Targeted Activation of Molecular Transportation by Visible Light - ACS

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Targeted Activation of Molecular Transportation by Visible Light Ammathnadu S. Amrutha, K. R. Sunil Kumar, Takashi Kikukawa, and Nobuyuki Tamaoki ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b06059 • Publication Date (Web): 10 Nov 2017 Downloaded from http://pubs.acs.org on November 13, 2017

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Targeted Activation of Molecular Transportation by Visible Light Ammathnadu S. Amrutha,§ K. R. Sunil Kumar,§† Takashi Kikukawa,ǁ‡ and Nobuyuki Tamaoki*§ § Research Institute for Electronic Science, Hokkaido University, N20, W10, Kita-ku, Sapporo, Hokkaido, 001-0020, Japan ǁ Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810, Japan ‡ Global Station for Soft Matter, Global Institution for Collaborative Research and Education, Hokkaido University, Sapporo 060-0810, Japan E-mail: [email protected] KEYWORDS: kinesin, microtubule, photoswitches, azo-peptides, push-pull azobenzene, bionanodevice.

ABSTRACT: Regulated transportation of nanoscale objects with a high degree of spatiotemporal precision is a prerequisite for the development of targeted molecular delivery. In vitro integration of the kinesin-microtubule motor system with synthetic molecules offers opportunities to develop controllable molecular shuttles for lab-on-a-chip applications. We attempted a combination of the kinesin-microtubule motor system with push-pull type azobenzene tethered inhibitory peptides (azo-peptides) through which reversible, spatiotemporal control over the

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kinesin motor activity was achieved locally by a single, visible wavelength. The fast thermal relaxation of the cis isomers of azo-peptides offered us quick and complete resetting of the trans state in the dark, circumventing the requirement of two distinct wavelengths for two-way switching of kinesin-driven microtubule motility. Herein, we report the manipulation of selected, single microtubule movement while keeping other microtubules at complete rest. The photoresponsive inhibitors discussed herein would help in realizing complex bionanodevices.

Active intracellular transportation of cellular cargos, at the expense of ATP hydrolysis energy, is an intriguing cellular function performed by the motor protein kinesin in association with cytoskeletal filaments called microtubules.1,2 The kinesin exhibits attractive force production abilities and high fuel efficiency. As a result, it is receiving great attention for potential applications in the development of hybrid bionanodevices.3,4 The gliding motility assay or the inverted geometry, where surface bound kinesin motors propel microtubules is convenient to actualize directional transport and offers opportunities to control the gliding movement of microtubules in the artificial milieu.5 Through traditional bioconjugation techniques6, different cargos like DNA,7 quantum dots,8,9 viruses,10 catalysts,11 etc. can be loaded on the microtubules and such loaded molecular shuttles need to be guided to achieve site-specific delivery, separation, and sorting of the cargo. Complete and reversible spatiotemporal control over the movement of any such cargo carrying shuttle in an ensemble is an essential requirement for the development of fully functional, biomimetic nanotransportation systems. Kinesin activity was shown to be locally regulated by employing visible light in a ‘light-toheat’ converting layer of carbon grafted with thermoresponsive polymer chains. However, detachment of the microtubules from the adsorbed kinesin motors was observed, which is undesirable for the applications requiring site-specific delivery of the cargo.12 Furthermore,


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selective and electrical control over translocation of microtubules was reported using a microfabricated channel on functionalized graphene electrodes.13 Similarly, manipulation and organization of microtubules by applying an AC voltage was also realized.14 Further, taking advantage of an electric field, a single microtubule was steered in microchannels.15 The works involving electrical control required careful monitoring of the motility buffer pH and prevention of undesired electrolysis. The higher field strengths applied could result in significant heating of the buffer, influencing the gliding velocity.16 The microtubule movements were controlled by magnetic field through partial functionalization of microtubules with magnetic nanoparticles17,18 and microtubule movements were dynamically altered by applying programmable, micropatterned magnetic fields.19 The methods employing magnetic fields use complicated microfabrication techniques and require prefunctionalization of the microtubules. Caged ATP20,21 and caged inhibitors22 were used to switch the microtubule motility ON and OFF, respectively upon photoirradiation. Still, localizing their activities into a particular region of interest was not possible, due to the one-way switching properties of these photoswitches and the associated fast diffusion of the active molecules in the solution. It is found that two-way regulation of biological functions23 or material properties24,25 could be realized by incorporating reversible photoswitches into biological molecules or molecular assemblies, respectively. Recently, we have reported some significant research findings on the photoregulation of kinesin-driven microtubule motility. We chose azobenzene - a reversible, robust photoswitch and attempted to couple its photoisomerization event to optically manipulate the kinesin activity with a high degree of spatiotemporal control. A non-nucleoside ATP analog containing azobenzene (AzoTP) reported by our group, reversibly regulated the microtubule velocity with a change up to 79% in its two isomeric


