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Reversible and Quantitative Photoregulation of Target Proteins
Article
Reversible and Quantitative Photoregulation of Target Proteins Sitao Xie, Liping Qiu, Liang Cui, ..., Zhou Chen, Xiaobing Zhang, Weihong Tan [email protected] (L.Q.) [email protected] (W.T.)
HIGHLIGHTS A DNA-based, noninvasive, photoresponsive nanoplatform of protein manipulation Achieving reversible and quantitative regulation of target protein activity This nanoplatform can be easily generalized to study different proteins
Noninvasive, precise, and reversible regulation of specific proteins is essential to elucidating fundamental biological processes. To achieve this goal, Tan and colleagues have developed a universal, aptamer-based, photoresponsive nanoplatform. By modulating light irradiation, they were able to reversibly and quantitatively manipulate the activity of target proteins in both complex serum and living bacteria samples.
Xie et al., Chem 3, 1021–1035 December 14, 2017 ª 2017 Elsevier Inc. https://doi.org/10.1016/j.chempr.2017.11.008
Article
Reversible and Quantitative Photoregulation of Target Proteins Sitao Xie,1 Liping Qiu,1,* Liang Cui,1 Honglin Liu,1 Yang Sun,1 Hao Liang,1 Ding Ding,1 Lei He,1 Huixia Liu,3 Jiani Zhang,3 Zhou Chen,1 Xiaobing Zhang,1 and Weihong Tan1,2,4,*
SUMMARY
The Bigger Picture
Essential to elucidating fundamental biological processes is first gaining precise and reversible external control over proteins without altering their natural structure. To achieve this goal, we developed an aptamer-based, noninvasive, photoresponsive nanoplatform capable of reversible and quantitative regulation of target protein activity. Protein-specific aptamers equipped with several light-responsive molecules were functionalized onto the surfaces of nanoparticles. Adjusting light irradiation allowed the light-responsive molecules to drive the conformational switch of aptamer between the blocked status and liberated status. In this way, the target proteins were captured around the nanoparticle or released into the medium, thus leading to inhibition or activation of the protein, respectively. Moreover, modulating light irradiation allowed quantitative control of protein activity.
Temporally precise, noninvasive control of target protein activity at the single-molecule level is essential to understanding many protein-dominant biological processes. Here, we report a DNA-based, noninvasive, photoresponsive nanoplatform capable of reversible and quantitative regulation of target protein activity. Adjusting light irradiation allows the target proteins to be captured around the nanoparticle or released into the medium, thus leading to inhibition or activation of the protein, respectively. Moreover, modulating irradiation time allows quantitative control of protein release. Consequently, this nanoplatform can be a potent tool for intensive and precise investigation and manipulation of specific proteins without altering their natural structures, which is significant for studying complex biological signaling networks, identifying new therapeutic targets, and developing intelligent strategies for precision medicine.
INTRODUCTION Proteins, as key components of organisms, are involved in virtually all biological processes. Protein dysregulation can lead to biological dysfunction and disease. Thus, precise and reversible regulation of protein activity at the single-molecule level is essential to understanding complex biological signaling networks or identifying new therapeutic targets.1,2 Photons, as efficient and controllable external triggers, have been used to perform precise temporal manipulation over many protein systems.3,4 For example, by covalently attaching a synthetic photoisomerizable small molecule to endogenous ion channels, Kramer and colleagues have developed a light-activated chemical gate for regulating neuronal activity.5 Lin and co-workers proposed a fluorescent-inducible protein through genetically fusing photochromic Dronpa domains to both termini of the protein, enabling precise optical manipulation of both protease domains and guanine nucleotide exchange factor.6 Heo and co-workers developed a light-activated reversible inhibition strategy to inhibit protein function by sequestering targets into large assembled protein structures.7 Although all of these methods can rapidly and reversibly control protein behavior at a defined time and location, they all involve chemical or genetic protein modification. This process is complicated and time consuming, and it potentially affects protein activity, as well as related biological events, thus limiting practical applications. Moreover, none of these methods has been able to achieve quantitative activation of target proteins. Alternative strategies with simple design, convenient operation, little influence on natural protein structure, and potent capability of regulation at the single-molecule level are being sought. The advancement of nanotechnology has allowed the construction of many versatile nanoplatforms for biological applications,8,9 especially protein regulation.10,11 To
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Scheme 1. Schematic Illustration of the Aptamer/AZO-Based Nanoplatform for Reversible and Quantitative Manipulation of Protein Protein-specific aptamers modified with several light-responsive molecules are functionalized onto the surface of nanoparticles. By adjusting light irradiation, the light-responsive molecules can drive the conformational switch of aptamer between the blocked status and liberated status. In this way, the target proteins are captured around the nanoparticle or released into the medium, thus leading to inhibition or activation of the protein, respectively. Moreover, by modulating light irradiation, protein activity can be quantitatively controlled.
perform targetable control on proteins of interest, specific recognition units are always required. Particularly suited to this task, aptamers are artificial oligonucleotides selected via an in vitro method12 on the basis of their specific binding affinity13,14 to the target molecules. Accordingly, they have been applied as promising alternatives to protein antibodies, owing to their intrinsic advantages15,16 of easy synthesis, convenient modification with various functional groups, and flexible design. The combination of aptamers with nanomaterials is expected to offer unlimited opportunities for protein research.17,18 Based on many reports, consensus would seem to hold that the formation of high-order protein clusters has the potential to trap and, hence, inactivate protein function.7,19 The hypothesis is plausible because the active sites of proteins are expected to be trapped and blocked within the assembled protein architectures. Therefore, in the present work, we developed an aptamer-functionalized nanoplatform to reversibly control the formation and dissociation of protein clusters with the expectation of first inhibiting and then restoring protein activity without affecting natural protein structure. As shown in Scheme 1, aptamers able to specifically recognize a target protein were functionalized onto the surface of nanoparticles, and photoresponsive azobenzene (AZO) molecules were incorporated into the aptamer probe, together with a short cDNA sequence. Subsequently, light-driven conformational change of the aptamer controls the capture and release of proteins. More specifically, irradiation with UV light drives a trans-to-cis isomerization of AZO, leading to dehybridization of the aptamer/cDNA duplex, allowing the aptamer sequence to capture the protein of interest, thus leading to inactivation. On the other hand, under illumination with visible light, AZO undergoes a cis-totrans conversion, and the strong affinity between aptamer and cDNA, compared with that between aptamer and protein, again forms, blocking the aptamer and liberating protein, leading to restoration of its activity. Importantly, by modulating the irradiation time, protein release can be quantitatively controlled. Consequently, this nanoplatform can be a potent tool for intensive and precise investigation and manipulation of specific proteins.
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1Molecular
Science and Biomedicine Laboratory, State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Life Sciences, Aptamer Engineering Center of Hunan Province, Hunan University, Changsha, Hunan 410082, China
2Center
for Research at the Bio/Nano Interface, Department of Chemistry and Department of Physiology and Functional Genomics, Health Cancer Center, UF Genetics Institute, and McKnight Brain Institute, University of Florida, Gainesville, FL 32611-7200, USA
3Division
of Geriatrics, Xiangya Hospital, Central South University, Changsha, Hunan 410008, China
4Lead
Contact
*Correspondence: [email protected] (L.Q.), [email protected] (W.T.) https://doi.org/10.1016/j.chempr.2017.11.008
Figure 1. Feasibility of Forming High-Order Protein Assembly for Inhibition of Thrombin Activity (A) Real-time light-scattering spectra of mixtures containing fibrinogen (1.14 mM) and thrombin (2.5 nM) treated with different amounts of TBA-AuNPs. (B) Real-time light-scattering spectra of mixtures containing fibrinogen (1.14 mM) and thrombin (2.5 nM) treated with 0.125 nM AuNPs (a), 0.125 nM rDNA 29 -AuNPs (b), 400 nM TBA (c), and 0.125 nM TBA-AuNPs (d). (C–E) AFM characterization of thrombin only (C), the mixture of thrombin and rDNA 29 -Au NPs (D), and the mixture of thrombin and TBA-AuNPs (E).
