Construction of Tetrahedral DNA-Quantum Dot Nanostructure with the

Jun 10, 2019 - Three-dimensional (3D) DNA scaffolds with well-defined structure and high controllability hold promising potentials for biosensing and ...
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Construction of Tetrahedral DNA-Quantum Dot Nanostructure with the Integration of Multistep Förster Resonance Energy Transfer for Multiplex Enzymes Assay Downloaded via BUFFALO STATE on July 17, 2019 at 07:50:30 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

Juan Hu,‡ Ming-hao Liu,‡ and Chun-yang Zhang* College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University, Jinan 250014, China S Supporting Information *

ABSTRACT: Three-dimensional (3D) DNA scaffolds with well-defined structure and high controllability hold promising potentials for biosensing and drug delivery. However, most of 3D DNA scaffolds can detect only a single type of molecule with the involvement of complex logic operations. Herein, we develop a 3D DNA nanostructure with the capability of multiplexed detection by exploiting a multistep Förster resonance energy transfer (FRET). The tetrahedron-structured DNA is constructed by four oligonucleotide strands and is subsequently conjugated to a streptavidin-coated quantum dot (QD) to obtain a QD-Cy3-Texas Red-Cy5 tetrahedron DNA. This QDCy3-Texas Red-Cy5 tetrahedral DNA nanostructure has well-defined dye-to-dye spacing and high controllability for energy transfer between intermediary acceptors and terminal acceptors, enabling the generation of multistep FRET between the QD and three dyes (i.e., Cy3, Texas Red, and Cy5) for simultaneous detection of multiple endonucleases and methyltransferases even in complex biological samples as well as the screening of multiple enzyme inhibitors. KEYWORDS: quantum dot, Förster resonance energy transfer, DNA nanostructure, multiplexed detection, self-assembly, tetrahedral DNA nanoparticles,18 and the organic dyes-encapsulated silica nanoparticles19) have been exploited to overcome these limitations. The QDs are ideal energy donors for the fabrication of multiplex FRET configurations due to their long fluorescence lifetime, broad absorption spectra from ultraviolet to infrared region, narrow and size-tunable emission spectra, good resistance to chemical and photodegradation, and the capability of coupling more than one dye as the FRET acceptor under a single light excitation.20−23 The QD-based FRET with the QD as the donor involves neither the washing nor the separation steps and has been applied for homogeneously multiplexed detection of various biomolecules including nucleic acids, miRNAs, and enzymes.24−30 The multiple QD-based FRET can be achieved by the integration of multiple single donor−acceptor pair,30 the surrounding of a central QD by pendant dyes in a sequential order,25,26 and the symmetrical arranging of multiple dye acceptors around a central QD.27,28 On the basis of Förster modeling, the

A

DNA molecule can be viewed as a prime selfassembled molecule, forming a duplex based solely on two complementary sequences by following Watson− Crick base pairing rules. The complex three-dimensional (3D) DNA origami nanostructures can be simply designed by utilizing the biological features of staple ssDNA, with wide applications in biosensing, photonics, drug delivery, and gene regulation.1−4 Among these, the 3D DNA nanostructure-based molecular detection systems enable the detection of DNAs, RNAs, proteins, and even intracellular temperature sensing.3−6 However, most of these researches focused on only one kind of molecule.3−5 DNA may act as a well-defined scaffold for arraying multiple fluorophores at precise positions and distances,7−10 in which the light energy can be controlled on the nanoscale by manipulating alternative energy-transfer paths based on Förster resonance energy transfer (FRET). The fluorescent organic dyes are usually used in FRET strategies, but they suffer from easy photobleaching, short fluorescence lifetimes (in the lower nanoseconds range), spectral cross-talk, and high background fluorescence in biological matrices.11 Alternatively, a variety of luminescent nanomaterials (e.g., quantum dot (QD),12−16 barcoded microparticles,17 polymeric © 2019 American Chemical Society

Received: April 8, 2019 Accepted: June 10, 2019 Published: June 10, 2019 7191

DOI: 10.1021/acsnano.9b02679 ACS Nano 2019, 13, 7191−7201

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Figure 1. (A) Schematic illustration of the assembly of the 525QD-Cy3-Texas Red-Cy5 tetrahedron DNA nanostructure for multiple enzymes assay. (B) Biotin-Cy3-Texas Red-Cy5 tetrahedron DNA contains the endonuclease incision sites positioned at 7 bp away from Cy3 end for PvuII, 8 bp away from Texas Red end for EcoRV, and 8 bp away from Cy5 end for HaeIII. (C) Emissions in the 525QD-Cy3-Texas Red-Cy5 tetrahedron DNA under a single laser excitation. The 525QD can be excited under an excitation wavelength of 392 nm; Cy3 emits fluorescence as a result of FRET between the 525QD and Cy3; Texas Red emits fluorescence as a result of FRET between the 525QD/Texas Red pair and the Cy3/Texas Red pair; Cy5 emits fluorescence as a result of FRET between the 525QD/Cy5 pair, the Cy3/Cy5 pair, and the Texas Red/Cy5 pair. Consequently, the emissions of 525QD, Cy3, Texas Red, and Cy5 can be observed simultaneously under a single laser excitation.

of 392 nm, the fluorescence signals of four fluorophores (i.e., 525QD, Cy3, Texas Red, and Cy5) can be observed simultaneously as a result of multiple interacting FRET pathways among 525QD, Cy3, Texas Red, and Cy5 (Figure 1C). In addition to the FRET from the 525QD to Cy3 (the 525QD/Cy3 pair), to Texas Red (the 525QD/Texas Red pair), and to Cy5 (the 525QD/Cy5 pair) acceptors, such multiple interacting FRET pathways also involve the FRET from Cy3 to Texas Red (the Cy3/Texas Red pair), from Cy3 to Cy5 (the Cy3/Cy5 pair), and from Texas Red to Cy5 (the Texas Red/Cy5 pair). Consequently, the FRET between the 525QD/Cy3 pair contributes to the Cy3 fluorescence emission, the FRET between the 525QD/Texas Red pair and the Cy3/Texas Red pair contributes to the Texas Red fluorescence emission, and the FRET between the 525QD/ Cy5 pair, the Cy3/Cy5 pair, and the Texas Red/Cy5 pair contributes to the Cy5 fluorescence emission. We designed the 525QD-Cy3-Texas Red-Cy5 tetrahedron DNA, with a 16bp double-stranded DNA (dsDNA) on each side as the enzyme substrate, with the catalytic site at nucleotide positions 7 bp away from the Cy3 end for PvuII assay, 8 bp away from the Texas Red end for EcoRV assay, and 8 bp away from the Cy5 end for HaeIII assay, respectively (Figure 1B). In the presence of only one endonuclease (e.g., HaeIII), the HaeIII-mediated cleavage reaction leads to the release of the Cy5-labeled DNA fragment from the biotin-Cy3Texas Red-Cy5 tetrahedron DNA. Even though the biotin− streptavidin interaction between the 525QD and biotin-labeled DNA nanostructure still exists, the spatial separation of Cy5 from the 525QD-labeled DNA nanostructure results in the

maximum FRET efficiency can be obtained via multiple interacting FRET pathways rather than independent channels by which excitons travel from initial donor(s) to final acceptor.31 Herein, we demonstrate the development of a 3D DNA nanostructure with the capability of multiplexing detection by exploiting multiple interacting FRET pathways with the QD as the donor.

