This work was supported by the National Institutes of Health [R01GM121847]; the Memorial Sloan Kettering Cancer Center (MSKCC) Support Grant/Core Grant (P30 CA008748); and MSKCC Functional Genomics Initiative.

1. Generation of reporter mRNA
<ol>
	<li>Modify a DNA transcription template that encodes a bulk-assay &#8220;untagged&#8221; reporter mRNA by inserting an N-terminus epitope tag-encoding sequence to generate a DNA transcription template for a &#8220;tagged&#8221; 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 pair...

In comparison to typical in vitro TIRF single-molecule experiments, single-molecule imaging with the assay described here is additionally complex due to the use of cell extract and a high concentration of fluorescently labeled antibody. Compared to the more common practice of one round of surface PEGylation, a second round of PEGylation (step 2) greatly reduces nonspecific antibody binding to detection surface15. The high concentration of diffusing fluorescent antibodies causes an extreme...

Translation in eukaryotic systems occurs predominantly through 7-methylguanosine (m7G) cap-dependent pathways1. Studies indicate that the initiation step of eukaryotic translation is rate-limiting and a common target for regulation2,3,4. Mechanisms of cap-dependent translation have been extensively studied using genetic5, biochemical6,7,8, structural9, and genomic10 bulk approaches. Although these methods have identified diverse mechanisms that regulate cap-dependent initiation, their resolution limits them to ensemble averaging of signals from heterogeneous and asynchronous initiation events. More recently, individual in vivo translation events have been visualized by methods that measure fluorescent antibody binding to epitopes on nascent polypeptides11,12,13,14. However, these new approaches are also limited in their ability to resolve individual initiation events because multiple fluorescent antibodies must bind a nascent peptide to allow single translation events to be resolved from a high intracellular fluorescence background. In many biological interactions, resolved individual kinetic events have provided critical insights into understanding complex multistep and repetitive biological processes that are not possible to synchronize at the molecular level. New methods that can track the dynamics of individual translation events are needed for a better understanding of cap-dependent initiation and regulation.
We recently developed an in vitro assay that measures cap-dependent initiation kinetics with single-molecule resolution15. Considering the large number of known and unknown protein factors involved in this initiation pathway3,16, the single-molecule assay was developed to be compatible with existing in vitro cell-free translation systems to benefit from their preservation of cellular factors and robust translation activity17,18,19,20,21,22,23,24,25. Furthermore, the use of cell-free translation systems allows more compatible comparisons between single-molecule observations and previous bulk results. This approach provides a straight-forward integration of new single-molecule kinetic insights into the existing mechanistic framework of cap-dependent initiation. To establish the single-molecule assay, the traditional cell-free translation system is modified in three ways: an epitope-encoding sequence is inserted at the beginning of the open reading frame (ORF) of a reporter mRNA; the 3&#8242; end of the reporter mRNA is biotinylated to facilitate mRNA end-tethering to single-molecule detection surface; and fluorescently-labeled antibodies are supplemented to the translation extract. These modifications require only basic molecular biology techniques and commonly available reagents. Furthermore, these modifications and the single-molecule imaging conditions preserve the translation kinetics of bulk cell-free translation reactions15.
In this assay (Figure 1), 5&#8242;-end capped and 3&#8242;-end biotinylated reporter mRNA is immobilized to a streptavidin-coated detection surface in a flow chamber. The flow chamber is then filled with a cell-free translation mixture supplemented with fluorescently labeled antibodies. After mRNA translation has occurred for approximately 30-40 codons downstream of the epitope sequence26,27, the epitope emerges from the ribosome exit tunnel and becomes accessible to interact with fluorescently-labeled antibody. This interaction is rapid and its detection by single-molecule fluorescence imaging techniques enables tracking of translation kinetics with single-molecule resolution during active cell-free translation. This assay should broadly benefit in vitro studies of cap-dependent translation kinetics and its regulation, particularly for systems with a working bulk in vitro assay.
A prerequisite for establishing this single-molecule assay is a working bulk cell-free translation assay, which can be achieved using translation extract that is either commercially available or prepared following previously described methods28. Eukaryotic translation extract can be obtained from diverse cells, including fungal, mammalian, and plant28. For imaging, this assay requires a TIRF microscope equipped with tunable laser intensity and incident angle, a motorized sample stage, a motorized fluidics system, and sample temperature control device. Such requirements are generic for modern in vitro single-molecule TIRF experiments and may be achieved differently. The experiment presented here uses an objective-type TIRF system made up of commercially available microscope, software, and accessories all listed in the Table of Materials.