RESULTS Construction and Optimization of the Nanoplatform With the aim of developing a simple, universal, and potent protein regulation nanoplatform with high potential for practical applications, we chose gold nanoparticles (AuNPs; diameter = 13 nm)—which are among the most well-studied nanoparticles with advantages of easy synthesis in aqueous solution, uniform size and shape, low biotoxicity,20 and efficient functionalization with oligonucleotides21,22—as the ‘‘core’’ for protein assembly. Thrombin, a key protein implicated in blood coagulation23 and one whose activity can be easily evaluated by common measurement techniques, was used as the model protein. A 29-mer thrombin-binding aptamer24 (TBA) able to specifically recognize thrombin but with little inhibition on thrombin activity was used to functionalize AuNPs via a gold-thiol bond, and the resultant product was termed TBA-AuNPs. In principle, active thrombin can catalyze the conversion of fibrinogen to insoluble fibrin, resulting in a rapid increase of scattered light intensity.25 Figure 1A shows dose-dependent inhibition of thrombin activity by TBA-AuNPs, achieving complete inhibition at the TBA-AuNP concentration of 0.125 nM (green line in Figures 1A and 1B). However, as shown in Figure 1B, negligible thrombin inhibition was observed in the samples containing excess TBA molecules (400 nM, which was about 50 times higher than the TBA concentration of the TBA-AuNPs). In addition, neither equivalent AuNPs nor rDNA29-AuNPs (AuNPs modified with 29-mer random DNA sequences) induced observable changes on thrombin activity, indicating that the thrombin inhibition originates from the TBA-specific thrombin assembly around the AuNP core, which is consistent with a previous report.19 To examine the interaction between TBA-AuNPs and thrombin, atomic force microscopy (AFM) was performed. Larger nanostructures were observed in the mixtures of thrombin and TBA-AuNPs (Figure 1E); neither
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thrombin only (Figure 1C) nor the mixture of rDNA29-AuNPs and thrombin (Figure 1D) showed any assembled morphology. To demonstrate the universality of this aptamer-based nanoassembly strategy for inhibiting target protein activity, another protein (lysozyme)-aptamer26 system was tested. Theoretically, active lysozyme can destroy the cell walls of Micrococcus lysodeikticus, which can be monitored via a decrease in absorbance at 450 nm.27 As shown in Figure S3, the activity of lysozyme was efficiently inhibited by LA (lysozyme aptamer)-AuNPs in a concentration-dependent manner. However, little inhibitory impact on lysozyme was caused by excess LA molecules (400 nM), AuNPs (0.3 nM), or rDNA40-AuNPs (0.3 nM), indicating specific lysozyme inhibition induced by LA-AuNPs. Moreover, AFM was performed to confirm the protein cluster morphology obtained in the LA-AuNPs + lysozyme sample, but not in the lysozyme only or rDNA40-AuNPs + lysozyme samples (Figure S4). These results demonstrated the high potential of the current aptamer-based nanoplatform for specific and efficient inhibition of protein activity. To exert temporal and reversible control over target proteins, the 50 end of TBA was extended with a short cDNA sequence, and AZO molecules, which have been widely introduced into DNA and subsequently shown to change the conformation of DNA via photons,28,29 were incorporated into this cDNA sequence. Initially, part of the TBA sequence was blocked through hybridization with cDNA, preventing the interaction between TBA and thrombin. However, irradiation by UV light (365 nm) could drive a trans-to-cis isomerization of the azobenzene moiety. This led to dehybridization of the TBA/cDNA duplex, thus releasing the intact functional TBA sequence to capture thrombin around the AuNP core to form a protein cluster, leading to inhibition of thrombin activity. Next, by illumination with visible light at around 450 nm, azobenzene underwent a cis-to-trans conversion. Because the binding affinity between TBA and cDNA was stronger than that between TBA and thrombin, as proved by a competition experiment (Figure S5), TBA/cDNA duplex was again formed and blocked the functional structure of TBA. Thus, thrombin was liberated and its activity restored. Consequently, by adjusting the light illumination, thrombin activity can be reversibly controlled. To verify the light-driven conformational change of TBA, a carboxyfluorescein (FAM) fluorophore was modified on one of its termini. To optimize the photoregulation efficiency, three aptamer probes (see sequences in Table S1) containing different numbers of AZO were separately synthesized and modified on AuNPs. The resultant aptamer-modified AuNPs were termed TBA-AZOn-AuNPs, where n represents the number of AZO moieties. When TBA partially hybridizes with cDNA, a hairpin structure forms, and the FAM fluorophore approaches the surface of AuNPs, resulting in fluorescence quenching. However, upon illumination with UV light, AZO undergoes a trans-to-cis conversion, leading to dehybridization of the TBA/cDNA duplex. The fluorophore then withdraws from the surface of AuNPs, and FAM fluorescence is, to some extent, restored. In this light-driven design, enhancement of fluorescence intensity was associated with the time of UV illumination (Figures 2A, 2B, and S6A–S6C). On the other hand, irradiation with visible light triggered cis-to-trans isomerization of AZO, accompanied by the formation of DNA hairpin structure and a decrease in fluorescence intensity (Figure 2A, 2B, and S6D–S6F), indicating light-dependent switching of DNA conformation. By setting the fluorescence intensity of FAM-TBA-AuNPs at 100%, the fluorescence ratiometric changes of TBA-AZO3,4,5-AuNPs between UV and visible irradiation were
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Figure 2. Light-Driven Conformational Switch of Aptamer/AZO Probe on the Surface of AuNPs (A) Schematic illustration of light-driven conformational switch of TBA-AZOn-AuNPs. (B) Fluorescence changes (l em = 520 nm) of TBA-AZO 3 -AuNPs (a), TBA-AZO 4 -AuNPs (b), and TBA-AZO5 -AuNPs (c) in response to light irradiation for different lengths of time. (C) Reversible probe conformational switch of TBA-AZO 4 -AuNPs in response to alternating irradiation by UV (10 min) and visible light (5 min). The corresponding concentration of cTBA was 400 nM.
4.12%, 56.06%, and 6.36%, respectively (Figure S7). TBA-AZO4-AuNPs achieved the best photoregulation of the aptameric conformation and were therefore used as the optimal design in subsequent experiments. With fluorescence spectrometry following a reported protocol,30 the average number of DNA molecules (TBA-AZO4) immobilized on each AuNP surface was quantified as 58.34 G 4.82. The reversible conformation switches of TBA-AZO4-AuNPs in response to alternate illumination with UV (10 min) and visible (5 min) light were also investigated for multiple cycles. As shown in Figure 2C, in each cycle, FAM fluorescence intensity increased upon UV-light irradiation and decreased upon visible-light irradiation. Of note, because intact TBA forms a G-quadruplex structure, which can partially quench FAM fluorescence, the restored fluorescence intensity of TBA-AZO4-AuNPs after illumination with UV light was lower than that induced by addition of cTBA. Also, FAM moves farther away from the AuNP surface by complete cTBA/TBA hybridization. Meanwhile, negligible attenuation was observed, even after alternating seven cycles of light irradiation, and little negative impact of UV irradiation on DNA integrity was proven with gel electrophoresis (Figure S8), indicating excellent reversibility and stability of this nanoplatform. Reversibly Regulating Protein Activity Having demonstrated the reversible conformational changes of this aptamer/ AZO-based nanoplatform, we proceeded to test its ability to photoregulate protein activity. As shown in Figure S9A, the inhibition of thrombin activity was gradually enhanced with incremental addition of TBA-AZO4-AuNPs and approached a plateau at 0.125 nM, corresponding well with the results for the TBA-AuNP samples (Figure 1A). To test whether the visible-light-driven conformational change of the TBA
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Figure 3. Visible-Light-Induced Thrombin Activation The kinetic light-scattering spectra of samples containing fibrinogen (1.14 mM), thrombin (2.5 nM), and TBA-AZO4 -AuNPs (0.125 nM) pretreated with visible light for different lengths of time.
probe could restore the activity of thrombin, UV-pretreated TBA-AZO4-AuNPs were irradiated with visible light for different lengths of time, followed by immediate mixing with the substrate solution to test their influence on thrombin activity. As shown in Figure 3, thrombin activity gradually recovered with the extension of time of visible-light irradiation. The ability of TBA-AZO3-AuNPs and TBA-AZO5AuNPs to reversibly photoregulate thrombin activity was also evaluated (Figure S10), and the results were well correlated with those of the fluorescence measurements just described (Figure S6). Meanwhile, different light treatments induced little change in thrombin activity in the samples containing TBA-AZO0-AuNPs with the same design as TBA-AZO4-AuNPs, but with no AZO incorporated (Figure S9B), indicating that regulation of thrombin activity originates from light-directed AZO isomerization. The ability of this aptamer/AZO-based nanoplatform to photoregulate thrombin activity was further confirmed by monitoring the thrombin-catalyzed conversion process from fibrinogen to fibrin with confocal laser scanning microscopy. To perform this experiment, an Alexa-Fluor-546-labeled fibrinogen, whose fluorescence can be significantly intensified after conversion into fibrin, was used as the substrate. For fibrinogen only, no obvious fluorescence signal was observed, even after 66 min of incubation (Figure S11). However, upon thrombin catalysis (Figures 4A and S12), fibrinogen rapidly turned into small fluorescent fibrin units (t = 3 min) and gradually formed larger fluorescent aggregates (t = 11 min), indicating a feasible system for evaluating thrombin activity. When treated with UV-pretreated TBA-AZO4-AuNPs, in which the functional TBA sequence was unblocked (Figures 4B and S13), thrombin activity was efficiently inhibited with no observable morphological change. However, upon illumination with visible light, dynamic fluorescence enhancement was observed to be compatible with that of the thrombin-only sample, indicating effective recovery of thrombin activity. The reversible ON/OFF regulation of protein activity was further verified by alternating UV- and visible-light illumination (Figure 4C, 4D, S14, and S15). The fluorescent morphological difference among these samples could be clearly observed through 3D projection of the z stack image at 66 min. The average fluorescence intensity of each image (Figure S16), calculated with ImageJ software, was consistent with the above results. Together, these results verify that the aptamer/AZO-based nanoplatform can be used to reversibly manipulate the activity of target proteins. Quantitative Activation of the Target Protein Although challenging, quantitative activation of specific proteins, especially at the single-molecule level, is essential for understanding their functional behavior. To demonstrate whether this nanoplatform has the potential to precisely activate target proteins, UV-pretreated TBA-AZO4-AuNPs were first used to capture and inactivate thrombin, and then different visible-light irradiations were performed during the
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Figure 4. Kinetic Fluorescence Imaging of the Reaction Process Catalyzed by Thrombin under Different Conditions (A) Thrombin only. (B) Thrombin treated with pretreated (UV 10 min) TBA-AZO4 -AuNPs. (C) Thrombin treated with pretreated (UV 10 min / visible 5 min) TBA-AZO 4 -AuNPs. (D) Thrombin treated with pretreated (UV 10 min / visible 5 min / UV 10 min) TBA-AZO4 -AuNPs. All 3D projections of z stack images were obtained at the time point of 66 min.
catalysis reaction. As shown in Figures 5A and S17, the scattered light intensity rose abruptly after each exposure to visible light. The initial catalysis reaction rate (V0) was calculated according to the slope of the kinetic curve (Figure 5B). A sharp increase in V0 was induced by each pulse of light, indicating sudden release and activation of thrombin. Moreover, V0 was proportional to visible-light exposure time. To further investigate the quantitative relationship between thrombin activation and visible-light irradiation, TBA-AZO4-AuNP-pretreated thrombin was irradiated with visible light for different times, and the thrombin released was collected and mixed with fibrinogen. The catalytic reaction was monitored by reading the kinetic absorption spectrum (at 450 nm) with a 96-well plate reader, which can measure multiple samples at one time (Figure S18). The kinetic absorption spectrum of thrombin at different concentrations was also collected, and a standard curve was prepared by plotting V0 versus the given thrombin concentration (Figure S19). As shown in Figures 5C and S20, a good linear relationship between amount of activated thrombin and visible irradiation time was obtained, ranging from 0 to 240 s. Meanwhile, 86.42% G 0.40% of the captured thrombin could be released with 240-s visible irradiation, indicating high efficiency of protein activation. Moreover, on the basis of these calculations, 20 thrombin molecules were initially captured per TBA-AZO4AuNP, and activation of one thrombin molecule could be achieved by visible irradiation of 14.4 s (Figure 5D), indicating the strong potency of this aptamer/AZO-based nanoplatform for quantitative protein regulation at the single-molecule level. Moreover, protein detection methods with higher sensitivity and proper light devices could potentially provide the current system with the ability to activate one protein at a time, thus providing a powerful tool for studying elusive biological processes.
