This work was supported financially by the Netherlands Organization for Scientific Research (NWO-CW), the European Research Council (ERC; advanced grant no. 694345 to B.L.F.), and the Ministry of Education, Culture and Science (Gravitation program no. 024.001.035).

NOTE: Organic synthesis is the major core of this project. Figure 1 shows the key steps and how to obtain the target molecule.
1. Preparation of 1b
NOTE: Solvents were purchased in analytical grade.
<ol>
	<li>Synthesis of ketone (3) 
	NOTE: At first, a five-member ring ketone 2 with an iodo group is functionalized because it is more reactive. This reaction is carried out under argon atmosphere.
	<ol>
		<li>In a sealed tube (100 mL) containing 2 (640 mg, 2.3 mmol), CuI (219 mg, 1.1 mmol), and NaI (3.44 g, 23 mmol), add 1,4-dioxane (50 mL) and N,N&#39;-dimethyl ethylenediamine (202 mg, 2.3 mmol).</li>
		<li>Set the reaction temperature to 140 &#176;C and stir for 24 h.</li>
		<li>Cool the mixture to room temperature (RT), remove the solvent in vacuo, and purify the remaining material by flash chromatography: SiO2 (40 g); eluent: pentane:EtOAc = 10:1 (total = 550 mL). The product should be obtained as a yellow, sticky oil (642 mg, 91%).</li>
	</ol>
	</li>
	<li>Synthesis of motor 5 
	NOTE: Use the Barton-Kellogg reaction to form the central double bond. This reaction is carried out under argon.
	<ol>
		<li>In a 50 mL round-bottom flask, add Lawesson&#39;s reagent (415 mg, 1.1 mmol), ketone 3 (219 mg, 0.68 mmol), and toluene (10 mL).</li>
		<li>Heat the mixture at reflux for 2 h and subsequently evaporate the solvent.</li>
		<li>Purify the residue by flash column: SiO2 (24 g), eluent: pentane:ethyl acetate = 30:1 (total = 155 mL) to obtain the corresponding thioketone, which is subsequently redissolved in 20 mL of THF to yield a blue solution.</li>
		<li>Add a THF solution (20 mL) of diazo compound 4&#160;(476 mg, 1.37 mmol) to the blue solution, and stir the newly mixed solution at 50 &#176;C overnight. 
		NOTE: This is the most crucial step of the entire synthesis. The diazo compound and thioketone solution must be fresh and should be made prior to the reaction.</li>
		<li>Evaporate the solvent and purify the residue by chromatography: SiO2 (24 g), eluent: pentane:CH2Cl2 = 10:1 (total = 220 mL) to yield motor 5 (250 mg, 50%) as a red solid.</li>
	</ol>
	</li>
	<li>Synthesis of motor 6 
	NOTE: When the central double bond is formed, replace the iodo group by an acetylene moiety.
	<ol>
		<li>To a 20 mL Schlenk tube, add 3 (165 mg, 0.26 mmol), Pd(PPh3)2Cl2 (2.5 mole %), CuI (5 mole %). Then, add THF (10 mL) and (i-Pr)2NH (2 mL) that should be bubbled with argon for 10 min before.</li>
		<li>Stir the mixture at RT for 10 min. Then, add triisopropylsilyl acetylene (42 mg, 0.27 mmol).</li>
		<li>Stir the mixture for 15 h, then pour it into an aqueous saturated NH4Cl solution (25 mL).</li>
		<li>Extract the mixture with CH2Cl2 (3 x 20 mL). Further wash the combined organic layers with saturated brine and dry (Na2SO4).</li>
		<li>Remove the solvent and purify the residue by flash chromatography: SiO2 (12 g), eluent: pentane:CH2Cl2 = 10:1 (220 mL) to yield 8 as a brown oil (171 mg, 99%).</li>
	</ol>
	</li>
	<li>Synthesis of motor 8 
	NOTE: This reaction is carried out under argon.
	<ol>