RESULTS AND DISCUSSION Principles of Multiple FRET-Based DNA Structures. As a proof of concept, we integrated the tetrahedral DNA with the 525 nm emission QD (525QD)-based multistep FRET for simultaneous detection of multiple nucleases including PvuII, EcoRV, and HaeIII endonucleases. Figure 1A shows the principle of the construction of FRET-based 525QD-Cy3Texas Red-Cy5 tetrahedron DNA for multiplexed detection of nucleases. The tetrahedron-structured DNA is assembled by four oligonucleotide strands (i.e., biotin-labeled probe (I), Cy3-labeled probe (II), Texas Red-labeled probe (III), and Cy5-labeled probe (IV)) (Table S1) via a simple annealing process. The resultant biotin-Cy3-Texas Red-Cy5 tetrahedron DNA may subsequently conjugate to a streptavidin-coated QD (525QD) through the specific biotin−streptavidin interaction (Kd = 10−15)21 to obtain a 525QD-Cy3-Texas Red-Cy5 tetrahedron DNA with well-defined dye-to-dye spacing due to the stable and highly rigid scaffold property of tetrahedral structure.32,33 Moreover, this system encapsulates the 525QD inside the tetrahedron, which may reduce the QD-dye distances and the variability. Under the excitation wavelength 7192

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Texas Red, and Cy5 signal can be observed simultaneously upon the excitation of 525QD (Figure 1C). Validation by Gel Electrophoresis. The assembly of tetrahedron DNA is confirmed by the nondenaturating polyacrylamide gel electrophoresis analysis (PAGE). As shown in Figure 2A and B, the products before (Figure 2A)

decrease/disappearance of Cy5 signal due to the elimination of FRET between the 525QD/Cy5 pair, the Cy3/Cy5 pair, and the Texas Red/Cy5 pair. In the presence of two endonucleases (e.g., HaeIII + EcoRV), HaeIII-mediated cleavage reaction leads to the release of Cy5-labeled DNA fragment from the biotin-labeled DNA nanostructure, and the EcoRV-mediated cleavage reaction leads to the release of Texas Red-labeled DNA fragment from the biotin-labeled DNA nanostructure. Consequently, no distinct Cy5 signal is observed as a result of the elimination of FRET between the 525QD/Cy5 pair, the Cy3/Cy5 pair, and the Texas Red/Cy5 pair, and no distinct Texas Red signal is observed as a result of the elimination of FRET between the 525QD/Texas Red pair and the Cy3/Texas Red pair. In the presence of HaeIII + EcoRV + PvuII, the cleavage reactions mediated by HaeIII, EcoRV, and PvuII lead to the release of Cy5-, Texas Red-, and Cy3-labeled DNA fragments from the biotin-labeled DNA nanostructure, and consequently no distinct Cy5, Texas Red and Cy3 signal can be observed as a result of the elimination of FRET between the 525QD/Cy5 pair, the Cy3/Cy5 pair, the Texas Red/Cy5 pair, the 525QD/Texas Red pair, the Cy3/Texas Red pair, and the 525QD/Cy3 pair. This strategy enables multiplexed detection of three enzymes simultaneously, with the decrease/disappearance of Cy5 signal indicating the presence of HaeIII, the decrease/disappearance of Texas Red signal indicating the presence of EcoRV, and the decrease/disappearance of Cy3 signal indicating the presence of PvuII. In the 525QD-Cy3-Texas Red-Cy5 tetrahedral DNA, the 525QD may function as a donor to couple multiple acceptors based on following factors: (1) significant spectral overlaps between the emission spectrum of the 525QD and the absorption spectra of Cy3, Texas Red, and Cy5 acceptor (Figure S1A), suggesting the potential FRET between the 525QD/Cy3 pair, the 525QD/Texas Red pair, the 525QD/ Cy5 pair, the Cy3/Texas Red pair, the Cy3/Cy5 pair, and the Texas Red/Cy5 pair; (2) each FRET pair has large Förster distance (R0) (Table S4),34 with the R0 value being 6.7 nm for the 525QD/Cy3 pair, 5.4 nm for the 525QD/Texas Red pair, 5.2 nm for the 525QD/Cy5 pair, 5.1 nm for the Cy3/Texas Red pair, 5.7 nm for the Cy3/Cy5 pair, and 6.8 nm for the Texas Red/Cy5 pair, guaranteeing efficient FRET between them. In the 525QD-Cy3-Texas Red-Cy5 tetrahedron DNA with four 16 bp edges, the separation distance is estimated to be 5.4 nm for the Cy3/Texas Red pair, 5.4 nm for the Cy3/ Cy5 pair, and 5.4 nm for the Texas Red/Cy5 pair based on the assumption that the length between the adjacent bases in dsDNA is 0.34 nm,35 within the efficient distance of 2R0 for the Cy3/Texas Red pair (2R0 = 10.2 nm), the Cy3/Cy5 pair (2R0 = 11.4 nm), and the Texas Red/Cy5 pair (2R0 = 13.6 nm). When the biotin-Cy3-Texas Red-Cy5 tetrahedron DNA is bound to the streptavidin-coated 525QD, the separation distance is estimated to be 9.2−12.9 nm for the 525QD/Cy3 pair, 9.2−12.9 nm for the 525QD/Texas Red pair, and 9.2− 12.9 nm for the 525QD/Cy5 pair (Figure S1B),36 within the efficient distance of 2R0 for the 525QD/Cy3 pair (2R0 = 13.4 nm), the 525QD/Texas Red pair (2R0 = 10.8 nm), and the 525QD/Cy5 pair (2R0 = 10.4 nm); (3) on the basis of the assembly of multiple streptavidins per 525QD and three available biotin-binding sites per streptavidin, multiple biotinCy3-Texas Red-Cy5 tetrahedron DNA may be assembled onto a single 525QD surface to obtain a single-donor/multipleacceptor nanostructure for improved FRET efficiency according to the eq in ref 37. Consequently, distinct 525QD, Cy3,

Figure 2. (A, B) Characterization of the biotin-Cy3-Texas Red-Cy5 tetrahedron DNA (A) before and (B) after staining with SYBR Gold by polyacrylamide gel electrophoresis. Lane M is the DNA marker. (C) Agarose gel electrophoresis analysis of the cleavage of the 525QD-Cy3-Texas Red-Cy5 tetrahedron DNA by multiple exonucleases. Lanes 1−3 represent the biotin-Cy3-Texas Red-Cy5 tetrahedron DNA (lane 1), streptavidin-coated 525QD (lane 2), and the 525QD-Cy3-Texas Red-Cy5 tetrahedron DNA (lane 3). Lanes 4−7 represent the products in the presence of HaeIII (lane 4), EcoRV (lane 5), PvuII (lane 6), and HaeIII + EcoRV + PvuII (lane 7). The gel images are pseudocolored, with blue indicating SYBR Gold and the 525QD, green indicating Cy3 and Texas Red, and red indicating Cy5.