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Figure 5. Quantitative Photoregulation of Thrombin Activity (A) Kinetic light-scattering spectra of the samples catalyzed with thrombin only (control), thrombin treated with UV-pretreated TBA-AZO4 -AuNPs (a), and thrombin treated with UV-pretreated TBA-AZO4 -AuNPs with visible-light irradiation applied at three kinetic catalysis reaction points (100, 200, and 300 s) (b and c). (B) Corresponding initial catalysis reaction rates (V 0 ) from (A). (C) The concentration and ratio of activated thrombin under different visible treatments. (D) Relationship between the molecular number of visible-activated thrombin per AuNP and visible illumination time. All data in (C) and (D) were collected from three independent experiments. Error bars represent standard deviations.
Photoregulation of Blood Clotting in Human Plasma After demonstrating the excellent capability of this aptamer/AZO-based nanoplatform for precisely regulating thrombin activity in buffer solution, we next tested its performance in human plasma by measuring thrombin clotting time (TCT). As shown in Figure 6A, TCT induced by thrombin only is 17.1 s, which is within the normal test range (14.0–21.0 s) provided by the manufacturer. After treatment with the UV-pretreated TBA-AZO4-AuNPs, however, the TCT value showed a dose-dependent increment and even reached 83.2 s (at 10 nM), which is 3.9 times longer than that induced by thrombin only, indicating that UV-pretreated TBA-AZO4-AuNPs can efficiently inhibit the coagulation process of human plasma samples. Next, we examined whether the current nanoplatform could be used to reversibly regulate the coagulation of human plasma samples. As shown in Figure 6B, by alternating irradiation with UV (10 min) and visible light (5 min), the TCT value of the plasma samples was switched accordingly. In addition, the attenuation of photo-driven conversion efficiency was negligible, even after nine cycles of alternating treatment, demonstrating the excellent performance of this nanoplatform for reversibly regulating the activity of target proteins in complex physiological fluids. Photoregulation of Protein Activity in Complex Living Systems To further evaluate the potential of this aptamer/AZO-based nanoplatform for regulating protein activity in more complex living systems, live Bacillus subtilis, a wellstudied Gram-positive bacterium that exists in the gastrointestinal tract of ruminants and humans, was used as a model. The cell wall of B. subtilis can be digested by
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Figure 6. Photoregulation of Blood Clotting Time of Human Plasma (A) Linear relationship between TCT and the concentration of UV-pretreated (10 min) TBA-AZO4-AuNPs. (B) Reversible regulation of TCT by TBA-AZO 4 -AuNPs in response to alternating UV- and visiblelight irradiation. All data were collected from three independent experiments. Error bars represent standard deviations.
active lysozyme, leading to cell death. To demonstrate lysozyme activity on living B. subtilis, a LIVE/DEAD BacLight Bacterial Viability Kit containing SYTO 9 (green fluorescence) and propidium iodide (PI, red fluorescence) was applied. Generally, SYTO 9 can stain both live and dead B. subtilis, whereas PI can only penetrate the damaged membrane of dead B. subtilis and then replace intracellular SYTO 9, causing a reduction in the SYTO 9 stain fluorescence. With treatment of SYTO 9/PI dyes, live B. subtilis showed a strong green fluorescence even after incubation for 70 min (Figure S21A). In contrast, with the addition of active lysozyme, most B. subtilis displayed red fluorescence after a 20-min incubation (Figure S21C), indicating the feasibility of this SYTO 9/PI strategy for analyzing the viability of B. subtilis and the activity of lysozyme. Meanwhile, rDNA40-AuNP- and LA-AuNP-treated active lysozyme samples (Figures S21D and S21E) showed red and green fluorescence, respectively, further demonstrating that the proposed mechanism to inactivate protein worked in this system. Large protein-AuNP aggregation architectures (as indicated by yellow arrows in the bright channel) were observed only in the LA-AuNPs-treated lysozyme sample (Figures S21D and S21E), consistent with the AFM results (Figures S4B and S4C) and indicating aptamer-specific lysozyme aggregation around the AuNP core. To exert photoregulation on lysozyme activity, the 50 end of LA was extended with a cDNA sequence incorporated with five AZO molecules (Table S1). The thiolated probe was modified onto the AuNP surface and the resultant product was termed LA-AZO5-AuNP. The live SYTO9/PI-stained B. subtilis were mixed with LA-AZO5AuNP-pretreated lysozymes and then irradiated with visible light for different times (Figure 7A). As shown in Figure 7B, the red fluorescence signal (as indicated with yellow arrows) gradually increased, and the green fluorescence decreased when extending the visible irradiation time, indicating visible-light-induced activation of lysozyme. The death rate of B. subtilis was increased by extending the visible irradiation time (Figure S22), corresponding well with the red-to-green fluorescence ratio calculated with ImageJ software. Specifically, with visible irradiation for 5 min, the percentage of dead B. subtilis reached 38.09% G 9.27% (Figure 7C). However, with no visible-light treatment, only 3.69% G 1.12% dead B. subtilis was observed. Of note, the visible-light irradiation itself had no negative influence on the viability of B. subtilis (Figure S21B). All these results demonstrate that our aptamer/AZO-based nanoplatform can be used to precisely photoregulate target protein activity in complex biological systems.
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Figure 7. Photoregulation of Lysozyme Activity in a Living B. subtilis System (A) Schematic illustration of photoregulation of lysozyme activity in a living B. subtilis system. (B) Fluorescence imaging of living B. subtilis treated with LA-AZO 5 -AuNP-captured lysozymes irradiated by visible light for 0 min (a), 1 min (b), 2 min (c), and 5 min (d). The concentration of lysozyme and LA-AZO 5 -AuNPs was 1,200 U/(mg$mL) and 10 nM, respectively. All the images were obtained 20 min after visible-light irradiation. Scale bar represents 10 mm. (C) Relationship between the percentage of dead B. subtilis and visible illumination time. All data were collected from three independent experiments. Error bars represent standard deviations.
DISCUSSION In summary, we have developed an aptamer/AZO-based nanoplatform for quantitative and reversible regulation of target protein activity. Unlike conventional photoregulation strategies, no modification of proteins is required in our design. By using thrombin and its TBA aptamer as demonstration models, we proved that TBA/AZO-functionalized AuNPs can reversibly regulate thrombin activity in both buffer solution and human plasma, indicating a promising strategy for intelligent anticoagulation therapy. Of note, this protein inactivation strategy was based on aptamer-specific protein capture around the AuNP core without a need for involving special aptamers that themselves are capable of protein inhibition. Because it is much more common and easier to select aptamers that can bind only with their target proteins but with no interference on the protein activity, this design is expected to provide a more universal strategy for protein regulation. Meanwhile, modulating visible-light irradiation achieved quantitative activation of thrombin, providing a potent tool for the precise and dynamic regulation of target protein, which is essential for studying the myriad of protein-dominant biological processes. With a proper light source and highly sensitive detection technique, this photoresponsive nanoplatform is expected to enable activation of one protein molecule at a time. Moreover, another protein (lysozyme)/ aptamer-AuNP system was successfully constructed and applied to a complex living B. subtilis system, revealing that this aptamer-based nanoplatform can be potentially expanded for studying and manipulating different proteins in more complex biological systems.
