Hairpin-Constrained Structure Enables

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A Programmable DNA Ring/Hairpin-Constrained Structure Enables LigationFree Rolling Circle Amplification for Imaging mRNAs in Single Cells Wenjiao Zhou, Daxiu Li, Ruo Yuan, and Yun Xiang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05613 • Publication Date (Web): 08 Feb 2019 Downloaded from http://pubs.acs.org on February 9, 2019

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Analytical Chemistry

A Programmable DNA Ring/Hairpin-Constrained Structure Enables Ligation-Free Rolling Circle Amplification for Imaging mRNAs in Single Cells Wenjiao Zhou, Daxiu Li, Ruo Yuan and Yun Xiang* Key Laboratory of Luminescent and Real-Time Analytical Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China * Corresponding author. E-mail: [email protected] (Y. Xiang). ABSTRACT Self-assembled functional DNA structures have proven to be excellent materials for designing and implementing a variety of nanoscale devices. We demonstrate here that a rationally designed and programmable DNA ring/hairpin-constrained structure can achieve in situ ligation-free rolling circle amplification (RCA), which further leads to highly specific, sensitive and multi-color imaging of mRNA molecules in single cells. Such a structure aims at addressing current challenges in terms of simplicity, sensitivity, and multiplexing capability related to the detection and imaging of intracellular mRNA sequences. With this new DNA ring/hairpin-RCA approach, we are able to detect the target mRNAs with high sensitivity at the sub-picomolar levels in vitro. Besides, the multiplexing capability of the DNA structures can be readily realized by barcoding the DNA rings and hairpins with distinct sequences. Due to the excellent sequence recognition ability of the hairpins, the DNA structures exhibit single-base variation discrimination capability for the target mRNA and can be used to image trace amounts of down-expressed mRNAs in single cells. Moreover, drug-dependent mRNA expression variations can also be clearly differentiated by these DNA structures, highlighting the great potential of such structures for early disease diagnosis and for screening possible therapeutic drugs. ACS Paragon Plus Environment

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KEYWORDS: Constrained DNA structure; Rolling circle amplification; Messenger RNA; Multiplex; Bioimaging; INTRODUCTION DNA has been an ideal material to use for constructing complex 2D/3D architectures and versatile nanomechanical devices, due to its highly specific Watson-Crick base pairing properties.1 The construction of DNA dynamic devices is particularly important as it can enable the DNA devices to perform complex tasks. Various DNA dynamic devices, including interlocked DNA nanostructures, 2-4 walkers,5,6 tweezers,7,8 shuttles9 and logic circuits,10 have been reported. For instance, the interlocked DNA nanostructures, which are composed of DNA components that are connected to one another by interlocks, can allow for operating nanomechanical DNA devices with highly specific and controlled motions after being triggered by external stimuli, such as pH,11 light,12 ions,13 or nucleic acid strands.14 Although the dynamic functions of the interlocked DNA nanostructures that undergo transition between different states have been well demonstrated, the assembly of new dynamic DNA nanostructures with exquisite designs that can be programmed into robust in situ sensing probes (e.g., for imaging intracellular species) has not been previously established. Messenger RNA (mRNA), which is produced by the transcription of DNA and serves as the subsequent template for protein synthesis through translation, plays a central role in biology. Detecting the expression levels of mRNA is important for understanding gene regulation in cells. The expression levels of mRNA also provides insights to uncovering the molecular mechanisms of the initiation and progression of certain diseases, as well as identifying therapeutic and diagnostic targets. 15-17 Common approaches, including real-time reverse transcription polymerase chain reaction,18,19 DNA microarrays,20,21 northern blotting,22,23 and recently reported nanomaterial/enzyme-based amplification assays,24,25 can achieve sensitive detection of mRNA. However, the mRNA target molecules involved in these methods are basically isolated from groups of cells, despite the fact that mRNA expression in cells

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is often stochastic and varies from cell to cell.26,27 The mRNA expression heterogeneity thus highlights the need to detect mRNA at the single cell level.28,29 Fluorescence in situ hybridization (FISH) assays have proven to be useful methods for detecting mRNA in cells.30,31 This technique, however, encounters the limitation of resolving highly similar sequences among gene family members and is difficult for detecting trace copy numbers of mRNA among a relatively high background noise. Recent efforts have thus been directed toward developing new methods to meet these challenges.32-36 One approach was to integrate hybridization chain reaction (HCR) signal amplification into mRNA expression assay to improve the detection sensitivity.37 The DNA probe hybridizes with the target mRNA and initiates the HCR assembly of two fluorescent hairpins into long DNA polymers to produce enhanced signals for in situ detection of mRNA. Although sensitive, such a method requires a 36-hour long assay time and has an increased background noise. In another attempt, Nilsson et al. applied a well-established rolling circle amplification (RCA) strategy for sensitively detecting mRNA in cells.38 The mRNA molecules were first converted into single stranded cDNAs through reverse transcription and RNase digestion. These cDNAs further served as primers to ligate the padlock probes for subsequent RCA, resulting in an enhanced signal for mRNA detection in situ. This approach, however, needs extra target molecule conversion steps and the low padlock probe ligation efficiency represents another challenge for amplified in situ detection of mRNA. Here, we provide a proof-of-concept demonstration addressing the aforementioned limitations by using a newly designed topologically constrained DNA ring/hairpin dynamic structure that can be programmed into a robust ligation-free RCA sensor, to create a simple and efficient in situ detection method for monitoring multiple mRNAs in single cells. The target mRNA molecules bind to and open the stem-part of the hairpins thus release the topological constraints from the DNA structures. This opening of the interlocked hairpin on the DNA ring converts the DNA rings and the hairpins into suitable templates and primers, respectively, to automatically initiate the RCA reaction. The resulting amplicons with multiple repeating sequences further bind and displace the short fluorescent labeled DNA from the fluorescently quenched dsDNA signal probes through a toehold-mediated strand ACS Paragon Plus Environment