and after (Figure 2B) staining with SYBR Gold are analyzed by direct excitation of Cy3, Texas Red, Cy5, and SYBR Gold to compare the fully assembled biotin-Cy3-Texas Red-Cy5 tetrahedron DNA (Figure 2A, lane 14, and Figure 2B, lane 4) with the constituent ssDNA oligonucleotides (Figure 2A, lanes 1−3, and Figure 2B, lane 1). The band of the biotin-Cy3Texas Red-Cy5 tetrahedron DNA (Figure 2A, lane 14, and Figure 2B, lane 4) migrates more slowly than those of the constituent ssDNA oligonucleotide (Figure 2A, lanes 1−3, and Figure 2B, lane 1), with the intermediate migration bands being observed for the assemblies of both two oligonucleotides (Figure 2A, lanes 4−9, and Figure 2B, lane 2) and three oligonucleotides (Figure 2A, lanes 10−13, and Figure 2B, lane 3). In addition, the assembly of different dye-labeled probes can be verified by the colocalization of Cy3/Texas Red and Cy5 fluorophores (Figure 2A). In the absence of Cy5-labeled probe, only the Cy3/Texas Red fluorescence signal is detected (Figure 2A, lanes 2−5, 7, 13, green color). In the absence of both Cy3-labeled probe and Texas Red-labeled probe, only the Cy5 fluorescence signal is detected (Figure 2A, lanes 1, 6, red color). In contrast, in the presence of Cy5-labeled probe + Cy3-labeled probe, Cy5-labeled probe + Texas Red-labeled 7193

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Figure 3. (A) Fluorescence emission spectra collected from the 525QD-Cy3-Texas Red-Cy5 tetrahedron DNA with four 16bp edges. The fluorescence emission spectra of the 525QD donor (black line), the 525QD-Cy3-sp-sp tetrahedron DNA (green line), the 525QD-Cy3-Texas Red-sp tetrahedron DNA (blue line), and the 525QD-Cy3-Texas Red-Cy5 tetrahedron DNA (red line) are shown. The sp represents the unlabeled spacer oligonucleotide. The inset in the panel A zooms in on the acceptor (i.e., Cy3, Texas Red, and Cy5) emission range. (B) Comparison of FRET efficiency of the 525QD-Cy3-Texas Red-Cy5 tetrahedral DNA with four 16bp edges with those of other tetrahedral DNAs with four 16bp edges (i.e., the double/triple-fusion fluorophore FRET systems). Error bars represent the standard deviation of three independent experiments. ∗ indicates that FRET efficiency of the 525QD-Cy3-Texas Red-Cy5 tetrahedral DNA show a significantly higher FRET efficiency than the indicated complex (∗P < 0.05. ∗∗P < 0.01). (C) Measurements of the fluorescence emission spectra of the 525QD, Cy3, Texas Red, Cy5 in the 525QD-Cy3-Texas Red-Cy5 tetrahedron DNA with four 16bp edges. The composite spectrum is shown in black. The fitted spectrum is shown in navy color, and individual contributions to the fit are shown by red for the 525QD, blue for Cy3, magenta for Texas Red, and green for Cy5. (D) Comparison of the fluorescence emission spectra of the 525QD-Cy3-Texas Red-Cy5 tetrahedral DNA with four 16 bp edges (blue line), the 525QD-TAMRA-Texas Red-Cy5 tetrahedral DNA with four 16 bp edges (red line), and the 525QDCy3-Texas Red-Cy5 tetrahedral DNA with four 20 bp edges (black line). The 525QD concentration is 5 nM. The concentration of constituent DNA oligonucleotides is 0.12 μM for the 525QD-Cy3-Texas Red-Cy5 tetrahedron DNA with four 16 bp edges, 0.12 μM for the 525QD-TAMRA-Texas Red-Cy5 tetrahedron DNA with four 16 bp edges, and 0.1 μM for the 525QD-Cy3-Texas Red-Cy5 tetrahedron DNA with four 20 bp edges, respectively.

probe, and Cy5-labeled probe + Cy3-labeled probe + Texas Red-labeled probe, both the Cy3/Texas Red and Cy5 fluorescence signals can be observed simultaneously with perfect colocalization (Figure 2A, lanes 8−12, 14, orange/ yellow color). Moreover, the Cy3/Texas Red, Cy5 and the incorporated SYBR Gold fluorescence signals can be observed simultaneously with perfect colocalization (Figure 2B, lane 4), indicating the successful assembly of the biotin-Cy3-Texas Red-Cy5 tetrahedron DNA. The construction of the 525QD-Cy3-Texas Red-Cy5 tetrahedron DNA was investigated by agarose gel electrophoresis (Figure 2C). The colocalization of Cy3/Texas Red and Cy5 results in a yellow single band (Figure 2C, lane 1), indicating the successful assembly of the biotin-Cy3-Texas Red-Cy5 tetrahedron DNA. A blue single band results from the streptavidin-coated 525QD alone (Figure 2C, lane 2). The conjugation of the biotin-Cy3-Texas Red-Cy5 tetrahedron DNA with the streptavidin-coated 525QD leads to a purple band due to the colocalization of the 525QD, Cy3/Texas Red, and Cy5 emission (Figure 2C, lane 3), indicating the successful assembly of the biotin-Cy3-Texas Red-Cy5 tetrahedron DNA onto the 525QD. Upon the addition of HaeIII (Figure 2C, lane 4), EcoRV (Figure 2C, lane 5), PvuII (Figure 2C, lane 6), and HaeIII + EcoRV + PvuII (Figure 2C, lane 7) enzymes to the biotin-Cy3-Texas Red-Cy5 tetrahedral DNA, short fragments carrying Cy5, Texas Red, Cy3, and Cy5 + Texas Red + Cy3 are

generated, respectively, accompanied by the decrease of the 525QD-Cy3-Texas Red-Cy5 tetrahedron DNA. Specifically, the presence of HaeIII induces the appearance of a red band corresponding to the Cy5-labeled DNA fragment (Figure 2C, lane 4, red color); the presence of EcoRV induces the appearance of a green band corresponding to the Texas Redlabeled DNA fragment (Figure 2C, lane 5, green color); the presence of PvuII induces the appearance of a green band corresponding to the Cy3-labeled DNA fragment (Figure 2C, lane 6, green color); the presence of HaeIII + EcoRV + PvuII induces the appearance of both red and green bands corresponding to the Cy5- and Cy3/Texas Red-labeled DNA fragments, respectively (Figure 2C, lane 7, red and green color). Detection of Multiple FRET Efficiencies. We investigated the FRET between the 525QD and three acceptor dyes (i.e., Cy3, Texas Red, and Cy5) in the 525QD-Cy3-Texas RedCy5 tetrahedron DNA. As shown in Figure 3, the tetrahedron DNA are obtained by either the unlabeled spacers (sp) or being labeled with the acceptor dyes at their ends. Only the 525QD signal is observed in the presence of 525QD (Figure 3A, black line) at the excitation of 392 nm (Figure S2). In contrast, the 525QD-Cy3-sp-sp tetrahedron DNA induces the decrease of 525QD fluorescence intensity accompanied by the increase of Cy3 acceptor fluorescence intensity (Figure 3A, green line), indicating the FRET from the 525QD to Cy3. The 7194