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EXPERIMENTAL PROCEDURES Materials and Reagents DNA synthesis reagents were purchased from Glen Research (Sterling, VA). KCl, CaCl2, KH2PO4, K2HPO4$3H2O, sucrose, and mercaptoethanol were purchased from Sinopharm Chemical Reagent Co. (Shanghai, China). NaCl, MgCl2$6H2O, human a-thrombin, fibrinogen from human plasma, and lysozyme were obtained from Sigma-Aldrich (Shanghai, China). Sodium citrate was purchased from Tianjin Fengchuan Chemical Reagent Science and Technology (Tianjin, China). Tween 20 was obtained from Beyotime Institute of Biotechnology (China). PBS 103 (pH 7.4) and Alexa-Fluor-546-labeled fibrinogen were purchased from Life Technologies. Chloroauric acid, tris-(2-carboxyethyl)-phosphine hydrochloride (TCEP), and 1 M Tris-buffer sterile solution (pH 7.4) were purchased from Sangon Biotech (Shanghai, China). B. subtilis was obtained from the China General Microbiological Culture Collection Center (Beijing, China). Milli-Q water (resistance >18 MU cm) was used to prepare all solutions. DNA Synthesis All DNA sequences (Table S1) were synthesized on a PolyGen DNA synthesizer. Synthesis and deprotection were performed according to the instructions provided by the manufacturers of the reagents. Deprotected DNA was then precipitated by adding 40 mL (1/10 volume) of 3 M NaCl and 1 mL (2.53 volume) of cold ethanol. The mixtures were placed in a freezer at 20 C for 30 min and then centrifuged at 4,000 rpm at 4 C for 30 min. After removing the supernatants, the DNA products were dissolved in 400 mL of 0.1 M triethylamine/acetate (TEAA) and purified by high-performance liquid chromatography (Agilent 1260) with a C18 column (5 mm, 4.6 3 250 mm, 100 A˚; GL Science, Inertsil ODS-3). The purified DNA was dried, detritylated by dissolving in 80% acetic acid (200 mL) for 20 min, and then precipitated with 20 mL of 3 M NaCl and 500 mL of cold ethanol. The mixtures were chilled at 20 C for another 30 min, collected through centrifugation at 14,000 rpm at 4 C for 5 min, and then resolved with ultrapure water. The final DNA products were desalted with illustra NAP-5 columns (GE Healthcare), and their concentration was determined by detecting the UV absorption at 260 nm on a BioSpec-nano (Shimadzu). Synthesis of AuNPs AuNPs with an average diameter of 13 nm were synthesized by reduction of HAuCl4 with sodium citrate.31 Chloroauric acid solution (0.01 wt %, 100 mL) was added to a clean round-bottom flask and heated with stirring and refluxing until boiling. Then, sodium citrate solution (3 wt %, 1 mL) was added rapidly to the solution, which was heated with continuous stirring for another 30 min. The solution was then cooled to room temperature and stored in the dark for subsequent experiments. The size of AuNPs (Figure S1) was characterized with a JEM 2100 transmission electron microscope (Hitachi). A UV-2450 spectrophotometer (Shimadzu) was used to measure the absorption of the AuNPs (Figure S2A). The hydrodynamic diameter of AuNPs (Figure S2B) was measured with a Zetasizer Nano ZS90 DLS system (Malvern Instruments, Worcestershire, UK). The concentration of AuNPs was determined by UV-visible absorbance via Beer’s law (A = εbc). Aptamer Modification of AuNPs Thiolated DNAs were attached to AuNPs as described in previous literature.32,33 First, the disulfide bond of thiolated DNAs was cleaved by treatment with TCEP (503) at pH 5.0 for 1 hr, and then the DNAs were desalted with illustra NAP-5 columns. TBA-AZO0, TBA-AZO3, TBA-AZO4, TBA-AZO5, and LA-AZO5 were dissolved
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in 13 PBS, heated at 95 C for 5 min, and then immediately cooled to 4 C for 2 min to form a hairpin structure. Other DNA sequences (Table S1) were dissolved directly with ultrapure water with no heating/cooling treatment. Then, 500 mL of 13-nm AuNP solution (2.5 nM), 5 mL of Tween 20 (1% v/v), 10 mL of thiolated DNA (100 mM) and 10 mL of citrate-HCl buffer (pH 7.0, 0.5 M) were mixed in a centrifuge tube. After that, NaCl solution (3 M) was slowly added to the mixture to reach a final concentration of 0.1 M. After salt aging at room temperature for 6 hr, the mixtures were centrifuged at 16,200 3 g at 4 C for 15 min. After removal of the supernatants, the precipitate was washed with 5 mM Tris-HCl buffer (containing 0.01% v/v Tween 20, pH 7.4) three times via centrifugation. The resultant DNA-AuNPs were resuspended in PBT solution (25 mM Tris-HCl [pH 7.4], 0.01% v/v Tween 20, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, and 1 mM CaCl2) and stored at room temperature in the dark for subsequent experiments. A UV-2450 spectrophotometer (Shimadzu) was used to measure the absorption of the DNA-AuNPs (Figure S2A). The hydrodynamic diameter of DNA-AuNPs (Figure S2B) was measured with a Zetasizer Nano ZS90 DLS system (Malvern Instruments, Worcestershire, UK). Fluorescence Measurements UV light at 365 nm was provided by a Spectroline Model SB-100P/FA UV lamp (100 W), and visible light at 450 nm was provided by a 250 W high-pressure mercury lamp (Shanghai Jiguang) with a 450 nm glass filter (Shenzhen Zhonglai Technology). The distance between the samples and the UV-visible light source was fixed at 5 cm. To evaluate the light-driven conformational switch of the TBA/AZO probes, FAM fluorophores were modified on one of their termini. Generally, the TBA-AZOn-AuNP samples (200 mL, 2.5 nM) were treated with UV or visible light for a certain length of time, and the fluorescence spectra were recorded with a Fluoromax-4 spectrofluorometer (HORIBA JobinYvon, Edison, NJ). The fluorescence peak intensity at 520 nm was used to analyze the performance of this system. The fluorescence intensity of equivalent AuNPs and FAM-TBA-AuNPs was used as the baseline and the internal standard, respectively. All fluorescence data were calculated by subtracting the baseline and normalized according to the internal standard. Real-Time Monitoring of Thrombin Catalytic Reaction Active thrombin can convert fibrinogen into insoluble fibrin, leading to an increase in scattering light intensity (650 nm) and the light absorbance value at about 450 nm. In brief, the samples were pretreated with or without UV-visible light, as described above, and mixed with thrombin for 10 min. The mixtures were added to the fibrinogen solution, which was dissolved in physiological buffer (25 mM Tris-HCl [pH 7.4] containing 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, and 1 mM CaCl2). The thrombin catalytic reaction was monitored by measuring the intensity change of the scattered light with a Fluoromax-4 spectrofluorometer (HORIBA JobinYvon, Edison, NJ). The final concentrations of thrombin and fibrinogen in the mixtures (V = 200 mL) were 2.5 and 1.14 nM, respectively. The excitation and emission wavelengths were both set at 650 nm with a bandwidth of 5 nm for excitation and emission. For calibration of systematic error, the catalytic reaction induced by thrombin only was always performed as an internal standard in each independent experiment. In some experiments, to guarantee that most TBAs would be liberated to capture thrombin, the functional AuNPs were pre-irradiated with UV light for 10 min. Meanwhile, the pretreated AuNPs were mixed and incubated with thrombin (2.5 nM) for 10 min before the fibrinogen substrate was added to initiate the catalytic reaction.
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Real-Time Monitoring of Lysozyme Activity Lysozyme is an enzyme that can catalyze the hydrolysis of Micrococcus lysodeikticus, resulting in decreased absorbance at 450 nm (A450). In the detection assay, Micrococcus lysodeikticus (Huich Bio-tech) was suspended in the reaction buffer (66 mM potassium phosphate [pH 6.2]), and its working concentration was adjusted according to the A450 value of 0.6–0.8. Initially, a certain concentration of free lysozyme—or lysozyme pretreated with LA, AuNPs only, LA-AuNPs, or rDNA40-AuNPs—was added into the Micrococcus lysodeikticus solution, and the kinetic absorbance spectra were immediately recorded with a UV-2450 spectrophotometer (Shimadzu). The concentration of LA and AuNPs was 400 and 0.3 nM, respectively. AFM Assay For AFM assay, 10 mL of the prepared sample was dropped onto a mica surface. After 5 min at room temperature, the mica was washed with 200 mL of Milli-Q water four times and dried under nitrogen. All samples were imaged on a Multimode 8 (Bruker, USA) with ScanAsyst mode at 0.977 Hz scan rate. In Situ Imaging of the Thrombin Catalytic Reaction For the fluorescence imaging assay, thrombin (2.5 nM) was treated with different TBA-AZO4-AuNP samples. Then, Alexa-Fluor-546-labeled fibrinogen (1.14 mM) was added. Subsequently, 30 mL of the mixtures was rapidly transferred to a 35-mm Petri dish and immediately imaged with an inverted microscope (Nikon Ti-E, Nikon, Japan) at different reaction time points. The excitation wavelength was 561 nm. The z stack images of the mixtures were scanned at 66 min. The images were recorded with a 203 dry objective. Quantitative Activation of Thrombin In this experiment, we changed to a 96-well reader, which can read multiple samples at a time, to test the activity of thrombin by measuring the kinetic absorbance at 450 nm. To set up a standard curve, pure thrombin at different concentrations was mixed with fibrinogen (1.14 mM), and the kinetic absorption spectra were measured immediately with a Synergy 2 multimode reader (BioTek, USA). The initial catalytic reaction rate (V0) was calculated and plotted versus the thrombin concentration. To guarantee that most TBAs would be liberated to capture thrombin in experiments of quantitative thrombin activation, TBA-AZO4-AuNPs were pre-irradiated with UV light for 10 min. Then, the pretreated AuNPs were mixed and incubated with thrombin (2.5 nM) for 10 min. The mixtures were irradiated by visible light for different lengths of time and then centrifuged at 16,200 3 g at 4 C for 15 min. The supernatants were collected and mixed with the fibrinogen substrate, and the kinetic absorbance spectra (at 450 nm) were recorded immediately. The active amount of thrombin was calculated according to the standard curve. Human Plasma Tests We studied the ability of the aptamer/AZO-based nanoplatform to reversibly regulate thrombin activity in the process of blood clotting of human plasma. To accomplish this, healthy human blood samples were collected with the permission of Xiangya Hospital and processed according to a standard procedure to extract plasma for subsequent analysis. Thrombin with different treatments was added to the human plasma, and the TCTs of samples were measured with an Automated Coagulation Blood Analyzer CS-2000i (Sysmex Shanghai, Shanghai, China).
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Imaging of Live and Dead B. subtilis B. subtilis was cultured with liquid nutrient medium at 37 C. After growing for 24 hr, living B. subtilis was stained with the LIVE/DEAD BacLight Bacterial Viability Kits (Invitrogen) according to the instructions provided by the manufacturer. To test the capability of LA-AZO5-AuNPs to photoregulate the lysozyme activity in the living B. subtilis system, LA-AZO5-AuNPs were pre-irradiated with UV light for 10 min and then mixed with active lysozyme for 10 min. The mixture was incubated with living B. subtilis. The resultant B. subtilis samples were irradiated with visible light for different times and subsequently imaged with an FV1000 confocal microscope (Olympus). The images were recorded with a 603 oil objective.
SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, 22 figures, and 1 table and can be found with this article online at https://doi.org/10. 1016/j.chempr.2017.11.008.
ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China grants 21505039, 2013CB932702, 21405041, 21221003, and 21327009. It was also supported by NIH grants GM079359 and CA133086. We sincerely thank Prof. Chaoyong Yang’s lab at Xiamen University for helping with DNA synthesis.
AUTHOR CONTRIBUTIONS Conceptualization, S.X., L.Q., W.T.; Methodology, L.C., Honglin Liu, Y.S., H. Liang, D.D., J.Z.; Investigation, Huixia Liu; Resources, Huixia Liu, Z.C., and X.Z.; Supervision and Funding Acquisition, L.Q. and W.T. Received: May 14, 2017 Revised: September 19, 2017 Accepted: November 21, 2017 Published: December 14, 2017
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Reversible and Quantitative Photoregulation of Target Proteins Sitao Xie, Liping Qiu, Liang Cui, ..., Zhou Chen, Xiaobing Zhang, Weihong Tan [email protected] (L.Q.) [email protected] (W.T.)
