Article pubs.acs.org/IECR
Multienzyme Immobilization and Colocalization on Nanoparticles Enabled by DNA Hybridization Feng Jia, Surya K. Mallapragada,* and Balaji Narasimhan* Department of Chemical and Biological Engineering, Iowa State University, Ames, Iowa 50011, United States ABSTRACT: Multienzyme complexes (MECs) in nature exhibit highly efficient catalytic mechanisms in reaction cascades. Different strategies have been developed to colocalize enzymes on nanocarriers to improve multienzyme catalytic efficiency by mimicking MEC structure and function. Numerous studies have indicated that the spatial arrangement and orientation of multiple enzymes in confined spaces are critical in facilitating cooperative enzymatic activity in multienzyme colocalization. Biomolecule scaffolds based on DNA hybridization have attracted great attention because of their unique effective control of the relative positions of different enzymes in multienzyme colocalization. To demonstrate this concept, glucose oxidase (GOX) and horseradish peroxidase (HRP) were colocalized onto polystyrene nanoparticles via specific DNA hybridization. Colocalization of GOX and HRP was evidenced by Förster resonance energy transfer studies of the dyes labeling the two tag DNAs. Finally, it was observed that colocalization of GOX and HRP via DNA hybridization significantly improved both the overall reaction efficiency and the storage shelf life compared with those of the single enzyme immobilization mixture control. In summary, DNA-directed colocalization of multiple enzymes on nanoparticles is an effective way to control the relative positioning of enzymes to mimic MECs and enhance catalytic activity.
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INTRODUCTION Enzymes, which are nature’s catalysts, are involved in many reactions that take place in living organisms and have evolved to catalyze various chemical reactions, including multistep reactions.1−3 Multienzyme complexes (MECs) are composed of multiple enzyme subunits or large polypeptides with defined tertiary and quaternary structures containing compact multiple catalytic centers that are in close proximity to each other.4,5 When enzymatic catalytic active sites are brought together, reaction intermediates can be transported rapidly among the active sites via a “substrate channeling” effect, which can reduce diffusion losses typically observed in free-enzyme catalytic processes, enabling the maintenance of high local concentrations of intermediates, which is especially critical for unstable intermediates.6,7 In addition, MECs are known to significantly increase the overall reaction turnover efficiency.8 These benefits of MECs have inspired researchers to design artificial MECs to mimic the structure and functionalities of MECs. A number of methodologies have been proposed to design artificial MECs, as reported in several recent studies and reviews.9−15 In this context, enzyme-immobilization-based strategies exhibit great potential because of their economical reusability, enhanced kinetic performance, and higher stability under harsh operating conditions (e.g., extreme pH and temperature).16,17 In particular, nanoparticles have attracted much attention because of their large surface areas for immobilization and no internal diffusion resistance, in contrast to porous materials. More specifically, polymeric nanoparticles are relatively inexpensive and provide flexibility in terms of material selection and architecture design compared with inorganic materials. The solution behavior of nanoparticles provides increased mobility of biocatalysts in solution, which could potentially enhance the catalytic performance.18 It is widely recognized that the spatial orientation of multiple enzymes plays a significant role in mimicking MECs found in nature.6 For example, it has been © XXXX American Chemical Society
demonstrated that enzymes colocalized in the same layers on nanoparticles have enhanced kinetic performance compared with those in separate layers.16,17 We have designed several strategies to synthesize biomimetic artificial MECs by colocalizing multiple enzymes on multifunctional nanoparticles11 or Pluronic quantum dot (QD) micelles.12 In both cases, the activity of each individual enzyme was retained in single-component immobilization and the colocalized enzymes exhibited superior catalytic performance compared with free enzymes in catalyzing cascade reactions.11 DNA-conjugated nanoparticles have shown great potential in biotechnology.19 More recently, researchers have used DNAbased biomolecular scaffolds to direct the colocalization of multiple enzymes in cascade reactions.20−24 The hybridization of complementary DNA provides precise control over the spatial arrangement of the enzymes, in contrast to other colocalization strategies.25,26 The relative positions of multiple enzymes can be controlled by hybridization of the tag DNA with the complementary capture DNA as well as by varying the DNA sequence length. DNA-directed colocalization has also been shown to provide exceptionally high stability for DNA−enzyme conjugates by preserving the enzymatic activity.27 Niemeyer et al. reported multienzyme colocalization using DNA hybridization catalyzed cascaded reactions on planar surfaces, resulting in enhanced enzymatic activity.20 Likewise, Müller and Niemeyer employed protein engineering combined with DNA-hybridization-directed evolution to develop supramolecular complexes using DNA as a scaffold for enzyme assembly.21 They attached Special Issue: Doraiswami Ramkrishna Festschrift Received: April 15, 2015 Revised: July 11, 2015 Accepted: August 13, 2015
A
DOI: 10.1021/acs.iecr.5b01423 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX
Article
Industrial & Engineering Chemistry Research
Figure 1. Schematic approach for colocalization of SHRP and AGOX on PS nanoparticles by hybridization of short tag DNA and respective segments on a long carrier DNA chain. The figure is drawn approximately to scale.