		<li>Stir a mixture of 6 (161 mg, 0.24 mmol), pinacol ester 7 (240 mg, 0.71 mmol), K3PO4 (300 mg, 1.44 mmol), and Pd(PPh3)4 (98 mg, 0.096 mmol) in 1,4dioxane (20 mL) at 90 &#176;C in a 50 mL Schlenk tube for 16 h.</li>
		<li>Cool the mixture to RT, dilute it with ethyl acetate (30 mL), and perform the filtration with a glass filter.</li>
		<li>Remove the solvent. Purify the residue by flash column chromatography: SiO2 (12 g), eluent: pentane: CH2Cl2= 1:6 (total = 122 mL) to yield ester 8 as a brown oil (156 mg, 56%).</li>
	</ol>
	</li>
	<li>Synthesis of motor 9
	<ol>
		<li>Add TBAF (0.1 mL) to a solution of 8 (120 mg, 0.13 mmol) in THF (10 mL) in a 20 mL Schlenk tube at 0 &#176;C.</li>
		<li>Stir the mixture at 0 &#176;C for 1 h, then pour it into an aqueous saturated NH4Cl solution (20 mL).</li>
		<li>After extraction with CHCl3 (3 x 10 mL), wash the combined organic layers with saturated brine and dry (Na2SO4).</li>
		<li>Remove the solvent and purify the residue by flash chromatography: SiO2 (12 g), eluent: pentane:ethyl acetate = 1:3 (124 mL) to yield 9 as a dark red oil (116 mg, 95%).</li>
	</ol>
	</li>
	<li>Synthesis of motor 12 
	NOTE: This reaction is carried out under argon.
	<ol>
		<li>To a 20 mL Schlenk tube, add motor 9 (75 mg, 0.10 mmol), PBI 11 (68 mg, 0.10 mmol) Pd(PPh3)2Cl2 (2.5 mol%), CuI (5 mol%). Then, add THF (10 mL) and (i-Pr)2NH (2 mL) that should be bubbled with argon for 10 min before.</li>
		<li>Stir the mixture overnight and pour it into an aqueous saturated NH4Cl solution.</li>
		<li>After extraction with CHCl3 (3 x 20 mL), wash the combined organic layers with saturated brine and dry (Na2SO4).</li>
		<li>Remove the solvent and purify the residue by flash chromatography: SiO2 (12 g), eluent: CHCl3 (100 mL) to yield motor 12 as a dark red solid (66 mg, 57%).</li>
	</ol>
	</li>
	<li>Synthesis of motor 1b 
	NOTE: When the ester compound is obtained, hydrolyze it to make the final target molecule.
	<ol>
		<li>Dissolve the ester 12 (90 mg, 0.038 mmol) in THF (5 mL), MeOH (5 mL), and NaOH(aq.) (1 M, 5 mL) in a 50 mL flask and heat the mixture to 75 &#176;C for 16 h.</li>
		<li>Cool the mixture to RT and add 5 mL of water. Remove the THF and MeOH by rotary evaporation.</li>
		<li>Titrate the mixture with HCl(aq.) (1 M) until reaching a pH of 1 to form a brown precipitate. Filtrate the mixture and wash the brown solid with cold water (10 mL) and dry in vacuo. This brown solid is the target molecule 1b (55 mg, 85%).</li>
	</ol>
	</li>
</ol>
2. Preparation of motor functionalized monolayer MS-1b
<ol>
	<li>Clean quartz slides by immersing them in a piranha solution (3:7 ratio of 30% H2O2 in H2SO4) at 90 &#176;C for 1 h. Rinse copiously with doubly distilled water 3x, then rinse with MeOH. Dry the slides under a stream of N2 before surface modification.</li>
	<li>Silanize the piranha-cleaned quartz slides by immersing in a 1 mM solution of 3-aminopropyl(diethoxy)methylsilane in freshly distilled toluene at RT for 12 h. Rinse copiously with toluene and MeOH.</li>
	<li>Sonicate the quartz first in toluene, then in MeOH, and dry them under a stream of argon.</li>
	<li>Immerse the amine-coated slides in DMF solution of 1b (10-4 M) at RT for 12 h.</li>
	<li>Wash the slides with DMF, water, and MeOH, then dry them under a stream of argon. After drying, the slides are ready for use.</li>
</ol>