HIGHLIGHTS A DNA-based, noninvasive, photoresponsive nanoplatform of protein manipulation Achieving reversible and quantitative regulation of target protein activity This nanoplatform can be easily generalized to study different proteins
Noninvasive, precise, and reversible regulation of specific proteins is essential to elucidating fundamental biological processes. To achieve this goal, Tan and colleagues have developed a universal, aptamer-based, photoresponsive nanoplatform. By modulating light irradiation, they were able to reversibly and quantitatively manipulate the activity of target proteins in both complex serum and living bacteria samples.
Xie et al., Chem 3, 1021–1035 December 14, 2017 ª 2017 Elsevier Inc. https://doi.org/10.1016/j.chempr.2017.11.008
Article
Reversible and Quantitative Photoregulation of Target Proteins Sitao Xie,1 Liping Qiu,1,* Liang Cui,1 Honglin Liu,1 Yang Sun,1 Hao Liang,1 Ding Ding,1 Lei He,1 Huixia Liu,3 Jiani Zhang,3 Zhou Chen,1 Xiaobing Zhang,1 and Weihong Tan1,2,4,*
SUMMARY
The Bigger Picture
Essential to elucidating fundamental biological processes is first gaining precise and reversible external control over proteins without altering their natural structure. To achieve this goal, we developed an aptamer-based, noninvasive, photoresponsive nanoplatform capable of reversible and quantitative regulation of target protein activity. Protein-specific aptamers equipped with several light-responsive molecules were functionalized onto the surfaces of nanoparticles. Adjusting light irradiation allowed the light-responsive molecules to drive the conformational switch of aptamer between the blocked status and liberated status. In this way, the target proteins were captured around the nanoparticle or released into the medium, thus leading to inhibition or activation of the protein, respectively. Moreover, modulating light irradiation allowed quantitative control of protein activity.
Temporally precise, noninvasive control of target protein activity at the single-molecule level is essential to understanding many protein-dominant biological processes. Here, we report a DNA-based, noninvasive, photoresponsive nanoplatform capable of reversible and quantitative regulation of target protein activity. Adjusting light irradiation allows the target proteins to be captured around the nanoparticle or released into the medium, thus leading to inhibition or activation of the protein, respectively. Moreover, modulating irradiation time allows quantitative control of protein release. Consequently, this nanoplatform can be a potent tool for intensive and precise investigation and manipulation of specific proteins without altering their natural structures, which is significant for studying complex biological signaling networks, identifying new therapeutic targets, and developing intelligent strategies for precision medicine.
INTRODUCTION Proteins, as key components of organisms, are involved in virtually all biological processes. Protein dysregulation can lead to biological dysfunction and disease. Thus, precise and reversible regulation of protein activity at the single-molecule level is essential to understanding complex biological signaling networks or identifying new therapeutic targets.1,2 Photons, as efficient and controllable external triggers, have been used to perform precise temporal manipulation over many protein systems.3,4 For example, by covalently attaching a synthetic photoisomerizable small molecule to endogenous ion channels, Kramer and colleagues have developed a light-activated chemical gate for regulating neuronal activity.5 Lin and co-workers proposed a fluorescent-inducible protein through genetically fusing photochromic Dronpa domains to both termini of the protein, enabling precise optical manipulation of both protease domains and guanine nucleotide exchange factor.6 Heo and co-workers developed a light-activated reversible inhibition strategy to inhibit protein function by sequestering targets into large assembled protein structures.7 Although all of these methods can rapidly and reversibly control protein behavior at a defined time and location, they all involve chemical or genetic protein modification. This process is complicated and time consuming, and it potentially affects protein activity, as well as related biological events, thus limiting practical applications. Moreover, none of these methods has been able to achieve quantitative activation of target proteins. Alternative strategies with simple design, convenient operation, little influence on natural protein structure, and potent capability of regulation at the single-molecule level are being sought. The advancement of nanotechnology has allowed the construction of many versatile nanoplatforms for biological applications,8,9 especially protein regulation.10,11 To
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Scheme 1. Schematic Illustration of the Aptamer/AZO-Based Nanoplatform for Reversible and Quantitative Manipulation of Protein Protein-specific aptamers modified with several light-responsive molecules are functionalized onto the surface of nanoparticles. By adjusting light irradiation, the light-responsive molecules can drive the conformational switch of aptamer between the blocked status and liberated status. In this way, the target proteins are captured around the nanoparticle or released into the medium, thus leading to inhibition or activation of the protein, respectively. Moreover, by modulating light irradiation, protein activity can be quantitatively controlled.
perform targetable control on proteins of interest, specific recognition units are always required. Particularly suited to this task, aptamers are artificial oligonucleotides selected via an in vitro method12 on the basis of their specific binding affinity13,14 to the target molecules. Accordingly, they have been applied as promising alternatives to protein antibodies, owing to their intrinsic advantages15,16 of easy synthesis, convenient modification with various functional groups, and flexible design. The combination of aptamers with nanomaterials is expected to offer unlimited opportunities for protein research.17,18 Based on many reports, consensus would seem to hold that the formation of high-order protein clusters has the potential to trap and, hence, inactivate protein function.7,19 The hypothesis is plausible because the active sites of proteins are expected to be trapped and blocked within the assembled protein architectures. Therefore, in the present work, we developed an aptamer-functionalized nanoplatform to reversibly control the formation and dissociation of protein clusters with the expectation of first inhibiting and then restoring protein activity without affecting natural protein structure. As shown in Scheme 1, aptamers able to specifically recognize a target protein were functionalized onto the surface of nanoparticles, and photoresponsive azobenzene (AZO) molecules were incorporated into the aptamer probe, together with a short cDNA sequence. Subsequently, light-driven conformational change of the aptamer controls the capture and release of proteins. More specifically, irradiation with UV light drives a trans-to-cis isomerization of AZO, leading to dehybridization of the aptamer/cDNA duplex, allowing the aptamer sequence to capture the protein of interest, thus leading to inactivation. On the other hand, under illumination with visible light, AZO undergoes a cis-totrans conversion, and the strong affinity between aptamer and cDNA, compared with that between aptamer and protein, again forms, blocking the aptamer and liberating protein, leading to restoration of its activity. Importantly, by modulating the irradiation time, protein release can be quantitatively controlled. Consequently, this nanoplatform can be a potent tool for intensive and precise investigation and manipulation of specific proteins.
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1Molecular
Science and Biomedicine Laboratory, State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Life Sciences, Aptamer Engineering Center of Hunan Province, Hunan University, Changsha, Hunan 410082, China
2Center
for Research at the Bio/Nano Interface, Department of Chemistry and Department of Physiology and Functional Genomics, Health Cancer Center, UF Genetics Institute, and McKnight Brain Institute, University of Florida, Gainesville, FL 32611-7200, USA
3Division
of Geriatrics, Xiangya Hospital, Central South University, Changsha, Hunan 410008, China
4Lead
Contact
*Correspondence: [email protected] (L.Q.), [email protected] (W.T.) https://doi.org/10.1016/j.chempr.2017.11.008
Figure 1. Feasibility of Forming High-Order Protein Assembly for Inhibition of Thrombin Activity (A) Real-time light-scattering spectra of mixtures containing fibrinogen (1.14 mM) and thrombin (2.5 nM) treated with different amounts of TBA-AuNPs. (B) Real-time light-scattering spectra of mixtures containing fibrinogen (1.14 mM) and thrombin (2.5 nM) treated with 0.125 nM AuNPs (a), 0.125 nM rDNA 29 -AuNPs (b), 400 nM TBA (c), and 0.125 nM TBA-AuNPs (d). (C–E) AFM characterization of thrombin only (C), the mixture of thrombin and rDNA 29 -Au NPs (D), and the mixture of thrombin and TBA-AuNPs (E).