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displacement reaction (TSDR).39,40 This results in the generation of substantially enhanced fluorescence responses, enabling the monitoring of the mRNA expressions in single cells. This method has many distinct advantages over the current available assays, including the elimination of the target conversion and ligation steps in RCA, a significant reduction in the background noise, as well as the enhancement of the detection sequence selectivity with the use of hairpin recognition probes. EXPERIMENTAL SECTION Chemicals

and

Materials:

Tris-HCl,

MgCl2,

Acrylamide/bis-acrylami

(29:1,

40%),

N,N,Nʹ,Nʹ-Tetramethylethylenediamine (TEMED), 4% paraformaldehyde in PBS buffer, 20 × SSC buffer (pH 7.4), DEPC-treated water and formamide were purchased from Shanghai Sangon Biological Engineering Technology and Services Co., Ltd. (Shanghai, China). RNase Inhibitor, T4 DNA Ligase, deoxyribonucleotides mixture (dNTPs), exonuclease III, exonuclease I, 4’,6-diamidino-2-phenylindole (DAPI) and phi29 DNA polymerase were all ordered from New England Biolabs Inc. (Beijing, China, NEB). Tamoxifen and β-estradiol were purchased from Sigma Aldrich Chemical Co. Ltd (St. Louis, MO). Human breast cancer cells (MCF-7) and human cervical carcinoma cells (HeLa) were purchased from the cell bank of the type culture collection of the Chinese Academy of Sciences (Shanghai, China). The following HPLC-purified oligonucleotide sequences were custom-synthesized by Invitrogen Biotechnology Co., Ltd. (Shanghai, China). TK1 mRNA: 5’-CUG AGU UUC UGU UCU CCC UGG-3’; S-TK1 mRNA: 5’-CUG AGU UUC UAU UCU CCC UGG-3’ (The underlined and bold letter indicated the single- mismatched base.); GalNAc-T mRNA: 5’-GCU UUC ACU AUC CGC AUA AGA-3’; Hairpin DNA (TK1): 5’-CCA GGG AGA ACA GAA ACT CAG CAA TTT TTT TTT TCT GAT TGA GGT GAG TGT GAG CAG GTT TTT TTT TTT TTT TTT TTT TAA TTT CTC CCT GG-3’; Hairpin DNA (GalNAc-T): 5’-TCT TAT GCG GAT AGT GAA AGC ATT TTT TTT TTT TCT GAT TGA GGT GAG TGT GAG CAG GTT TTT TTT TTT TTT TTT TTT TTT ACC GCA TAA GA-3’; Random hairpin: 5’-CTT GGC TAC GCC CTG CTC TGT GCA TTT TTT TTT TCT GAT TAC CGT GAC TGT TAG CAT TTT TTT TTT TTT TTT TTT TTT TTT TCG TAG CCA ACS Paragon Plus Environment

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AG-3’; Linear DNA (TK1): 5’-Phos-CAC CTC AAT CAG CTT TCT TTC TTT CTT TCT TTC TTT TCC TAC GCC TAC GTT CAC TAT GTC TTT CTT TCT TTC TTT CTT TCC CTG CTC ACA CT-3’; Linear DNA (GalNAc-T): 5’-Phos-CAC CTC AAT CAG TTT CTT TCT TTC TTT CTT TCT TTT CTG TTC GCT GAC TCC TCA CAT CTT CTT TCT TTC TTT CTT TCT TTC CTG CTC ACA CT-3’; Template DNA: 5’-CTG ATT ACC GTG ACT GTT AGC ATT-3’; Cy5-FISH signal probe (TK1): 5’-CCA GGG AGA ACA GAA ACT CAG C-Cy5-3’; Cy5-DNA: 5’-TCC TAC GCC TAC GTT CAC TAT G-Cy5-3’; BHQ-2-DNA: 5’-BHQ-2-C ATA GTG AAC GTA-3’; Cy3-DNA: 5’-CTG TTC GCT GAC TCC TCA CAT C-Cy3-3’; BHQ-1-DNA: 5’-BHQ-1-GAT GTG AGG AGT C-3’; TK1 mRNA inhibitor: 5’-CCA GGG AGA ACA GAA ACT CAG-3’; GalNAc-T mRNA inhibitor: 5’-TCT TAT GCG GAT AGT GAA AGC-3’. Apparatus: The confocal fluorescence images were obtained on an Olympus FluoView 500 confocal scanning system with an Olympus IX-70 inverted microscope. All fluorescence spectra were recorded using a RF-6000 Spectro Fluorophotometer (Shimadzu, Tokyo, Japan) at room temperature. Gel electrophoresis: Native polyacrylamide (12%) and agarose gels (1.5%) were prepared in a 1 × TBE/Mg2+ (10 mM) buffer. The sample solutions were mixed with a 6 × loading buffer and loaded into the gels. The polyacrylamide gel was run at a constant voltage of 120 V for 90 min and the agarose gel was run at 60 V for 120 min. Both gels were stained with an ethidium bromide (0.5 μg mL-1) solution for about 30 min and then washed with a 1 × TBE/Mg2+ (10 mM) buffer three times. Gels were then imaged with a Bio-Rad Gel Doc XR + System (Bio-Rad, Hercules, CA). Preparation of the ring DNA: The ring DNA was prepared by mixing the linear DNA and the template DNA in equal molar concentrations (10 µM) in a Tris-HCl buffer. After that, the mixture was heated to 95 °C for 5 min and then gradually cooled down to room temperature over 2 h. Then, 5 µL of T4 DNA ligase (5 U µL-1) and 10 × T4 DNA ligase buffer (400 mM Tris-HCl, 100 mM MgCl2, 100 mM DTT, 5 mM ATP, pH 7.8) were mixed together and incubated at room temperature for 8 h to seal the nicks. After heating the mixture at 70 °C for 5 min to denature the T4 DNA ligase, exonuclease III (100 U), ACS Paragon Plus Environment

