Reversible Regulation of Enzyme Activity by pH-Responsive Encapsulation in DNA Nanocages Seong Ho Kim,†,∥ Kyoung-Ran Kim,† Dae-Ro Ahn,†,∥ Ji Eun Lee,† Eun Gyeong Yang,† and So Yeon Kim*,†,∥ †
Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul, Republic of Korea 02792 ∥ Division of Bio-Medical Science and Technology, KIST School, Korea University of Science and Technology (UST), Seoul, Republic of Korea 02792 S Supporting Information *
ABSTRACT: Reversible regulation of enzyme activity by chemical and physical stimuli is often achieved by incorporating stimuli-responsive domains in the enzyme of interest. However, this method is suitable for a limited number of enzymes with well-defined structural and conformational changes. In this study, we present a method to encapsulate enzymes in a DNA cage that could transform its conformation depending on the pH, allowing reversible control of the accessibility of the enzyme to the surrounding environment. This enabled us to regulate various properties of the enzyme, such as its resistance to protease-dependent degradation, binding affinity to the corresponding antibody, and most importantly, enzyme activity. Considering that the size and pH responsiveness of the DNA cage can be easily adjusted by the DNA length and sequence, our method provides a broadimpact platform for controlling enzyme functions without modifying the enzyme of interest. KEYWORDS: DNA tetrahedron, conformational change, enzyme encapsulation, reversible control, enzyme activity interest11 because the introduction of these domains may impair the enzyme activity or proper folding in some cases. A more universal way to control enzyme activity can be devised by caging an enzyme in a nanostructure to prevent or enhance its contact with interacting molecules.12−15 In this context, it has recently been reported that multiple enzymes required for a chain reaction could be precisely positioned in a DNA nanostructure, and the efficiency of the chain reaction could be enhanced compared with that of enzymes freely present in a solution.16 On the other hand, DNA nanostructures have been used to compartmentalize proteins in their interior spaces to inhibit the function of the protein and to restore the function of the protein by releasing it from the structure.17−19 However, these previous methods to regulate protein functions using cage-like DNA nanostructures are irreversible and thus have limitations in comparison with the reversible regulation observed in nature.
E
nzymes are essential molecules that mediate various biological functions in organisms. In nature, the functions of enzymes can be controlled by conformational changes caused by physical and chemical signals. For example, it is well-known that post-translational modifications that are basically chemical functionalization of the amino acid residues in an enzyme can alter its conformation and thereby regulate not only the enzyme activity but also the cell fate.1,2 In addition, it has also been reported that physical stimuli such as light can change the binding affinity between light-sensitive domains, resulting in modification of the enzyme function and signaling pathway.3−5 Moreover, a protein that is naturally irresponsive to a physicochemical signal can become responsive to a stimulus when an extra domain responsive to the stimulus is fused with the protein of interest.6,7 The DNA nanostructure has been utilized as an extra scaffold to adjust the distance between an enzyme and a cofactor (or inhibitor) to control the enzyme activity.8−10 To this end, it is necessary to precisely incorporate a limited range of stimuli-sensitive domains or extra scaffold DNA into the proper locations in the protein of © 2017 American Chemical Society
Received: July 7, 2017 Accepted: August 28, 2017 Published: August 28, 2017 9352
DOI: 10.1021/acsnano.7b04766 ACS Nano 2017, 11, 9352−9359
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Scheme 1. Schematic Presentation of the Reversible Regulation of RA Activity Based on pH-Sensitive DNA Td Structural Changes
Figure 1. Characterization of DNA Td. (A) Schematic presentation of i-motif formation. (B) The DNA Td assembly was verified by native PAGE analysis. To examine the efficiency of the Td assembly, a SYBR Gold stained image of the same gel was obtained (left). Cy3-S3 (acceptor, A channel, right) and S2-AX488 (donor, D channel, middle) oligonucleotides were used to visualize the assembled structure by their fluorescence. Lane 1: Cy3-S3; Lane 2: Cy3-S3 and S1; Lane 3: Cy-S3, S1, and S4; Lane 4: Cy3-S3, S1, S4, and S2-AX488. The fluorescence from the same gel was recorded with either acceptor excitation at 532 nm (right) or donor excitation at 488 nm (middle). Then, the same gel was further stained with SYBR Gold, and the fluorescence from SYBR Gold was obtained (left). (C) The normalized FRET ratio plotted as a function of pH. (D) The hydrodynamic size distribution of DNA Td in the open (acidic, pH 6.3) and closed (basic, pH 8.3) state.