<ol>
	<li>Berg, J. M., Tynoczko, J. L., Styer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biochemistry 5th ed. , (2006).</li><li>Erbas-Cakmak, S., Leigh, D. A., McTernan, C. T., Nussbaumer, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+Molecular+Machines.">Artificial Molecular Machines.</a> Chemical Reviews. 115, 10081-10206 (2015).</li><li>Feringa, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Art+of+Building+Small:+From+Molecular+Switches+to+Motors.">The Art of Building Small: From Molecular Switches to Motors.</a> Angewandte Chemie: International Edition. 56, 11059-11078 (2017).</li><li>Stoddart, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Machines.">Molecular Machines.</a> Accounts of Chemical Research. 34, 410-411 (2001).</li><li>Kinbara, K., Aida, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+Intelligent+Molecular+Machines:+Directed+Motions+of+Biological+and+Artificial+Molecules+and+Assemblies.">Toward Intelligent Molecular Machines: Directed Motions of Biological and Artificial Molecules and Assemblies.</a> Chemical Reviews. 105, 1377-1400 (2005).</li><li>Kay, E. R., Leigh, D. A., Zerbetto, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+Molecular+Motors+and+Mechanical+Machines.">Synthetic Molecular Motors and Mechanical Machines.</a> Angewandte Chemie: International Edition. 46, 72-191 (2007).</li><li>Kottas, G. S., Clarke, I. L., Horinek, D., Michl, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+Molecular+Rotors.">Artificial Molecular Rotors.</a> Chemical Reviews. 105, 1281-1376 (2005).</li><li>Watson, M. A., Cockroft, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Man-made+Molecular+Machines:+Membrane+Bound.">Man-made Molecular Machines: Membrane Bound.</a> Chemical Society Reviews. 45, 6118-6129 (2016).</li><li>Kassem, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+Molecular+Motors.">Artificial Molecular Motors.</a> Chemical Society Reviews. 46, 2592-2621 (2017).</li><li>Sauvage, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Machines and Motors. , (2001).</li><li>van Delden, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unidirectional+molecular+motor+on+a+gold+surface.">Unidirectional molecular motor on a gold surface.</a> Nature. 437, 1337-1340 (2005).</li><li>Noji, H., Yasuda, R., Yoshida, M., Kinosita, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+observation+of+the+rotation+of+F1-ATPase.">Direct observation of the rotation of F1-ATPase.</a> Nature. 386, 299-302 (1997).</li><li>Hutchison, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+surface-bound+molecule+that+undergoes+optically+biased+Brownian+rotation.">A surface-bound molecule that undergoes optically biased Brownian rotation.</a> Nature Nanotechnology. 9, 131-136 (2014).</li><li>Krajnik, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defocused+Imaging+of+UV-Driven+Surface-Bound+Molecular+Motors.">Defocused Imaging of UV-Driven Surface-Bound Molecular Motors.</a> Journal of the American Chemical Society. 139, 7156-7159 (2017).</li></ol>

The authors have nothing to disclose.

This project involves a significant amount of synthetic work; therefore, the most critical step is organic synthesis towards the final molecule. Among the total synthesis, the Barton-Kellogg reaction is the key step, since it is the reaction in which the central double bond of the molecular motor is formed. Currently, several methods have been used to form these types of structures. Here, diazo-thioketone coupling is used, and the upper and lower halves have been prepared as the diazo and thioketone compounds, respectively. Thioketone and diazo compounds are usually not stable under air; therefore, the reaction requires fast operating under a strictly inert atmosphere. 
Existing methods to confine molecular motors on the surfaces are mostly based on bipodal systems. However, the isomerization processes of previously designed bipodal motors were obstructed due to intermolecular interactions. In addition, some of the bipodal examples requires further activation prior to attachment. The current method accomplishes this in a tetrapodal manner, which provides robust attachment of the motor on surfaces with sufficient isolated space. 
A limitation of this method is the choice of fluorescent tag. Only dyes with specific wavelengths are allowed, as the rotation of motor is triggered by the 365 nm wavelength and thus should not be overlapped. In addition, the synthetic route employed in the described protocol towards the target molecule requires several steps in which harsh conditions are needed for completion of the reaction. In the future, a more facile synthetic design is probably needed if a more advanced molecule for single molecular imaging is required. 
In conclusion, the design and synthesis of a highly functionalized light-driven molecular motor is described for the first time. Some details of the synthetic effort are discussed, as well. Furthermore, methods to graft the motor onto a quartz slide surface are demonstrated, and the sample can be further tested for visualization of single molecular motion14.