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are involved in the 525QD-Cy3-Texas Red-sp tetrahedron DNA. Similar results are observed for the 525QD-Cy3-sp-Cy5 tetrahedron DNA with FRET among the 525QD/Cy3 pair, the 525QD/Cy5 pair, and the Cy3/Cy5 pair (Figure S3E). For the 525QD-Cy3-sp-Cy5 tetrahedron DNA, EQD‑Cy3‑Cy5 = 53.3% (Figure 3B); for the 525QD-Cy3-sp-sp tetrahedron DNA, EQD‑Cy3 = 41.5% (Figure 3B); and for the 525QD-sp-sp-Cy5 tetrahedron DNA, EQD‑Cy5 = 33.6%, suggesting that energy from 525QD is transferred not only to Cy3 but also directly to Cy5 in the 525QD-Cy3-sp-Cy5 tetrahedron DNA. The theoretical total efficiency for the 525QD-Cy3-sp-Cy5 tetrahedron DNA is 54.8%, consistent with the experimental data (53.3%) obtained from the 525QD-Cy3-sp-Cy5 tetrahedron DNA (Table S6). Similar results are observed for the 525QD-sp-Texas Red-Cy5 tetrahedron DNA (EQD‑TR‑Cy5 = 45.7%, Figure 3B). The total FRET efficiency for the 525QDsp-Texas Red-Cy5 tetrahedron DNA is theoretically calculated to be 51.5% (Table S6), in agreement well with the value (45.7%) experimentally obtained (Table S6). Similar results are observed for the 525QD-Cy3-Texas Red-Cy5 tetrahedron DNA (EQD‑Cy3‑TR‑Cy5(16bp) = 61.7%, Figure 3B). The total FRET efficiency for the 525QD-Cy3-Texas Red-Cy5 tetrahedron DNA is theoretically calculated to be 63.9% (Table S6), consistent with the value (61.7%) experimentally obtained (Table S6). For the 525QD-Cy3-Texas Red-Cy5 tetrahedron DNA (Figure 3A, red line), multistep FRET may occur among the 525QD/Cy3 pair, the 525QD/Texas Red pair, the 525QD/Cy5 pair, the Cy3/Texas Red pair, the Cy3/Cy5 pair, and the Texas Red/Cy5 pair. We analyzed the individual contributions of each fluorophore (i.e., 525QD, Cy3, Texas Red, and Cy5) to the composite spectrum (Figure 3C), with the individual contribution being shown in red (525QD), blue (Cy3), magenta (Texas Red), and green (Cy5), respectively. The experimentally measured composite spectrum curve of the 525QD-Cy3-Texas Red-Cy5 tetrahedron DNA (Figure 3C, black line) consists well with the fitted spectrum (Figure 3C, navy line). We further investigated the FRET of the 525QD-Cy3-Texas Red-Cy5 tetrahedron DNA with four 20 bp edges (Table S3), whose fluorophore spacing increases by 1.4 nm for the Cy3/ Texas Red pair, the Cy3/Cy5 pair, and the Texas Red/Cy5 pair, and by 0.9−1.4 nm for the 525QD/Cy3 pair, the 525QD/ Texas Red pair, and the 525QD/Cy5 pair compared with that of the 525QD-Cy3-Texas Red-Cy5 tetrahedron DNA with four 16 bp edges. On the basis of the fluorescence spectra of 525QD-Cy3-Texas Red-Cy5 tetrahedron DNA with four 20 bp (Figure 3D, black line, and Figure S4A), the FRET efficiency is calculated to be 41.3% (EQD‑Cy3‑TR‑Cy5(20bp) = 41.3%, Figure S4B). In addition, we measured the FRET of the 525QDTAMRA-Texas Red-Cy5 tetrahedron DNA with four 16bp edges (Table S2), whose overlap integral decreases by ∼46% compared with that of the 525QD-Cy3-Texas Red-Cy5 tetrahedron DNA with four 16bp edges (Table S4). On the basis of the fluorescence spectra of the 525QD-TAMRA-Texas Red-Cy5 tetrahedron DNA with four 16bp edges (Figure 3D, red line, and Figure S4C), the FRET efficiency is calculated to 55.5% (EQD‑TAM‑TR‑Cy5(16bp) = 55.5%, Figure S4D). Notably, the FRET efficiency of the 525QD-Cy3-Texas Red-Cy5 tetrahedron DNA with four 16 bp edges (EQD‑Cy3‑TR‑Cy5(16bp) = 61.7%) improves by 20.4% compared with that of the 525QD-Cy3Texas Red-Cy5 tetrahedron DNA with four 20 bp edges (EQD‑Cy3‑TR‑Cy5(20bp) = 41.3%), and by 6.2% compared with that

525QD-Cy3-Texas Red-sp tetrahedron DNA and the 525QDCy3-Texas Red-Cy5 tetrahedron DNA induce the greater decrease of 525QD fluorescence intensity accompanied by the increase of corresponding Texas Red (Figure 3A, blue line) and Cy5 fluorescence intensity (Figure 3A, red line), respectively, indicating the multistep FRET in the 525QDCy3-Texas Red-Cy5 tetrahedron DNA. We further demonstrate that Cy3, Texas Red, and Cy5 are efficient acceptors for the 525QD donor. The assembly of the 525QD-sp-sp-Cy5 tetrahedron DNA, the 525QD-sp-Texas Red-sp tetrahedron DNA, and the 525QD-Cy3-sp-sp tetrahedron DNA leads to the decrease of 525QD fluorescence intensity and the simultaneous increase of Cy5, Texas Red, and Cy3 fluorescence intensity, respectively (Figure S3A−C, red line), indicating the efficient FRET between the 525QD/Cy5 pair, the 525QD/Texas Red pair, and the 525QD/Cy3 pair. Experimentally, the FRET efficiency (E) can be derived from the quenching of the QD donor fluorescence intensity based on eq 1: E=1−

FDA FD

(1)

where FD and FDA are the fluorescence intensity of the 525QD donor in the absence and presence of acceptors, respectively. The E is calculated to be EQD‑Cy5 = 33.6% for the 525QD/Cy5 pair in the 525QD-sp-sp-Cy5 tetrahedron DNA, EQD‑TR = 35.8% for the 525QD/Texas Red pair in the 525QD-sp-Texas Red-sp tetrahedron DNA, and EQD‑Cy3 = 41.5% for the 525QD/Cy3 pair in the 525QD-Cy3-sp-sp tetrahedron DNA (Figure 3B), respectively. We further investigated the FRET in the tetrahedron DNA containing triple-fusion fluorophores (i.e., the 525QD-spTexas Red-Cy5 tetrahedron DNA, the 525QD-Cy3-sp-Cy5 tetrahedron DNA, and the 525QD-Cy3-Texas Red-sp tetrahedron DNA, Figure S3D−F). In the 525QD-Cy3-Texas Red-sp tetrahedron DNA, FRET may occur among the 525QD/Cy3 pair, the 525QD/Texas Red pair, and the Cy3/ Texas Red pair (Figure S3F). The FRET efficiency of the 525QD-Cy3-Texas Red-sp tetrahedron DNA is calculated to be 58.0% (EQD‑Cy3‑TR = 58.0%, Figure 3B), much higher than that of the 525QD-Cy3-sp-sp tetrahedron DNA (EQD‑Cy3 = 41.5%) and that of the 525QD-sp-Texas Red-sp tetrahedron DNA (EQD‑TR = 35.8%) (Figure 3B), suggesting that energy transfers from the 525QD not only to Cy3 but also to Texas Red in the 525QD-Cy3-Texas Red-sp tetrahedron DNA. We calculated the FRET efficiency for the 525QD/Cy3 pair and the 525QD/Texas Red pair in the 525QD-Cy3-Texas Red-sp tetrahedron DNA (Table S5) as the result of the competition between two pathways for FRET from the 525QD to two different acceptors (i.e., the 525QD/Cy3 pair and the 525QD/ Texas Red pair).24,38 The total FRET efficiency for the 525QD-Cy3-Texas Red-sp tetrahedron DNA is theoretically calculated to be 55.9% (Table S6),24,39 in agreement well with the value (58.0%) experimentally obtained (Table S6). Moreover, the greater decrease of Cy3 is observed in the 525QD-Cy3-Texas Red-sp tetrahedron DNA (Figure S3I, red line) than in the 525QD-Cy3-sp-sp tetrahedron DNA (Figure S3I, black line), indicating the energy transfer from Cy3 to Texas Red. Above all, these results demonstrate that three mutually dependent FRET processes (i.e., the 525QD/Cy3 pair, the 525QD/Texas Red pair, and the Cy3/Texas Red pair) 7195