20 and 21 for review). We chose the specific i-motif sequence (labeled as pink in the S3 strand, Scheme 1and Figure 1A; see Table S1 for detailed sequence) showing a fast response time with a pH-sensitive regime of 7.0−7.5.22 The self-assembly of Td constructed with the previously reported three strands (S1, S2, and S4),23 and one i-motif-embedded strand (S3) was verified by 5% native polyacrylamide gel electrophoresis (PAGE) analysis (Figure 1B). The bands visualized by Cy3 labeled at the 5′-end of S3 clearly demonstrated the strand-wise assembly as indicated by the retarded gel mobility of the assembled structures. Td was properly formed when all four strands were hybridized with one another, indicated by two fluorescent bands visualized by either the AlexaFluor (AX) 488 dye attached on the 3′-end of S2 or Cy3 at S3. Importantly, the SYBR Gold stained image of the same gel (Figure 1B) clearly showed that no other DNA oligomeric band was observed, suggesting that all the DNA strands were efficiently assembled into the DNA Td in our experimental conditions. To probe the structural transition of the DNA Td resulting from pH changes, we measured the
To gain reversible enzyme activity control in a universal nanostructure platform, we attempted here to covalently attach an enzyme inside of a DNA tetrahedron (Td) cage, whose conformation can be reversibly switched by pH changes (Scheme 1). The opening and closing of the DNA Td cage was verified by measuring the distance change between two vertexes. We demonstrated that the enzyme accessibility to the surrounding environment, the binding affinity to the specific antibody, and even the enzyme activity can be reversibly controlled via pH-dependent opening and closing of the DNA Td cage.
RESULTS AND DISCUSSION We first explored whether DNA oligonucleotides with a pHsensitive i-motif could properly assemble into the Td structure and whether the assembled DNA Td could show structural transitions in response to a pH change. The i-motif exists as a linear DNA strand at physiological pH and forms a more compact secondary structure based on the noncanonical C−C+ base-pairing upon exposure to an acidic pH (Figure 1A, see refs 9353
DOI: 10.1021/acsnano.7b04766 ACS Nano 2017, 11, 9352−9359
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Figure 2. Characterization of the RA attached DNA Td. (A) Schematic presentation of Td-IN-RA and Td-OUT-RA. Depending on the position of the RA attachment in the DNA double helix, RA is located either inside the Td (left) or outside the Td (right). (B) Native PAGE analysis of DNA Td and RA-attached DNA Td (Td-IN-RA and Td-OUT-RA). To examine the efficiency of the Td assembly, the SYBR Gold stained image of the same gel was included (left). Cy3-S3 oligonucleotides were used to visualize the assembled structure by their fluorescence (middle). Finally, the presence of RA protein was confirmed by Western blot analysis with RA Ab (right). Lane 1: Cy3-S3; Lane 2: Cy3-S3 and S1; Lane 3: Cy-S3, S1, and S4; Lane 4: Cy3-S3, S1, S4, and S2. Lane 5: S1-IN-RA, S2, Cy3-S3, and S4; Lane 6: S1-OUT-RA, S2, Cy3-S3, and S4. (C) The hydrodynamic size distribution of DNA Td and RA-attached DNA Td (Td-IN-RA and Td-OUT-RA).
