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.

The authors have nothing to disclose.

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 extremely high fluorescent background that masks single-molecule detection of antibody binding. To reduce this fluorescent background, the laser incident angle is increased well above the critical angle (step 3.4.). Antibody detection is also diminished by excessive fluorophore instability, which can limit the capability to quantify dwell time. When encountering this problem, replace the fluorophore with a more photostable fluorophore, such as the ones conjugated to a triplet-state quencher47, or lower the laser illumination intensity. Although oxygen scavenging systems are commonly used in in vitro single-molecule experiments to suppress fluorophore photobleaching48,49, the assay described here can provide a very generous detection window without implementing any oxygen scavenging systems15. Furthermore, oxygen scavenging systems can greatly reduce eukaryotic cell-free translation efficiency. Therefore, oxygen scavenging systems should be carefully evaluated prior to their use with this assay.
It is important to point out that small variations in reagent preparation are detected by the assay due to its single-molecule resolution. For example, replicate translation extract preparations can display different activities that may or may not be detectable by bulk methods. Furthermore, freeze-thaw cycles of translation extract and other reagents can rapidly impair translation activity. We recommend doing small-scale pilot experiments with readily accessible material to test conditions before planning for a systematic study. For a systematic study, large-scale preparations of translation reagents, single-use aliquots for freeze/thaw-sensitive reagents, and consistency in performing protocol steps are recommended. Translation extract that is flash frozen in liquid nitrogen and stored at -80 &#730;C shows minimal activity loss for at least two years. Application of these measures can effectively suppress experimental variations and increase the robustness of the single-molecule assay.
As this method combines existing bulk cell-free translation systems and in vitro single-molecule imaging techniques, troubleshooting of generic problems in these two areas are not discussed here. Examples of such generic issues include low quality preparations of reporter mRNA or PEGylated coverslip and low translation activity of cell extract. Here, we will focus on issues specific for this method. In addition to the several technical issues that have been discussed in the protocol, another potential problem may be low or no antibody binding in the flow channel for a translation condition that is expected to have good activity. There are several possibilities to troubleshoot. The efficiency of biotinylated mRNA immobilization to the detection surface can be assessed by annealing complementary and fluorophore-conjugated DNA oligomers to the immobilized mRNA and imaging the fluorescent DNA:RNA duplexes. The quality of the translation mix and biotinylated reporter mRNAs can be checked by running the bulk translation assay. Furthermore, after carrying out a translation reaction in an mRNA-immobilized flow channel, the translation reaction solution can be pipetted out of the channel and used in a reporter activity assay to confirm the occurrence of in-channel translation. If necessary, an excessively high mRNA surface density can be used for the in-channel translation reaction to allow easier detection by the reporter activity assay.
The major limitation of this assay is its resolution for initiation kinetics. In this assay, the direct experimental output, first arrival time, contains both the first-round initiation time and the peptide elongation time for translating the epitope and 35 additional codons. Although the contribution of the peptide elongation time can be corrected (step 6.9.), this assay consequently is most sensitive for measuring initiation kinetics that occur on the order of minutes, which appears to be a generic property of cap-dependent initiation15. Experimentally, it is easy to adapt this assay to study other initiation mechanisms. However, if an initiation mechanism occurs significantly fast on the order of seconds, this assay is unlikely to resolve the initiation kinetics.
The single-molecule approach described here combines single-molecule imaging and extract-based translation to enable measurements of individual cap-dependent translation events in real-time and during active cell-free translation. The assay allows easy transitioning from a bulk translation assay to a single-molecule assay, while preserving the bulk translation kinetics. Thus, single-molecule kinetic observations can be interpreted in direct reference to corresponding bulk results. Previous methods, including recently developed in vivo approaches11,12,13,14, lack the resolution for kinetic measurements of individual translation initiation events and require assumptions of translation mechanisms to extract initiation time averages from experimental observables. The single-molecule method presented here provides the first hypothesis-free approach for quantifying the average initiation time of active cap-dependent translation. Furthermore, although bulk kinetic measurements with a luminescence assay have been utilized to provide the most quantitative and systematic bulk characterizations of cap-dependent translation kinetics to date46,50, the single-molecule assay described here measures average initiation kinetic changes with greater sensitivity than the bulk assay (Figure 7). In addition, the single-molecule data preserves single mRNA translation stochasticity and heterogeneity, properties that are averaged out in bulk measurements. The single-molecule resolution of translation kinetics can be utilized for systematic kinetic analyses to reveal insights of translation mechanisms unattainable with other methods.
This assay is compatible with existing biochemical and genetic approaches that are commonly used for translation studies. For example, the translational function of a specific factor can be studied by generating factor-depleted extract and supplementing it with wild-type or mutant factor. In addition, by supplementing fluorescently labeled factor, studies can potentially investigate the kinetic relationship between factor binding and the progression of translation. With the use of two distinct epitope/antibody pairs, this protocol could potentially be expanded from a one-color assay to a two-color assay that enables simultaneous detection of two distinct coding sequences. This adaptation would allow tracking the translation of different reading frames and could be applied to frameshifting and start site selection studies. Alternatively, a two-color system could be used to detect antibody interactions with tagged polypeptides encoded by upstream and downstream open reading frames to monitor both upstream and downstream translation events on individual mRNAs. These expansions can enable a wide range of mechanistic studies that investigate cap-dependent translation and its regulation.