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Figure 4. (A−C) Fluorescence images of the 525QD-Cy3-Texas Red-Cy5 tetrahedral DNA in the (A) absence of any endonucleases and presence of (B) HaeIII and (C) HaeIII + EcoRV + PvuII. (a−c) Fuorescence images of the same field in the 525QD channel (500−550 nm) and the Cy3/Texas Red channel I (573−617 nm) are acquired simultaneously by using dual-view optics at an excitation wavelength of 405 nm. Then (d−f) fluorescence images in the Cy3/Texas Red channel II (565−605 nm) and Cy5 channel (672−712 nm) are acquired simultaneously. Fluorescence spots shown in the Cy3/Texas Red channel I/II (b, d, green color) result from FRET from the 525QD to Cy3/ Texas Red (yellow arrows); fluorescence spots shown in the Cy5 channel (e, red color) result from FRET from the 525QD to Cy5 (white arrows). Fluorescence images with blended colors in (c) cyan indicate the colocalization of 525QD and Cy3/Texas Red; fluorescence images with blended colors in (f) yellow indicate the colocalization of Cy3/Texas Red and Cy5. The scale bar is 2 μm. (D, F, H) Variance of fluorescence emission spectra of the 525QD-Cy3-Texas Red-Cy5 tetrahedral DNA before (blue line) and after (red line) incubation with (D) HaeIII, (F) EcoRV at a fixed concentration of 0.5 U/μL HaeIII, and (H) PvuII at a fixed concentration of 0.5 U/μL EcoRV and 0.5 U/μL HaeIII, respectively. The insets in panels D, F, and H zoom in on the (D) Cy5, (F) Texas Red, and (H) Cy3 emission range, respectively. (E) Reduction of Cy5 fluorescence intensity as a function of HaeIII concentration. The inset shows the log−linear correlation between the reduction of Cy5 fluorescence intensity and the concentration of HaeIII in the range from 0.025 to 0.5 U/μL. (G) Reduction of Texas Red fluorescence intensity as a function of EcoRV concentration. The inset shows the log−linear correlation between the reduction of Texas Red fluorescence intensity and the concentration of EcoRV in the range from 0.025 to 0.5 U/μL. (I) Reduction of FRET efficiency as a function of PvuII concentration. The inset shows the log−linear correlation between the reduction of FRET efficiency and the concentration of PvuII in the range from 0.025 to 0.5 U/μL. The 525QD concentration is 5 nM.

pair in the 525QD-TAMRA-Texas Red-Cy5 tetrahedron DNA with four 16 bp edges (Table S4), and (2) the smaller fluorophore spacing for each FRET pair in the tetrahedron DNA with four 16 bp edges than the tetrahedron DNA with 20 bp edges. To confirm the energy-transfer process involved, we measured the excited-state fluorescence lifetimes of different

of the 525QD-TAMRA-Texas Red-Cy5 tetrahedron DNA with four 16 bp edges (EQD‑TAM‑TR‑Cy5(16bp) = 55.5%). The increase in FRET efficiency can be ascribed to (1) the higher overlap integral (8.5 × 1015 cm3/M) for the 525QD/Cy3 pair in the 525QD-Cy3-Texas Red-Cy5 tetrahedron DNA with four 16 bp edges than that (4.6 × 1015 cm3/M) for the 525QD/TAMRA 7196

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simultaneously with perfect colocalization (Figure 4B(c), cyan color), without Cy5 fluorescence signal being observed (Figure 4B(e)). When HaeIII + EcoRV + PvuII is present, only the 525QD fluorescence signals (Figure 4C(a), blue color) can be detected, without either the Cy3/Texas Red (Figure 4C(b,d)) or the Cy5 fluorescence signal (Figure 4C(e)) being observed. The HaeIII-, EcoRV-, and PvuII-mediated cleavage reaction can be monitored as a function of enzyme concentration. In the absence of any endonucleases, the 525QD-Cy3-Texas RedCy5 tetrahedral DNA exhibits four peaks at 530 nm (525QD), 565 nm (Cy3), 613 nm (Texas Red), and 670 nm (Cy5) (Figure 4D blue line, and Figure S9A), indicating the occurrence of multistep FRET among the 525QD/Cy3 pair, the 525QD/Texas Red pair, the 525QD/Cy5 pair, the Cy3/ Texas Red pair, the Cy3/Cy5 pair, and the Texas Red/Cy5 pair. In contrast, the presence of HaeIII induces the decrease of Cy5 fluorescence intensity, accompanied by the increase of 525QD fluorescence intensity (Figure 4D, red line, and Figure S9B). Moreover, the higher the HaeIII concentration, the greater the reduction of Cy5 fluorescence intensity (Figure 4E). A good linear relationship is obtained between the reduction of Cy5 fluorescence intensity and the logarithm of HaeIII concentration in the range from 0.025 to 0.5 U/μL (insert of Figure 4E), and the corresponding equation is ΔI = 107.96 + 51.85 log10 C (R2 = 0.990), where C is the concentration of HaeIII and ΔI is the reduction of Cy5 intensity (ΔI = I0 − I, where I0 and I are the Cy5 fluorescence intensity in the absence and presence of HaeIII, respectively). The detection limit is calculated to be 0.0112 U/μL by evaluating the average response of control plus three-times the standard deviation. The proposed method exhibits high sensitivity compared with those of cationic polyfluorene/YDNA assembly based assay (0.05 U/μL),44 with potential applications in the fields of nucleases and drug development. The 525QD-Cy3-Texas Red-Cy5 tetrahedral DNA can be used for multiplexed assay. Figure 4F shows the fluorescence emission spectra in response to different concentrations of EcoRV at a fixed concentration of HaeIII (0.5 U/μL). In the absence of EcoRV, distinct Texas Red signal is observed (Figure 4F, blue line, and Figure S9C), indicating the occurrence of FRET from the 525QD to Texas Red. In contrast, the presence of EcoRV induces the decrease of Texas Red fluorescence intensity, accompanied by the increase of 525QD fluorescence intensity (Figure 4F, red line, and Figure S9D). Moreover, the higher the EcoRV concentration, the greater the reduction of Texas Red fluorescence intensity (Figure 4G). A good linear relationship is obtained between reduction of Texas Red fluorescence intensity and the logarithm of EcoRV concentration in the range from 0.025 to 0.5 U/μL (insert of Figure 4G), and the linear fitting equation is ΔI = 78.13 + 42.43 log10 C (R2 = 0.998), where C is the concentration of EcoRV and ΔI is the reduction of the Texas Red intensity (ΔI = I0 − I, where I0 and I are the Texas Red fluorescence intensity in the absence and presence of EcoRV, respectively). The detection limit is calculated to be 0.0233 U/μL. The sensing capability of the 525QD-Cy3-Texas Red-Cy5 tetrahedral DNA was tested by multiplexed detection of PvuII, EcoRV, and HaeIII. Figure 4H shows the fluorescence emission spectra in response to different concentrations of PvuII at a fixed concentration of EcoRV (0.5 U/μL) and HaeIII (0.5 U/μL). In the absence of PvuII, distinct Cy3 signal is observed (Figure 4H, blue line, and Figure S9E), indicating