RESULTS Construction and Optimization of the Nanoplatform With the aim of developing a simple, universal, and potent protein regulation nanoplatform with high potential for practical applications, we chose gold nanoparticles (AuNPs; diameter = 13 nm)—which are among the most well-studied nanoparticles with advantages of easy synthesis in aqueous solution, uniform size and shape, low biotoxicity,20 and efficient functionalization with oligonucleotides21,22—as the ‘‘core’’ for protein assembly. Thrombin, a key protein implicated in blood coagulation23 and one whose activity can be easily evaluated by common measurement techniques, was used as the model protein. A 29-mer thrombin-binding aptamer24 (TBA) able to specifically recognize thrombin but with little inhibition on thrombin activity was used to functionalize AuNPs via a gold-thiol bond, and the resultant product was termed TBA-AuNPs. In principle, active thrombin can catalyze the conversion of fibrinogen to insoluble fibrin, resulting in a rapid increase of scattered light intensity.25 Figure 1A shows dose-dependent inhibition of thrombin activity by TBA-AuNPs, achieving complete inhibition at the TBA-AuNP concentration of 0.125 nM (green line in Figures 1A and 1B). However, as shown in Figure 1B, negligible thrombin inhibition was observed in the samples containing excess TBA molecules (400 nM, which was about 50 times higher than the TBA concentration of the TBA-AuNPs). In addition, neither equivalent AuNPs nor rDNA29-AuNPs (AuNPs modified with 29-mer random DNA sequences) induced observable changes on thrombin activity, indicating that the thrombin inhibition originates from the TBA-specific thrombin assembly around the AuNP core, which is consistent with a previous report.19 To examine the interaction between TBA-AuNPs and thrombin, atomic force microscopy (AFM) was performed. Larger nanostructures were observed in the mixtures of thrombin and TBA-AuNPs (Figure 1E); neither
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thrombin only (Figure 1C) nor the mixture of rDNA29-AuNPs and thrombin (Figure 1D) showed any assembled morphology. To demonstrate the universality of this aptamer-based nanoassembly strategy for inhibiting target protein activity, another protein (lysozyme)-aptamer26 system was tested. Theoretically, active lysozyme can destroy the cell walls of Micrococcus lysodeikticus, which can be monitored via a decrease in absorbance at 450 nm.27 As shown in Figure S3, the activity of lysozyme was efficiently inhibited by LA (lysozyme aptamer)-AuNPs in a concentration-dependent manner. However, little inhibitory impact on lysozyme was caused by excess LA molecules (400 nM), AuNPs (0.3 nM), or rDNA40-AuNPs (0.3 nM), indicating specific lysozyme inhibition induced by LA-AuNPs. Moreover, AFM was performed to confirm the protein cluster morphology obtained in the LA-AuNPs + lysozyme sample, but not in the lysozyme only or rDNA40-AuNPs + lysozyme samples (Figure S4). These results demonstrated the high potential of the current aptamer-based nanoplatform for specific and efficient inhibition of protein activity. To exert temporal and reversible control over target proteins, the 50 end of TBA was extended with a short cDNA sequence, and AZO molecules, which have been widely introduced into DNA and subsequently shown to change the conformation of DNA via photons,28,29 were incorporated into this cDNA sequence. Initially, part of the TBA sequence was blocked through hybridization with cDNA, preventing the interaction between TBA and thrombin. However, irradiation by UV light (365 nm) could drive a trans-to-cis isomerization of the azobenzene moiety. This led to dehybridization of the TBA/cDNA duplex, thus releasing the intact functional TBA sequence to capture thrombin around the AuNP core to form a protein cluster, leading to inhibition of thrombin activity. Next, by illumination with visible light at around 450 nm, azobenzene underwent a cis-to-trans conversion. Because the binding affinity between TBA and cDNA was stronger than that between TBA and thrombin, as proved by a competition experiment (Figure S5), TBA/cDNA duplex was again formed and blocked the functional structure of TBA. Thus, thrombin was liberated and its activity restored. Consequently, by adjusting the light illumination, thrombin activity can be reversibly controlled. To verify the light-driven conformational change of TBA, a carboxyfluorescein (FAM) fluorophore was modified on one of its termini. To optimize the photoregulation efficiency, three aptamer probes (see sequences in Table S1) containing different numbers of AZO were separately synthesized and modified on AuNPs. The resultant aptamer-modified AuNPs were termed TBA-AZOn-AuNPs, where n represents the number of AZO moieties. When TBA partially hybridizes with cDNA, a hairpin structure forms, and the FAM fluorophore approaches the surface of AuNPs, resulting in fluorescence quenching. However, upon illumination with UV light, AZO undergoes a trans-to-cis conversion, leading to dehybridization of the TBA/cDNA duplex. The fluorophore then withdraws from the surface of AuNPs, and FAM fluorescence is, to some extent, restored. In this light-driven design, enhancement of fluorescence intensity was associated with the time of UV illumination (Figures 2A, 2B, and S6A–S6C). On the other hand, irradiation with visible light triggered cis-to-trans isomerization of AZO, accompanied by the formation of DNA hairpin structure and a decrease in fluorescence intensity (Figure 2A, 2B, and S6D–S6F), indicating light-dependent switching of DNA conformation. By setting the fluorescence intensity of FAM-TBA-AuNPs at 100%, the fluorescence ratiometric changes of TBA-AZO3,4,5-AuNPs between UV and visible irradiation were
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Figure 2. Light-Driven Conformational Switch of Aptamer/AZO Probe on the Surface of AuNPs (A) Schematic illustration of light-driven conformational switch of TBA-AZOn-AuNPs. (B) Fluorescence changes (l em = 520 nm) of TBA-AZO 3 -AuNPs (a), TBA-AZO 4 -AuNPs (b), and TBA-AZO5 -AuNPs (c) in response to light irradiation for different lengths of time. (C) Reversible probe conformational switch of TBA-AZO 4 -AuNPs in response to alternating irradiation by UV (10 min) and visible light (5 min). The corresponding concentration of cTBA was 400 nM.
4.12%, 56.06%, and 6.36%, respectively (Figure S7). TBA-AZO4-AuNPs achieved the best photoregulation of the aptameric conformation and were therefore used as the optimal design in subsequent experiments. With fluorescence spectrometry following a reported protocol,30 the average number of DNA molecules (TBA-AZO4) immobilized on each AuNP surface was quantified as 58.34 G 4.82. The reversible conformation switches of TBA-AZO4-AuNPs in response to alternate illumination with UV (10 min) and visible (5 min) light were also investigated for multiple cycles. As shown in Figure 2C, in each cycle, FAM fluorescence intensity increased upon UV-light irradiation and decreased upon visible-light irradiation. Of note, because intact TBA forms a G-quadruplex structure, which can partially quench FAM fluorescence, the restored fluorescence intensity of TBA-AZO4-AuNPs after illumination with UV light was lower than that induced by addition of cTBA. Also, FAM moves farther away from the AuNP surface by complete cTBA/TBA hybridization. Meanwhile, negligible attenuation was observed, even after alternating seven cycles of light irradiation, and little negative impact of UV irradiation on DNA integrity was proven with gel electrophoresis (Figure S8), indicating excellent reversibility and stability of this nanoplatform. Reversibly Regulating Protein Activity Having demonstrated the reversible conformational changes of this aptamer/ AZO-based nanoplatform, we proceeded to test its ability to photoregulate protein activity. As shown in Figure S9A, the inhibition of thrombin activity was gradually enhanced with incremental addition of TBA-AZO4-AuNPs and approached a plateau at 0.125 nM, corresponding well with the results for the TBA-AuNP samples (Figure 1A). To test whether the visible-light-driven conformational change of the TBA
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Figure 3. Visible-Light-Induced Thrombin Activation The kinetic light-scattering spectra of samples containing fibrinogen (1.14 mM), thrombin (2.5 nM), and TBA-AZO4 -AuNPs (0.125 nM) pretreated with visible light for different lengths of time.
probe could restore the activity of thrombin, UV-pretreated TBA-AZO4-AuNPs were irradiated with visible light for different lengths of time, followed by immediate mixing with the substrate solution to test their influence on thrombin activity. As shown in Figure 3, thrombin activity gradually recovered with the extension of time of visible-light irradiation. The ability of TBA-AZO3-AuNPs and TBA-AZO5AuNPs to reversibly photoregulate thrombin activity was also evaluated (Figure S10), and the results were well correlated with those of the fluorescence measurements just described (Figure S6). Meanwhile, different light treatments induced little change in thrombin activity in the samples containing TBA-AZO0-AuNPs with the same design as TBA-AZO4-AuNPs, but with no AZO incorporated (Figure S9B), indicating that regulation of thrombin activity originates from light-directed AZO isomerization. The ability of this aptamer/AZO-based nanoplatform to photoregulate thrombin activity was further confirmed by monitoring the thrombin-catalyzed conversion process from fibrinogen to fibrin with confocal laser scanning microscopy. To perform this experiment, an Alexa-Fluor-546-labeled fibrinogen, whose fluorescence can be significantly intensified after conversion into fibrin, was used as the substrate. For fibrinogen only, no obvious fluorescence signal was observed, even after 66 min of incubation (Figure S11). However, upon thrombin catalysis (Figures 4A and S12), fibrinogen rapidly turned into small fluorescent fibrin units (t = 3 min) and gradually formed larger fluorescent aggregates (t = 11 min), indicating a feasible system for evaluating thrombin activity. When treated with UV-pretreated TBA-AZO4-AuNPs, in which the functional TBA sequence was unblocked (Figures 4B and S13), thrombin activity was efficiently inhibited with no observable morphological change. However, upon illumination with visible light, dynamic fluorescence enhancement was observed to be compatible with that of the thrombin-only sample, indicating effective recovery of thrombin activity. The reversible ON/OFF regulation of protein activity was further verified by alternating UV- and visible-light illumination (Figure 4C, 4D, S14, and S15). The fluorescent morphological difference among these samples could be clearly observed through 3D projection of the z stack image at 66 min. The average fluorescence intensity of each image (Figure S16), calculated with ImageJ software, was consistent with the above results. Together, these results verify that the aptamer/AZO-based nanoplatform can be used to reversibly manipulate the activity of target proteins. Quantitative Activation of the Target Protein Although challenging, quantitative activation of specific proteins, especially at the single-molecule level, is essential for understanding their functional behavior. To demonstrate whether this nanoplatform has the potential to precisely activate target proteins, UV-pretreated TBA-AZO4-AuNPs were first used to capture and inactivate thrombin, and then different visible-light irradiations were performed during the
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Figure 4. Kinetic Fluorescence Imaging of the Reaction Process Catalyzed by Thrombin under Different Conditions (A) Thrombin only. (B) Thrombin treated with pretreated (UV 10 min) TBA-AZO4 -AuNPs. (C) Thrombin treated with pretreated (UV 10 min / visible 5 min) TBA-AZO 4 -AuNPs. (D) Thrombin treated with pretreated (UV 10 min / visible 5 min / UV 10 min) TBA-AZO4 -AuNPs. All 3D projections of z stack images were obtained at the time point of 66 min.
catalysis reaction. As shown in Figures 5A and S17, the scattered light intensity rose abruptly after each exposure to visible light. The initial catalysis reaction rate (V0) was calculated according to the slope of the kinetic curve (Figure 5B). A sharp increase in V0 was induced by each pulse of light, indicating sudden release and activation of thrombin. Moreover, V0 was proportional to visible-light exposure time. To further investigate the quantitative relationship between thrombin activation and visible-light irradiation, TBA-AZO4-AuNP-pretreated thrombin was irradiated with visible light for different times, and the thrombin released was collected and mixed with fibrinogen. The catalytic reaction was monitored by reading the kinetic absorption spectrum (at 450 nm) with a 96-well plate reader, which can measure multiple samples at one time (Figure S18). The kinetic absorption spectrum of thrombin at different concentrations was also collected, and a standard curve was prepared by plotting V0 versus the given thrombin concentration (Figure S19). As shown in Figures 5C and S20, a good linear relationship between amount of activated thrombin and visible irradiation time was obtained, ranging from 0 to 240 s. Meanwhile, 86.42% G 0.40% of the captured thrombin could be released with 240-s visible irradiation, indicating high efficiency of protein activation. Moreover, on the basis of these calculations, 20 thrombin molecules were initially captured per TBA-AZO4AuNP, and activation of one thrombin molecule could be achieved by visible irradiation of 14.4 s (Figure 5D), indicating the strong potency of this aptamer/AZO-based nanoplatform for quantitative protein regulation at the single-molecule level. Moreover, protein detection methods with higher sensitivity and proper light devices could potentially provide the current system with the ability to activate one protein at a time, thus providing a powerful tool for studying elusive biological processes.
