I-Motif-Based in situ Bipedal Hybridization Chain Reaction for

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I-Motif-Based in situ Bipedal Hybridization Chain Reaction for Specific Activatable Imaging and Enhanced Delivery of Antisense Oligonucleotides Wenjie Ma, Biao Chen, Shanzi Zou, Ruichen Jia, Hong Cheng, Jin Huang, Huizhen Wang, Xiaoxiao He, and Kemin Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b03420 • Publication Date (Web): 03 Sep 2019 Downloaded from pubs.acs.org on September 3, 2019

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Analytical Chemistry

I-Motif-Based in situ Bipedal Hybridization Chain Reaction for Specific Activatable Imaging and Enhanced Delivery of Antisense Oligonucleotides Wenjie Ma, Biao Chen, Shanzi Zou, Ruichen Jia, Hong Cheng, Jin Huang, Huizhen Wang, Xiaoxiao He∗ and Kemin Wang∗ State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, College of Chemistry and Chemical Engineering, Hunan University, Key Laboratory for Bio-Nanotechnology and Molecule Engineering of Hunan Province, Changsha 410082, China ABSTRACT: The efficient and precise delivery of antisense oligonucleotides (ASOs) to target cells is of great value in gene silencing. However, the specificity and packaging capacity of delivery system still remains challenging. Here, we designed an imotif forming-initiated in situ bipedal hybridization chain reaction (pH-Apt-BiHCR) amplification strategy for specific target cells imaging and enhanced gene delivery of ASOs. As a proof of concept, an 8-nt ASO modified with locked nucleic acid (LNA) which is complementary to the seed region of microRNA21 (miR-21) was used for gene silencing studies. Benefiting from the design of hairpin-contained i-motif, the stimuli-responsive assembly of pH-Apt-BiHCR was successfully achieved on MCF-7 cells surface based on the specific recognition of aptamer. Using this strategy, the pH-Apt-BiHCR not only contains repeated fluorescence resonance energy transfer (FRET) units for activatable tumor imaging with high contrast, but also arrays plenty of LNA ASOs as interference molecules for cancer cells inhibition. An in vitro assay showed that this strategy presented an excellent response ability in buffer within a narrow pH range (6.0-7.0) with a transition midpoint (pHT) of 6.44 ± 0.06. Moreover, live cell studies revealed that it realized a specific activatable imaging of target cells, while the ASOs arrayed pH-Apt-BiHCR exhibited improved internalization via an endocytosis pathway and enhanced gene silencing to MCF-7 cells compared to single ASO alone. We believe that this design will inspire the development of novel probes for early diagnosis and therapy of cancer cells.

Gene silencing, a molecular process involved in the down regulation of specific genes which leads to the inactivation of previously active genes in an organism, is a promising approach for the treatment of various diseases, such as cancers, Alzheimer's, cardiovascular disease and neurodegenerative diseases.1-4 In recent years, a series of oligonucleotide drugs include Ribozymes, DNAzymes, siRNAs and antisense oligonucleotides (ASOs) have been widely used as potential therapeutic agents for cancer treatment, due to their potent and directed silencing of a gene of interest.5-8 Compared to various oligonucleotide drugs, ASO, an 8-50 nucleotides singlestranded sequence which can intracellularly bind to the target RNA (miRNA, piRNA, mRNA, etc) by means of standard Watson-Crick base pairing, has been proven to be a powerful and selective method for gene expression modulation owing to the advantages of automated synthesis and low cost.9,10 The traditional approach to gene delivery is direct transfer with naked plasmid DNA.11 However, naked genes showed inefficient cellular uptake and susceptibility to degradation by nucleases,12 which largely limits its applications. In addition, viral vectors have been widely used for their efficient gene transfection, there are, nevertheless, concerns and limitations in its further clinical transformation as well, including mutagenic toxicity, immunogenicity, limited DNA packaging capacity and difficulty of vector production.13 Therefore, the

development of safe nonviral vectors capable of efficient transport and elevated therapeutics is of high significance and demand. In this regard, a great deal of nonviral vectors have been developed for their ASOs delivery potential, such as nanoparticles,14 polymers,15 dendrimers,16 hydrogel,17 lipid,18 and DNA/RNA nanostructures.19-22 Among them, DNA nanostructures have been explored as strong delivery vector candidates because of their biocompatibility, flexibility and spatial addressability. For example, Sleiman et al. reported a method to generate a set of 3D DNA prisms with integrated ASO binding regions.19 It showed that ASOs scaffolded on DNA cages can readily induce gene silencing in mammalian cells, which is significantly greater than that using singlestranded ASO alone. Bermudez et al. designed a DNA pyramid displaying antisense motifs, which is able to specifically degrade mRNA and inhibit protein expression in vitro, and they showed improved cell uptake and gene silencing when compared to linear DNA.20 Nevertheless, these strategies need to integrate ASOs into DNA nanostructures, the limited ASOs payload capacity and sophisticated design have hampered the transition to clinical application. As an alternative, hybridization chain reaction (HCR), a powerful amplification strategy, has attracted enormous attention in