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 (LUCFLAG with a stable stem-loop in the 5&#39; leader region) (Figure 2C). The use of luciferase-encoding mRNA enables comparisons between single-molecule observations and bulk luminescence measurements. Integrity of 3&#39; biotinylated mRNA was assessed by denaturing polyacrylamide gel electrophoresis (PAGE) and RNA staining. Good quality mRNA shows a single clean band and should not show additional faster migrating signals (Figure 3A). Saccharomyces cerevisiae translation extract was used here and prepared following a protocol that was previously described30 and modified15. Bulk measurements of luciferase activity in cell-free translation reactions with the yeast extract and biotinylated LUCFLAG mRNA that either lacks or contains an m7G cap shows cap stimulation of LUCFLAG mRNA translation (Figure 3B).
For single-molecule imaging, translation extract was supplemented with 67 nM of Cy3-labeled anti-FLAG antibody (Cy3-&#945;FLAG). This antibody had a high labeling ratio of 2-7 dyes per antibody. The 532 nm laser was set to 10 &#956;W at the objective. The laser incident angle was set to 71.5&#730;, which was greater than the critical angle of 63.8&#730; for our set up. A low level of fluorescence was imaged from detection surfaces that contain mRNA but lack translation mix (Figure 4A). For channel dimensions of approximately 2 mm (width) x 50 &#956;m (depth) x 24 mm (length), a flow delivery rate of 150 &#956;l/min yielded a reagent exchange time of 3 &#8211; 4 s. Within 5 s of translation mix delivery, the background fluorescence level increases due to the diffusing fluorescent antibodies. Approximately 2 min after translation mix delivery to LUCFLAG mRNA-containing channels, Cy3 spots began to appear in a field of view and continued to accumulate for ~30 min (Figure 4B). Nonspecific antibody binding to surface, measured using the 3xFLAG-lacking LUC mRNA (Figure 2C), remained at a very low level15. The signals from mRNA/ribosome/peptide-bound Cy3- &#945;FLAG were clearly visible with the optimized laser incident angle but could be masked by a high fluorescence background with lower laser incident angles (Figure 4C).
To analyze Cy3-&#945;FLAG binding events, the fluorescence intensities of selected Cy3-&#945;FLAG spots (Figure 5A) were calculated frame by frame for the entire movie and then connected to form an intensity trajectory (Figure 5B). Raw trajectories can appear noisy and may require refinement with a non-linear forward-backward filter to reduce background noise41. Further refinement could be achieved using step-detection algorithms to digitize trajectories42. For all trajectories, a universal background fluorescence level increase appears during reagent exchange due to the diffusing fluorescent antibodies in the translation mix (Figure 5B). Antibody binding to a nascent polypeptide caused an instantaneous increase in fluorescence whereas antibody/polypeptide dissociation from mRNA causes an instantaneous fluorescence decrease (Figure 5B).