In living organisms, there are abundant molecular motors functioning to sustain daily life. They are able to perform various tasks such as fuel production, transport, mobility, etc.1. Drawing from the inspiration of these fascinating examples in Nature, scientists have developed a series of artificial molecular motors over the last several decades to convert different types of energy into controlled motion at the molecular level2,3,4,5,6,7,8,9,10. The Nobel Prize in Chemistry in 2016 was awarded to three pioneers in this field. Ben Feringa, one of the laureates, has developed the light-driven molecular motor that is able to undergo continuous unidirectional rotary motion.
However, at a molecular level, Brownian motion, also known as the random motion due to molecular collisions and vibrations, is usually the major obstacle for further application of these molecular motors. Brownian motion can disrupt any directed motion. Confining the molecular motors on surfaces may be one of the options to overcome this problem. By doing so, the relative rotation of one part of the molecule with respect to the other is converted to the absolute rotation of the rotor relative to the surface11. In addition, use of the single molecular imaging technique may help visualize the motion. Therefore, the results obtained by this work will help to gain more insight into the synthetic molecular motor.
The pioneering work of Yoshida and Kinosita (Figure 2a)12,13 has served as the inspiration for the design in the current work, shown in Figure 2b. The lower half of a light-driven molecular motor is attached to a surface to serve as the stator. The rotor part is functionalized with a rigid arm and fluorescent label. When applying two different irradiation wavelengths to the system, one will trigger the rotation of the motor, while the other will excite the fluorescent tag. In principle, the rotary motion of the rotor part triggers the rotation of the fluorescent group. Therefore, the rotation of the fluorescent tag can be followed by defocused wide-field fluorescence microscopy. This method provides, for the first time, a method to convert the relative rotation of a molecular motor into absolute rotation, and therefore a way to visualize the rotation of a synthetic motor.
This article provides details on the design, total synthesis, and solution isomerization studies of a molecular motor that is used for single molecular imaging. The molecular structure is shown in Figure 3. In addition, the method to attach molecular motors on quartz surface is described.

<table><tbody><tr><td>NMR spectrometer</td><td>Varian</td><td>AMX400</td><td>for proton nmr study</td></tr><tr><td>Reagent for organic reactions</td><td>Sigma</td><td>analytical grade</td><td>reagent for organic reactions</td></tr><tr><td>Silica gel&amp;nbsp;</td><td>Merck</td><td>230-400 mesh ASTM</td><td>Flash chromatography&amp;nbsp;</td></tr><tr><td>Solvent</td><td>Acros</td><td>spectrophotometric grade&amp;nbsp;</td><td>Flash chromatography&amp;nbsp;</td></tr><tr><td>UV lamp</td><td>ENB</td><td>280C</td><td>for UV-vis irradation</td></tr><tr><td>UV-vis absorption spectrophotometer</td><td>JASCO</td><td>V-630&amp;nbsp;</td><td>UV-vis measurment</td></tr></tbody></table>

light-driven molecular motors on surfaces for single molecular imaging

The design and synthesis of a synthetic system that aims for the direct visualization of a synthetic rotary motor at the single molecule level on surfaces are demonstrated. This work requires careful design, considerable synthetic effort, and proper analysis. The rotary motion of the molecular motor in solution is shown by 1H NMR and UV-vis absorption spectroscopy techniques. In addition, the method to graft the motor onto an amine-coated quartz is described. This method helps to gain more insight into molecular machines.