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states.26 Also, azobenzene tethered inhibitory peptides (azo-peptides) earlier designed by us stopped the microtubule motility completely in their trans-state and allowed their motility in the cis-rich state, which was generated upon the UV light irradiation.27,28 With these two photoswitches, we achieved the local regulation of kinesin motor activity by simultaneous irradiation with UV and visible light using a simple, in-house built optical set-up.29 By reducing the light beam size, we were able to select a microtubule and manipulate its movement specifically. Though our method involved no surface patterning and prefunctionalization, optimization of the two light intensities to achieve an appropriate isomer ratio at the specific region of interest was challenging. Also, while moving a specific, single microtubule, slow movements of neighboring microtubules were observed. Moreover, continuous UV light irradiation used could be detrimental to proteins. Herein, we report a range of azo-peptides whose photoresponsive azobenzene units are designed to show a reduced HOMO-LUMO energy gap by the introduction of electron releasing (push) and electron withdrawing (pull) substituent pairs at the p and p' positions. These substitutions not only enhanced the inhibitory activities of the azo-peptides but also shifted the ππ* transition band into the visible wavelength region and reduced the lifetime of cis azopeptides. By taking advantage of the quick thermal relaxation behavior of the cis azo-peptides, we successfully achieved the complete and reversible spatiotemporal control over the kinesin motor activity by a single, visible wavelength. The high degree of precision obtained, allowed the manipulation of a particular, single microtubule movement without disturbing the adjacent filaments. We emphasize that employing visible wavelength in the local regulation of kinesin activity is advantageous over the UV light, being innocuous to proteins and offers easy implementation into the fluorescence optical microscope.


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Results Synthesis of azo-peptides The general protocol for solid phase peptide synthesis based on the Fmoc strategy was adopted in the synthesis of azo-peptides.27 Synthesized azo-peptides are presented in Figure 1. Azopeptides 1a, 2a, and 3a bear -Ile-Pro-Lys-Ala-Ile-Arg-OH and 1b, 2b, and 3b bear -Arg-Ile-ProLys-Ala-Ile-Arg-OH peptide units. These two peptides have been reported to possess the vital amino acids responsible for the complete inhibition of kinesin motor activity.28 Azo-peptides 2a and 2b were obtained by coupling the azobenzene carboxylic acid derivatives at the N-termini of the peptides. Azo-peptides 1a, 1b, 3a, and 3b were procured by coupling the intermediates formed by the reaction between azobenzene amine derivatives and N,N'-Carbonyldiimidazole at the N-termini of the peptides. Synthesized azo-peptides were purified by reversed-phase HPLC and characterized by ESI-TOF mass spectra. Mass spectra (Figure S1-S5) and HPLC chromatograms (Figure S6) affirming the purity of azo-peptides are displayed in the supporting information. The mass spectra and HPLC chromatogram of the azo-peptide 2a are excluded from the supporting information since they are presented in our previous report.28 Azobenzene amine derivatives were synthesized by following the earlier reported procedures.30,31 Reversible isomerization of azo-peptides The trans isomers of azo-peptides presented here undergo photoisomerization by absorbing visible light forming cis isomers whereas the cis isomers formed showed quick thermal relaxation behavior forming the trans isomers as depicted in the Scheme 1. Figure 2a presents the UV-Visible absorption spectra of azo-peptides 1b, 2b, and 3b in BRB-80 buffer at 25 oC before visible light (510 nm) irradiation. The absorption maxima (λmax) corresponding to the π-


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π* transition band were observed in the visible wavelength region. Upon visible light irradiation, significant changes in the spectral profiles corresponding to the cis isomer formation were not detected with our conventional spectrophotometer, indicating the quick thermal relaxation property of the cis isomer generated by photoirradiation with the rate exceeding the acquisition speed of the spectrophotometer which is ca. 1 s. The different azobenzene chromophores presented here are ‘push-pull’ type azobenzenes. It is well known that in ‘push-pull’ type azobenzenes, the cis (Z) to trans (E) thermal isomerization occurs faster than in regular azobenzenes.32 Therefore we adopted a laser flash photolysis technique to determine the thermal relaxation rates and lifetimes of the cis isomers. The flash photolysis experiments were carried out in BRB-80 buffer at 25 ºC. The change in optical density (OD) at 461 nm when the solution of azo-peptide 1b was flashed with the laser (λ= 532 nm) is presented in Figure 2b. Upon laser flash for 7 ns, we observed a sudden decrement in the OD. Within ca. 2 ms the change in OD was recovered. The flash photolysis experiment indicated the generation of cis isomer by visible light irradiation and fast thermal relaxation of the photogenerated cis isomer into the trans isomer in the dark. Thermal relaxation rate constants and the lifetimes of cis isomers were determined by fitting the curve to the exponential form of the first order rate equation. UV-Visible absorption spectra and flash photolysis curves of all the remaining azo-peptides are presented in the supporting information (Figure S7 - S12). Table 1 shows the thermal relaxation rate constant (k) and lifetime (τ) values of cis isomers obtained from the flash photolysis experiments. In vitro gliding motility assays were performed to investigate the concentration dependent inhibitory activities of the azo-peptides in BRB-80 buffer at 25 ºC in the presence of 1 mM ATP.