The dwell time of antibody binding can be used to measure the total decoding time of an open reading frame. The dwell time histogram for LUCFLAG mRNA fits to a log-normal distribution (Figure 6A) and yielded a peptide synthesis rate of 2.5 &#177; 0.1 amino acids per second15. The first arrival time histogram fits to a shifted (3-parameter) log-normal function (Figure 6B). The wide spread of the first arrival time histogram shows the high degree of heterogeneity in single mRNA translation activity. The histogram indicated that the first peptide synthesis event on single LUCFLAG mRNA molecules could begin as early as 2 min, and as late as 20 min, after translation mix delivery (Figure 6B). Compared to translation with LUCFLAG mRNA, the first arrival time histogram with hp-LUCFLAG mRNA translation showed slower antibody binding (Figure 7A). Using bulk luminescence measurements from cell-free translation reactions with these same mRNAs, however, does not resolve differences in translation kinetics (Figure 7B,C). These results demonstrate the higher resolution of our method compared to bulk kinetic luciferase activity measurements for detecting small changes in initiation kinetics.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61648/61648fig01.jpg" /> 
Figure 1: Protocol flow chart.&#160;Schematic representations of coverslip and slide preparation, single-molecule chamber assembly, TIRF imaging and data acquisition, and data analysis steps are shown. The TIRF imaging step includes schematic depictions of mRNA immobilization and translation in a flow channel. Detection surface components, fluorescently labeled antibody, and cell extract components are indicated. <a href="https://www.jove.com/files/ftp_upload/61648/61648fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61648/61648fig2v2.jpg" /> 
Figure 2: mRNA designs for the single-molecule assay.&#160;(A) To adapt a bulk translation assay to the single-molecule assay, the DNA template of the bulk reporter ORF was modified with an N-terminal epitope tag sequence insertion to generate a tagged reporter-encoding ORF. ORF and N-terminal tag-coding regions are indicated. (B) mRNAs were 5&#8242;-m7G capped to allow cap-dependent translation and 3&#8242;-biotinylated for their immobilization to single-molecule detection surface. 5&#8242;-m7G cap and 3&#8242;-biotin are indicated on untagged and tagged ORF RNAs. (C) Luciferase-encoding mRNAs were used to demonstrate the single-molecule assay. LUC and LUCFLAG mRNAs encode luciferase that lacks and contains an N-terminal 3xFLAG tag, respectively. hp-LUCFLAG mRNA contains an additional insertion that introduces a hairpin secondary structure in the LUCFLAG mRNA 5&#8242;-leader. The hairpin thermostability, 3&#8242;-poly(A) tail, and 3&#8242;-biotin are indicated. All three mRNAs included a 30 nt 3&#8242;-poly(A) tail for more efficient cap-dependent translation. <a href="https://www.jove.com/files/ftp_upload/61648/61648fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61648/61648fig3v2.jpg" /> 
Figure 3: Single-molecule reporter mRNA integrity and bulk translation.&#160;(A) Representative denaturing PAGE imaging of single-molecule reporter mRNA. 5&#8242;-m7G capped and 3&#8242;-biotinylated LUCFLAG mRNA was loaded onto a denaturing 10% polyacrylamide gel next to an RNA ladder. (B) Single-molecule reporter mRNA preserved 5&#39; cap-dependence in bulk cell-free translation reactions. 3&#8242;-biotinylated LUCFLAG mRNA that either lacked (&#8211;cap) or contained (+ cap) a 5&#8242;-m7G cap was translated in S. cerevisiae translation extract for 30 min at 25 &#730;C. Luciferase activity was measured from two independent reactions and normalized to the activity with &#8211;cap mRNA, which is arbitrarily set to 1.0. Standard deviations from mean values are indicated by error bars. <a href="https://www.jove.com/files/ftp_upload/61648/61648fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61648/61648fig04.jpg" /> 