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Figure 5. Quantitative Photoregulation of Thrombin Activity (A) Kinetic light-scattering spectra of the samples catalyzed with thrombin only (control), thrombin treated with UV-pretreated TBA-AZO4 -AuNPs (a), and thrombin treated with UV-pretreated TBA-AZO4 -AuNPs with visible-light irradiation applied at three kinetic catalysis reaction points (100, 200, and 300 s) (b and c). (B) Corresponding initial catalysis reaction rates (V 0 ) from (A). (C) The concentration and ratio of activated thrombin under different visible treatments. (D) Relationship between the molecular number of visible-activated thrombin per AuNP and visible illumination time. All data in (C) and (D) were collected from three independent experiments. Error bars represent standard deviations.
Photoregulation of Blood Clotting in Human Plasma After demonstrating the excellent capability of this aptamer/AZO-based nanoplatform for precisely regulating thrombin activity in buffer solution, we next tested its performance in human plasma by measuring thrombin clotting time (TCT). As shown in Figure 6A, TCT induced by thrombin only is 17.1 s, which is within the normal test range (14.0–21.0 s) provided by the manufacturer. After treatment with the UV-pretreated TBA-AZO4-AuNPs, however, the TCT value showed a dose-dependent increment and even reached 83.2 s (at 10 nM), which is 3.9 times longer than that induced by thrombin only, indicating that UV-pretreated TBA-AZO4-AuNPs can efficiently inhibit the coagulation process of human plasma samples. Next, we examined whether the current nanoplatform could be used to reversibly regulate the coagulation of human plasma samples. As shown in Figure 6B, by alternating irradiation with UV (10 min) and visible light (5 min), the TCT value of the plasma samples was switched accordingly. In addition, the attenuation of photo-driven conversion efficiency was negligible, even after nine cycles of alternating treatment, demonstrating the excellent performance of this nanoplatform for reversibly regulating the activity of target proteins in complex physiological fluids. Photoregulation of Protein Activity in Complex Living Systems To further evaluate the potential of this aptamer/AZO-based nanoplatform for regulating protein activity in more complex living systems, live Bacillus subtilis, a wellstudied Gram-positive bacterium that exists in the gastrointestinal tract of ruminants and humans, was used as a model. The cell wall of B. subtilis can be digested by
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Figure 6. Photoregulation of Blood Clotting Time of Human Plasma (A) Linear relationship between TCT and the concentration of UV-pretreated (10 min) TBA-AZO4-AuNPs. (B) Reversible regulation of TCT by TBA-AZO 4 -AuNPs in response to alternating UV- and visiblelight irradiation. All data were collected from three independent experiments. Error bars represent standard deviations.
active lysozyme, leading to cell death. To demonstrate lysozyme activity on living B. subtilis, a LIVE/DEAD BacLight Bacterial Viability Kit containing SYTO 9 (green fluorescence) and propidium iodide (PI, red fluorescence) was applied. Generally, SYTO 9 can stain both live and dead B. subtilis, whereas PI can only penetrate the damaged membrane of dead B. subtilis and then replace intracellular SYTO 9, causing a reduction in the SYTO 9 stain fluorescence. With treatment of SYTO 9/PI dyes, live B. subtilis showed a strong green fluorescence even after incubation for 70 min (Figure S21A). In contrast, with the addition of active lysozyme, most B. subtilis displayed red fluorescence after a 20-min incubation (Figure S21C), indicating the feasibility of this SYTO 9/PI strategy for analyzing the viability of B. subtilis and the activity of lysozyme. Meanwhile, rDNA40-AuNP- and LA-AuNP-treated active lysozyme samples (Figures S21D and S21E) showed red and green fluorescence, respectively, further demonstrating that the proposed mechanism to inactivate protein worked in this system. Large protein-AuNP aggregation architectures (as indicated by yellow arrows in the bright channel) were observed only in the LA-AuNPs-treated lysozyme sample (Figures S21D and S21E), consistent with the AFM results (Figures S4B and S4C) and indicating aptamer-specific lysozyme aggregation around the AuNP core. To exert photoregulation on lysozyme activity, the 50 end of LA was extended with a cDNA sequence incorporated with five AZO molecules (Table S1). The thiolated probe was modified onto the AuNP surface and the resultant product was termed LA-AZO5-AuNP. The live SYTO9/PI-stained B. subtilis were mixed with LA-AZO5AuNP-pretreated lysozymes and then irradiated with visible light for different times (Figure 7A). As shown in Figure 7B, the red fluorescence signal (as indicated with yellow arrows) gradually increased, and the green fluorescence decreased when extending the visible irradiation time, indicating visible-light-induced activation of lysozyme. The death rate of B. subtilis was increased by extending the visible irradiation time (Figure S22), corresponding well with the red-to-green fluorescence ratio calculated with ImageJ software. Specifically, with visible irradiation for 5 min, the percentage of dead B. subtilis reached 38.09% G 9.27% (Figure 7C). However, with no visible-light treatment, only 3.69% G 1.12% dead B. subtilis was observed. Of note, the visible-light irradiation itself had no negative influence on the viability of B. subtilis (Figure S21B). All these results demonstrate that our aptamer/AZO-based nanoplatform can be used to precisely photoregulate target protein activity in complex biological systems.
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Figure 7. Photoregulation of Lysozyme Activity in a Living B. subtilis System (A) Schematic illustration of photoregulation of lysozyme activity in a living B. subtilis system. (B) Fluorescence imaging of living B. subtilis treated with LA-AZO 5 -AuNP-captured lysozymes irradiated by visible light for 0 min (a), 1 min (b), 2 min (c), and 5 min (d). The concentration of lysozyme and LA-AZO 5 -AuNPs was 1,200 U/(mg$mL) and 10 nM, respectively. All the images were obtained 20 min after visible-light irradiation. Scale bar represents 10 mm. (C) Relationship between the percentage of dead B. subtilis and visible illumination time. All data were collected from three independent experiments. Error bars represent standard deviations.
DISCUSSION In summary, we have developed an aptamer/AZO-based nanoplatform for quantitative and reversible regulation of target protein activity. Unlike conventional photoregulation strategies, no modification of proteins is required in our design. By using thrombin and its TBA aptamer as demonstration models, we proved that TBA/AZO-functionalized AuNPs can reversibly regulate thrombin activity in both buffer solution and human plasma, indicating a promising strategy for intelligent anticoagulation therapy. Of note, this protein inactivation strategy was based on aptamer-specific protein capture around the AuNP core without a need for involving special aptamers that themselves are capable of protein inhibition. Because it is much more common and easier to select aptamers that can bind only with their target proteins but with no interference on the protein activity, this design is expected to provide a more universal strategy for protein regulation. Meanwhile, modulating visible-light irradiation achieved quantitative activation of thrombin, providing a potent tool for the precise and dynamic regulation of target protein, which is essential for studying the myriad of protein-dominant biological processes. With a proper light source and highly sensitive detection technique, this photoresponsive nanoplatform is expected to enable activation of one protein molecule at a time. Moreover, another protein (lysozyme)/ aptamer-AuNP system was successfully constructed and applied to a complex living B. subtilis system, revealing that this aptamer-based nanoplatform can be potentially expanded for studying and manipulating different proteins in more complex biological systems.