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nucleic acids delivery because of its high payload capacity and simple protocols.23,24 Motivated by these advantages, we presented a DNA nanocentipede as multivalent vehicles for enhanced delivery of CpG oligonucleotides and successively initiated immune responses.25 However, such a strategy need to synthesize HCR products in advance, which was even left at room temperature for 24 h. The time-consuming synthesis process and nonspecific adsorption are the major obstacles to their widespread use. Taking all these into account, it is of great significance to develop an in situ stimuli-responsive delivery system with high payload capacity and high specificity. DNA i-motif, as one of the promising stimuli-responsive molecule tools, which can fold into tetraplex structure via a stack of intercalating hemiprotonated C-neutral C base pairs (C+ : C) in slightly acidic environment of the tumor, has sparked a great deal of interest of researchers.26-29 Thus far, several i-motif based strategies have been proposed for pHtriggered in situ tumor imaging.30-32 These encouraging attempts at stimuli-responsive imaging highlight their unique advantages of low-background and high-contrast. Moreover, the widespread use of i-motif structure offers a good opportunity for constructing new types of responsive systems and providing more precise and controllable DNA-based building blocks. We thereupon envisage that it might significantly improve imaging contrast and payload capacity if the i-motif could be used as potential “triggers” for in situ HCR amplification imaging and intracellular genes delivery. With this in mind, taking advantage of the design concept of hairpin-contained i-motif we have reported previously,33 and combining the programmability of DNA nanostructures,34,35 here for the first time, a novel aptamer-targeted, i-motif forming-initiated in situ bipedal hybridization chain reaction (pH-Apt-BiHCR) amplification strategy was developed for specific imaging and enhanced gene delivery of ASOs. As illustrated in Scheme 1, three single-stranded DNA were custom-designed. One is Apt-T-R consisting of three different DNA fragments, the aptamer sequence targeting MCF-7 cells, the complementary of T-L and the trigger sequence of HCR. Another is T-L, composed of a short DNA fragment complementary to Apt-T-R partially and another trigger sequence of HCR. The third one is I-M6 strand, the i-motif sequence embedding an internal hairpin. By engineering hairpin-contained i-motif structures rationally, the pH response ranges can be precisely tuned. At neutral pH, these three single-stranded DNA self-assembled into a stable Yshaped DNA structure (named as pH-Apt-Y6), resulting in two blocked trigger fragments of HCR, thus causing a very weak HCR signal of background. Once encountering target cancer cells, the aptamer domain of pH-Apt-Y6 can recognize the target receptor on the cell surface, while the I-M6 tends to form an intact intramolecular i-motif and falls down from pHApt-Y6 due to the low extracellular pH,36 thus leading to the liberation of triggers. As a result, in response to the specific target-i-motif interaction, a pH-initiated in situ BiHCR (pHApt-BiHCR) was achieved. In brief, the exposed fragment in Apt-T-R initiates the HCR of H1 and H2 (H1 first), while another HCR initiated from H2 first by fragment in T-L. Here the H1 and H2 were skillfully designed and modified with Cy5

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and Cy3, respectively. Each H1-H2 pair hybridization event leads to fluorescence resonance energy transfer (FRET) signal generation, resulting in an activatable cell imaging with high contrast. Particularly, an 8-nt locked nucleic acid (LNA) was extended at the end of H1 (LNA-H1) as antisense agent for gene silencing.21,22 So that the pH-Apt-BiHCR could also array a mass of ASOs modified with LNAs, which was internalized into cells through receptor-mediated endocytosis after a period of incubation, and induced gene silencing to tumor cells via complementation of the seed region of microRNA21 (miR21).21,22,37,38 As an activatable probe, the pH-Apt-BiHCR provides a potential platform for the amplified imaging and enhanced delivery of oligonucleotide drugs.

Scheme 1. Structure and working principle of the pH-Apt-BiHCR strategy for specific activatable imaging and enhanced delivery of antisense oligonucleotides.

EXPERIMENTAL SECTION Chemicals and reagents. All the oligonucleotides used in this study were custom-designed and then synthesized by Sangon Biotechnology Co., Ltd. (Shanghai, China). Before use, all the DNA probes were purified with high performance liquid chromatography (HPLC) and further identified using mass spectrometry. The sequences of oligonucleotides were listed in Table S1 (Supporting Information). SYBR Gold was purchased from Thermo Fisher Scientific (MA, USA). 35 mm glass bottom dishes were purchased from MatTek (MA, USA). MTS Cell Proliferation Colorimetric Assay Kit was purchased from Promega (WI, USA). Annexin V-FITC/PI Apoptosis Detection Kit and Trizol reagent were obtained from Sangon Biotechnology Co., Ltd. (Shanghai, China). The buffer for in vitro experiments was phosphate buffer saline (PBS, 10 mM) at a given pH, which was prepared by mixing 100 mM NaH2PO4·2H2O and 100 mM Na2HPO4·12H2O in a proper ratio, then added with 130 mM NaCl, 4.6 mM KCl and 5 mM MgCl2. The pH of all solutions was calibrated with a San-Xin MP511 miniature pH meter (Shanghai, China). All reagents were used as received without further purification. All solutions were prepared using deionized water, obtained by