Figure 4: Imaging of detection surfaces containing immobilized mRNA.&#160;(A) Background fluorescence in the absence of translation mix. (B) Cy3 fluorescent spots in a field of view 30 min after the translation mix delivery and with an optimized laser incident angle. (C) Imaged field of view 30 min after translation mix delivery and with a low laser incident angle. <a href="https://www.jove.com/files/ftp_upload/61648/61648fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61648/61648fig05.jpg" /> 
Figure 5: Image analysis.&#160;(A) Cy3 spots in a field of view without (left panel) or with (right panel) spot selection. (B) Example single-molecule trajectory for a selected Cy3 spot. The black arrow indicates the rise in background fluorescence due to diffusing antibody during translation mix delivery. The green arrow indicates the sudden increase in fluorescence intensity due to Cy3-&#945;FLAG binding. The red arrow indicates the sudden decrease in fluorescence intensity due to Cy3-&#945;FLAG/nascent peptide dissociation from mRNA. The dwell time of a Cy3-&#945;FLAG binding event is indicated with a double-headed arrow. Raw, filtered, and digitized data are indicated. <a href="https://www.jove.com/files/ftp_upload/61648/61648fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61648/61648fig06.jpg" /> 
Figure 6: Kinetic analysis of Cy3-&#945;FLAG binding with LUCFLAG mRNA translation.&#160;(A) The Cy3-&#945;FLAG binding dwell time histogram fits to a log-normal distribution. (B) The Cy3-&#945;FLAG binding first arrival time histogram fits to a shifted (3-parameter) log-normal function. Adapted from Wang et al.15 with permission. <a href="https://www.jove.com/files/ftp_upload/61648/61648fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61648/61648fig7v3.jpg" /> 
Figure 7: The single-molecule assay has a higher resolution for initiation kinetics than the bulk luminescence assay.&#160;(A) First arrival time histograms for Cy3-&#945;FLAG binding with translation of LUCFLAG and hp-LUCFLAG mRNAs revealed slower initiation caused by a small hairpin structure in the mRNA 5&#8242;-leader. N = 10650 trajectories (LUCFLAG), combined from 3 data sets and N = 4079 trajectories (hp-LUCFLAG), combined from 2 data sets. (B,C) Bulk luciferase activity assay kinetic measurements did not resolve differences in initiation kinetics for LUCFLAG (N = 9 data sets) and hp-LUCFLAG (N = 6 data sets) mRNA translation. (B) Bulk luminescence kinetics. (C) Scatter plots of first round translation times determined by Gaussian fitting to the second derivative of the luminescence kinetics curves in (B), as described by Vassilenko et al.46. The red circles and bars in (C) represent the mean value and standard deviation of the calculated first round translation time, respectively. Adapted from Wang et al.15 with permission. <a href="https://www.jove.com/files/ftp_upload/61648/61648fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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Nanyang Technological University