<table>
	<tbody>
		<tr>
			<td>100X oil objective, N.A. 1.49</td>
			<td>Olympus</td>
			<td>UAPON 100XOTIRF</td>
			<td></td>
		</tr>
		<tr>
			<td>Acryamide/bis (40%, 19:1)</td>
			<td>Bio-Rad</td>
			<td>161-0144</td>
			<td></td>
		</tr>
		<tr>
			<td>Alkaline liquid detergent</td>
			<td>Decon</td>
			<td>5332</td>
			<td></td>
		</tr>
		<tr>
			<td>Aminosilane (N-(2-Aminoethyl)-3-Aminopropyltrimethoxysilane)</td>
			<td>UCT Specialties, LLC</td>
			<td>A0700</td>
			<td></td>
		</tr>
		<tr>
			<td>Andor ixon Ultra DU 897V EMCCD</td>
			<td>Andor</td>
			<td>DU-897U-CSO-#BV</td>
			<td></td>
		</tr>
		<tr>
			<td>Andor Solis software</td>
			<td>Andor</td>
			<td></td>
			<td>For controlling the Andor EMCCD</td>
		</tr>
		<tr>
			<td>Band-pass filter</td>
			<td>Chroma</td>
			<td>532/640/25</td>
			<td></td>
		</tr>
		<tr>
			<td>Band-pass filter</td>
			<td>Chroma</td>
			<td>NF03-405/488/532/635E-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin-PEG-SVA</td>
			<td>Laysan Bio Inc</td>
			<td>Biotin-PEG-SVA</td>
			<td></td>
		</tr>
		<tr>
			<td>Coenzyme A free acid</td>
			<td>Prolume</td>
			<td>309-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Coolterm software</td>
			<td></td>
			<td></td>
			<td>For controlling the syringe pump</td>
		</tr>
		<tr>
			<td>Desktop computer</td>
			<td>Dell</td>
			<td></td>
			<td>For controlling the microscope, camera, stage, and pump.</td>
		</tr>
		<tr>
			<td>Dichroic mirror</td>
			<td>Semrock</td>
			<td>R405/488/532/635</td>
			<td></td>
		</tr>
		<tr>
			<td>Direct-zol RNA microprep 50RNX</td>
			<td>Fisher Scientific</td>
			<td>NC1139450</td>
			<td></td>
		</tr>
		<tr>
			<td>Dual-Luciferase Reporter Assay System</td>
			<td>Promega</td>
			<td>E1910</td>
			<td></td>
		</tr>
		<tr>
			<td>Epoxy</td>
			<td>Devcon</td>
			<td>14250</td>
			<td></td>
		</tr>
		<tr>
			<td>Firefly luciferin D-Luciferin free acid</td>
			<td>Prolume</td>
			<td>306-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>Fisher Scientific</td>
			<td>BP1185500</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen perioxide</td>
			<td>Sigma-Aldrich</td>
			<td>216763-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Immersion oil</td>
			<td>Olympus</td>
			<td>Z-81226A</td>
			<td>Low auto-fluorescence</td>
		</tr>
		<tr>
			<td>Luciferase Assay System</td>
			<td>Promega</td>
			<td>E1500</td>
			<td></td>
		</tr>
		<tr>
			<td>MEGASCRIPT T7 Transcription Kit</td>
			<td>Thermo fisher</td>
			<td>AM1334</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>MMX04751</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>IX83</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slide</td>
			<td>Thermo Scientific</td>
			<td>3048</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal anti-FLAG M2-Cy3</td>
			<td>Sigma-Aldrich</td>
			<td>A9594</td>
			<td></td>
		</tr>
		<tr>
			<td>mPEG-SVA</td>
			<td>Laysan Bio Inc</td>
			<td>mPEG-SVA-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>MS(PEG)4</td>
			<td>Thermo Scientific</td>
			<td>22341</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl (5M)</td>
			<td>Thermo Scientific</td>
			<td>AM9760G</td>
			<td></td>
		</tr>
		<tr>
			<td>No 1.5 microscope Cover glass</td>
			<td>Fisherband</td>
			<td>12-544-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus Laser, 532nm 100mM</td>
			<td>Olympus digital Laser system</td>
			<td>CMR-LAS 532nm 100mW</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus TirfCtrl software</td>
			<td>Olympus</td>
			<td></td>
			<td>For controlling the laser intensity and incident angle</td>
		</tr>
		<tr>
			<td>Optical table</td>
			<td>TMC vibration control</td>
			<td>63-563</td>
			<td>With vibration isolation</td>
		</tr>
		<tr>
			<td>Phenol chloroform isoamyl alcohol mix</td>
			<td>Sigma-Aldrich</td>
			<td>77617-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce RNA 3&#39; End Biotinylation Kit</td>
			<td>Thermo Scientific</td>
			<td>20160</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium hydroxide pellets</td>
			<td>Sigma-Aldrich</td>
			<td>P1767-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Prior motorized XY translation stage</td>
			<td>Prior</td>
			<td>PS3J100</td>
			<td></td>
		</tr>
		<tr>
			<td>Prior PriorTest software</td>
			<td>Prior</td>
			<td></td>
			<td>For controlling the Prior motorized stage</td>
		</tr>
		<tr>
			<td>Recombinant RNasin RNase Inhibitor</td>
			<td>Promega</td>
			<td>N2515</td>
			<td></td>
		</tr>
		<tr>
			<td>Stage top Incubator</td>
			<td>In vivo scientific (world precision Instruments)</td>
			<td>98710-1</td>
			<td>With a custom built acrylic cage</td>
		</tr>
		<tr>
			<td>Staining jar</td>
			<td>Fisher Scientific</td>
			<td>08-817</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin</td>
			<td>Thermo Scientific</td>
			<td>43-4301</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfuric acid</td>
			<td>Fisher Scientific</td>
			<td>A300212</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR green II</td>
			<td>Fisher Scientific</td>
			<td>S7564</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Hamilton</td>
			<td>1725RN</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Harvard apparatus</td>
			<td>55-3333</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris (1M), pH = 7.0</td>
			<td>Thermo Scientific</td>
			<td>AM9850G</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic Bath</td>
			<td>Branson</td>
			<td>CPX1800H</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Sigma-Aldrich</td>
			<td>U5378-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Vaccinia Capping system</td>
			<td>New England Biolabs</td>
			<td>M2080S</td>
			<td></td>
		</tr>
		<tr>
			<td>Zymo-Spin IC Columns</td>
			<td>Zymo Research</td>
			<td>C1004</td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vitro single-molecule imaging assay for the analysis of cap-dependent translation kinetics