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and exonuclease I (100 U) were mixed together and incubated at 37 °C for 60 min to degrade the linear DNA components. Exonuclease III and I were further inactivated by heating the mixture at 80 °C for 15 min. The resulting ring DNA was stored at 4 °C for further use. Preparation of the DNA ring/hairpin-constrained structure: The mixture of the ring DNA (5 μM) and hairpin DNA (5 μM) in the reaction buffer was first incubated at 95 °C for 5 min, then slowly cooled down to and maintained at 65 °C for 30 min. Then, the mixture was further cooled down to 25 °C over 40 min to form the interlocked DNA ring/hairpin structure. The DNA ring/hairpin-constrained structures were loaded into a 12% native PAGE gel and run at room temperature in 1 × TBE/Mg2+ (10 mM) buffer at 120 V for 90 min, the band of DNA ring/hairpin structures were cut from the PAGE gel and eluted by using the crush and soak method. Finally, the DNA ring/hairpin structures were concentrated with Amicon Ultra 0.5 mL filters (30 K) and quantified spectroscopically. In vitro mRNA detection. The DNA ring/hairpin structure (200 nM) was first incubated with various concentrations of the target mRNA at 37 °C for 2 h. Subsequent RCA reaction was performed in 1 × phi29 reaction buffer (50 mM Tris-HCl, 4 mM DTT, 10 mM (NH4)2SO4, 10 mM MgCl2, pH 7.5) with phi29 (8 U) and dNTPs (500 μM) at 37 °C for 2 h, followed by heating the mixture at 65 °C for 10 min to stop the RCA reaction. After cooling down to 37 °C, the dsDNA signal probe (2 µM) was added and further incubated at 37 °C for 60 min. Finally, the fluorescence of the solution was obtained by the RF-6000 Spectro Fluorophotometer. Cell culture and mRNA imaging with the FISH method. The MCF-7 and HeLa cells were seeded in a 10 mm well on a 35 mm × 35 mm Petri dish (MatTek, U.S.A.) at a density of 1.0 × 10 5 cells and maintained for 48 h under a 5% CO2 environment, respectively. Then, the cells were fixed with 4% (w/v) paraformaldehyde in 1 × PBS for 15 min at room temperature and washed three times with 1 × PBS (each for 3 min). After fixation, the cells were permeabilized with 1 × PBS containing 0.5% v/v Triton-X 100 at room temperature for 5 min. This step was then followed by washing the cells three ACS Paragon Plus Environment

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times with 1 × PBS (each for 3 min). The hybridization of the FISH probes with the TK1 mRNA was conducted in a 100 μL mixture at 37 °C for 1 h. This mixture contained the Cy5-conjugated FISH signal probe (2 μM), 2 × SSC, 15% formamide, and a RNase inhibitor (100 U). After washing the cells three times with 1 × PBS, DAPI was used to stain the cells for 5 min prior to confocal imaging. In situ sensitive and multi-color imaging of mRNAs in single cells. The Petri dishes with fixed cells were permeabilized with 1 × PBS containing 0.5% v/v Triton-X 100 at room temperature for 5 min. After washing the cells three times with 1 × PBS (each for 3 min), the mixture (100 μL) of the DNA ring/hairpin structures (200 nM), 20% formamide, and 2 × SSC were added and incubated for 2 h in a humidified 37 °C incubator. This step was then followed by washing three times with 2 × SSC. Next, the RCA reaction was performed in 100 μL of the reaction solution containing a 1 × phi 29 reaction buffer (50 mM Tris-HCl, 4 mM DTT, 10 mM (NH4)2SO4, 10 mM MgCl2, pH 7.5), a RNase inhibitor (100 U), a dNTPs mixture (500 µM) and a phi 29 DNA polymerase (8 U). The reaction solution was maintained in a humidified incubator at 37 °C for 2 h. The cells were then subjected to another round of washing with 1 × PBS (each for 3 min) for three times and 2 × SSC (each for 2 min) twice. After that, 100 μL of the signal probe mixture, which was consisted of Cy5/BHQ-2 dsDNA (2 μM), Cy3/BHQ-1 dsDNA (2 μM), 2 × SSC, and 20% formamide, was added to the cells and incubated in a humidified incubator at 37 °C for 1 h. This step was followed by washing the cells with 1 × PBS (each for 3 min) for three times. The cells were then stained with a DAPI solution for 10 min and washed three times with 1 × PBS before imaging. RESULTS AND DISCUSSION


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Figure 1. A schematic illustration of the DNA ring/hairpin-constrained structures for highly sensitive and multiplexed imaging of two types of mRNAs in single cells via mRNA-initiated in situ RCA. (A) The stepwise assembly process required to form the DNA ring/hairpin-constrained structure; (B) Simultaneous sensitive detection and imaging of intracellular TK1 and GalNAc-T mRNAs in single cells. Scale bars: 20 μm. Design principle for the DNA ring/hairpin structures and the multiplexed mRNA imaging in single cells. The assembly of the topologically constrained DNA ring/hairpin structures and the subsequent usage of these structures for multiplexed imaging of the two types of mRNAs in single cells are illustrated in Figure 1. The DNA ring/hairpin structure is assembled by interlocking a hairpin and a ssDNA ring together through a programmed two-step cooling process. The stem length of the hairpin was first theoretically analyzed and optimized from 8- to 12-bp by using the IDT OligoAnalyzer online software (https://www.idtdna.com/). As listed in Table 1, the corresponding melting temperatures (Tm) of the hairpins were estimated to be 38.1 °C, 47.3 °C, 51.6 °C, 53.7 °C and 56.2 °C, respectively, while the Tm between the target GalNAc-T mRNA and the hairpin DNA was about 57.7 °C. Considering the fact that a short stem of the hairpin may reduce its stability and cause a high background noise signal, while a long stem may lead to a higher stability, which is difficult to be opened by the target mRNA, the stem length of the hairpin of 10-bp was used in our experiments. Such a hairpin probe contains a 21-nt ACS Paragon Plus Environment