multiple enzymes by covalent binding to short single-stranded biotinylated and thiolated DNA oligonucleotides and brought them together using DNA-directed hybridization of short DNA with long complementary capture DNA to form a scaffold. The activity of the multienzyme complex was significantly enhanced compared with that of enzymes immobilized on separated strands, and it was demonstrated that the efficiency of the selfassembly significantly depended on the position and the steric factor of the DNA−enzyme conjugates.21 In this work, DNA hybridization was coupled with the use of nanoparticles with the hypothesis that spherical nanoscale particles can be used to efficiently colocalize multiple enzymes, leading to enhanced catalytic performance.20,21 The goal was to develop a biomimetic strategy to colocalize multiple enzymes on nanoparticles using DNA-directed hybridization, as shown in Figure 1. Specifically, the colocalization of two tag DNAs as well as tag DNA−enzyme conjugates was demonstrated. Next, singleenzyme immobilization by DNA hybridization was optimized. Finally, multienzyme colocalization was demonstrated with various configurations, and significant enhancements in catalytic performance were observed.
Table 1. DNA Sequences Used in This Work abbreviation A B c(AB) c(BA) c(AA) c(BB)
function
sequences
tag DNA 5′-biotin-GG TCC GGT CAT AAA GCG ATA AG-3′ tag DNA 5′-biotin-GT GGA AAG TGG CAA TCG TGA AG-3′ carrier 5′-biotin-CT TAT CGC TTT ATG ACC GGA DNA CCCT TCA CGA TTG CCA CTT TCC AC-3′ carrier 5′-biotin-CT TCA CGA TTG CCA CTT TCC DNA ACCT TAT CGC TTT ATG ACC GGA CC-3′ carrier 5′-biotin-CT TAT CGC TTT ATG ACC GGA DNA CCCT TAT CGC TTT ATG ACC GGA CC-3′ carrier 5′-biotin-CT TCA CGA TTG CCA CTT TCC DNA ACCT TCA CGA TTG CCA CTT TCC AC-3′
with 500 μL of 0.5 mg/mL SHRP or 120 μL of 5 mg/mL AGOX solution, and the mixture was incubated for 1 h at room temperature. The unbound tag DNAs were removed by centrifugation using a 30 kDa molecular weight cutoff membrane (Amicon Bioseparations, Millipore). To attach the carrier DNA onto PS-STV nanoparticles, 150 μL of 1% PS-STV nanoparticle suspension was mixed with an appropriate amount of 0.1 nmol/ mL carrier DNA solution using different molar ratios (the biotin binding capability (in nmol/mg of nanoparticles) was provided by the manufacturer on the basis of biotin−FITC binding efficiency). Specifically, the PS-STV particles were mixed with the carrier DNA solution and incubated for 1 h using a rotoshaker at room temperature. Carrier DNA labeled with Alexa Fluor 594 dye was used to investigate the effect of reaction time. After the reaction, the mixture was microcentrifuged at 16000g for 6 min to separate the nanoparticles from the original supernatants, and the particles at the bottom were resuspended in Tris buffer. The sample was dispersed using sonication for 10 s and separated again by microcentrifugation for 6 min at 16000g. The supernatant was collected, and the wash step was repeated until no intensity was detected. The concentration of DNA in the supernatant was quantified by its fluorescence intensity. Colocalization of DNA Hybridization. To evaluate how the two short tag DNA strands colocalized by hybridization with the corresponding segments on the long carrier DNA strand, two fluorescent dyes, Alexa Fluor 594 and 647, were conjugated to the tag DNA to afford Alexa Fluor 594-conjugated B (AF594-B) and Alexa Fluor 647-conjugated A (AF647-A). The dye−DNA conjugates were synthesized by Integrated DNA Technologies.