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed extensive studies of cap-dependent translation and its regulation, high resolution kinetic characterization of this translation pathway is still lacking. Recently, we developed an in vitro assay to measure cap-dependent translation kinetics with single-molecule resolution. The assay is based on fluorescently labeled antibody binding to nascent epitope-tagged polypeptide. By imaging the binding and dissociation of antibodies to and from nascent peptide&#8211;ribosome&#8211;mRNA complexes, the translation progression on individual mRNAs can be tracked. Here, we present a protocol for establishing this assay, including mRNA and PEGylated slide preparations, real-time imaging of translation, and analysis of single molecule trajectories. This assay enables tracking of individual cap-dependent translation events and resolves key translation kinetics, such as initiation and elongation rates. The assay can be widely applied to distinct translation systems and should broadly benefit in vitro studies of cap-dependent translation kinetics and translational control mechanisms.

Following the protocol described enables the imaging of individual antibody interactions with nascent N-terminal-tagged polypeptides with single-molecule resolution during active cell-free translation of 3&#39; end-tethered reporter mRNA (Figure 1). A minimal demonstration experiment is reported with the use of three synthetic mRNAs: LUC (encoding untagged luciferase), LUCFLAG (encoding 3xFLAG-tagged luciferase), and hp-LUCFLAG...

Watch this Scientific Journal Video about An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics at JoVE.com

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

Tracking individual translation events allows for high-resolution kinetic studies of cap-dependent translation mechanisms. Here we demonstrate an in vitro single-molecule assay based on imaging interactions between fluorescently labeled antibodies and epitope-tagged nascent peptides. This method enables single-molecule characterization of initiation and peptide elongation kinetics during active in vitro cap-dependent translation.

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

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed ...