Irradiation of the molecular motor is performed with UV light (&#955;max = 365 nm). Upon irradiation, a photo-induced E-Z isomerization around the central double bond occurs. During this process, the molecule is transformed from a stable to an unstable isomer. A thermally activated helix inversion step then follows to release the strain of the whole molecule. This results in the original stable state. 1H NMR spectroscopy is then employed to evaluate the rotary process (Figure 4a). A solution of the sample is prepared in an NMR tube, then a lamp of UV-light (&#955;max = 365 nm) is placed next to the tube. After 2 h of irradiation, distinct changes can be found in the 1H NMR spectrum. These changes indicate the generation of a new isomer that is considered to be unstable-1b (Figure 4b). It is seen in the 1H NMR spectroscopy that Ha shifts from 2.9 ppm (doublet) to 3.3 ppm (double doublet). The signal at 1.4 ppm can be assigned as the absorption of the methyl group, and it downfield shifts from 1.4 ppm to 1.6 ppm. When the sample is kept overnight at room temperature in the dark, the original spectrum can be recovered (Figure 4a). It indicates the process of the thermal helix inversion that converts unstable-1b to stable-1b.
In order to study the rotary motion of motor 1b on surfaces, the surface-attached motor assemblies MS-1b (MS = motor on surfaces) are prepared. The quartz slides are first functionalized with amine. After this step, the quartz is immersed in a DMF solution (10-4 M) of 1b at RT overnight. The resultant quartz is rinsed with DMF, water, and MeOH. The prepared quartz slides are then submitted for UV/vis studies. A UV/vis absorption spectrum of MS-1b (solid line) is shown in Figure 5b. As seen in the spectrum, the major absorption band and absorption profile are similar to that observed in solution (Figure 5a). It also shows the characteristic absorptions for motor (420 nm) and PBI (456 nm, 490 nm, 524 nm). These peaks suggest the successful attachment of motor 1b to the amine-coated surfaces. In addition, the quartz slide is irradiated for 15 min, and spectral changes are observed similar to that of the solution, indicating generation of the unstable MS-1b.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58750/58750fig1.jpg" /> 
Figure 1: Synthetic scheme towards the preparation of target molecule 1b. The scheme shows the reagents, solvents, and reaction conditions that are used in each step.&#160;
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58750/58750fig2.jpg" /> 
Figure 2: (a) Schematic illustration of the structure of F0F1-ATPase grafted on a surface for visualization of unidirectional rotation (reproduced with permission12). (b) Conceptual design of a synthetic surface-bound light-driven molecular motor for single molecule imaging.&#160;
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58750/58750fig3.jpg" /> 
Figure 3: Structure of a surface-bound molecular motor 1b, bearing a rigid long arm between the motor core and PBI label.&#160;
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58750/58750fig4.jpg" /> 
Figure 4: Aliphatic region of the 1H-NMR spectra of motor 1b (CD2Cl2, -20 &#176;C, c = 10-3 M) (a) stable-1b, before irradiation (365 nm). (b) Photo stationary state mixture after irradiation. <a href="https://www.jove.com/files/ftp_upload/58750/58750fig4large.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/58750/58750fig5v2.jpg" /> 
Figure 5: UV/vis absorption spectra. UV/vis absorption spectra of (a) motor 1b (CH2Cl2, 0 &#176;C), stable isomer (solid line) and unstable isomer (dashed line) at PSS. (b) MS-1b (quartz, 4 &#176;C) before (solid line) and after (dashed line) irradiation.&#160;

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Light-driven Molecular Motors on Surfaces for Single Molecular Imaging

The manuscript describes how to synthesize and graft a molecular motor on surfaces for single molecular imaging.

The design and synthesis of a synthetic system that aims for the direct visualization of a synthetic rotary motor at the single molecule level on surfaces ...

light-driven-molecular-motors-on-surfaces-for-single-molecular-imaging

Research

JoVE Journal

Chemistry

11.3K Views.  University of Groningen. Dit protocol geeft een eerste voorbeeld van het visualiseren van de rotatie van een door de mens gemaakte lichtaangedreven moleculaire motor op enkel moleculair niveau. De synthetische techniek die hier wordt gebruikt is ontworpen om de synthese van een complexe moleculaire machine met verschillende functionele groepen mogelijk te maken. Ten eerste, onder een argon atmosfeer, plaats 640 milligram van de rotor precursor verbinding twee, 219 milligram koperjodide, en 3,44 gram natriumjodide in ...