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JoVE Journal

Biochemistry

3.0K Views.  Memorial Sloan Kettering Cancer Center. 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.

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

Single-molecule Super-resolution Imaging of Phosphatidylinositol 4,5-bisphosphate in the Plasma Membrane with Novel Fluorescent Probes

Phosphoinositides in the cell membrane are signaling lipids with multiple cellular functions. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a determinant phosphoinositide of the plasma membrane (PM), and it is required to modulate ion channels, actin dynamics, exocytosis, endocytosis, intracellular signaling, and many other cellular processes. However, the spatial organization of PI(4,5)P2 in the PM is controversial, and its nanoscale distribution is poorly understood due to the technical limitations of research approaches. Here by utilizing single molecule localization microscopy and the Pleckstrin Homology (PH) domain based dual color fluorescent probes, we describe a novel method to visualize the nanoscale distribution of PI(4,5)P2 in the PM in fixed membrane sheets as well as live cells.

PI(4,5)P2 regulates various cellular functions, but its nanoscale organization in the cell plasma membrane is poorly understood. By labeling PI(4,5)P2 with a dual-color fluorescent probe fused to the Pleckstrin Homology domain, we describe a novel approach to study the PI(4,5)P2 spatial distribution in the plasma membrane at the nanometer scale.

Phosphoinositides in the cell membrane are signaling lipids with multiple cellular functions. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a ...

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA processing, and telomere elongation. During these processes, various proteins bind and resolve G4 structures to perform their function. As the function of G4 often depends on the stability of its folded structure, it is important to investigate how G4 binding proteins regulate the stability of G4. This work presents a method to manipulate single G4 molecules using magnetic tweezers, which enables studies of the regulation of G4 binding proteins on a single G4 molecule in real time. In general, this method is suitable for a wide scope of applications in studies for proteins/ligands interactions and regulations on various DNA or RNA secondary structures.

A single-molecule magnetic tweezers platform to manipulate G-quadruplexes is reported, which allows for the study of G4 stability and regulation by various proteins.

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA ...

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation of protein synthesis is the key to cellular stress-response, and dysregulation is central to many disease states, such as cancer. For instance, although cellular stress leads to the inhibition of global translation by attenuating cap-dependent initiation, certain stress-response proteins are selectively translated in a cap-independent manner. Discreet RNA regulatory elements, such as cellular internal ribosome entry sites (IRESes), allow for the translation of these specific mRNAs. Identification of such mRNAs, and the characterization of their regulatory mechanisms, have been a key area in molecular biology. Toeprinting is a method for the study of RNA structure and function as it pertains to translation initiation. The goal of toeprinting is to assess the ability of in vitro transcribed RNA to form stable complexes with ribosomes under a variety of conditions, in order to determine which sequences, structural elements, or accessory factors are involved in ribosome binding&#8212;a pre-cursor for efficient translation initiation. Alongside other techniques, such as western analysis and polysome profiling, toeprinting allows for a robust characterization of mechanisms for the regulation of translation initiation.

Toeprinting aims to measure the ability of in vitro transcribed RNA to form translation initiation complexes with ribosomes under a variety of conditions. This protocol describes a method for toeprinting mammalian RNA and can be used to study both cap-dependent and IRES-driven translation.

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.

This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed &#8216;labeling occupancy&#8217; (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample at the single molecule level.

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Production of Dynein and Kinesin Motor Ensembles on DNA Origami Nanostructures for Single Molecule Observation

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and others. Many of these functions require motors to operate in ensembles. Despite a wealth of knowledge about the mechanisms of individual cytoskeletal motors, comparatively less is known about the mechanisms and emergent behaviors of motor ensembles, examples of which include changes to ensemble processivity and velocity with changing motor number, location, and configuration. Structural DNA nanotechnology, and the specific technique of DNA origami, enables the molecular construction of well-defined architectures of motor ensembles. The shape of cargo structures as well as the type, number and placement of motors on the structure can all be controlled. Here, we provide detailed protocols for producing these ensembles and observing them using total internal reflection fluorescence microscopy. Although these techniques have been specifically applied for cytoskeletal motors, the methods are generalizable to other proteins that assemble in complexes to accomplish their tasks. Overall, the DNA origami method for creating well-defined ensembles of motor proteins provides a powerful tool for dissecting the mechanisms that lead to emergent motile behavior.

The goal of this protocol is to form ensembles of molecular motors on DNA origami nanostructures and observe the ensemble motility using total internal reflection fluorescence microscopy.

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and ...