configurations. The greatest decrease in QD donor excitedstate lifetime is observed for the 525QD-Cy3-Texas Red-Cy5 tetrahedron DNA with four 16 bp edges (Figure S5). Furthermore, we measured the number of acceptors per 525QD based on the variation of FRET efficiency with the tetrahedron DNA-to-525QD ratio. For the 525QD-Cy3-Texas Red-Cy5 tetrahedron DNA with four 16 bp edges, the FRET efficiency improves with the increasing tetrahedron DNA-to525QD ratio and reaches a plateau at the ratio of 24 (Figure S6A), suggesting that a maximum of 24 tetrahedron DNAs can be assembled on the surface of a single 525QD and contribute to the FRET efficiency experimentally. This is also supported by the measurement of the number of tetrahedra units per 525QD using fluorescence emission spectra (Figure S7). Similarly, the maximum tetrahedron DNA-to-525QD ratio of 24 is obtained for the 525QD-TAMRA-Texas Red-Cy5 tetrahedron DNA with four 16 bp edges (Figure S6B), and the maximum tetrahedron DNA-to-525QD ratio of 21 is obtained for the 525QD-Cy3-Texas Red-Cy5 tetrahedron DNA with four 20 bp edges (Figure S6C). The discrepancy can be explained by the smaller steric hindrance of the 525QDCy3-Texas Red-Cy5 tetrahedron DNA with four 16 bp edges than the 525QD-Cy3-Texas Red-Cy5 tetrahedron DNA with four 20 bp edges (the volume is 18.6 nm3 for the biotin-Cy3Texas Red-Cy5 tetrahedron DNA with four 16 bp edges versus 37.1 nm3 for the biotin-Cy3-Texas Red-Cy5 tetrahedron DNA with four 20 bp edges). Analysis of Multiple Nucleases and Methyltransferase Activity. We employed the 525QD-Cy3-Texas Red-Cy5 tetrahedral DNA with four 16 bp edges to simultaneously measure the activities of three endonucleases including HaeIII, EcoRV, and PvuII. HaeIII can specifically recognize and cleave the sequence 5′-GGCC-3′ to release the Cy5-labeled DNA fragment; EcoRV can specifically recognize and cleave the sequence 5′-GATATC-3′ to release the Texas Red-labeled DNA fragment; PvuII can specifically recognize and cleave the sequence 5′-CAGCTG-3′ to release the Cy3-labeled DNA fragment. We used the single-molecule imaging40−42 to measure the enzyme cleavage events in the 525QD-Cy3Texas Red-Cy5 tetrahedral DNA. When the 525QD-Cy3Texas Red-Cy5 tetrahedral DNA is excited at an excitation wavelength of 405 nm, the fluorescence images at 525 nm (for the 525QD) and 595 nm (for Cy3/Texas Red) are acquired simultaneously, and meanwhile, the fluorescence images at 585 nm (for Cy3/Texas Red) and 692 nm (for Cy5) can be obtained simultaneously by using dual-view optics. In the absence of any enzymes, the colocalization of the 525QD fluorescence signals (Figure 4A(a), blue color) and the Cy3/ Texas Red fluorescence signals (Figure 4A(b), green color) leads to the appearance of cyan-color signals (Figure 4A(c), cyan color), indicating the FRET from the 525QD to Cy3/ Texas Red. Similarly, the colocalization of the Cy3/Texas Red fluorescence signals (Figure 4A(d), green color) and the Cy5 fluorescence signals (Figure 4A(e), red color) indicates the FRET from the 525QD to Cy5 (Figure 4A(f), yellow color). The colocalization was further investigated by calculating the colocalization ratio of different fluorescence channels (Figure S8).43 The higher colocalization ratios of 525QD channel with Cy3/Texas Red channel I, 525QD channel with Cy5 channel, and Cy3/Texas Red channel II with Cy5 channel are observed in the absence of any enzymes. When HaeIII is present, both the 525QD (Figure 4B(a), blue color) and the Cy3/Texas Red (Figure 4B(b), green color) fluorescence signals are observed 7197

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Figure 5. (A) Measured FRET efficiency in response to (a) HaeIII + EcoRV + PvuII, (b) heat-treated HaeIII + EcoRV + PvuII, XhoI, KpnI, and the reaction buffer (control). (B) Variance of fluorescence emission spectra of the 525QD-Cy3-Texas Red-Cy5 tetrahedral DNA before (blue line) and after (red line) treatment with HaeIII methyltransferase (MTase) at a fixed concentration of 0.5 U/μL HaeIII. (C) Enhancement of Cy5 fluorescence intensity as a function of HaeIII MTase concentration. The inset shows the log−linear correlation between the enhancement of Cy5 fluorescence intensity and the concentration of HaeIII MTase in the range from 0.01 to 1 U/μL. (D) Variance of the relative activity of HaeIII (black line), EcoRV (red line), and PvuII (blue line) with different concentrations of EDTA. The concentration of each enzyme is 0.5 U/μL.

Texas Red-Cy5 tetrahedral DNA toward the tested enzymes. In addition, the 525QD-Cy3-Texas Red-Cy5 tetrahedral DNA can be used for the detection of various enzymes in complex samples containing 10% fetal bovine serum, and the recoveries are measured to be 98.1−106.9% with an RSD of 1.24−4.14% (Table S7), suggesting the feasibility of the 525QD-Cy3-Texas Red-Cy5 tetrahedral DNA for complex sample analysis. The 525QD-Cy3-Texas Red-Cy5 tetrahedral DNA can be further employed to detect HaeIII methyltransferase activity. The HaeIII methyltransferase may catalyze the methylation of the sequence 5′-GGCC-3′ to generate the methylation product 5′-GGmCC-3′. The subsequent addition of HaeIII enzyme cannot cleave the methylated product, and Cy5 is still caged in the 525QD-Cy3-Texas Red-Cy5 tetrahedral DNA. Consequently, efficient FRET may occur in the 525QD-Cy3-Texas Red-Cy5 tetrahedral DNA, resulting in the decrease of 525QD emission and the increase of Cy5 emission (Figure 5B, red line). The higher the concentration of HaeIII methyltransferase, the greater the enhancement of Cy5 fluorescence intensity (Figure 5C). A good linear relationship is obtained between the enhancement of Cy5 fluorescence intensity and the logarithm of HaeIII methyltransferase concentration (insert of Figure 5C), and the linear fitting equation is ΔI = 70.47 + 31.45 log10 C (R2 = 0.993), where C is the concentration of HaeIII methyltransferase and ΔI is the enhancement of Cy5 fluorescence intensity (ΔI = I − I0, where I0 and I are the Cy5 fluorescence intensity in the absence and presence of HaeIII methyltransferase, respectively). The detection limit is estimated to be 0.01 U/μL. In contrast, in the absence of target HaeIII methyltransferase, the sequence 5′-GGCC-3′ in the biotin-Cy3-Texas Red-Cy5 tetrahedral DNA remains unmethylated and it can be cleaved by HaeIII, and thus, no distinct Cy5 signal is observed (Figure 5B, blue line).