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Figure 3 shows the gliding velocities of microtubules at different concentrations of the azopeptides in dark and under continuous visible light (488 nm; intensity ca. 8 × 10-1 W/cm2) irradiated conditions. In the trans state of the azo-peptide 1a (Figure 3a), the gliding velocity sharply decreased at a concentration of 0.5 mM and became zero at a 0.75 mM concentration. Motility was observed only under continuous 488 nm light irradiation and stopped immediately after the removal of light irradiation. At a 0.75 mM concentration, the gliding motility was switched between ON and OFF states completely and reversibly for several cycles by a single, visible wavelength of light (Movie S1 demonstrates the ON/OFF switching of gliding motility by 488 nm light in the presence of a 0.3 mM concentration of the azo-peptide 1b). The IC50 value calculated for the trans isomer of azo-peptide 1a is 0.29 mM. Table 2 presents the IC50 values calculated for the different trans azo-peptides and the minimum azo-peptide concentration displaying complete ON/OFF switching of the microtubule movement. Dependence of gliding velocity on the actinic light intensity Further, we studied the effect of irradiated light intensity on the gliding velocity of microtubules with azo-peptides 1b (0.3 mM), 2b (0.75 mM), and 3b (0.3 mM) in the presence of 1 mM ATP by measuring the gliding velocities of the microtubules at different intensities of the irradiated light (488 nm). The intensity of the laser beam directly emerging from the source ca. 8 × 10-1 W/cm2 was considered as 100% and ND filters having different transmittance were introduced to alter this light intensity. ND50, ND25, ND12, ND6, and ND1 are the filters used which transmitted 50% (ca. 4 × 10-1 W/cm2), 25% (ca. 2 × 10-1 W/cm2), 12% (ca. 1 × 10-1 W/cm2), 6% (ca. 5 × 10-2 W/cm2), and 1% ca. 8 × 10-3 W/cm2) of the source light intensity respectively. Figure 4 presents the changes in gliding velocity upon changing the light intensity.


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Local regulation of the microtubule movement We used the same optical set-up developed by our group earlier to translocate the microtubule and to manipulate its movement selectively.29 Here we used only one wavelength of light, unlike our previous report where we used UV and visible wavelengths simultaneously. The schematic representation of the optical set-up used is provided in the supporting information (Figure S13). We prepared a flow cell with surface adsorbed kinesin (0.125 mg/mL), fluorescent dye-labeled microtubules (0.025 µM), casein, oxygen scavengers, ATP (1 mM) and azo-peptide 1b (0.3 mM). The flow cell was analyzed under fluorescence optical microscope. Initially, all the microtubules were in the resting state. We irradiated a selected microtubule with the 488 nm laser light (diameter of the circular light beam = 5 µm), which initiated the microtubule movement. When the microtubule started moving, the position of the light beam was slowly changed, and the microtubule was translocated to the other region of the imaging area (see Movie S2). The microtubule moved with the velocity equal to 0.47 ± 0.01 µm/s. Figure 5 presents the selected single microtubule movement under continuous 488 nm light irradiation (intensity ca. 3 × 101 W/cm2). Adjacent microtubules remained in the same position throughout the experiment. In another experiment, we prepared the flow cell as mentioned above. A selected microtubule was irradiated at its trailing end with the 488 nm laser light (intensity ca. 3 × 101 W/cm2; diameter of the circular light beam = 5 µm). Irradiation at the trailing end induced bending of the microtubule initially and prolonged irradiation at the trailing end finally resulted in breaking of the microtubule at the position of bending (see Movie S3). Figure 6 presents the sequential images showing bending and breaking of the selected single microtubule and translocation of the newly formed two microtubules by light.