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MATERIALS AND METHODS Chemicals. Streptavidin-conjugated polystyrene (PS-STV) nanoparticles (300−390 nm) were purchased from Spherotech (Lake Forest, IL). Streptavidin-coated microplates were purchased from Pierce (Rockford, IL). Streptavidin-conjugated horseradish peroxidase (SHRP) and avidin-conjugated glucose oxidase (AGOX) were purchased from BioLegend (San Diego, CA) and Vector Lab (Burlingame, CA), respectively. Amplex Red was purchased from Invitrogen (Carlsbad, CA). Hydrogen peroxide, sodium chloride, and trisodium phosphate were purchased from Fisher Scientific (Hampton, NH). The tag DNA oligonucleotides (defined as A and B) and the carrier DNA oligonucleotides (defined as c(AB), c(BA), c(AA), and c(BB))21 were synthesized by Integrated DNA Technologies (Coralville, IA), and the corresponding sequences are shown in Table 1. All of the aqueous solutions were prepared using purified water from a Thermo Scientific Barnstead Nanopure ultrapure water system. Attachment of Biotinylated DNA to SHRP, AGOX, and PS-STV Nanoparticles. To attach tag DNA A or B to SHRP or AGOX to afford A-SHRP, B-SHRP, A-AGOX, or B-AGOX, respectively, 27 μL of 0.1 nmol/mL DNA solution was mixed B
DOI: 10.1021/acs.iecr.5b01423 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX
Article
Industrial & Engineering Chemistry Research
Enzyme Kinetics Assay. For all of the samples and controls, 90 μL of substrate solution containing hydrogen peroxide and Amplex Red was loaded into 96-well plates. For the colocalized bienzyme samples, 10 μL of enzyme−particle solution was added to the substrate solution. For the mixture control sample, 5 μL of each immobilized single enzyme was added to the corresponding substrate solution. For all of the samples, in the final 100 μL of solution volume, the concentrations of H2O2, Amplex Red, and PS-STV were 10 μM, 55 μM, and 5 μg/mL, respectively. The reaction was initiated by addition of the enzyme samples and immediately monitored using a fluorescent microplate reader (SynergyMx, Biotek, Winooski, VT).
The dye-conjugated tag DNA was hybridized with c(AB)conjugated PS-STV nanoparticles. Specifically, in a reaction tube, 6 μL of 0.1 nmol/μL AF594-B was first mixed with 100 μL of 1.4 nmol/mg carrier DNA−PS-STV, and 6 μL of 0.1 nmol/μL AF647-A was added. The emission fluorescence from 604 to 750 nm was monitored as a function of time after excitation at 594 nm. A Tris buffer control was used. Colocalization of DNA−Enzyme Conjugates. To investigate the colocalization of the DNA−enzyme conjugates on the long carrier DNA chain, the tag DNA was conjugated with fluorescent dyes that can be paired to enable Förster resonance energy transfer (FRET). The conjugated DNAs AF594-B and AF647-A were attached to AGOX and SHRP, respectively, as described above, to obtain AF594-B−AGOX and AF647-A− SHRP. Capture DNA was conjugated with PS-STV as described above. Specifically, 40 μL of AF594-B−AGOX was first mixed with 50 μL of 1.4 nmol/mg carrier DNA conjugated with PSSTV, and 160 μL of AF647-A−SHRP was added. The emission fluorescence from 604 to 750 nm was monitored as a function of time after excitation at 594 nm. A Tris buffer control was used. Immobilization of SHRP and AGOX on PS-STV Nanoparticles via DNA Hybridization. SHRP was immobilized onto the PS-STV nanoparticles via hybridization of the short tag DNA and the long carrier DNA strands. Each sample containing 50 μL of 10 mg/mL nanoparticles with 1.4 nmol of carrier DNA per mg of PS-STV in Tris buffer was mixed with the appropriate volume of A-SHRP, B-SHRP, or both to obtain a 1:1 reaction ratio of tag DNA and carrier DNA. The samples were incubated for 3 h at room temperature. After the reaction, each sample was separated by microcentrifugation for 6 min at 16000g. The supernatants were removed, and this step was repeated until no protein was detected in the supernatant. Similarly, AGOX was immobilized by hybridization of the short tag DNA and the long carrier DNA strands. Colocalization of SHRP and AGOX on PS-STV Nanoparticles via DNA Hybridization. Prior to colocalization of SHRP and AGOX on the PS-STV nanoparticles, SHRP-tag DNA, AGOX-DNA, and carrier DNA-conjugated nanoparticles were prepared as described above. The two