the occurrence of FRET from the 525QD to Cy3. In contrast, the presence of PvuII induces the decrease of Cy3 fluorescence intensity, accompanied by the increase of 525QD fluorescence intensity (Figure 4H, red line, and Figure S9F). Moreover, the reduction of FRET efficiency becomes greater with the increase of PvuII concentration (Figure 4I). A good linear relationship is obtained between the reduction of FRET efficiency and the logarithm of PvuII concentration in the range from 0.025 to 0.5 U/μL (insert of Figure 4I), and the linear fitting equation is ΔE = 7.69 + 3.92 log10 C (R2 = 0.997), where C is the concentration of PvuII and ΔE is the reduction of the FRET efficiency (ΔE = E0 − E, where E0 and E are the FRET efficiency in the absence and presence of PvuII, respectively). The detection limit value is calculated to be 0.0204 U/μL. In addition, this 525QD-Cy3-Texas Red-Cy5 tetrahedral DNA performs well in the combination of HaeIII + EcoRV + PvuII with varying nuclease amounts (Figure S10). To investigate the specificity of the 525QD-Cy3-Texas RedCy5 tetrahedral DNA for nucleases assays, we used XhoI and KpnI as the negative controls. XhoI catalyzes the cleavage of the 5′-CTCGAG-3′ sequence, and KpnI specifically cleaves the 5′-GGTACC-3′ sequence. Neither XhoI nor XhoI can recognize and cleave the 525QD-Cy3-Texas Red-Cy5 tetrahedral DNA. As expected, no significant reduction of FRET efficiency is observed in response to either XhoI or KpnI (Figure 5A). Interestingly, the heat-treated nucleases including HaeIII + EcoRV + PvuII induce the decrease of FRET efficiency (Figure 5A(b)) compared with the control group (Figure 5A, control). This can be explained by the factor that heat-treatment can deactivate HaeIII and EcoRV instead of PvuII. In contrast, extremely lower FRET efficiency is obtained in response to HaeIII + EcoRV + PvuII (Figure 5A(a)). These results demonstrate the good specificity of the 525QD-Cy37198

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New England Biolabs Inc. (Beverly, MA, U.S.A.). Streptavidin-coated 525 nm emission QDs (525QDs) and fetal bovine serum were obtained from Thermo Fisher Scientific (Carlsbad, CA, U.S.A.). Formation of Tetrahedral DNA. To form the tetrahedral DNA, equal concentrations (1 μM) of four constituent ssDNA oligonucleotides with the dye-labeled (e.g., biotin-labeled probe (I), Cy3-labeled probe (II), Texas Red-labeled probe (III), and Cy5-labeled probe (IV) for the construction of the 525QD-Cy3-Texas Red-Cy5 tetrahedron DNA with four 16 bp edges) and the unlabeled spacer (sp) segments (e.g., biotin-labeled probe (I), Cy3-labeled probe (II), the unlabeled spacer oligo (iii), and the unlabeled spacer oligo (iv) for the construction of the 525QD-Cy3-sp-sp tetrahedron DNA with four 16 bp edges) were added to 1× CutSmart buffer solution. The mixture was heated at 95 °C for 10 min, followed by placing on ice immediately for 1 h. Enzyme Reactions. The 0.12 μM products were added into 1× CutSmart buffer containing various concentrations of HaeIII, EcoRV, and PvuII. The mixture was incubated at 37 °C for 1 h. Subsequently, 5 nM streptavidin-coated 525QDs were added to the mixture and incubated at room temperature for 15 min to obtain the 525QD-Cy3Texas Red-Cy5 tetrahedron DNA nanostructures. HaeIII methyltransferase may catalyze the methylation of the sequence 5′-GGCC-3′ to generate the methylation product 5′GGmCC-3′. The methylation of biotin-Cy3-Texas Red-Cy5 tetrahedral DNA was performed at 37 °C for 1 h in 1× CutSmart buffer containing 0.12 μM biotin-Cy3-Texas Red-Cy5 tetrahedral DNA, 60 μM SAM, and varied concentrations of HaeIII methyltransferase. Then HaeIII digestion was performed in the presence of 0.5 U/μL HaeIII at 37 °C for 1 h. After digestion, the streptavidin-coated 525QDs (5 nM) were added to the resultant solutions for the measurement of emission spectra. For the enzyme assay in biological fluid, the sample mixture containing 10% fetal bovine serum, 0.12 μM biotin-Cy3-Texas Red-Cy5 tetrahedral DNA, 1× CutSmart buffer, various concentrations of endonucleases were incubated at 37 °C for 1 h. For the inhibition assay, various concentrations of inhibitor were mixed with the reaction solution containing 0.12 μM tetrahedral DNA (i.e., the 525QD-Cy3-Texas Red-Cy5 tetrahedral DNA for HaeIII, the 525QD-Cy3-Texas Red-sp tetrahedral DNA for EcoRV, and the 525QD-Cy3-sp-sp tetrahedral DNA for PvuII), 1× CutSmart buffer, 0.5 U/μL enzyme, and incubated at 37 °C for 1 h. Electrophoresis Analysis. The biotin-Cy3-Texas Red-Cy5 tetrahedral DNAs before and after staining with SYBR Gold were loaded onto 8% polyacrylamide gel in TBE buffer (44.5 mM Trisboric acid, 1 mM EDTA), followed by electrophoresis for 45 min. After incubating with 5 nM 525QD, the biotin-Cy3-Texas Red-Cy5 tetrahedral DNAs were loaded onto a 1% agarose gel in TAE buffer (40 mM Tris-acetic acid, 2 mM EDTA), followed by electrophoresis for 30 min. The images were taken with a Bio-Rad ChemiDoc MP imaging system (California, U.S.A.). The 525QD-Cy3-Texas Red-Cy5 tetrahedral DNAs were analyzed using an illumination source of Epiblue (460−490 nm excitation) and a 518−546 nm filter for the 525QD, an illumination source of Epi-green (520−545 nm excitation) and a 577−613 nm filter for the Cy3/Texas Red fluorophores, and an illumination source of Epi-red (625−650 nm excitation) and a 675− 725 nm filter for the Cy5 fluorophore. Fluorescence Measurements. The fluorescence spectra were recorded by a Hitachi F-7000 fluorometer (Tokyo, Japan) at an excitation wavelength of 392 nm. The fluorescence intensity at the emission wavelength of 530 nm (525QD), 565 nm (Cy3), 613 nm (Texas Red), and 670 nm (Cy5) was used for data analysis, respectively. Single-Molecule Imaging with Total Internal Reflection Microscopy. Single-molecule imaging was performed on a total internal reflection (TIR) fluorescence microscope based on an inverted microscope (IX-73, Olympus) equipped with an oilimmersion objective (100×, NA 1.49). To obtain the images of single molecules by TIR fluorescence microscopy, the reaction products were diluted 100-fold in buffer (100 mM Tris-HCl, 10 mM (NH4)2SO4, 3 mM MgCl2, pH 8.0). After the dilution, 10 μL of 525QD-Cy3-Texas Red-Cy5 tetrahedral DNA complex (50 pM) was