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specific target mRNA recognition sequence (blue/orange) that covers the stem and part of the unpaired loop region of the hairpin, and the ring DNA with a unique barcode sequence in the middle of the unpaired region of the ring (red/green) is used for signal production. The hairpin and the ring DNA are also designed to serve as the RCA primer and template, respectively. However, the stable interlocked structure prevents the initiation of the RCA reaction. The ring DNA was first prepared by circularizing a 5’-phosphorylated linear DNA with a ligation template DNA and T4 DNA ligase. The 24-nt complementary sequences between the hairpin loop and the ring DNA were designed to form a linking duplex. The corresponding melting temperature (Tm) was estimated to be 70 °C (verified by IDT OligoAnalyzer online software). To assemble the topologically constrained DNA ring/hairpin structure, the annealing temperature of the mixture was steadily lowered from 95 °C down to 65 °C and maintained at 65 °C for 30 min. During this process, the hairpin strand unfolded first and then hybridized with the ring DNA to form a stable 24-bp linked duplex. By continually lowering the temperature down below 50 °C, the unfolded termini of the hairpin strand tended to hybridize to form the stem part of the hairpin, resulting in the formation of the constrained DNA ring/hairpin structure. In the absence of the target mRNA, the topologically constrained DNA ring/hairpin structure remains stable, and the initiation of RCA is inhibited. This minimizes the fluorescence background noise as the fluoreophore-quencher pair labeled dsDNA signal probe remained intact. In the presence of the target mRNA, the target mRNA sequence specifically binds to the recognition region of the hairpin. This process was performed in a two-step protocol: the target mRNA was firstly hybridized with the ring/hairpin complex, and then the phi29 was added for RCA reaction. An enthalpy-driven strand displacement reaction opens the hairpin stem and frees the 3’-terminus. The freed 3’-terminus is recognized and digested by the 3’ to 5’ single-stranded DNA exonucleolytic activity of phi29 DNA polymerase along the strand up to the duplex region.41,42 In this case, the bound ring DNA acts as the RCA template, the opened and 3’-end digested hairpin strand is converted into a suitable primer to allow RCA initiation. The RCA extension of the primer is catalyzed by phi29 polymerase to generate a long DNA amplicon of many repeated segments with the barcoded sequence, which is designed to be ACS Paragon Plus Environment

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fully complementary to the fluorescently labeled strand in the dsDNA signal probe. This complementarity causes the displacement of the quencher labeled strand, generating significantly amplified and distinct fluorescence signal. This enables a highly sensitive monitoring of the target mRNAs in single cells. Multiplexed and simultaneous monitoring of different mRNA targets (e.g. TK1 and GalNAc-T mRNAs) can be made possible by designing different sequences in the hairpin target recognition region and the signal probe pairing region in the ring DNA, and using multi-colored fluorephores for labeling the signal probes. Table 1 Structure analysis of hairpins (GalNAc-T) with five different stem lengths. 8-bp

9-bp

10-bp

11-bp

12-bp

ΔG = -4.83 KCal/mol





Tm = 38.1 °C

Tm = 47.3 °C

Tm = 51.6 °C

Tm = 53.7 °C

Tm = 56.2 °C

Formation of the DNA ring/hairpin-constrained structures and in vitro mRNA detection. The step-by-step assembly process for the constrained DNA ring/hairpin structures and the subsequent mRNA-initiated RCA process were first verified with gel electrophoresis. The artificially synthesized TK1 mRNA was chosen as the model target mRNA in order to trigger the RCA process. The gel images (Figure 2A) showed that the electrophoretic mobility of the hairpin (92-nt) and the ring DNA (92-nt) was much slower in comparison to that of the linear DNA (92-nt) (Lane 2, 3 vs. Lane 1), due to the increased steric hindrance caused by the stem-loop secondary structure of the hairpin and the circular conformation of the ring DNA. The incubation of phi29 with the linear DNA and the hairpin, respectively, resulted in the disappearance of the linear DNA band (Lane 4) and the intact hairpin band ACS Paragon Plus Environment

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(lane 5), which was consistent with the fact that phi29 has a specific single-stranded DNA exonucleolytic activity but is not active for double stranded blunt ends or circular DNA. A new band with a much slower mobility (Lane 6) was observed after a programmed cooling of the ring DNA and hairpin mixture. This new band indicated that a DNA ring/hairpin complex with a larger molecular weight had been successfully assembled. Further incubation of the assembled structure with phi29 and dNTPs had no effect on the mobility of the structure (Lane 7), suggesting that the DNA ring/hairpin structure was resistant to phi29 digestion and that the RCA was inhibited in the absence of the TK1 target mRNA. This result confirms the correct formation of the interlocked ring/hairpin structure. In contrast, after the incubation of the ring/hairpin complex with TK1 mRNA with the addition of dNTPs and phi29, a bright band with a significantly slower gel mobility (i.e. increased molecular weight) was observed at the top of the gel (Lane 8), which was accompanied with the disappearance of the DNA ring/hairpin band (in comparison with Lane 7). Further analysis of the reaction mixture with a 1.5% agarose gel led to consecutive bands of various sizes (Lane 9), corresponding to typical RCA amplicons of varied lengths of repeating sequences. These results demonstrate that the TK1 mRNA triggered resolution of the topological constrains of the interlocked ring/hairpin structure, allowing for RCA initiation.

Figure 2. (A) A 12% native polyacrylamide gel (Lane M-8) and a 1.5% agarose gel (Lane 9) electrophoresis characterization of the mRNA-triggered transition of the DNA ring/hairpin structures for RCA. Lane M, dsDNA ladder, 50-500 bp; Lane 1: The linear DNA (200 nM); Lane 2: The hairpin ACS Paragon Plus Environment