fluorescence resonance energy-transfer (FRET) ratio between AX488 (donor) and Cy3 (acceptor) at varying pH values. Before any FRET measurements, we first confirmed that the fluorescence intensities of both AX488 and Cy3 were maintained regardless of the pH changes from 6.0 to 8.0 (Figure S1). In our case, the distance between the two fluorophores decreased upon the i-motif formation with the lowering pH, and therefore the FRET ratio would be expected to increase. When we measured the emission spectrum of the DNA Td with donor excitation at 488 nm, increased acceptor fluorescence by FRET (at 580 nm) was evident in the open state (acidic, pH 6.0) compared to the closed state (basic, pH 8.3, Figure S2). The pH-dependent changes in the relative FRET ratio calculated by the equation described in the Methods illustrated a sigmoidal decrease with a sensitive pH range from 6.4 to 7.3 (Figure 1C), reasonably consistent with the previous report.22 On the other hand, the hydrodynamic sizes of the DNA Td estimated by dynamic light scattering (DLS) measurements were 11.9 ± 0.8 nm when open (pH 6.4) and 12.0 ± 0.6 nm closed (pH 8.3), respectively (Figure 1D). These data indicated that the pH-dependent conformational transition of the i-motif does not cause a significant change in size, which is consistent with the findings of others.24 Based on the observation of the opening/closing of the DNA Td upon pH changes, we investigated whether the accessibility and activity of the encapsulated enzyme could be reversibly regulated by the pH-responsive caging and uncaging. RNase A (RA) was chosen as the encapsulated enzyme because (1) it is small (∼13.4 kDa, ca. 2 nm)25 enough to be embedded in the inner space of the DNA Td cage17 and (2) its enzyme activity is well maintained in a broad range of temperature and pH.26,27 The attachment positions of RA in the DNA oligonucleotide were carefully selected based on the previous report, which suggested that a protein can be located inside or outside of the
DNA cage depending on the position of the protein-conjugated nucleotide because of the helical nature of DNA.28 In our study, we compared two positions: one oriented toward the inside of the structure (Td-IN-RA with S1-IN-AZ strand, Figure 2A) and the other oriented outward from the structure (Td-OUT-RA with S1-OUT-AZ strand, Figure 2A, see Table S1 for full sequence). RA was first reacted with a heteroreactive linker, dibenzylcyclooctyne (DBCO)-PEG4-NHS ester using an amine-NHS ester reaction, and DBCO-labeled RA was further reacted with the azide-modified S1 (Figure S3A and B). The gel purified S1-RA (Figure S3C) was quantified by integrated band intensity (Figure S3D) and then mixed with equal amounts of S2, S3, and S4 for DNA Td-RA assembly. The assembled DNA Td-RA was first characterized by native PAGE analysis. The fluorescence images using SYBR Gold (left in Figure 2B) and Cy3 (middle in Figure 2B) clearly indicated that Td, Td-INRA, and Td-OUT-RA efficiently formed. When we calculated the assembly efficiency = ITd/∑IB using SYBR Gold stained images, where ITd is the integrated band intensity of Td or TdRA, and IB is the integrated band intensity of any band appearing in the same lane; the efficiency of Td was about 98%. However, RA attached Td showed decreased efficiency, probably due to the steric hindrance produced by RA. In this regard, it is plausible that the assembly of Td-IN-RA, where RA should be encapsulated inside of Td, was less efficient (∼86%) than Td-OUT-RA (∼94%). The presence of RA in Td-IN-RA and Td-OUT-RA was confirmed by the Western blot with RA-specific antibody (Ab, right in Figure 2B). First, all of RA was attached to Td, and no free RA was observed. While the Td-IN-RA (lane 5 in Figure 2B) and Td-OUT-RA (lane 6 in Figure 2B) had similar mobilities, both Td-RAs (lanes 5 and 6 in Figure 2B) were slightly retarded compared with Td itself (lane 4 in Figure 2B), probably due to the presence of the conjugated RA. Because 9354
DOI: 10.1021/acsnano.7b04766 ACS Nano 2017, 11, 9352−9359
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Figure 3. Measurements of RA accessibility to the RA Ab estimated by the resistance to the PK and single-molecule pull-down assay. (A) PK protection assay. Western blot analysis of RA and RA-attached DNA Td (Td-IN-RA and Td-OUT-RA) treated with/without PK at different pH values. (B) Schematic of the single-molecule assay concept and surface modification. (C) The number of pulled-down molecules in a given surface area. The number of captured molecules by the RA Ab was measured by counting each fluorescent spot from RA or RA-attached DNA Td. Representative fluorescence images from various samples are also illustrated. Scale bar, 4 μm. Data represent the mean ± S.D. **P