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Discussion We recently reported the local regulation of kinesin motor activity through azobenzene-based photoswitches.29 There, we selectively induced motility into a particular microtubule by irradiating the selected microtubule with one wavelength and the entire imaging area with the other wavelength. There, the irradiation of the entire imaging area with the second wavelength was essential to avoid the activation of motors present in the surrounding region by diffused active molecules. In this case, optimizing the intensities of two wavelengths to obtain clearly distinguishable kinesin motor activity between the locally irradiated area and the surrounding was important and challenging. Between the trans and cis forms of azobenzene, the cis form is thermally less stable than the trans and the lifetime of cis isomer largely depends upon the substitution pattern of the azobenzene.32 This property prompted us to go for the push-pull type azobenzenes. The short lifetime for cis isomers observed in the case of push-pull type azobenzenes was beneficial for us with which, use of two distinct wavelengths for two-way switching can be circumvented by the fast thermal relaxation process offering quick and complete resetting of the trans state in the dark. In the flash photolysis experiments, azo-peptides 3a and 3b showed the shortest lifetime values for cis isomers (Table 1) which are attributed to the presence of a strong push-pull substituent pair (electron-donor N'-ethyl-1,2-ethanediamino and electron-acceptor nitro groups) causing the asymmetric electron distribution. It is observed that the azobenzenes with large asymmetric electron distribution possess a low HOMO-LUMO energy gap which facilitates the fast thermal relaxation of the cis isomer into trans.33 Azo-peptides 2a and 2b showed longer lifetime than the azo-peptides 3a and 3b due to the presence of relatively weaker push-pull substituent pair (electron-donor dimethylamino group and electron acceptor carbonyl group). Azo-peptides 1a


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and 1b showed the longest lifetime for cis isomers because; the electron-acceptor carbonyl group was connected to the azobenzene ring through the amine –NH. Hence, the asymmetry in the electron distribution was reduced. Structure-property relationship studies of various azo-peptides performed by us earlier revealed that an additional Arg unit at the N-terminus of the peptide interacts through electrostatic interaction with the acidic amino acid residue of the kinesin motor domain and improves the affinity of the azo-peptide towards kinesin.28 Evidently, azo-peptides with the ArgIle-Pro-Lys-Ala-Ile-Arg-OH peptide unit (1b, 2b, and 3b) showed enhanced inhibition compared to the respective azo-peptides having a -Ile-Pro-Lys-Ala-Ile-Arg-OH peptide unit (1a, 2a, and 3a) in the in vitro gliding motility assays (Table 2 and Figure 3). On the other hand, substituent groups present on azobenzene also significantly affected the inhibitory activities of the azopeptides. Among azo-peptides 1a, 2a, and 3a, azo-peptide 3a showed complete inhibition at relatively lower concentration than the others (Table 2 and Figure 3a, 3c, and 3e). Here the N'ethyl-1,2-ethanediamine substituent on azobenzene acted as an additional moiety capable of interacting with the kinesin pocket through H-bonding interactions. This enhanced inhibition property was observed with the azo-peptide 3b also when compared with the azo-peptides 1b and 2b (Table 2 and Figure 3b, 3d, and 3f). Between 1a and 2a (Figures 3a and 3c), 1a showed more affinity towards kinesin owing to the presence of an additional -NH moiety along with the dimethylamino group, with which multiple H-bonding interactions are expected between the azobenzene unit and the kinesin pocket. Similar behavior was observed with the azo-peptides 1b and 2b (Figures 3b and 3d), where 1b showed stronger inhibition property than 2b. The introduction of push-pull substituent pair into the azobenzene unit shifted the π-π* transition band into the visible wavelength region (Figure 2a). Therefore a visible wavelength


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(488 nm) was chosen to elicit the photochemical conversion of trans azo-peptide into cis azopeptide. The kinesin-driven microtubule movement was observed only under the continuous irradiation of 488 nm light. The microtubule movement stopped immediately after removing the light irradiation due to the spontaneous conversion of cis azo-peptide into trans azo-peptide in the dark driven by the fast thermal relaxation. Thus we were able to regulate the kinesin-driven microtubule movement reversibly by the single wavelength of light. We believe that the gliding velocity of microtubules observed under the continuous light irradiation is the resultant of two factors. The first factor is the affinity of the cis form of azo-peptide for the kinesin motor domain. The higher the affinity the lower is the velocity observed. The second factor is the intensity of the irradiated light beam used for the trans to cis isomerization. Quick thermal relaxation obviously leads to a small steady state fraction of the cis isomers and the highintensity light is required to generate a large enough fraction of cis isomers to observe the kinesin-driven microtubule movement. The experiment performed to examine the dependence of gliding velocity over the intensity of irradiated beam (Figure 4) justified our hypothesis. A relatively higher intensity was needed to trigger the microtubule movement in the case of azo-peptide 3b whose cis form had the shortest lifetime. Also in the case of azo-peptide 3b, even at higher intensities, microtubules moved with lower velocities when compared with their velocities in the presence of azo-peptides 1b and 2b. The ascending order of the azo-peptides based on the minimum intensity of light required to trigger the microtubule movement is 1b < 2b < 3b. The ascending order of the azo-peptides based on the gliding velocities of microtubules observed at any intensity of light is 3b < 2b < 1b. Thus the azo-peptide 1b emerged as a more efficient inhibitor which completely inhibited the kinesin motor activity at relatively low concentration (0.3 mM). Also, with the azo-peptide 1b, we were able to trigger the microtubule