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probe (200 nM); Lane 3: The circularized DNA ring (200 nM); Lane 4: The mixture of linear DNA (200 nM) and phi29 (10 U); Lane 5: The mixture of the hairpin probes (200 nM) and phi29 (10 U); Lane 6: The DNA ring/hairpin structure (200 nM); Lane 7: The mixture of the DNA ring/hairpin structures (200 nM), phi29 (10 U) and dNTPs; Lane 8 and 9: The mixture of the DNA ring/hairpin structures (200 nM), TK1 mRNA (200 nM), phi29 (10 U) and dNTPs. (B) Fluorescence spectra of different reaction mixtures. (a) The dsDNA signal probe (2 μM); (b) The mixture of the DNA ring/hairpin structure (200 nM) and the dsDNA signal probe (2 μM); (c) The mixture of the DNA ring/hairpin structure (200 nM), phi29 (10 U), dsDNA signal probe (2 μM) and dNTPs; (d) The mixture of the DNA ring/hairpin structure (200 nM), phi29 (10 U), TK1 mRNA (target, 10 nM), the dsDNA signal probe (2 μM) and dNTPs; (e) The mixture of the DNA ring/hairpin structure (200 nM), phi29 (10 U), S-TK1 mRNA (non-target 10 nM), dsDNA signal probe (2 μM) and dNTPs. All reactions were performed at 37 °C for 180 min. The effects of (C) the amount of phi29 (with 180 min of RCA reaction time) and (D) the RCA reaction time (with 8 U of phi29) on the fluorescence signal gain. The potential application of the DNA ring/hairpin structure for detecting small amounts of TK1 mRNA was evaluated by introducing the fluorescently quenched partial dsDNA signal probes into the solutions after RCA. When the dsDNA signal probes were alone in the reaction buffer, they exhibited a small fluorescence emission peak (Figure 2B, curve a) at 665 nm due to the efficient quenching of Cy5 by BHQ-2. When the DNA ring/hairpin structures were mixed with the dsDNA signal probes, a negligible change of fluorescence intensity was observed (curve b vs. a), indicating that these two probes can co-exist without cross reactivity. Consistent with the gel electrophoresis results, in the absence of the TK1 mRNA target sequence, adding phi29 caused a negligible fluorescent signal change (curve c vs. b) due to the inhibition of the RCA. However, further addition of TK1 mRNA resulted in significantly enhanced fluorescence (curve d vs. c) due to the successful implementation of the RCA. In contrast, the replacement of the TK1 mRNA target with a single base-mismatched (S-TK1) mRNA as the negative control sequence led to a very small fluorescent signal change (curve e vs. c), which further verified the precise recognition ability of the ring/hairpin structure. These results therefore revealed the promising capability of this DNA ring/hairpin structure for highly selective and sensitive detection of specific mRNA sequences.


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Experimental optimizations for in vitro TK1 mRNA detection. After demonstrating the potential of the DNA ring/hairpin structure for TK1 mRNA detection, experimental conditions that affect the signal gain were further optimized. For this purpose, the amounts of phi29 used and the RCA reaction time were varied while keeping the DNA ring/hairpin structure (200 nM), dNTPs (500 µM), TK1 mRNA (10 nM) and the signal probe (2 µM) constant. The signal probe also affects the signal amplification. We have previously tried different concentrations, and the signal leveled off after 2 µM. The fluorescent signal increased with an increasing amount of phi29, from 2 to 8 U, followed by a plateau after 8 U, which indicated that within the reaction time, all reaction reached equilibrium, and the optimal amount of phi29 was 8 U (Figure 2C). Next, the dependence of the signal gain upon RCA reaction time was examined by varying RCA reaction time in the range from 30 min to 210 min with an increment of 30 min. A steady increase in the fluorescence intensity was observed (Figure 2D) when the reaction time was increased from 30 min to 120 min, and it remained almost unchanged after 120 min, suggesting that an optimal RCA reaction time is 120 min.

Figure 3. (A) The fluorescence spectra of the DNA ring/hairpin structures for potential multiplexed detection of GalNAc-T mRNA and TK1 mRNA at various concentrations (0 pM, 1 pM, 5 pM, 10 pM, 50 pM, 100 pM, 500 pM, 1 nM, and 5 nM). The calibration plots for the the fluorescence intensities vs. the logarithmic concentrations of GalNAc-T (B) and TK1 (C) respectively. Solutions were excited at 530 nm (Cy3 labeled probe for GalNAc-T) and 640 nm (Cy5 labeled probe for TK1), respectively. All fluorescence spectra were obtained after incubating the RCA amplicons with the dsDNA signal probes for 60 min at 37 °C. Error bars: SD, n = 3. ACS Paragon Plus Environment

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Feasibility of multiplexed detection of TK1 and GalNAc-T mRNAs in vitro. The dependence of the fluorescence intensity on the concentrations of the target mRNAs for multiplexed detection were examined by using two different sets of DNA ring/hairpin structures and the corresponding fluorescently quenched signal probes, Cy3 and Cy5 labeled, respectively. The resulting fluorescence intensities gradually increased at 566 and 665 nm, responding to the increasing concentration of the GalNAc-T and TK1 mRNA targets from 1 pM to 5 nM, respectively (Figure 3A). Two linear calibration curves were obtained by plotting the fluorescence intensities vs. the logarithmic concentrations of the GalNAc-T and the TK1 mRNA (Figure 3B and 3C), respectively. The detection limits were determined to be 0.48 pM for GalNAc-T and 0.56 pM for TK1, respectively, on the basis of the standard 3σ rule. Such sub-picomolar detection limits for the mRNA targets can be attributed to the integrated capability of the RCA to substantially amplify the signal with a high degree of specificity, due to the recognition of the DNA ring/hairpin for the corresponding mRNA sequences.

Figure 4. Confocal fluorescence imaging of TK1 mRNA in MCF-7 and HeLa cells obtained with the conventional FISH method (a, b) and the DNA ring/hairpin structure-based in situ RCA method (c-f). The RCA amplicons are shown in red while the cell nuclei are shown in blue, respectively; Scale bars: 20 μm. Highly sensitive and multi-color imaging of mRNAs in cells. To evaluate the feasibility of the DNA structures for imaging the intracellular mRNA, MCF-7 (over-expressed) and HeLa (down-expressed) cells with distinct TK1 mRNA expression levels were studied with the use of the ACS Paragon Plus Environment

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conventional FISH method and our DNA ring/hairpin structure coupled with RCA, respectively. With the conventional FISH approach, MCF-7 cells that had an over-expressed TK1 level showed only a very weak fluorescence signal (Figure 4a), while HeLa cells with down-expressed level of the TK1 mRNA exhibited no fluorescence (Figure 4b). This low level of fluorescence responses reveals that the FISH procedure has a limited sensitivity for imaging low to high copy numbers of intracellular target sequences. However, with the DNA ring/hairpin structure-based in situ RCA method, a significantly stronger fluorescence signal in the MCF-7 cells was observed (Figure 4c) and obvious fluorescence was also observed in the HeLa cells (Figure 4d), which indicated that our method is highly sensitive and could reliably detect and distinguish the different mRNA expression levels in the cells. To confirm that the fluorescence signals were a result of the TK1 mRNA target sequences, a control experiment that used a DNA ring/random hairpin structure (which was unable to hybridize with the TK1 mRNA) was then performed. As expected, no fluorescence signal could be observed from either the MCF-7 (Figure 4e) or the HeLa cells (Figure 4f), suggesting that the DNA ring/hairpin structure was sequence-specific to TK1 mRNA and would not react to other non-target mRNAs.