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk population methods (Northern blot, PCR, and RNAseq) that measure mRNA levels lack the ability to provide information on cell-to-cell variation in responses. Precise single cell and allelic visualization and quantification is possible via single molecule RNA fluorescence in situ hybridization (smFISH). RNA-FISH is performed by hybridizing target RNAs with labeled oligonucleotide probes. These can be imaged in medium/high throughput modalities, and, through image analysis pipelines, provide quantitative data on both mature and nascent RNAs, all at the single cell level. The fixation, permeabilization, hybridization and imaging steps have been optimized in the lab over many years using the model system described herein, which results in successful and robust single cell analysis of smFISH labeling. The main goal with sample preparation and processing is to produce high quality images characterized by a high signal-to-noise ratio to reduce false positives and provide data that are more accurate. Here, we present a protocol describing the pipeline from sample preparation to data analysis in conjunction with suggestions and optimization steps to tailor to specific samples.&#160;

Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs.

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for hundreds of individual restriction endonuclease (REase) molecules in one experiment. This assay does not require protein labeling and can be carried out with a single imaging channel. In addition, the results of multiple individual experiments can be pooled to generate well-populated dwell-time distributions. Analysis of the resulting dwell-time distributions can help elucidate the DNA cleavage mechanism by revealing the presence of kinetic steps that cannot be directly observed. Example data collected using this assay with the well-studied REase, EcoRV - a dimeric Type IIP restriction endonuclease that cleaves the palindromic sequence GAT&#8595;ATC (where &#8595; is the cut site) - are in agreement with prior studies. These results suggest that there are at least three steps in the pathway to DNA cleavage that is initiated by introducing magnesium after EcoRV binds DNA in its absence, with an average rate of 0.17 s-1 for each step.

Using quantum-dot-labeled DNA and total internal reflection fluorescence microscopy, we can investigate the reaction mechanism of restriction endonucleases while using unlabeled protein. This single-molecule technique allows for massively multiplexed observation of individual protein-DNA interactions, and data can be pooled to generate well-populated dwell-time distributions.

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for ...

Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 (FET) family of fusion oncoproteins, is one of the most frequently involved fusion genes in Ewing sarcoma. These FET family fusion proteins typically harbor a low-complexity domain (LCD) of FET protein at their N-terminus and a DNA-binding domain (DBD) at their C-terminus. EWS-FLI1 has been confirmed to form biomolecular condensates at its target binding loci due to LCD-LCD and LCD-DBD interactions, and these condensates can recruit RNA polymerase II to enhance gene transcription. However, how these condensates are assembled at their binding sites remains unclear. Recently, a single-molecule biophysics method-DNA Curtains-was applied to visualize these assembling processes of EWS-FLI1 condensates. Here, the detailed experimental protocol and data analysis approaches are discussed for the application of DNA Curtains in studying the biomolecular condensates assembling on target DNA.

Here, we describe the use of the single-molecule imaging method, DNA Curtains, to study the biophysical mechanism of EWS-FLI1 condensates assembling on DNA.

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 ...

An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to measure protein phosphorylation cannot preserve the heterogeneity in phosphorylation across individual proteins. The single-molecule pull-down (SiMPull) assay was developed to investigate the composition of macromolecular complexes via immunoprecipitation of proteins on a glass coverslip followed by single-molecule imaging. The current technique is an adaptation of SiMPull that provides robust quantification of the phosphorylation state of full-length membrane receptors at the single-molecule level. Imaging thousands of individual receptors in this way allows for quantifying protein phosphorylation patterns. The present protocol details the optimized SiMPull procedure, from sample preparation to imaging. Optimization of glass preparation and antibody fixation protocols further enhances data quality. The current protocol provides code for the single-molecule data analysis that calculates the fraction of receptors phosphorylated within a sample. While this work focuses on phosphorylation of the epidermal growth factor receptor (EGFR), the protocol can be generalized to other membrane receptors and cytosolic signaling molecules.

The present protocol describes sample preparation and data analysis to quantify protein phosphorylation using an improved single-molecule pull-down (SiMPull) assay.

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to ...