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motility with a low-intensity laser beam (ca. 8 × 10-3 W/cm2) and the microtubules moved with the higher velocity in comparison with the velocities observed in the presence of other azopeptides. Selective manipulation of the single microtubule movement became easier than before with the azo-peptides reported here since they do not require two distinct wavelengths for the two-way switching. When a particular microtubule was irradiated with the visible light of suitable intensity sufficient concentration of cis isomers was generated which could induce the microtubule movement. The cis azo-peptides diffused from the irradiated region immediately undergo fast thermal relaxation to form the trans azo-peptide. As a result, only the kinesin motors presenting in the locally irradiated area were activated (Figure 5). Further, irradiating a microtubule at its trailing end initially bent the microtubule. When the kinesin motors of the irradiated region pushed the rear portion of microtubule forward, the nonmoving front end of the microtubule acted as an obstacle opposing the forward movement which resulted in the bending of the microtubule. Irradiation at the trailing end generated stress among the tubulin units of the microtubule. Thus, continued irradiation resulted in the breaking of the microtubule at the position of bending. The two microtubule segments generated acted as two free shuttles which were later translocated to different regions by light (Figure 6). The observation of the nonmoving part of the single microtubule in the distance of several micrometers from the edge of the laser beam allow us to conclude that deactivated peptide in the cis form cannot diffuse over several micrometers distance before it reverts to inhibitory. For local regulation of kinesin activity we used a high intensity laser light (intensity ca. 3 × 10 W/cm2) as the cis azo-peptides employed in this study are short-lived with their lifetimes in submilliseconds. Furthermore, in order to selectively manipulate a single microtubule we


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reduced the beam size. The highly focused light beam (diameter of the circular light beam = 5 µm) covered only around 10-20% of the microtubule length. Consequently, the number of active kinesin motors propelling the microtubule was considerably less. Above all, we needed to overcome the inhibitory effect caused by the trans isomers rapidly diffusing from the nonirradiated region. In the case of azo-peptide 3b we used the highest laser intensity possible with our laser source (intensity ca. 8 × 103 W/cm2) to achieve maximum gliding velocity of microtubules as 3b exhibited strong inhibition property relative to other azo-peptides and possessed shortest lifetime for cis isomer (0.028 ms). Although we could observe the movement of microtubules at this intensity of laser light, prolonged irradiation (ca. 1 min) resulted in serious photobleaching (Figure S14). In our earlier report we had used a 365 nm light (intensity ca. 2 × 102 W/cm2) to locally irradiate a selected microtubule and the entire flow cell area was irradiated with the 510 nm light (intensity ca. 2.6 × 10-2 W/cm2). Though the cis azo-peptide used in our earlier report posessed longer lifetimes (in the order of hours), considerably high concentration (2 mM) of the azo-peptide used and rapid diffusion of inhibitory trans isomers generated under 510 nm light irradiation from the surrounding prompted us to make use of high intensity UV light to selectively drive the single microtubule.29 It must be beneficial to compare the needed light intensity in the present experiment to regulate the motility of single microtubules with those used in other related optical methods. In optogenetics34 - a tool used to optically regulate the functioning of genetically modified cells, the photoactive units are covalently bound to the proteins and therefore diffusion of the other isomer is not observed. In addition, the cis isomers of the chromophores present (eg. cis-p-coumaric acid present in photoactive yellow protein35) exhibited lifetime in the order of seconds. As a result a lower intensity light (< 0.1 W/cm2) could afford the desired regulation of the protein function.34


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The super resolution microscopy (FPALM) uses high intensity light (ca. 1 × 102 W/cm2), which is comparable with that for our present technique, for imaging the small number of photoactivatable fluorescent protein molecules.36 These intensities of light is stronger than the intensity of excitation light (ca. 2 W/cm2 at 640 nm with 60x objective lens) for our normal imaging of microtubules. In our earlier reported experiments performed under simultaneous irradiations of UV and visible lights,29 we had observed slow movements of neighboring microtubules owing to the longer lifetime of diffused cis azo-peptides. In addition, the method required careful optimization and monitoring of the light intensities, since slight variation in the intensities could result in the enhancement in the velocities of neighboring microtubules. The single microtubule manipulation experiments demonstrated in this work using push-pull azobenzene tethered inhibitory peptides and single wavelength attempts controlled nano transportation, besides overcoming the challenges faced earlier. Conclusion We developed a series of photoresponsive inhibitors for kinesin motor by modifying the lightresponsive azobenzene units tethered to the inhibitory peptides. We introduced different pushpull substituent pairs at the p and p' positions of the azobenzene unit to reduce the HOMOLUMO energy gap. The substitutions significantly shifted the π-π* transition band into the visible wavelength region and reduced the lifetime of the cis isomer as a consequence of the lowered energy barrier. The shorter lifetime of the cis isomer was advantageous allowing the reversible regulation of the kinesin activity by a single, visible wavelength. With these inhibitors, we achieved specific, single microtubule translocation, bending, and breaking, while keeping the


