is not recommended because the dwell time distributions are typically skewed with long tails.
   3. Determine the number of amino acids that are translated during the antibody binding dwell time by subtracting the sum of the epitope amino acid length and 35 (the average amino acid length of nascent peptide within the ribosome exit tunnel) from the total number of ORF codons.
   4. To determine the average peptide synthesis rate, divide the number of translated amino acids by the average dwell time.
9. Optional: Calculation of the average first-round initiation time (<t_{1_I}>).
   NOTE: Initiation and elongation conditions, such as the initiation site sequence context and the coding sequence, are generally preserved deliberately for studies of initiation kinetics. Therefore, the average first arrival time <t₁> from step 6.7. can be used directly to calculate the change in first round initiation kinetics between different conditions. The following correction is only necessary for determining the actual value of first-round initiation time.
   1. Divide the sum of the epitope length and 35 (the average amino acid length of nascent peptide within the ribosome exit tunnel) by the average peptide synthesis rate (from step 6.8.4.) to calculate the average peptide elongation time of this N-terminal region (<t_{1_tag}>).
   2. As the first arrival time is the sum of the first-round initiation time and t_{1_tag}, subtract <t_{1_tag}> from the average first arrival time <t₁> to calculate the average first-round initiation time <t_{1_I}>: <t_{1_I}> = <t₁> – <t_{1_tag}>

7. Assay calibration

NOTE: Perform the following calibration steps when initially establishing the assay.

1. To examine the antibody binding specificity, perform the protocol steps in sections 4 and 5 separately for untagged and tagged mRNAs (Figure 2). The antibody binding levels in channels with these respective mRNAs represent the extent of nonspecific antibody binding to the detection surface and specific antibody binding to nascent peptide.
   NOTE: The specific to nonspecific binding ratio is recommended to be >10 and may be increased with different surface passivation strategies, lower antibody concentration, or higher mRNA surface density.
2. To measure the rate of antibody binding, perform the protocol with an extra step between step 4 and step 5.
   1. After step 4, incubate the channel with translation mixture that lacks antibody to pre-generate mRNA/ribosome/peptide complexes.
   2. Then proceed to step 5 and flow antibody-supplemented translation mixture into the channel.
   3. Quantify the average antibody binding rate to pre-generated nascent peptide with exponential fitting to the first arrival time histogram.
      NOTE: Antibody binding to pre-generated peptide should be rapid, on the order of seconds, and should be faster than the translation kinetics. A higher antibody concentration may be used to increase the antibody binding rate.
3. Photostability of fluorophore-labeled antibody.
   1. Prepare a slide according the protocol in step 2 but skip the PEGylation step. Assemble the flow chamber after the salinization step. The silane coating allows efficient nonspecific antibody binding to the detection surface.
   2. Add the fluorescently-labeled antibodies into the channel to achieve single-molecule density of nonspecific antibody binding to the detection surface. Begin by adding fluorescently-labeled antibody that is highly diluted in T50 wash buffer and gradually increase the antibody concentration until the desired single-molecule density is achieved.
   3. Image the detection surface until most of the antibody fluorescence is no longer detectable.
   4. Plot the total number of visible antibodies over time to assess the rate of antibody fluorescence loss due to fluorophore photobleaching.
      NOTE: Antibody labeling typically yields multiple fluorophores per antibody. Individual antibodies remain detectable until all conjugated fluorophores photobleach.