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EXPERIMENTAL PROCEDURES Materials and Reagents DNA synthesis reagents were purchased from Glen Research (Sterling, VA). KCl, CaCl2, KH2PO4, K2HPO4$3H2O, sucrose, and mercaptoethanol were purchased from Sinopharm Chemical Reagent Co. (Shanghai, China). NaCl, MgCl2$6H2O, human a-thrombin, fibrinogen from human plasma, and lysozyme were obtained from Sigma-Aldrich (Shanghai, China). Sodium citrate was purchased from Tianjin Fengchuan Chemical Reagent Science and Technology (Tianjin, China). Tween 20 was obtained from Beyotime Institute of Biotechnology (China). PBS 103 (pH 7.4) and Alexa-Fluor-546-labeled fibrinogen were purchased from Life Technologies. Chloroauric acid, tris-(2-carboxyethyl)-phosphine hydrochloride (TCEP), and 1 M Tris-buffer sterile solution (pH 7.4) were purchased from Sangon Biotech (Shanghai, China). B. subtilis was obtained from the China General Microbiological Culture Collection Center (Beijing, China). Milli-Q water (resistance >18 MU cm) was used to prepare all solutions. DNA Synthesis All DNA sequences (Table S1) were synthesized on a PolyGen DNA synthesizer. Synthesis and deprotection were performed according to the instructions provided by the manufacturers of the reagents. Deprotected DNA was then precipitated by adding 40 mL (1/10 volume) of 3 M NaCl and 1 mL (2.53 volume) of cold ethanol. The mixtures were placed in a freezer at 20 C for 30 min and then centrifuged at 4,000 rpm at 4 C for 30 min. After removing the supernatants, the DNA products were dissolved in 400 mL of 0.1 M triethylamine/acetate (TEAA) and purified by high-performance liquid chromatography (Agilent 1260) with a C18 column (5 mm, 4.6 3 250 mm, 100 A˚; GL Science, Inertsil ODS-3). The purified DNA was dried, detritylated by dissolving in 80% acetic acid (200 mL) for 20 min, and then precipitated with 20 mL of 3 M NaCl and 500 mL of cold ethanol. The mixtures were chilled at 20 C for another 30 min, collected through centrifugation at 14,000 rpm at 4 C for 5 min, and then resolved with ultrapure water. The final DNA products were desalted with illustra NAP-5 columns (GE Healthcare), and their concentration was determined by detecting the UV absorption at 260 nm on a BioSpec-nano (Shimadzu). Synthesis of AuNPs AuNPs with an average diameter of 13 nm were synthesized by reduction of HAuCl4 with sodium citrate.31 Chloroauric acid solution (0.01 wt %, 100 mL) was added to a clean round-bottom flask and heated with stirring and refluxing until boiling. Then, sodium citrate solution (3 wt %, 1 mL) was added rapidly to the solution, which was heated with continuous stirring for another 30 min. The solution was then cooled to room temperature and stored in the dark for subsequent experiments. The size of AuNPs (Figure S1) was characterized with a JEM 2100 transmission electron microscope (Hitachi). A UV-2450 spectrophotometer (Shimadzu) was used to measure the absorption of the AuNPs (Figure S2A). The hydrodynamic diameter of AuNPs (Figure S2B) was measured with a Zetasizer Nano ZS90 DLS system (Malvern Instruments, Worcestershire, UK). The concentration of AuNPs was determined by UV-visible absorbance via Beer’s law (A = εbc). Aptamer Modification of AuNPs Thiolated DNAs were attached to AuNPs as described in previous literature.32,33 First, the disulfide bond of thiolated DNAs was cleaved by treatment with TCEP (503) at pH 5.0 for 1 hr, and then the DNAs were desalted with illustra NAP-5 columns. TBA-AZO0, TBA-AZO3, TBA-AZO4, TBA-AZO5, and LA-AZO5 were dissolved
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in 13 PBS, heated at 95 C for 5 min, and then immediately cooled to 4 C for 2 min to form a hairpin structure. Other DNA sequences (Table S1) were dissolved directly with ultrapure water with no heating/cooling treatment. Then, 500 mL of 13-nm AuNP solution (2.5 nM), 5 mL of Tween 20 (1% v/v), 10 mL of thiolated DNA (100 mM) and 10 mL of citrate-HCl buffer (pH 7.0, 0.5 M) were mixed in a centrifuge tube. After that, NaCl solution (3 M) was slowly added to the mixture to reach a final concentration of 0.1 M. After salt aging at room temperature for 6 hr, the mixtures were centrifuged at 16,200 3 g at 4 C for 15 min. After removal of the supernatants, the precipitate was washed with 5 mM Tris-HCl buffer (containing 0.01% v/v Tween 20, pH 7.4) three times via centrifugation. The resultant DNA-AuNPs were resuspended in PBT solution (25 mM Tris-HCl [pH 7.4], 0.01% v/v Tween 20, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, and 1 mM CaCl2) and stored at room temperature in the dark for subsequent experiments. A UV-2450 spectrophotometer (Shimadzu) was used to measure the absorption of the DNA-AuNPs (Figure S2A). The hydrodynamic diameter of DNA-AuNPs (Figure S2B) was measured with a Zetasizer Nano ZS90 DLS system (Malvern Instruments, Worcestershire, UK). Fluorescence Measurements UV light at 365 nm was provided by a Spectroline Model SB-100P/FA UV lamp (100 W), and visible light at 450 nm was provided by a 250 W high-pressure mercury lamp (Shanghai Jiguang) with a 450 nm glass filter (Shenzhen Zhonglai Technology). The distance between the samples and the UV-visible light source was fixed at 5 cm. To evaluate the light-driven conformational switch of the TBA/AZO probes, FAM fluorophores were modified on one of their termini. Generally, the TBA-AZOn-AuNP samples (200 mL, 2.5 nM) were treated with UV or visible light for a certain length of time, and the fluorescence spectra were recorded with a Fluoromax-4 spectrofluorometer (HORIBA JobinYvon, Edison, NJ). The fluorescence peak intensity at 520 nm was used to analyze the performance of this system. The fluorescence intensity of equivalent AuNPs and FAM-TBA-AuNPs was used as the baseline and the internal standard, respectively. All fluorescence data were calculated by subtracting the baseline and normalized according to the internal standard. Real-Time Monitoring of Thrombin Catalytic Reaction Active thrombin can convert fibrinogen into insoluble fibrin, leading to an increase in scattering light intensity (650 nm) and the light absorbance value at about 450 nm. In brief, the samples were pretreated with or without UV-visible light, as described above, and mixed with thrombin for 10 min. The mixtures were added to the fibrinogen solution, which was dissolved in physiological buffer (25 mM Tris-HCl [pH 7.4] containing 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, and 1 mM CaCl2). The thrombin catalytic reaction was monitored by measuring the intensity change of the scattered light with a Fluoromax-4 spectrofluorometer (HORIBA JobinYvon, Edison, NJ). The final concentrations of thrombin and fibrinogen in the mixtures (V = 200 mL) were 2.5 and 1.14 nM, respectively. The excitation and emission wavelengths were both set at 650 nm with a bandwidth of 5 nm for excitation and emission. For calibration of systematic error, the catalytic reaction induced by thrombin only was always performed as an internal standard in each independent experiment. In some experiments, to guarantee that most TBAs would be liberated to capture thrombin, the functional AuNPs were pre-irradiated with UV light for 10 min. Meanwhile, the pretreated AuNPs were mixed and incubated with thrombin (2.5 nM) for 10 min before the fibrinogen substrate was added to initiate the catalytic reaction.
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Real-Time Monitoring of Lysozyme Activity Lysozyme is an enzyme that can catalyze the hydrolysis of Micrococcus lysodeikticus, resulting in decreased absorbance at 450 nm (A450). In the detection assay, Micrococcus lysodeikticus (Huich Bio-tech) was suspended in the reaction buffer (66 mM potassium phosphate [pH 6.2]), and its working concentration was adjusted according to the A450 value of 0.6–0.8. Initially, a certain concentration of free lysozyme—or lysozyme pretreated with LA, AuNPs only, LA-AuNPs, or rDNA40-AuNPs—was added into the Micrococcus lysodeikticus solution, and the kinetic absorbance spectra were immediately recorded with a UV-2450 spectrophotometer (Shimadzu). The concentration of LA and AuNPs was 400 and 0.3 nM, respectively. AFM Assay For AFM assay, 10 mL of the prepared sample was dropped onto a mica surface. After 5 min at room temperature, the mica was washed with 200 mL of Milli-Q water four times and dried under nitrogen. All samples were imaged on a Multimode 8 (Bruker, USA) with ScanAsyst mode at 0.977 Hz scan rate. In Situ Imaging of the Thrombin Catalytic Reaction For the fluorescence imaging assay, thrombin (2.5 nM) was treated with different TBA-AZO4-AuNP samples. Then, Alexa-Fluor-546-labeled fibrinogen (1.14 mM) was added. Subsequently, 30 mL of the mixtures was rapidly transferred to a 35-mm Petri dish and immediately imaged with an inverted microscope (Nikon Ti-E, Nikon, Japan) at different reaction time points. The excitation wavelength was 561 nm. The z stack images of the mixtures were scanned at 66 min. The images were recorded with a 203 dry objective. Quantitative Activation of Thrombin In this experiment, we changed to a 96-well reader, which can read multiple samples at a time, to test the activity of thrombin by measuring the kinetic absorbance at 450 nm. To set up a standard curve, pure thrombin at different concentrations was mixed with fibrinogen (1.14 mM), and the kinetic absorption spectra were measured immediately with a Synergy 2 multimode reader (BioTek, USA). The initial catalytic reaction rate (V0) was calculated and plotted versus the thrombin concentration. To guarantee that most TBAs would be liberated to capture thrombin in experiments of quantitative thrombin activation, TBA-AZO4-AuNPs were pre-irradiated with UV light for 10 min. Then, the pretreated AuNPs were mixed and incubated with thrombin (2.5 nM) for 10 min. The mixtures were irradiated by visible light for different lengths of time and then centrifuged at 16,200 3 g at 4 C for 15 min. The supernatants were collected and mixed with the fibrinogen substrate, and the kinetic absorbance spectra (at 450 nm) were recorded immediately. The active amount of thrombin was calculated according to the standard curve. Human Plasma Tests We studied the ability of the aptamer/AZO-based nanoplatform to reversibly regulate thrombin activity in the process of blood clotting of human plasma. To accomplish this, healthy human blood samples were collected with the permission of Xiangya Hospital and processed according to a standard procedure to extract plasma for subsequent analysis. Thrombin with different treatments was added to the human plasma, and the TCTs of samples were measured with an Automated Coagulation Blood Analyzer CS-2000i (Sysmex Shanghai, Shanghai, China).
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Imaging of Live and Dead B. subtilis B. subtilis was cultured with liquid nutrient medium at 37 C. After growing for 24 hr, living B. subtilis was stained with the LIVE/DEAD BacLight Bacterial Viability Kits (Invitrogen) according to the instructions provided by the manufacturer. To test the capability of LA-AZO5-AuNPs to photoregulate the lysozyme activity in the living B. subtilis system, LA-AZO5-AuNPs were pre-irradiated with UV light for 10 min and then mixed with active lysozyme for 10 min. The mixture was incubated with living B. subtilis. The resultant B. subtilis samples were irradiated with visible light for different times and subsequently imaged with an FV1000 confocal microscope (Olympus). The images were recorded with a 603 oil objective.
SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, 22 figures, and 1 table and can be found with this article online at https://doi.org/10. 1016/j.chempr.2017.11.008.
ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China grants 21505039, 2013CB932702, 21405041, 21221003, and 21327009. It was also supported by NIH grants GM079359 and CA133086. We sincerely thank Prof. Chaoyong Yang’s lab at Xiamen University for helping with DNA synthesis.
AUTHOR CONTRIBUTIONS Conceptualization, S.X., L.Q., W.T.; Methodology, L.C., Honglin Liu, Y.S., H. Liang, D.D., J.Z.; Investigation, Huixia Liu; Resources, Huixia Liu, Z.C., and X.Z.; Supervision and Funding Acquisition, L.Q. and W.T. Received: May 14, 2017 Revised: September 19, 2017 Accepted: November 21, 2017 Published: December 14, 2017
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