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Analytical Chemistry the Milli-Q ultrapure water system (Barnstead/Thermolyne NANO pure, Dubuque, IA, USA). Preparation of pH-Apt-Y6. The oligonucleotides of AptT-R, T-L and I-M6 were firstly mixed at a molar ratio of 1:1:1 in PBS (pH 7.4), and heated at 95 °C for 5 min. Then, cooled to room temperature slowly to construct pH-Apt-Y6 probe. Gel electrophoresis analysis. 2% agarose gel electrophoresis was employed to verify the stepwise assembly of pH-Apt-Y6 in pH 7.4, and 12% native polyacrylamide gel electrophoresis (PAGE) was used for determining the feasibility of HCR. Firstly, the hairpin probes of H1 and H2 were annealed at 95 °C for 5 min and then cooled down to room temperature over hours to enable the probe to perfectly fold into a hairpin structure. Next, the pH-Apt-BiHCR (125 nM pH-Apt-Y6 + 1 μM H1 + 1 μM H2) was incubated at 37 °C for 2 h for electrophoretic characterization. The gels were performed at a constant potential of 100 V in 1 × TBE buffer (8.9 mM Tris-Boric acid, 0.2 mM EDTA, pH 8.3) at room temperature for 1 h to agarose gel electrophoresis and 2 h to PAGE, respectively. All the samples were stained with 100 × SYBR Gold and then photographed by using an Azure C600 Imaging Biosystems (California, USA). UV-vis spectrum measurement. Generally, the I-M6 and pH-Apt-Y6 were both diluted to 250 nM in 10 mM PBS buffer with different pH. Then, UV-vis absorption spectra of each probe were collected in the 220-320 nm range with a data interval of 1 nm by a Biospec-nano UV-vis spectrophotometer (Kyoto, Japan). CD measurement. Circular dichroism (CD) spectra were acquired in the range of 220-320 nm at room temperature on a Bio-Logic MOS-500 CD spectrophotometer (Claix, France). The pH-Apt-Y6 and I-M6 were diluted to 500 nM and 2 μM in 10 mM PBS with different pH, respectively. In each case, the background of the buffer solution was subtracted from the CD data and processed using Sigma Plot software. Fluorescence analysis. The pH-Apt-BiHCR was performed in 100 μL of reaction mixture containing 25 nM pH-Apt-Y6, 200 nM H1 and 200 nM H2 at different PBS buffer (pH 5.68.0) to characterize its pH response performance. Before the reaction, each hairpin probe of H1 and H2 was annealed at 95 °C for 5 min and then slowly cooled to room temperature. After annealing, the hairpin probes were mixed with pH-AptY6 and incubated at 37 °C for 2 h. Then, the fluorescent spectra were collected from 550 to 750 nm in 1 nm increments while excited at 535 nm using a QuantaMaster™ fluorescence spectrophotometer (PTI, Canada), and the FRET spectra were normalized to the maximum Cy3 donor peak around 565 nm. For the stability of fluorophores in different pH solutions, we investigated the effect of pH value (5.0-8.0) on the fluorescence intensity of Cy5 and Cy3 labeled on the hairpin probes of H1 and H2, respectively. The excitation wavelength of Cy3 was 535 nm while the emission spectra were collected from 550 to 650 nm in 1 nm increments. The excitation wavelength of Cy5 was 635 nm while the emission spectra were collected from 650 to 750 nm in 1 nm increments. Besides, for the real-time fluorescence intensity investigation of HCR under different triggers, the excitation wavelength was set at 535 nm, and the emission spectra were collected at