This protocol is significant because it enables the utilization of existing cell-free translation systems for the single molecule analysis of active eukaryotic translation. The main advantages of this technique are that it preserves bulk translation properties, provide single molecule resolution, and is relatively simple to adapt from other bulk translation assays. This method can be applied to any cell-free translation system that has been verified to enable efficient cap-dependent translation under bulk conditions.Demonstrating the procedure will be Hongyun Wang, a research associate, and Anthony Gaba, a senior research scientist from Xiaohui Qu laboratory. At least three hours 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 the experiment. Then set the incubator temperature to the optimal temperature specific for the cell-free translation system.At least one hour before the experiment, press the laser power button behind the laser box, turn the laser safety key, and press the start button to turn the laser on. Turn on the microscope and select the imaging mode, objective, and filter set. Turn on the EMCCD camera, the stage controller, the syringe pump and the computer and open the camera, laser, motorized stage, and syringe pump software.In the laser control software, click connect to set the laser intensity, uncheck the shutter box and click control to drag the intensity slide bar in the laser control pop-up window. Enter the penetration depth to set the laser to the desired angle and confirm that the shutter box is closed to maintain the laser in the off mode when not imaging. When the camera has cooled and stabilized to its designated working temperature, in the camera software, select kinetic for the acquisition mode and enter the exposure time, kinetic series length, and electron multiplying gain value parameters.Select the directory and assign filenames for saving the data image file. Set the syringe pump to a modest to fast flow rate and add 500 microliters of 70%ethanol to a microfuge tube. Insert the syringe pump tubing end into the ethanol solution and use the syringe pump software to toggle the syringe pump between the withdrawal and dispense modes to wash the tubing three times with ethanol and three times with RNase-free water.After the final water wash, dab the tubing end on a clean tissue wipe to remove any residual water from the inside of the tubing and place the end of the tubing into a clean microfuge tube. For three prime end biotinylated mRNA immobilization, place the flow chamber into the microscope sample holder with the coverslip side facing down and secure the chamber with tape. Use the tape to cover the flow channel inlets and outlets that are not in use and add 1.5 times the volume of a 200 microgram per milliliter streptavidin solution to the flow channel.After 10 minutes at room temperature, wash and wipe the channel three times with three times the volume of T50 wash buffer per wash and the folded laboratory tissue. After the last wash, place a drop of immersion oil onto the microscope objective and place the holder and chamber onto the microscope stage. Then bring the objective close to the coverslip until the immersion oil on the objective contacts the coverslip and move the sample stage so that the position of the flow channel is directly above the objective lens.On the microscope control touchscreen, select the focus tab and click focus search and start. A beep will be heard when the focus plane is successfully achieved. In the camera software, select the live mode.In the laser control software, open the laser shutter and adjust the laser intensity level. Observe the real-time fluorescence image in live mode and in the microscope control touchscreen using the fine step control to adjust the objective position. In the camera software, move the stage to evaluate the level of fluorescence background at various areas of the flow channel detection surface.If the fluorescence is low, stop the live mode and close the laser shutter in the laser control software and replace the wash buffer from the flow channel with two times the volume of the biotinylated mRNA solution. After 15 minutes, wash the channel three times with three times the volume of translation compatible buffer per wash. For single molecule detection, assemble three times the volume of freshly thawed translation mix without mRNA and supplemented with an optimized concentration of fluorescently labeled antibodies in a microcentrifuge tube on ice and briefly spin the tube to collect the mix at the bottom of the tube.Transfer the translation mix to the inside of a 700 microliter microfuge tube cap and set the syringe pump to the withdraw mode. Insert the pump tubing end into the translation mix and start the syringe withdrawal. When the translation mix has completely entered the pump tubing, stop the withdrawal, reverse the syringe pump settings to the dispensing mode, and set the flow to an optimal rate for translation mix delivery.Start and stop the syringe to allow the translation mixture to reach end of tubing as necessary and insert the pump tubing end into the inlet of the flow channel, taking care to avoid bubbles. Screw the custom-made metal bracket onto the microscope sample holder plate to stabilize the connection and assess the microscope focus and imaging area fluorescence. Confirm that the movie acquisition parameters are correct and that the appropriate filename and directory have been entered for saving the files.Move the stage to allow imaging of an area in the center of the flow channel detection surface. And under the continuous AF tab, click the start buttons. A beep will be heard when a focus plane has been successfully achieved.After reducing the ambient light, open the laser shutter and click the take signal button to start recording. After five seconds, enter the run command to deliver the translation mix into the flow channel. Then record the data for up to two hours at a 0.5 to two seconds per frame time resolution.To analyze Cy3 ANTI-FLAG binding events, the fluorescence intensities of selected Cy3 alpha flag spots were calculated frame by frame for the entire movie and connected to form an intensity trajectory. Raw trajectories can appear noisy and may require refinement with a non-linear forward/backward filter to reduce background noise. Further refinement can be achieved using step detection algorithms to digitize trajectories.Antibody binding to a nascent polypeptide causes an instantaneous increase in fluorescence, whereas antibody polypeptide dissociation causes an instantaneous fluorescence decrease. The dwell time histogram for luciferase flag mRNA fits to a log normal distribution and yields a peptide synthesis rate of 2.5 plus or minus 0.1 amino acids per second. The first arrival time histogram fits to a shifted log normal function, illustrating the high degree of heterogeneity in single mRNA translation activity.As the histogram indicates, the first peptide synthesis event on single luciferase flag mRNA molecules can begin as early as two minutes and as late as 20 minutes after translation mix delivery. Compared to translation with luciferase flag mRNA, the first arrival time histogram with hairpin luciferase flag mRNA translation showed slower antibody binding. Using bulk luminescence measurements from cell-free translation reactions with these same mRNAs, however, does not resolve differences in translation kinetics.The single molecule resolution of this assay provides more kinetic insights of cap-dependent translation that are not easily attainable with other approaches. Eukaryotic cell-free translation is sensitive to small variations and experimental conditions. Therefore, attention to detail and consistency are critical when using this assay to accurately detect small differences in translation.

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

Abstract