Figure 5. Confocal fluorescence images of the simultaneous monitoring of the TK1 and the GalNAc-T mRNA in the MCF-7 and the HeLa cells with the use of the DNA ring/hairpin structure-coupled RCA method. The cell nuclei are shown in blue, and the RCA amplicons corresponding to the TK1 and the GalNAc-T mRNA are in red and green, respectively. Scale bars: 20 μm. Further verification of the DNA ring/hairpin structure-coupled RCA method for simultaneous, sensitive and multi-color monitoring of different mRNAs in cells, was performed by using two sets of DNA ring/hairpin structures for the TK1 and the GalNAc-T mRNAs in the MCF-7 and the HeLa cells, ACS Paragon Plus Environment

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respectively. After incubating the fixed cells with the DNA ring/hairpin structures along with the phi29 and dNTPs, the Cy5/BHQ-2 and Cy3/BHQ-1 partial duplex signal probes were introduced to generate fluorescence signals. Both red (Cy5 for the TK1 mRNA) and green (Cy3 for the GalNAc-T mRNA) fluorescence signals were clearly observable inside the MCF-7 and HeLa cells, suggesting that the expression of both of the two target mRNAs was occurring in both of the cells simultaneously (as seen in the confocal fluorescence images shown in Figure 5). In addition, the MCF-7 cells exhibited a significantly increased fluorescence over that of the HeLa cells, indicating that both the TK1 and the GalNAc-T mRNAs were expressed at a higher level in MCF-7 cells than in HeLa cells, which is consistent with previous findings.43 Control experiments, where the TK1 and the GalNAc-T mRNA recognition sites were first blocked by their inhibitors (the DNA sequences complementary to the mRNA recognition sites) were also performed. In these control experiments, no fluorescent signals were detected. This implies that only the target mRNAs could be specifically recognized by the corresponding DNA ring/hairpin structures to trigger the RCA for amplified fluorescent signal readouts. These comparisons clearly demonstrate the capability of the DNA ring/hairpin structures for highly sensitive and simultaneous discrimination of distinct mRNA expression levels in different cells.

Figure 6. Confocal fluorescence images for in situ monitoring of the TK1 mRNA in MCF-7 cells treated with/without tamoxifen and β-estradiol, respectively. The RCA amplicons and the cell nuclei are shown in red and blue, respectively. Scale bars: 20 μm. Potential for screening drugs for regulation of mRNA expression in cells. The use of this method for screening potential drugs that can regulate mRNA expression in cells was also investigated. In this regard, two drugs, tamoxifen and β-estradiol, which are known to decrease or increase TK1 mRNA


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expression levels in MCF-7 cells, respectively, were used to treat the cells. The DNA ring/hairpin structure-RCA method was applied to detect the expression levels of the TK1 mRNA in the MCF-7 cells with/without the drug treatments. According to the confocal fluorescence images shown in Figure 6, MCF-7 cells that had been treated with tamoxifen showed obviously reduced fluorescence, while those treated with β-estradiol exhibited a significant increase in fluorescence intensity. These results reveal distinct TK1 mRNA expression variations in the MCF-7 cells after treated with the two respective drugs. This method for monitoring cellular changes in mRNA expression levels in response to drugs can therefore be useful for discovering potential drugs for therapeutic purposes. CONCLUSIONS In conclusion, we have demonstrated the design and the application of a programmable DNA ring/hairpin-constrained structure for highly sensitive and multi-color imaging of intracellular mRNAs in single cells. The target mRNAs can sequence-specifically open the topologically constrained DNA hairpin/ring structures to trigger subsequent RCA, therefore achieve significant signal amplification for highly sensitive and selective detection of the target mRNAs. Precise manipulation of the sequences of the DNA rings and hairpins can also enable us to perform simultaneous multiplex imaging of distinct mRNAs in cells. Such a DNA structure/RCA system is also useful for discriminating drug-dependent mRNA expression levels in cells, thus enables the screening of therapeutic drugs. Compared with currently available methods, the new strategy has advantages including significantly enhanced selectivity, elimination of the complicated conventional target conversion and ligation steps, and the reduction of the background noise. This approach can be easily modified and extended for in situ monitoring of other nucleic acid targets, proteins, or small molecules (that can bind with an aptamer recognition sequence), thus potentially applicable for exploring a diversity of cell functions. Although highly sensitive and multiplexed imaging of mRNAs in single cells have been shown herein, it should also be worth noting that the expansion of such method for in situ quantification of mRNA in cells by