665 nm over time. The concentration of Apt-T-R + N-L, AptN-R + T-L and Apt-T-R + T-L was 25 nM, the concentration of H1 and H2 was 200 nM. For all the fluorescence experiments, the slit was set to be 10 nm for both of the excitation and emission. Cells culture. MCF-7 cells (human breast cancer cell line) were cultured in Dulbecco's modified Eagles medium (DMEM) supplemented with 12% (v/v) fetal bovine serum (FBS), and MCF-10A cells (normal human mammary epithelial cell line) were grown in Mammary Epithelia cell medium (MEpiCM). Then all cells were maintained at 37 °C in a 5% CO2 atmosphere in humidified HF90 CO2 incubator (Shanghai Lishen Scientific Equipment Co., Ltd.). Confocal fluorescence microscopy imaging. Cells were plated in 35 mm glass bottomed culture dishes and grown for 24 h to reach ~70% confluency. The dishes were washed three times with PBS before treatment. To investigate the specific activation and pH-responsiveness, cells were incubated with pH-Apt-BiHCR (50 nM pH-Apt-Y6 + 400 nM H1 + 400 nM H2) at 37 °C for 2 h. Subsequently, cells were washed three times with PBS and then resuspended in PBS (pH 7.4) for microscopic imaging. Control cell experiments were performed using the same procedure as above. For the investigation of cellular uptake ability, cells were incubated with pH-Apt-BiHCR (50 nM pH-Apt-Y6 + 400 nM H1 + 400 nM H2) at 37 °C for 0, 1, 2, 4, 5 and 6 h, respectively. Then, resuspended in PBS for microscopic observation after three times wash. Fluorescent images were acquired on an A1 + Ti2 confocal microscope (Nikon, Japan) with a 100 × oil immersion objective. Excitation and emission wavelength were described as follows. Cy3: Ex = 561 nm, Em = 570-620 nm bandpass (green fluorescence channel); Cy5: Ex = 561 nm, Em = 663-738 nm bandpass (red fluorescence channel). The Pearson’s correction coefficient was calculated by software Image Proplus 6.0. Cytotoxicity assays. For the viability assay, cells (1 × 104 cells/well) were cultivated in 200 μL media in each well of a 96-well plate for 12 h at 37 °C in a humidified atmosphere containing 5% CO2. After discarding the original media, each well was washed one time with PBS and then 100 μL of PBS with 1% FBS alone or PBS with 1% FBS containing different DNA samples was added to each well and cultured for 5 h. Then, removed the PBS solution and 200 μL fresh medium was added to each well and cultured for 43 h. Subsequently, 20 μL MTS and 100 μL fresh medium (without FBS) were added to each well with incubation at 37 °C for 2 h after removal of the old medium. Finally, the absorption of the solution for each well was measured at 490 nm using a microplate reader (BioTek Instrument, Inc., USA) to determine cell viability after another vibration for 10 min. The cell viability was calculated as follows: viability (%) = (OD treated - OD blank)/(OD control - OD blank) × 100, where OD treated was acquired from the cells treated with different DNA samples, OD control was obtained from the cells treated with PBS and OD blank was got from the well plates without cells or PBS. Apoptosis assays. Cell apoptosis was detected using the Annexin V-FITC/PI assay. Briefly, the MCF-7 cells were seeded in 1 mL media in each well of a 12-well plate for 12 h

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at 37 °C in a humidified atmosphere containing 5% CO2, then incubated with different samples for 5 h. After removing the old solution, 1 mL fresh medium was added to each well and cultured for 43 h. Next, the cells were harvested and treated with Annexin V-FITC/PI according to the manufacture’s instruction, and then subjected to flow cytometric analysis with a Gallios cytometer (Beckman Coulter, USA) by counting 20,000 events. RT-PCR quantification of miR-21 expression. To quantify the relative expression of miR-21 in MCF-7 cells, cells were treated with different samples (PBS with 1% FBS, 400 nM LNA-H1, 50 nM pH-Apt-Y6 + 400 nM LNA-H1 + 400 nM N-H2). Total cellular RNA was extracted from MCF7 cells using Trizol reagent according to its manual, then stored at -80 °C for the quantitative real time polymerase chain reaction detection (qRT-PCR, Sangon Co. Ltd., Shanghai, China). Western blot analysis of Bcl-2. Generally, cells were lysed in ice-cold RIPA buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1% SDS, 0.5% sodium deoxycholate) supplemented with protease inhibitor mixture after treated with different samples as described in the RT-PCR assay. The lysates were placed on ice for 30 min and then centrifuged for 15 min with the speed of 12,000 rpm to remove cell debris. Total cellular proteins were resolved on a 12% SDS-PAGE and transferred to a PVDF membrane, blocked with 5% skimmed milk in PBS, and incubated with antibody against the Bcl-2 at 4 °C for 12 h. The membrane was washed three times with PBS containing Tween-20 (0.1%) followed by incubation with horseradish peroxidase-conjugated secondary antibody diluted 1:2,000. Then, the membrane was washed three times and proteins were visualized with the SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, USA).

RESULTS AND DISCUSSION Design and characterization of BiHCR. Taking the advantage of favorable features of DNA, we designed and realized the construction of BiHCR strategy based on the same hairpins but different triggers for the first time. This approach is expected to solve the problem of low trigger efficiency of HCR to a certain extent, and it only requires a pair of hairpins, which simplifies the design. Electrophoresis was firstly preformed to verify the feasibility of BiHCR strategy. As shown in Figure 1A, in the absence of Apt-T-R or T-L, the mixture of H1 and H2 only showed one bright band, which was ascribed to the same mobility of H1 and H2. When the mixture was incubated with trigger Apt-T-R or T-L (Lane 4 or 5), various lengths of large molecular weight products appear as a bright band in this case, evidencing the assembly of many H1 and H2 to form chain-like DNA polymers. Interestingly, a larger HCR product appeared when the mixture was exposed to both triggers Apt-T-R and T-L (Lane 6), compared to any single HCR (Lane 4 or 5). The results clearly demonstrated the feasibility of our proposed BiHCR strategy. Further, the fluorescence intensity of different HCR were assessed in pH 7.4 by using pH-insensitive Cy3 and Cy5 as the FRET pairs (Figure S1 and S2). As illustrated in Figure 1B, the H1 and H2 could coexist stably in solution and merely gave a very