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surrounding microtubules at rest. These demonstrations showcase the easy, spatiotemporal control of the kinesin motor activity, which is achievable simply by light, circumventing the laborious fabrication and prefunctionalization processes involved in other methods. This work involving photoregulation of a specific, single microtubule movement in an ensemble is highly desirable in applications requiring controlled cargo manipulations, promising applications in bionanodevices. We aim to achieve specific microtubule translocation with the steering ability with the help of light in future. Experimental Section Chemicals The biochemical reagents and other chemicals were procured from commercial sources (Watanabe Chemical Industries; Wako Pure Chemical Industries; Tokyo Chemical Industry and Dojindo Molecular Technologies). They were used as such without further purification. Instrumentation Azo-peptides were synthesized using a Burrell Wrist Action Shaker (model 75). A Shimadzu reversed-phase (RP) HPLC system was used for the azo-peptide purification. Azo-peptides were freeze-dried using a EYELA FDU-2200 lyophilization system. Electrospray ionization time-offlight mass spectrometry (ESI-TOF-MS) was carried out using a JMS-T100CS (JEOL) instrument by operating it in positive-ion mode. An Agilent 8453 single-beam spectrophotometer was used to record the UV-Visible absorption spectra. A Hayasaka LED Controller (model CS_LED 3W_510) was used in the photoisomerization experiment for 510 nm light. Flash photolysis experiments were carried out using a Q-switched Nd:YAG laser (Surelite I-10;


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Continuum). In vitro motility experiments were carried out by using Olympus IX71, an inverted fluorescence optical microscope containing UPlan F1 100x/1.30 oil C1 and PlanApo 60x/1.45 NA oil objective lenses (Olympus) with 640 nm excitation light from a mercury lamp transmitted through appropriate filters. A Coherent Innova 90 argon-ion laser was used to irradiate the flow cell. The condenser lenses of magnifications 5x (0.55 N.A./27 mm W.D.) and 50x (0.42 N.A./4 mm focal length) were used to confine the laser beam. A handheld optical power meter (modelFM, Coherent) was used to measure the light power. Videos were recorded using an EMCCD digital camera from Andor Solis Technology (model DL-604M-0EM-H1). Recorded videos were processed using ImageJ software. Synthesis Synthesis of azobenzene derivatives Azobenzene amine derivatives were synthesized according to the earlier reported procedures.30,31 Synthesis of azo-peptides Azo-peptides were synthesized by following the standard protocol for solid phase peptide synthesis based on the Fmoc strategy as reported earlier.27 Fmoc-Ala-OH, Fmoc-Ile-OH, FmocArg(Pbf)-OH, Fmoc-Lys(Boc)-OH and Fmoc-Pro-OH were used to synthesize the peptide unit (Pbf: 2,2,4,6,7-pentamethyldihydrobenzofurane-5-sulfonyl; Boc: tert-butyloxycarbonyl). Firstly, the N-terminus Fmoc-amino group of the first amino acid preloaded on the alko-resin was deprotected using 20% piperidine solution prepared in N,N-dimethylformamide (DMF, 4 mL). After deprotection, coupling reactions were carried out with a solution of Fmoc amino acid (4


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eq.), O-(benzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (HBTU, 4 eq.), 1hydroxy-1H-benzotriazole monohydrate (HOBt•H2O, 4 eq.), and N,N-diisopropylethylamine (DIPEA, 8 eq.) in DMF for 1 h at room temperature. Reaction completion was confirmed by performing the Kaiser test. In the case of azo-peptides 2a and 2b, after completing the peptide elongation, an azobenzene carboxylic acid derivative was introduced directly at the N-terminus of the peptide. But in the synthesis of azo-peptides 1a, 1b, 3a, and 3b, the azobenzene amine derivatives were first treated with N,N'-Carbonyldiimidazole in DMF for 2 hours at room temperature. The intermediates obtained were later coupled at the N-terminus of the elongated peptide, adding DIPEA (8 eq.). Finally, the azo-peptide formed was cleaved from the resin by treating with reagent-K [trifluoroacetic acid (TFA)/ phenol/thioanisole/H2O/triisopropylsilane, 8.25:0.5:0.5:0.5:0.25] and precipitated using cold diethyl ether. Later, azo-peptide was purified through a preparative RP-HPLC system. Characterization of azo-peptides The purity of the azo-peptides was analyzed through a RP-HPLC system (column: 5C18-MS-II, 4.6 × 250 mm (Nacalai Tesque); eluent: CH3CN/H2O with 0.1% TFA; solvent gradient: 20–45% CH3CN in water, over 1 h; flow rate: 1 mL/min; injection volume: 20 µL). ESI-TOF mass spectra of the azo-peptides were obtained. In vitro gliding motility assay Gliding motility assays were performed at 25 °C by following the standard protocol described previously, using BRB-80 [pH 6.9; piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), 80 mM; MgCl2, 2 mM; O,O-bis(2-aminoethyl)ethyleneglycol-N,N,N´,N´-tetraacetic acid (EGTA), 1 mM] as the assay buffer.28 The flow cell was irradiated with the laser light (488 nm) of intensity ca. 8