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overcoming the challenge of establishing an internal calibration curve can be envisioned in the future, considering the central importance of in situ quantification of mRNA in cells for biomedical research. AUTHOR INFORMATION Corresponding Author *Tel./Fax: +86-23-68252277; E-mail: [email protected]. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work is supported by National Natural Science Foundation of China (No. 21675128) and Fundamental Research Funds for the Central Universities (XDJK2017D048). REFERENCES (1) Watson, J. D.; Crick, F. H. C. Molecular Structure of Nucleic Acids. Nature 1953, 171, 737-738. (2) Kristoffersen, E. L.; Givskov, A.; Jørgensen, L. A.; Jensen, P. W.; Byl, J. A. W.; Osheroff, N.;Andersen, A. H.; Stougaard, M.; Ho, Y. P.; Knudsen, B. R. Interlinked DNA Nano-Circles for Measuring Topoisomerase II Activity at the Level of Single Decatenation Events. Nucleic Acids Research, 2017, 45, 7855-7869. (3) Lu, C. H.; Cecconello, A.; Elbaz, J.; Credi, A.; Willner, I. A Three-station DNA Catenane Rotary Motor with Controlled Directionality. Nano Lett. 2013, 13, 2303-2308. (4) Wu, Z. S.; Shen, Z. F.; Tram, K.; Li, Y. F. Engineering Interlocking DNA Rings with Weak Physical Interactions. Nat. Commun. 2014, 5, 4279. (5) Valero, J.; Pal, N.; Dhakal, S.; Walter, N. G.; Famulok, M. A Bio-hybrid DNA Rotor-Stator Nanoengine That Moves Along Predefined Tracks. Nat. Nanotechnol. 2018, 13, 496-503. (6) Zhu, L. Y.; Liu, Q. H.; Yang, B. Y.; Ju, H. X.; Lei, J. P. Pixel Counting of Fluorescence Spots Triggered by DNA Walkers for Ultrasensitive Quantification of Nucleic Acid. Anal. Chem. 2018, 90, 6357-6361. (7) Liu, M. H.; Jiang, S. X.; Loza, O.; Fahmi, N. E.; Šulc, P.; Stephanopoulos, N. Rapid Photoactuation of a DNA Nanostructure Using an Internal Photocaged Trigger Strand. Angew. Chem. Int. Ed. 2018, 130, 9485-9489. (8) Zeng, S.; Liu, D.; Li, C. Y.; Yu, F.; Fan, L.; Lei, C. Y.; Huang, Y.; Nie, Z.; Yao, S. Z. Cell-Surface-Anchored Ratiometric DNA Tweezer for Real-Time Monitoring of Extracellular and Apoplastic pH. Anal. Chem. 2018, 90, 13459-13466. (9) Lohmann, F.; Ackermann, D.; Famulok, M. Reversible Light Switch for Macrocycle Mobility in a DNA Rotaxane. J. Am. Chem. Soc. 2012, 134, 11884-11887. ACS Paragon Plus Environment

18

Page 19 of 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(10) Li, T.; Lohmann, F.; Famulok, M. Interlocked DNA Nanostructures Controlled by a Reversible Logic Circuit. Nat. Commun. 2014, 5, 4940-4947. (11) Lu, C. H.; Cecconello, A.; Willner, I. Recent Advances in the Synthesis and Functions of Reconfigurable Interlocked DNA Nanostructures. J. Am. Chem. Soc. 2016, 138, 5172-5185. (12) You, M.; Huang, F.; Chen, Z.; Wang, R. W.; Tan, W. H. Building a Nanostructure with Reversible Motions Using Photonic Energy. ACS Nano 2012, 6, 7935-7941. (13) Zhang C.; Shen, L. J.; Liang, C.; Dong, Y. F.; Yang, J.; Xu, J. DNA Sequential Logic Gate Using Two-Ring DNA. ACS Appl. Mater. Interfaces 2016, 8, 9370-9376. (14) Elbaz, J.; Wang, Z. G.; Wang, F.; Willner, I. Programmed Dynamic Topologies in DNA Catenanes. Angew. Chem. Int. Ed. 2012, 51, 2349-2353. (15) Schwarzenbach, H. D.; Hoon, S. B.; Pantel, K. Cell-free Nucleic Acids as Biomarkers in Cancer Patients. Nat. Rev. Cancer 2011, 11, 426-437. (16) Deng, R. J.; Zhang, K. X.; Sun, Y. P.; Ren, X. J.; Li, J. H.; Highly Specific Imaging of mRNA in Single Cells by Target RNA Initiated Rolling Circle Amplification. Chem. Sci. 2017, 8, 3668-3675. (17) Wang, X. D.; Dai, J.; Min, X. H.; Yu, Z. H.; Cheng, Y.; Huang, K. X.; Yang, J. L.; Yi, X. Q.; Lou, X. D.; Xia, F. DNA-Conjugated Amphiphilic Aggregation-Induced Emission Probe for Cancer Tissue Imaging and Prognosis Analysis. Anal. Chem. 2018, 90, 8162-8169. (18) Nolan, T.; Hands, R. E.; Bustin, S. A. Quantification of mRNA Using Real-time RT-PCR. Nat. Protoc. 2006, 1, 1559-1582. (19) Tsotetsi, T. N.; Collins, N. E.; Oosthuizen, M. C.; Sibeko-Matjila, K. P.; Selection and Evaluation of Housekeeping Genes as Endogenous Controls for Quantification of mRNA Transcripts in Theileria Parva Using Quantitative Real-Time Polymerase Chain Reaction. Plos One 2018, 13, e0196715. (20) Brown, P. O.; Botstein, D. Exploring the New World of the Genome with DNA Microarrays. Nat. Genet. 1999, 21, 33-37. (21) Liu, B.; Yang, H.; Taher, L.; Denz, A.; Grützmann, R.; Pilarsky, C.; Weber, G. F. Identification of Prognostic Biomarkers by Combined mRNA and miRNA Expression Microarray Analysis in Pancreatic Cancer. Transl. Oncol. 2018, 11, 700-714. (22) Alwine, J. C.; Kemp, D. J.; Parker, B. A.; Reiser, J.; Renart, J.; Stark, G. R.; Wahl, M. G. Detection of Specific RNAs or Specific Fragments of DNA by Fractionation in Gels and Transfer to Diazobenzyloxymethyl Paper. Methods Enzymol. 1979, 68, 220-242. (23) Pall, G. S.; Codony-Servat, C.; Byrne, C. J.; Ritchie, L.; Hamilton, A. Carbodiimide-Mediated Cross-Linking of RNA to Nylon Membranes Improves the Detection of siRNA, miRNA and piRNA by Northern Blot. Nucleic Acids Res. 2007, 35, e60. (24) Latorre, A.; Posch, C.; Garcimartín, Y.; Ortiz-Urda, S.; Somoza, Á. Single-Point Mutation Detection in RNA Extracts Using Gold Nanoparticles Modified with Hydrophobic Molecular Beacon-Like Structures. Chem. Commun. 2014, 50, 3018-3020. (25) Cheng, H.; Liu, J. Q.; Ma, W. J.; Duan, S. D.; Huang, J.; He, X. X.; Wang, K. M. Low Background Cascade Signal Amplification Electrochemical Sensing Platform for Tumor-Related mRNA Quantification by Target-Activated Hybridization Chain Reaction and Electroactive Cargo Release. Anal. Chem. 2018, 90, 12544-12552. (26) Balagopal, V.; Parker, R. Polysomes, P. Bodies and Stress Granules: States and Fates of Eukaryotic mRNAs. Curr. Opin.Cell Biol. 2009, 21, 403-408.