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low background fluorescence signal. In the co-presence of Apt-T-R and T-L, the efficiency of FRET was higher than any one of the single trigger to Apt-T-R or T-L, which was in line with the result of electrophoresis. In addition, the real-time of fluorescence intensity changes of HCR under different triggers were performed to further conform the advantages of BiHCR. As displayed in Figure S3, N-L represents the sequence on the left side of DNA scaffold without the trigger, while Apt-N-R represents the sequence on the right side without the trigger. Obviously, the reaction kinetics of BiHCR was significantly faster than that of single HCR. These gave direct evidence for efficient amplification of the BiHCR strategy.

Figure 1 (A) 12% PAGE gel electrophoresis analysis of different DNA samples. Lane M, 20 bp DNA marker; Lane 1, 1 μM H1; Lane 2, 1 μM H2; Lane 3, 1 μM H1 + 1 μM H2; Lane 4, 250 nM Apt-T-R + 1 μM H1 + 1 μM H2; Lane 5, 250 nM T-L + 1 μM H1 + 1 μM H2; Lane 6, 125 nM Apt-T-R + 125 nM T-L + 1 μM H1 + 1 μM H2. (B) The fluorescence behavior of different DNA samples. Black line, 200 nM H1 + 200 nM H2; Red line, 50 nM Apt-T-R + 200 nM H1 + 200 nM H2; Green line, 50 nM T-L + 200 nM H1 + 200 nM H2; Blue line, 25 nM Apt-T-R + 25 nM TL + 200 nM H1 + 200 nM H2. All the samples were incubated at 37 °C for 2 h.

Probe construction and activation of pH-Apt-Y6. To screen an i-motif sequence with excellent responsive behavior to slight acidity on pH-Apt-Y6 at 37 °C, seven sets of different i-motif probes were firstly designed by varying the sequence of i-loops and the lengths of C-tracts. A series of electrophoresis and fluorescence experiments were used to optimize i-motif sequence. By examining the activation of imotif structure on pH-Apt-Y6 at different pH (pH 5.6 and 8.0) as well as the pH-initiated amplification of BiHCR, we found that the i-motif sequence of I-M6 assembled on pH-Apt-Y6 gave desired pH-responsiveness (Figure S4). Subsequently, the stepwise assembly of pH-Apt-Y6 was confirmed by 2% agarose gel electrophoresis. As shown in Figure S5, Lane 2 represented T-L, a trigger of BiHCR on the left of Y-shaped DNA scaffold, showing a greater mobility than Lane 3 (Apt-T-R, another trigger of BiHCR on the right of Y-shaped DNA scaffold with the extended aptamer recognition sequence). Lane 4 showed one brighter band, which was ascribed to the hybridization of T-L and Apt-T-R. It was worth noting that, in the presence of I-M6, a distinct band lag was observed in Lane 5, suggesting the successful and efficient self-assembly of pH-Apt-Y6 with increased mass and geometrical size. Then, we verified the conformational changes of the I-M6, which could be assembled on pH-Apt-Y6 at neutral pH value. With the pH decreasing from 8.0 to 5.6, the UV absorption at 295 nm increases gradually, presenting a

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Analytical Chemistry sharp pH transition from random coil to i-motif structure.39,40 Further, the pH-value-stimulated conformational activation of i-motif structure on pH-Apt-Y6 was also characterized by UV absorption spectra at different pH. As we can see, the result is consistent with the behavior of I-M6 in different pH values, indicating that changes in pH can cause the activation of imotif structure on pH-Apt-Y6 (Figure S6A and B). In addition, the pH-triggered conformational transition of i-motif structure on I-M6 was demonstrated by comparing the CD spectra at pH 5.6 and 8.0, as well as the activation of i-motif structure on pH-Apt-Y6. As shown in Figure S6C and D, both of them exhibited the same CD phenomenon under various pH values. With the pH changed from 8.0 to 5.6, the positive band near 275 nm and the negative band near 245 nm were redshifted to near 290 and 260 nm, respectively, suggesting the formation of intact i-motif structure in acidic environment. It is agreed with the characteristics of i-motif structure as reported previously.41,42 These data gave clear evidence for that the i-motif tetraplex structure can be well formed in acidic environment.