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× 10-1 W/cm2. The gliding velocity was determined from the average of 5 measurements using ImageJ software. To examine the effect of irradiated light intensity on the gliding velocity of microtubules, the light intensity was varied using the ND filters. We used ND1, ND6, ND12, ND25 and ND50 filters for our experiments. Experiments demonstrating the single microtubule manipulations were performed using high intensity laser lights (ca. 3 × 101 W/cm2 and ca. 8 × 103 W/cm2). Measurement and calculation of light intensity The laser light intensities used in different experiments are calculated as follows. (a) Dependence of microtubule gliding velocity on the concentration of azo-peptides The power (P) of laser light after passing through the three reflective mirrors (the schematic representation of the optical set-up is provided in the supporting information, Figure S13) was measured to be 4 × 10-3 W. The diameter of the circular aperture for the laser beam was ca. 3.97 × 10-1 cm. Thus, total area (A) of the laser beam calculated to be ca. 1.24 × 10-1 cm2 (A = πr2 = 3.14 × (1.99 × 10-1)2) assuming the uniform intensity of light across the diameter of the beam. Power divided by the total area gave the intensity (I) of the beam and it was found to be ca. 3 × 10-2 W/cm2 (I = P/A = 4 × 10-3 W/ 1.24 × 10-1 cm2 = 3 × 10-2 W/cm2). Further, the light beam was made to pass through a 5x condenser lens which reduced the beam size by 5 times and consequently enhanced the light intensity by 25 times. As a result, the light intensity obtained at the sample was determined to be ca. 8 × 10-1 W/cm2 (3 × 10-2 W/cm2 × 25 = 8 × 10-1 W/cm2) assuming the transmittance of the condenser lens as 100%. (b) Dependence of gliding velocity on actinic light intensity


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Intensity of the laser beam emerging from the third reflective mirror (ca. 3 × 10-2 W/cm2) was reduced further before passing it through the condenser lens using appropriate ND filter. For example, the light intensity was reduced to 50% (ca. 1.5 × 10-2 W/cm2) by using a ND50 filter. The light was later passed through a 5x condenser lens resulting in final intensity of ca. 4 × 10-1 W/cm2 at the sample. Similarly, ND25, ND12, ND6, and ND1 filters used resulted in ca. 2 × 10-1 W/cm2, ca. 1 × 10-1 W/cm2, ca. 5 × 10-2 W/cm2, and ca. 8 × 10-3 W/cm2 intensities of the light beam, respectively. (c) Local regulation of the microtubule movement Using azo-peptide 1b: The power (P) of laser light after passing through the three reflective mirrors was measured to be 2 × 10-2 W. Area of the laser beam was ca. 1.24 × 10-1 cm2. Thus intensity of the light beam determined to be ca. 2 × 10-2 W/cm2. The light beam was passed through a ND6 filter which transmitted only 6% of the light beam and reduced the light intensity into ca. 12 × 10-3 W/cm2. Subsequently, the light beam was passed through a pinhole of diameter ca. 300 µm followed by a 50x condenser lens. The condenser lens reduced the beam size by 50 times and consequently increased the intensity by 2500 times resulting in final intensity of ca. 30 W/cm2. Using azo-peptide 3b: The power of laser beam was measured to be 4 × 10-1 W. Area of the light beam was ca. 1.24 × 10-1 cm2. Hence, intensity of the light beam is ca. 3 W/cm2. Further, the light beam was made to pass through a pinhole of diameter 90 µm followed by a 50x condenser lens that resulted in the final intensity of ca. 8 × 103 W/cm2. Flash photolysis


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The experiments were performed using a computer-controlled flash photolysis system constructed according to the reported procedure.37 Sample solutions of the azo-peptides were prepared in BRB-80 buffer (pH 6.9) showing absorbance around 0.5 at the absorption maxima at 25 °C. Azo-peptide solution present in the sample cell (10 mm × 10 mm quartz cuvette) was flashed with the laser (λ = 532 nm, ~5 mJ pulse) for 7 ns which was the second harmonic of the fundamental beam of a Q-switched Nd:YAG laser. The temperature was maintained at 25 °C. A 100 W halogen lamp was used as the source of the monitoring light. FIGURES

Figure 1. Synthesized azo-peptides.