Video: Light-driven Molecular Motors on Surfaces for Single Molecular Imaging

Compact Quantum Dots for Single-molecule Imaging

Single-molecule imaging is an important tool for understanding the mechanisms of biomolecular function and for visualizing the spatial and temporal heterogeneity of molecular behaviors that underlie cellular biology 1-4. To image an individual molecule of interest, it is typically conjugated to a fluorescent tag (dye, protein, bead, or quantum dot) and observed with epifluorescence or total internal reflection fluorescence (TIRF) microscopy. While dyes and fluorescent proteins have been the mainstay of fluorescence imaging for decades, their fluorescence is unstable under high photon fluxes necessary to observe individual molecules, yielding only a few seconds of observation before complete loss of signal. Latex beads and dye-labeled beads provide improved signal stability but at the expense of drastically larger hydrodynamic size, which can deleteriously alter the diffusion and behavior of the molecule under study.
Quantum dots (QDs) offer a balance between these two problematic regimes. These nanoparticles are composed of semiconductor materials and can be engineered with a hydrodynamically compact size with exceptional resistance to photodegradation 5. Thus in recent years QDs have been instrumental in enabling long-term observation of complex macromolecular behavior on the single molecule level. However these particles have still been found to exhibit impaired diffusion in crowded molecular environments such as the cellular cytoplasm and the neuronal synaptic cleft, where their sizes are still too large 4,6,7.
Recently we have engineered the cores and surface coatings of QDs for minimized hydrodynamic size, while balancing offsets to colloidal stability, photostability, brightness, and nonspecific binding that have hindered the utility of compact QDs in the past 8,9. The goal of this article is to demonstrate the synthesis, modification, and characterization of these optimized nanocrystals, composed of an alloyed HgxCd1-xSe core coated with an insulating CdyZn1-yS shell, further coated with a multidentate polymer ligand modified with short polyethylene glycol (PEG) chains (Figure 1). Compared with conventional CdSe nanocrystals, HgxCd1-xSe alloys offer greater quantum yields of fluorescence, fluorescence at red and near-infrared wavelengths for enhanced signal-to-noise in cells, and excitation at non-cytotoxic visible wavelengths. Multidentate polymer coatings bind to the nanocrystal surface in a closed and flat conformation to minimize hydrodynamic size, and PEG neutralizes the surface charge to minimize nonspecific binding to cells and biomolecules. The end result is a brightly fluorescent nanocrystal with emission between 550-800 nm and a total hydrodynamic size near 12 nm. This is in the same size range as many soluble globular proteins in cells, and substantially smaller than conventional PEGylated QDs (25-35 nm).

We describe the preparation of colloidal quantum dots with minimized hydrodynamic size for single-molecule fluorescence imaging. Compared to conventional quantum dots, these nanoparticles are similar in size to globular proteins and are optimized for single-molecule brightness, stability against photodegradation, and resistance to nonspecific binding to proteins and cells.

Single-molecule imaging is an important tool for  understanding the mechanisms of biomolecular function and for visualizing the  spatial and temporal ...

Engineering

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 ...

Biochemistry

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Bioengineering

Assembling Molecular Shuttles Powered by Reversibly Attached Kinesins

This protocol describes how to create kinesin-powered molecular shuttles with a weak and reversible attachment of the kinesins to the surface. In contrast to previous protocols, in this system, microtubules recruit kinesin motor proteins from solution and place them on a surface. The kinesins will, in turn, facilitate the gliding of the microtubules along the surface before desorbing back into the bulk solution, thus being available to be recruited again. This continuous assembly and disassembly leads to striking dynamic behavior in the system, such as the formation of temporary kinesin trails by gliding microtubules.
Several experimental methods will be described throughout this experiment: UV-Vis spectrophotometry will be used to determine the concentration of stock solutions of reagents, coverslips will first be ozone and ultraviolet (UV) treated and then silanized before being mounted into flow cells, and total internal reflection fluorescence (TIRF) microscopy will be used to simultaneously image kinesin motors and microtubule filaments.

We present a protocol to build molecular shuttles, where surface-adhered kinesin motor proteins propel dye-labelled microtubules. Weak interactions of the kinesins with the surface enables their reversible attachment to it. This creates a nanoscale system which exhibits dynamic assembly and disassembly of its components while retaining its functionality.

This protocol describes how to create kinesin-powered molecular shuttles with a weak and reversible attachment of the kinesins to the surface. In contrast ...

Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, such as nucleic acids and proteins. Single molecule FRET (sm-FRET) measurements on immobilized molecules permit long observations of the system -effectively until both dyes photobleach- resulting in time-traces that report on biomolecular dynamics with a broad range of timescales from milliseconds to minutes. To facilitate the acquisition of large number of traces for statistical analyses, the process must be automated and the sample environment should be tightly controlled over the entire measurement time (~12 hours). This is accomplished using an automated scanning confocal microscope that allows the interrogation of thousands of single molecules overnight, and a microfluidic cell that permits the controlled exchange of buffer, with restricted oxygen content and maintains a constant temperature throughout the entire measuring period. Here we show how to assemble the microfluidic device and how to activate its surface for DNA immobilization. Then we explain how to prepare a buffer to maximize the photostability and lifetime of the fluorophores. Finally, we show the steps involved in preparing the setup for the automated acquisition of time-resolved single molecule FRET traces of DNA molecules.

In this article we describe how we obtain FRET traces from individual DNA molecules immobilized to a surface using an automated scanning confocal microscope.

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, ...

Biology

Myosin-Specific Adaptations of In vitro Fluorescence Microscopy-Based Motility Assays

Myosin proteins bind and interact with filamentous actin (F-actin) and are found in organisms across the phylogenetic tree. Their structure and enzymatic properties are adapted for the particular function they execute in cells. Myosin 5a processively walks on F-actin to transport melanosomes and vesicles in cells. Conversely, nonmuscle myosin 2b operates as a bipolar filament containing approximately 30 molecules. It moves F-actin of opposite polarity toward the center of the filament, where the myosin molecules work asynchronously to bind actin, impart a power stroke, and dissociate before repeating the cycle. Nonmuscle myosin 2b, along with its other nonmuscle myosin 2 isoforms, has roles that include&#160;cell adhesion, cytokinesis, and tension maintenance. The mechanochemistry of myosins can be studied by performing in vitro motility assays using purified proteins. In the gliding actin filament assay, the myosins are bound to a microscope coverslip surface and translocate fluorescently labeled F-actin, which can be tracked. In the single molecule/ensemble motility assay, however, F-actin is bound to a coverslip and the movement of fluorescently labeled myosin molecules on the F-actin is observed. In this report, the purification of recombinant myosin 5a from Sf9 cells using affinity chromatography is outlined. Following this, we outline two fluorescence microscopy-based assays: the gliding actin filament assay and the inverted motility assay. From these assays, parameters such as actin translocation velocities and single molecule run lengths and velocities can be extracted using the image analysis software. These techniques can also be applied to study the movement of single filaments of the nonmuscle myosin 2 isoforms, discussed herein in the context of nonmuscle myosin 2b. This workflow represents a protocol and a set of quantitative tools that can be used to study the single molecule and ensemble dynamics of nonmuscle myosins.

Presented here is a procedure to express and purify myosin 5a followed by a discussion of its characterization, using both ensemble and single molecule in vitro fluorescence microscopy-based assays, and how these methods can be modified for the characterization of nonmuscle myosin 2b.

Myosin proteins bind and interact with filamentous actin (F-actin) and are found in organisms across the phylogenetic tree. Their structure and enzymatic ...

Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells

The mitotic bipolar kinesin-5 motors perform essential functions in spindle dynamics. These motors exhibit a homo-tetrameric structure with two pairs of catalytic motor domains, located at opposite ends of the active complex. This unique architecture enables kinesin-5 motors to crosslink and slide apart antiparallel spindle microtubules (MTs), thus providing the outwardly-directed force that separates the spindle poles apart. Previously, kinesin-5 motors were believed to be exclusively plus-end directed. However, recent studies revealed that several fungal kinesin-5 motors are minus-end directed at the single-molecule level and can switch directionality under various experimental conditions. The Saccharomyces cerevisiae kinesin-5 Cin8 is an example of such bi-directional motor protein: in high ionic strength conditions single molecules of Cin8 move in the minus-end direction of the MTs. It was also shown that Cin8 forms motile clusters, predominantly at the minus-end of the MTs, and such clustering allows Cin8 to switch directionality and undergo slow, plus-end directed motility. This article provides a detailed protocol for all steps of working with GFP-tagged kinesin-5 Cin8, from protein overexpression in S. cerevisiae cells and its purification to in vitro single-molecule motility assay. A newly developed method described here helps to differentiate between single molecules and clusters of Cin8, based on their fluorescence intensity. This method enables separate analysis of motility of single molecules and clusters of Cin8, thus providing the characterization of the dependence of Cin8 motility on its cluster size.

Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells

The bi-directional mitotic kinesin-5 Cin8 accumulates in clusters that split and merge during their motility. Accumulation in clusters also changes the velocity and directionality of Cin8. Here, a protocol for motility assays with purified Cin8-GFP and analysis of motile properties of single molecules and clusters of Cin8 is described.

The mitotic bipolar kinesin-5 motors perform essential functions in spindle dynamics. These motors exhibit a homo-tetrameric structure with two pairs of ...

Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved single-molecule studies and has gained immense popularity in detecting and understanding the dynamic behavior of fluorescent-tagged molecules. Here, we describe a detailed method for&#160;in vitro single-molecule studies of kinesin-3 family motors using Total Internal Reflection Fluorescence (TIRF) microscopy. Kinesin-3 is a large family that plays critical roles in cellular and physiological functions ranging from intracellular cargo transport to cell division to development. We have shown previously that constitutively active dimeric kinesin-3 motors exhibit fast and superprocessive motility with high microtubule affinity at the single-molecule level using cell lysates prepared by expressing motor in mammalian cells. Our lab studies kinesin-3 motors and their regulatory mechanisms using cellular, biochemical and biophysical approaches, and such studies demand purified proteins at a large scale. Expression and purification of these motors using mammalian cells would be expensive and time-consuming, whereas expression in a prokaryotic expression system resulted in significantly aggregated and&#160;inactive protein. To overcome the limitations posed by bacterial purification systems and mammalian cell lysate, we have established a robust Sf9-baculovirus expression system to express and purify these motors. The kinesin-3 motors are C-terminally tagged with 3-tandem fluorescent proteins (3xmCitirine or 3xmCit) that provide enhanced signals and decreased photobleaching. In vitro single-molecule and multi-motor gliding analysis of Sf9 purified proteins demonstrate that kinesin-3 motors are fast and superprocessive akin to our previous studies using mammalian cell lysates. Other applications using these assays include detailed knowledge of oligomer conditions of motors, specific binding partners paralleling biochemical studies, and their kinetic state.

This study details purification of KIF1A(1-393LZ), a member of kinesin-3 family, using Sf9-baculovirus expression system. In vitro single-molecule and multi-motor gliding analysis of these purified motors exhibited robust motility properties comparable to motors from mammalian cell lysate. Thus, Sf9-baculovirus system is amenable to express and purify motor protein of interest.

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved ...

Use of Dual Optical Tweezers and Microfluidics for Single-Molecule Studies

Visual biochemistry is a powerful technique for observing the stochastic properties of single enzymes or enzyme complexes that are obscured in the averaging that takes place in bulk-phase studies. To achieve visualization, dual optical tweezers, where one trap is fixed and the other is mobile, are focused into one channel of a multi-stream microfluidic chamber positioned on the stage of an inverted fluorescence microscope. The optical tweezers trap single molecules of fluorescently labeled DNA and fluid flow through the chamber and past the trapped beads, stretches the DNA to B-form (under minimal force, i.e., 0 pN) with the nucleic acid being observed as a white string against a black background. DNA molecules are moved from one stream to the next, by translating the stage perpendicular to the flow to enable the initiation of reactions in a controlled manner. To achieve success, microfluidic devices with optically clear channels are mated to glass syringes held in place in a syringe pump. Optimal results use connectors permanently bonded to the flow cell, tubing that is mechanically rigid and chemically resistant, and which is connected to switching valves that eliminate bubbles that prohibit laminar flow.

Visual, single-molecule biochemistry studied through microfluidic chambers is greatly facilitated using glass barrel, gas-tight syringes, stable connections of tubing to flow cells, and elimination of bubbles by placing switching valves between the syringes and tubing. The protocol describes dual optical traps that enable visualization of DNA transactions and intermolecular interactions.

Visual biochemistry is a powerful technique for observing the stochastic properties of single enzymes or enzyme complexes that are obscured in the ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Light-driven Molecular Motors on Surfaces for Single Molecular Imaging

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