the bands in Lane 7, 8 and 9, with similar mobility but the brightness was increasing, indicating that the remaining amount of pH-Apt-Y6 in the system was gradually increasing. That is, the efficiency of HCR was gradually weakened with the increase of pH value. In addition, we verified this phenomenon with 8% PAGE gel electrophoresis under the same conditions (Figure S7). These results substantially manifested the feasibility of the pH-Apt- BiHCR can be achieved at acidic pH. Subsequently, we validated the pH responsiveness of pHApt-BiHCR, and the fluorescence profile of pH-Apt-BiHCR was assessed within the pH interval of 5.6-8.0 at 37 °C. As shown in Figure 3A, all spectra were obtained by irradiating solutions at 535 nm, corresponding to the excitation wavelength of the FRET donor, Cy3. Sequential increases in the fluorescence emission intensity of the acceptor (Cy5) was observed with decreasing pH, suggesting that the decrease of pH could protonate the I-M6 to tend to form an intact intramolecular i-motif and fall down from pH-Apt-Y6, thus leading to the cascaded hybridization of H1 with H2 yields the long DNA polymers carrying a large number of adjacent Cy3 and Cy5, producing a remarkably FRET signals. The results illustrated excellent FRET signal change according to different pH of the pH-Apt-Y6 exposed to H1 and H2. Notably, the plot of acceptor-to-donor ratios (FA/FD) of Cy5/Cy3 as a function of pH showed a characteristic sigmoidal decrease from pH 5.6 to 8.0, with the sharp signal transition within the range of pH 6.0-7.0 (Figure 3B), indicating an ultra-pH-responsiveness to slight acidity. Furthermore, the results showed that the transition midpoint (pHT) of pH-Apt-BiHCR was 6.44 ± 0.06, which might promise its application in early diagnosis of tumor cells.

Figure 2. 12% PAGE gel electrophoresis verifies the feasibility of pH-Apt-BiHCR under different conditions. Lane M, 20 bp DNA marker; Lane 1, 1 μM H1; Lane 2, 1 μM H2; Lane 3, 1 μM H1 + 1 μM H2; Lane 4, 250 nM Apt-T-R + 1 μM H1 + 1 μM H2; Lane 5, 250 nM T-L + 1 μM H1 + 1 μM H2; Lane 6, 125 nM Apt-T-R + 125 nM T-L + 1 μM H1 + 1 μM H2; Lane 7, 125 nM pH-AptY6 + 1 μM H1 + 1 μM H2, pH 5.6; Lane 8, 125 nM pH-Apt-Y6 + 1 μM H1 + 1 μM H2, pH 6.5; Lane 9, 125 nM pH-Apt-Y6 + 1 μM H1 + 1 μM H2, pH 7.4. All the samples were incubated at 37 °C for 2 h.

Feasibility of pH-initiated BiHCR (pH-Apt-BiHCR). Motivated by the above experimental phenomena, we next explored the feasibility of the pH-Apt-BiHCR amplification under different hybridization conditions, 12% PAGE gel electrophoresis analysis was performed to ascertain the formation of HCR. As depicted in Figure 2, Lane 4 and 5 represented different HCR triggered by Apt-T-R and T-L, respectively. Lane 6 represented the HCR exposed to both triggers Apt-T-R and T-L. Obviously, the mixture incubated with pH-Apt-Y6 displayed bright bands at pH 5.6 (Lane 7), which was similar to that exposed to both triggers Apt-T-R and T-L (Lane 6), suggesting HCR occurred efficiently at acidic pH. Moreover, it was worth noting that the mixture incubated with pH-Apt-Y6 displayed varied bright bands near the sample hole at different pH values. With a brighter band at pH 5.6 and gradually weakened bands at pH 6.5 and 7.4, suggesting that the feasibility of this HCR system could be achieved under acidic conditions. Meanwhile, we noted that

Figure 3. Investigation of pH-response performance. (A) Fluorescence profile of the pH-Apt-BiHCR (25 nM pH-Apt-Y6 + 200 nM H1 + 200 nM H2) responding to different PBS at pH 5.68.0, the spectra were normalized to the maximum Cy3 donor peak around 565 nm. (B) Plot of FA/FD as a function of pH in PBS. The excitation wavelengths of Cy3 was 535 nm while the emission spectra were collected from 550 to 750 nm. The error bars denote the standard deviation of three replicate measurements.