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Figure 2. (a) UV-Visible absorption spectra of the azo-peptides 1b (red line), 2b (blue line), and 3b (black line) measured in BRB-80 buffer at 25 ºC before irradiation (b) Change in the optical density (OD) profile of azo-peptide 1b at 461 nm after laser photoflash (λ = 532 nm). The solid red line is a fit with the exponential form of the first order rate equation [y = m1∗exp(−m2∗t); t = time (ms); fitting parameters m1 = -6.89, m2 = 1.702; R = 0.99].


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Figure 3. Gliding velocities of microtubules plotted against the concentrations of the azopeptides (black circles - gliding velocity under dark condition; red circles - gliding velocity under continuous visible light irradiated condition). Error bars present the standard deviation for five microtubules in one flow cell.


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Figure 4. Gliding velocities of microtubules plotted against the percentage intensity of 488 nm light. Error bars show the standard deviation for five microtubules in one flow cell. Note: 100% intensity is ca. 8 × 10-1 W/cm2.

Figure 5. Sequential fluorescence images presenting the translocation of the selected, single microtubule by light in the presence of azo-peptide 1b (0.3 mM) and ATP (1 mM). The images display the locations of the microtubule at 0 s, 1 min and 26 s, 2 min and 31 s, and 4 min and 37 s. White spots - 488 nm light beam; arrow marks - positions of the moving microtubule.


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Figure 6. Sequential fluorescence images presenting the bending and breaking of the selected microtubule by light in the presence of azo-peptide 1b (0.3 mM) and ATP (1 mM). (1) Initial state; (2) irradiation at the trailing end (inset: bending of the microtubule filament); (3) after 4 s of irradiation at the trailing end (inset: breaking of the microtubule at the position of bending); (4) formation of two new filaments; (5) - (8) translocation of the newly formed microtubules by light one by one. White spots - 488 nm light beam. SCHEMES Scheme 1. Reversible isomerization of azo-peptides.


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TABLES Table 1. Thermal relaxation rate and lifetime of cis azo-peptides. Thermal

Lifetime of cis

relaxation rate

azo-peptide (τ)

constant (k) (s-1)

(ms)

1a

1.53 × 103

0.652

1b

1.70 × 103

0.587

2a

1.71 × 104

0.058

2b

1.79 × 104

0.056

3a

3.79 × 104

0.026

3b

3.72 × 104

0.028

Azo-peptide

Table 2. Inhibitory activity and photoswitchability of azo-peptides. Azo-peptide

IC50 trans

Minimum

(mM)

concentration showing complete ON/OFF switching of motility (mM)


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1a

0.29

0.75

1b

0.21

0.30

2a

0.86

1.25

2b

0.27

0.75

3a

0.25

0.60

3b

0.17

0.30

ASSOCIATED CONTENT Supporting Information The following files are available free of charge via the Internet at http://pubs.acs.org. ESI+ mass spectra, HPLC chromatograms, UV-Visible absorption spectra, LASER flash photolysis graphs, schematic representation of the optical set-up, fluorescence images displaying photobleaching effect (PDF) Movie S1: Complete ON/OFF switching of microtubule motility by single wavelength Fluorescence optical microscopy video (AVI) Movie S2: Single microtubule translocation by light - Fluorescence optical microscopy video (AVI) Movie S3: Bending and breaking of the microtubule by light - Fluorescence optical microscopy video (AVI)


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The authors declare no competing financial interest. AUTHOR INFORMATION Corresponding Author *Email: tamaoki＠es.hokudai.ac.jp

Present Address † Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Tokyo 113-8656, Japan Author Contributions § N. T. conceived the idea. § A. S. A. synthesized the compounds and performed most of the experiments. §† K. R. S. performed the local regulation of microtubule movement and dependence of gliding velocity on actinic light intensity experiments. § A. S. A. and ǁ‡ T. K. performed the flash photolysis experiments. § A. S. A. and § N. T. analyzed all the experimental data and wrote the manuscript. Funding Sources Authors thank the Research Foundation for Opto-Science and Technology for the financial support. ACKNOWLEDGMENT We thank T. Kamei (Hokuriku University) for kindly providing the human kinesin-1 and tubulin labeled with the CF633 succinimidyl ester. We also thank H. Kumano (Institute of Science and Technology, Niigata University) for helping us in setting up the LASER source.


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