enzymes were colocalized on the nanoparticles by mixing the enzyme−DNA conjugate and the DNA−PS-STV conjugate simultaneously. In a single sample, 50 μL of c(AB)- or c(BA)-conjugated PS-STV was mixed with the appropriate volume of A- or B-conjugated SHRP or AGOX, and the mixture was incubated for 3 h. The nonconjugated enzyme/DNA mixtures were removed by microcentrifugation, and the process was repeated until no enzyme/DNA was detected in the supernatant. As controls, either AGOX or SHRP conjugates were mixed with c(AA) or c(BB) to result in single-enzyme-immobilized PS-STV nanoparticles. SHRP and AGOX Colocalization on StreptavidinCoated Microplates via DNA Hybridization. Each well in the microplate was washed thrice with washing buffer, and 200 μL of Tris buffer with an additional 20, 5, or 1.7 μL of 1 pmol carrier DNA solution was added to the respective well and incubated rotationally at room temperature for 1 h. The carrier DNA binding solution was discarded, and the wells were washed again three times using the washing buffer. Then 2-fold molar quantities of both types of enzyme−DNA conjugates were added to each well and allowed to colocalize for 3 h at room temperature. The unbound enzyme−DNA conjugates were removed, and each well was again washed three times with the washing buffer.
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RESULTS AND DISCUSSION Attachment of Carrier DNA on PS-STV Nanoparticles. The loading capacity of the carrier DNA on the PS-STV nanoparticles was investigated by varying the amount of carrier DNA (from 1 to 3 nmol) conjugated with fluorescent dye to 1.5 mg of PS-STV nanoparticles. The loading efficiency was estimated by subtracting the DNA in the collected supernatant from the initial amount. The DNA loading capacities and efficiencies are shown in Table 2. It was found that the maximal Table 2. Carrier DNA Density Parameters DNA quantity (nmol)
PS-STV (mg)
loading efficiency (%)
loading capacity (nmol/mg)
1 2 3
1.5 1.5 1.5
70.3 ± 2.9 62.0 ± 4.3 40.6 ± 0.4
0.47 0.83 0.81
DNA density obtained was 0.81 nmol per mg of nanoparticles using 2−3 nmol of DNA in the reaction. Further increases in the amount of carrier DNA did not improve the efficiency (data not shown). Therefore, in order to maximize the enzyme loading efficiency and reduce nonspecific adsorption of the enzyme− DNA conjugates on the PS-STV surface, 3 nmol of carrier DNA and 1.5 mg of PS-STV were used in all of the following experiments. Confirmation of DNA Hybridization. Prior to the use of DNA hybridization to direct enzyme immobilization, the DNA hybridization process and its kinetics were studied by FRET. Two different fluorescent dyes, AF594 and AF647, were respectively conjugated to the 5′ end of the tag DNA and the corresponding complementary segment on the 3′ end of the carrier DNA (Figure 2). When the tag DNA was paired with the carrier DNA, the excitation was transferred from the donor dye (AF594) to the acceptor fluorophore (AF647), as evidenced by the quenching of the donor. This observation is indicative of successful hybridization of the two DNA sequences. The further quenching of the donor dye and increase of the excited dye from different curves at various time points indicated that hybridization was completed within 1 h. In addition to hybridization of the free DNA, similar results were obtained for hybridization of the carrier DNA on the PS-STV nanoparticles (data not shown). This rapid hybridization process is also consistent with previous work on real-time monitoring of DNA hybridization kinetics.28 Colocalization of Tag DNA. To demonstrate the possibility of colocalization of two components by hybridization of two tag DNA fragments with the corresponding segments on the carrier DNA, FRET studies were carried out. Specially, the two fluorescent dyes AF594 and AF647 were conjugated to the 3′ ends of the two tag DNAs as illustrated in the cartoon in Figure C
DOI: 10.1021/acs.iecr.5b01423 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX
Article
Industrial & Engineering Chemistry Research
DNA c(BA), which was indicative of successful colocalization of the two DNAs on the carrier DNA via hybridization, as shown in Figure 2c. The colocalization was monitored approximately every minute and was rapidly completed (in