Extracellular pH-Apt-BiHCR initiation and imaging applications. In view of the ubiquitous acidic tumor microenvironment, we further inspected the feasibility of pHApt-BiHCR strategy for specific tumor cells imaging. MCF-7 cells were incubated with pH-Apt-BiHCR at different pH, then the cellular fluorescence signal was monitored using a laser confocal microscopy. When binding to the target cells, the pHApt-Y6 underwent structure switching due to the slightly acidic environment around the cells. Then the I-M6 folded into an intramolecular i-motif structure and fell down from pH-

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Apt-Y6, thus leading to the liberation of two single-stranded overhangs acted as initiator sequences, which could trigger a real-time BiHCR with probes H1 and H2. Hence, an amplified FRET signal was activated in pH-Apt-BiHCR due to the proximity of Cy3 and Cy5, enabling real-time in situ imaging of tumor cells. As shown in Figure 4A, after incubation for 2 h, gradually enhanced fluorescent intensities in Cy5 were observed on MCF-7 cells surface with the decrease of pH from 7.4 to 5.6, which was in line with the results in buffer. Whereas the fluorescence intensity of the Cy3 in extracellular was also increased gradually, which was not in agreement with that the fluorescence intensity of Cy3 weakened with decreasing pH in buffer. It was because that the donor of Cy3 and the acceptor of Cy5 was labeled on the hairpin probes, and the hairpin probes without HCR would be washed off before imaging. So that the fluorescence intensity of the acceptor and donor at acidic tumor microenvironment were both higher than that under alkaline conditions. Given this situation, the calibration curve of extracellular pH was obtained by calculating the average fluorescence intensity of Cy5 (FRET signal) only. Strikingly, as shown in Figure 4B, the fluorescence intensity of Cy5 showed a sigmoidal decrease against pH values from 5.6 to 7.4 on MCF-7 cells, which possessed the same sigmoid trends but a little difference between a cell free system because of a distinct working environment and apparatus.

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showed almost no signal response on the cell surface at different pH (Figure S8), further evidencing the specific response of pH-Apt-BiHCR strategy to the target cells under acidic condition. Taken together, the results thereupon confirmed the successful i-motif forming-induced imaging of our pH-Apt- BiHCR strategy with high contrast at different pH, making it a promising strategy for early diagnosis and therapy of cancer. Having demonstrated the ability of pH-Apt-BiHCR for specific imaging of MCF-7 cells, we further evaluated the internalization of pH-Apt-BiHCR as genes vector in target cells. Hence, the time-dependent confocal fluorescence imaging of MCF-7 cells was performed with pH-Apt-BiHCR at pH 6.0 in different incubation time (0, 1, 2, 4, 5 and 6 h). As shown in Figure S9, sequential increases in the fluorescence intensity of Cy5 while that of Cy3 remains constant with increasing incubation time was observed. In addition, it can be clearly observed that the fluorescence signal of Cy5 distributed to the whole cytoplasm after 5 h incubation. The images also provided evidence of the efficient binding and internalization of pH-Apt-BiHCR into MCF-7 cells through receptor mediated endocytosis, suggested that the pH-AptBiHCR is a competitive strategy for theranostic applications.

Figure 5. MTS assay of MCF-7 cells after treated with different formulations for 48 h. (A) Cell viability of MCF-7 cells after treated with different concentrations of pH-Apt-BiHCR. The details are as follows: PBS (1% FBS), 12.5 nM pH-Apt-Y6 + 100 nM LNA-H1 + 100 nM N-H2, 50 nM pH-Apt-Y6 + 400 nM LNA-H1 + 400 nM N-H2, 100 nM pH-Apt-Y6 + 800 nM LNAH1 + 800 nM N-H2. (B) Cell viability of MCF-7 cells after treated with different concentrations of LNA-H1.

Figure 4. Confocal fluorescence imaging of MCF-7 cells. (A) Fluorescent images of MCF-7 cells treated with pH-Apt-BiHCR (50 nM pH-Apt-Y6 + 400 nM H1 + 400 nM H2) at different pH (pH 5.6, 6.0, 6.2, 6.4, 6.8, 7.0 and 7.4). Ex = 561 nm, 570-620 nm emission band for Cy3 (green) and 663-738 nm emission band for Cy5 (red). (B) Intensity of the FRET signal versus extracellular pH (R2 = 0.9980).

Next, we investigated the specificity of pH-Apt-BiHCR strategy. The pH-Y6, a control probe for pH-Apt-Y6 designed without aptamer recognition sequence but sensitive to pH and BiHCR (pH-BiHCR), exhibited negligible fluorescence signal response to pH variation due to its poor affinity to target cells. In the meantime, the normal immortalized human mammary epithelial cell line MCF-10A, used as the negative control cell,

Cell apoptosis assays and gene silencing. As a proof of concept, an 8-nt ASO modified with LNA which is complementary to the miR-21 seed region was incorporated in the substrates of BiHCR as antisense agent for cancer cells inhibition. MTS assays were firstly conducted to assess the cell viability of ASOs arrayed pH-Apt-BiHCR with different concentrations. As displayed in Figure 5A, under acidic pH conditions, the pH-Apt-BiHCR resulted in a remarkable inhibition of cell proliferation in a dose-dependent manner, which was much more pronounced than that under alkaline conditions. It was suggested that the pH-Apt-BiHCR could be achieved under acidic conditions, thereby carrying ASOs into the cells and inhibiting cell proliferation activity by silencing miR-21. Of note, obvious inhibition with an efficiency up to 40% could be observed for 50 nM pH-Apt-Y6 initiated BiHCR (Figure S10). In contrast, there was negligible inhibition in MCF-7 cell growth after incubation with single LNA-H1 because of the inefficient cellular uptake (Figure 5B).43 Further, we investigated the effect of pure pH-Apt-