19


Page 20 of 22

(27) Jung, H.; Gkogkas, C. G.; Sonenberg, N.; Holt, C. E. Remote Control of Gene Function by Local Translation. Cell 2014, 157, 26-40. (28) Liu, Y.; Le, P.; Lim, S. J.; Ma, L.; Sarkar, S.; Han, Z. Y.; Murphy, S. J.; Kosari, F.; Vasmatzis, G.; Cheville, J. C.; Smith, A. M.; Enhanced mRNA FISH with Compact Quantum Dots. Nat. Commun. 2018, 9, 4461. (29) Park, H. Y.; Lim, H.; Yoon, Y. J.; Follenzi, A.; Nwokafor, C.; Lopez-Jones, M.; Meng, X. H.; Singer, R. H. Visualization of Dynamics of Single Endogenous mRNA Labeled in Live Mouse. Science 2014, 343, 422-424. (30) Kloosterman, W. P.; Wienholds, E.; de Bruijn, E.; Kauppinen, S.; Plasterk, R. H. In Situ Detection of miRNAs in Animal Embryos Using LNA-modified Oligonucleotide Probes. Nat. Methods 2006, 3, 27-29. (31) Politz, J. C.; Zhang, F.; Pederson, T. MicroRNA-206 Colocalizes with Ribosome-Rich Regions in Both the Nucleolus and Cytoplasm of Rat Myogenic Cells. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 18957-18962. (32) Deng, R. J.; Zhang, K. X.; Wang, L. D.; Ren, X. J.; Sun, Y. P.; Li, J. H. DNA-Sequence-Encoded Rolling Circle Amplicon for Single-Cell RNA Imaging. Chem 2018, 4, 1373-1386. (33) Yang, Y. J.; Huang, J.; Yang, X. H.; Quan, K.; Wang, H.; Ying, L.; Xie, N. L.; Ou, M.; Wang, K. M. FRET Nanoflares for Intracellular mRNA Detection: Avoiding False Positive Signals and Minimizing Effects of System Fluctuations. J. Am. Chem. Soc. 2015, 137, 8340-8343. (34) Huang, J.; Wang, H.; Yang, X. H.; Quan, K.; Yang, Y. J.; Ying, L.; Xie, N. L.; Ou, M.; Wang, K. M. Fluorescence Resonance Energy Transfer-based Hybridization Chain Reaction for in Situ Visualization of Tumor-Related mRNA. Chem. Sci. 2016, 7, 3829-3835. (35) Ou, M.; Huang, J.; Yang, X. H.; Quan, K.; Yang, Y. J.; Xie, N. L.; Wang, K. M. MnO2 Nanosheet Mediated “DD-A” FRET Binary Probes for Sensitive Detection of Intracellular mRNA. Chem. Sci. 2017, 8, 668-673. (36) Huang, J.; Wang, H.; Yang, X. H.; Yang, Y. J.; Quan, K.; Ying, L.; Xie, N. L.; Ou, M.; Wang, K. M. A Supersandwich Fluorescence in Situ Hybridization Strategy for Highly Sensitive and Selective mRNA Imaging in Tumor Cells. Chem. Commun. 2016, 52, 370-373. (37) Choi, H. M. T.; Beck, V. A.; Pierce, N. A. Next-Generation in Situ Hybridization Chain Reaction: Higher Gain, Lower Cost, Greater Durability. ACS Nano 2014, 8, 4284-4294. (38) Larsson, C.; Grundberg, I.; Söderberg, O.; Nilsson, M. In Situ Detection and Genotyping of Individual mRNA Molecules. Nat. Methods 2010, 7, 395-397. (39) Hu, P.; Li, M. M.; Wei, X. J.; Yang, B.; Li, Y.; Li, C. Y.; Du, J. Cooperative Toehold: A Mechanism to Activate DNA Strand Displacement and Construct Biosensors. Anal. Chem. 2018, 90, 9751-9760. (40) Zhang, N.; Shi, X. M.; Guo, H. Q.; Zhao, X. Z.; Zhao, W. W.; Xu, J. J.; Chen, H. Y. Gold Nanoparticle Couples with Entropy-Driven Toehold-Mediated DNA Strand Displacement Reaction on Magnetic Beads: Toward Ultrasensitive Energy-Transfer-Based Photoelectrochemical Detection of miRNA-141 in Real Blood Sample. Anal. Chem. 2018, 90, 11892-11898. (41) Blanco, L.; Salas, M. Characterization of a 3’-5’ Exonuclease Activity in the Phage ɸ29-Encoded DNA Polymerase. Nucleic Acids Res. 1985, 13, 1239-1249. (42) Liu, M.; Zhang, W.; Zhang, Q.; Brennan, J. D.; Li, Y. F. Biosensing by Tandem Reactions of Structure Switching, Nucleolytic Digestion, and DNA Amplification of a DNA Assembly. Angew. Chem. Int. Ed. 2015, 54, 9637-9641. ACS Paragon Plus Environment

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(43) Tang, P. T.; Zheng, J.; Tang, J. R.; Ma, D. D.; Xu, W. J.; Li, J. S.; Cao, Z.; Yang, R. H. Programmable DNA Triple-helix Molecular Switch in Biosensing Applications: From in Homogenous Solutions to in Living Cells. Chem. Commun. 2017, 53, 2507-2510.


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Hairpin-Constrained Structure Enables

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