The 525QD-Cy3-Texas Red-Cy5 tetrahedral DNA can be further used for the screening of enzyme inhibitors.45 We used ethylenediaminetetraacetic acid disodium salt dehydrate (EDTA) as a model inhibitor. The chelator EDTA is known to inhibit endonucleases.46 EDTA-induced inhibition of HaeIII, EcoRV, and PvuII reaction can be investigated by the measurement of Cy5 emission spectrum of the 525QDCy3-Texas Red-Cy5 tetrahedral DNA, Texas Red emission spectrum of the 525QD-Cy3-Texas Red-sp tetrahedral DNA, and Cy3 emission spectrum of the 525QD-Cy3-sp-sp tetrahedral DNA (Figure S11), respectively. The relative activity of HaeIII, EcoRV, and PvuII is monitored as a function of EDTA concentrations (Figure 5D). We used the IC50 value (the half-maximal inhibitory concentration) to evaluate the inhibition effect of EDTA upon different enzymes. The EDTA inhibitor exhibits the following trend in IC50 values: PvuII (26.02 mM) < HaeIII (33.23 mM) < EcoRV (49.65 mM).

CONCLUSIONS In conclusion, we demonstrate the construction of a 525QDCy3-Texas Red-Cy5 tetrahedral DNA for simultaneously homogeneous detection of multiple nucleases including HaeIII, EcoRV, and PvuII. The 525QD-Cy3-Texas Red-Cy5 tetrahedral DNA utilizes a single-color 525QD as the donor and three fluorescent dyes as the acceptors, with well-defined dye-to-dye spacing and precise controllability for energy transfer among the donor, intermediary acceptors, and terminal acceptor. The 525QD-Cy3-Texas Red-Cy5 tetrahedral DNA has significant advantages in the construction of multiple FRET systems: (1) it harnesses single light energy to preferentially excite the 525QD and transfer the light energy to Cy3, Texas Red, and Cy5; the total FRET efficiency is calculated theoretically by summing FRET efficiency of each implemented donor/ acceptor pairs, and the results obtained by both theoretical and experimental approaches are consistent; (2) the 525QDCy3-Texas Red-Cy5 tetrahedral DNA increases the capability of harvesting energy; (3) the array of 525QD, Cy3, Texas Red and Cy5 in a tetrahedral DNA enables the achievement of efficient FRET due to the multiple interacting FRET pathways in the 525QD-Cy3-Texas Red-Cy5 tetrahedral DNA, superior to the linear configurations with independent channels by which excitons travel from initial donors to the final acceptor;25,26 (4) in contrast to the use of multiple conventional QD-based FRET probes (each comprises a single donor/acceptor pair) for multiplexed assays,30 only one 525QD-Cy3-Texas Red-Cy5 tetrahedral DNA enables multiplexed detection of various enzymes including endonucleases and methyltransferases, and it can be extended to quantitative detection of various epigenetic modification and base excision repair. Importantly, this concept can be extended to the construction of various 3D DNA nanostructures (e.g., the triangular prism, the cube, the pentagonal prism, the hexagonal prism, the elongated square bipyramid, and triangular bipyramid),47,48 holding great potential in multiplexed sensing, imaging, and drug delivery. METHODS Materials. All HPLC-purified oligonucleotides (Tables S1−3) were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). HaeIII, EcoRV-HF, and PvuII-HF endonucleases, 10× CutSmart buffer (500 mM potassium acetate, 200 mM Tris-acetate, 100 mM magnesium acetate, 1000 μg/mL BSA, pH 7.9), HaeIII methyltransferase, and S-adenosylmethionine (SAM) were purchased from 7199

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placed on a glass coverslip above the objective. The lower concentration (20−100 pM) enabled the immobilization/dispersion at the desired single molecule density, and higher density increased the chance of overlapping neighboring molecules.42,49 The samples were illuminated by a 405 nm laser with an excitation power of 30 mW. The images were acquired by a chilled charge-coupled-device electron-multiplying camera (EMCCD). Fluorescence signals from the 525QD and Cy3/Texas Red were separated by the dichroic mirror and band-pass filters (500−550 nm for the 525QD, and 573− 617 nm for Cy3/Texas Red (channel I)) in the dual-view. Fluorescence signals from Cy3/Texas Red and Cy5 were separated by the dichroic mirror and band-pass filters (565−605 nm for Cy3/ Texas Red (channel II), and 672−712 nm for Cy5) in the dual-view. The four emission filters were marked by 525QD channel, Cy3/Texas Red channel I, Cy3/Texas Red channel II, and Cy5 channel, respectively (Figure 4A−C and Figure S8). Since their colocalization ratio is more than 0.95, there is no obvious difference between the 573−617 nm for Cy3/Texas Red (channel I) and 565−605 nm for Cy3/Texas Red (channel II). A region of interest of 70 × 70 pixels was selected for data analysis.

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.9b02679. Sequences of oligonucleotides, normalized emission and absorption spectra of fluorophores and modeling of 525QD-Cy3-Texas Red-Cy5 tetrahedron DNA structures, photophysical and FRET properties of fluorophores, calculation of energy-transfer efficiencies, timeresolved fluorescence data, calculation of number of tetrahedra units per 525QD, calculation of colocalization ratio, analysis of combination of PvuII + EcoRV + HaeIII with varying nuclease amounts, supplementary figures (PDF)

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. ORCID

Chun-yang Zhang: 0000-0002-8010-1981 Author Contributions ‡

These authors contributed equally.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grant Nos. 21527811, 21735003, and 21575152) and the Award for Team Leader Program of Taishan Scholars of Shandong Province, China. REFERENCES (1) Seeman, N. C. DNA in a Material World. Nature 2003, 421, 427−431. (2) Pinheiro, A. V.; Han, D.; Shih, W. M.; Yan, H. Challenges and Opportunities for Structural DNA Nanotechnology. Nat. Nanotechnol. 2011, 6, 763−772. (3) Pei, H.; Lu, N.; Wen, Y.; Song, S.; Liu, Y.; Yan, H.; Fan, C. A DNA Nanostructure-Based Biomolecular Probe Carrier Platform for Electrochemical Biosensing. Adv. Mater. 2010, 22, 4754−4758. (4) Zhang, Y.; Shuai, Z.; Zhou, H.; Luo, Z.; Liu, B.; Zhang, Y.; Zhang, L.; Chen, S.; Chao, J.; Weng, L.; Fan, Q.; Fan, C.; Huang, W.; Wang, L. Single-Molecule Analysis of MicroRNA and Logic 7200

DOI: 10.1021/acsnano.9b02679 ACS Nano 2019, 13, 7191−7201

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DOI: 10.1021/acsnano.9b02679 ACS Nano 2019, 13, 7191−7201