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Analytical Chemistry BiHCR (pH-Apt-Y6 + N-H1 + N-H2) on cell viability, where the H1 was used without any modification (denoted as N-H1). As shown in Figure S11, pure pH-Apt-BiHCR showed inappreciable cytotoxicity, indicating that the pH-Apt-BiHCR could be a safe and effective strategy for enhanced ASOs delivery. To further verify the suppressive efficiency of pH-AptBiHCR, cell apoptosis was characterized using the Annexin V-FITC/PI assay. As shown in Figure 6A, the cell viability was low to 77% for pH-Apt-BiHCR in pH 6.0, which was significantly lower than that treated with different samples in pH 7.4 (more than 90% viability). It possessed the same trends but a little difference between MTS assays because of a distinct post processing and instruments. Further, the relative expression of miR-21 in MCF-7 cells was measured using RTPCR analysis. In contrast to cells treated with PBS or single LNA-H1, the relative expression level of miR-21 was significantly suppressed in which treated with pH-Apt-BiHCR at pH 6.0 (Figure 6B), implying the effective miR-21 silence by improving delivery efficacy by BiHCR strategy. Moreover, Bcl-2 is a direct participant in the apoptosis pathway, it is reported that miR-21 may upregulate Bcl-2, inhibiting apoptosis.44,45 Therefore, the expression of Bcl-2 was examined by western blot assay at the protein level. As shown in Figure 6C, the expression of Bcl-2 deceased obviously for cells treated with pH-Apt-BiHCR at pH 6.0 compared to cells treated with PBS or single LNA-H1, suggesting that the downregulation of miR-21 will result in decreasing expression of Bcl-2 protein. These results demonstrated that BiHCR strategy has the potential to be applied for clinical applications as a targeted therapeutic delivery system to treat cancer in vivo.

PBS (1% FBS), 400 nM LNA-H1 and 50 nM pH-Apt-Y6 + 400 nM LNA-H1 + 400 nM N-H2.

CONCLUSION In summary, we have presented a novel pH-Apt-BiHCR strategy based on the specific recognition of aptamer while an i-motif formation triggered BiHCR on the cell surface, which realizes real-time activation for high-contrast fluorescence imaging and efficient gene silencing of cancer cells. To our knowledge, this is the first time that BiHCR amplification has been realized in situ initiated by i-motif formation. An in vitro assay showed that the pH-Apt-BiHCR could be initiated under acidic conditions and presented an excellent response ability within a narrow pH range 6.0-7.0 in buffer. Confocal imaging revealed that the stimuli-responsive assembly of pH-AptBiHCR could be successfully achieved on the surface of target MCF-7 cells with high imaging contrast. Meanwhile, plenty of 8-nt ASOs were arrayed on pH-Apt-BiHCR as antisense agent, which could be internalized into cells via an endocytosis pathway and complementary to the seed region of miR-21 for gene silencing. Collectively, the results suggested that this pH-Apt-BiHCR strategy showed improved cell uptake and gene silencing compared to single ASO alone, which holds great potential application in early diagnosis and therapy of cancer cells.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Additional information as noted in text: Oligonucleotide sequences used in the work, fluorescence spectra of Cy3 and Cy5, stability of Cy3 and Cy5, kinetic investigation of HCR under different triggers, sequence optimization of imotif, agarose gel electrophoresis analysis of pH-Apt-Y6, UV absorption and CD spectra, 8% PAGE gel electrophoresis verifies the feasibility of pH-Apt-BiHCR, specific imaging of pH-Apt-BiHCR strategy, timedependent CLSM images of MCF-7 cells incubated with pH-Apt-BiHCR at pH 6.0, apoptosis of MCF-7 cells, biosafety of the pure pH-Apt-BiHCR. (PDF)

AUTHOR INFORMATION Corresponding Author * Tel: 86-731-88821566; Fax: 86-731-88821566; E-mail: [email protected]. * Tel: 86-731-88821566; Fax: 86-731-88821566; E-mail: [email protected].

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes Figure 6. Cell apoptosis assays and gene silencing. (A) FITCAnnexin V and propidium iodide (PI) stained cell apoptosis assay via flow cytometry. (B) RT-PCR analysis of miR-21 and (C) western blot analysis of Bcl-2 in MCF-7 cells after treated with

The authors declare no competing financial interest.

ACKNOWLEDGMENT

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This work was supported by the Natural Science Foundation of China (21675046, 21735002, 21521063, 21806186, 21775036 and 21874035) and the Key Point Research and Invention Program of Hunan Province (2017DK2011).

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Analytical Chemistry

for TOC only

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I-Motif-Based in situ Bipedal Hybridization Chain Reaction for

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