Showcasing research from Kai Xu’s Laboratory/Department of Radiology, Affiliated Hospital of Xuzhou Medical College, Xuzhou, Jiangsu Province, PR China
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A “light-up” and “spectrum-shift” response of aptamer-functionalized silver nanoclusters for intracellular mRNA imaging A novel multifunctional DNA scaffold for the one-pot synthesis of fluorescent silver nanoclusters (Ag NCs) was designed. The obtained DNA/Ag NCs presented a “light-up” and “spectrum-shift” response to target DNA in vitro and could further image the tumor-related mRNA in living cells. See Kai Xu, Jun-Jie Zhu et al., Chem. Commun., 2014, 50, 7107.
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Published on 21 March 2014. Downloaded by Purdue University on 31/08/2014 19:19:29.
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Cite this: Chem. Commun., 2014, 50, 7107 Received 8th January 2014, Accepted 21st March 2014
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A ‘‘light-up’’ and ‘‘spectrum-shift’’ response of aptamer-functionalized silver nanoclusters for intracellular mRNA imaging† Jingjing Li,ab Jia You,b Yinping Zhuang,b Cuiping Han,ab Junfeng Hu,b Aming Wang,b Kai Xu*ab and Jun-Jie Zhu*c
DOI: 10.1039/c4cc00160e www.rsc.org/chemcomm
We have designed a novel multifunctional DNA scaffold for the synthesis of fluorescent silver nanoclusters (Ag NCs) using a onepot approach. The obtained DNA/Ag NCs presented a ‘‘light-up’’ and ‘‘spectrum-shift’’ response to target DNA in vitro and could further image the tumor-related mRNA in living cells.
Estimation of abnormalities in gene expression in living cells is an important way to identify cancer at the cellular level at an early stage.1 Tumor-related messenger RNA (mRNA) has been widely used as a specific marker to identify cancer cells.2 There are various techniques available for mRNA detection, for example, the northern blot (NB) technique, ribonuclease protection assays (RPA), the reverse-transcription quantitative polymerase chain reaction (RT-qPCR) and optical imaging. Because of its simplicity and visualization, optical imaging has been an area of great interest.3 The difference between the signal and background of this technique is critical for accurate target detection, especially for targets with a relatively low expression in living cells. In order to reduce the background signal, probes that produce fluorescence only in the presence of target mRNA have been designed based on fluorescence resonance energy transfer (FRET).4 Mirkin’s group developed nanoflares to generate an intracellular fluorescence signal proportional to the relative quantity of a specific intracellular RNA. In their study, Au NPs were functionalized with thiolated oligonucleotides complementary to target mRNA. The hybridization between thiolated oligonucleotides and a short cyanine (Cy 5) dye-terminated reporter sequence resulted in a weak fluorescence emission due to the proximity of the Au NP surface. In the presence of target mRNA, the reporter sequence was displaced and released from the Au NP, resulting in an increased fluorescence signal. Au NPs served as both a
Department of Radiology, Affiliated Hospital of Xuzhou Medical College, Xuzhou 221006, China. E-mail: [email protected] b School of Medical Imaging, Xuzhou Medical College, Xuzhou 221004, China c State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China. E-mail: [email protected] † Electronic supplementary information (ESI) available: Experimental details and additional figures and tables. See DOI: 10.1039/c4cc00160e
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the fluorescence quencher and the cellular transfection agent.5 They further designed multiplexed nano-flares for simultaneous detection of two distinct mRNA targets inside living cells.6 However, such cellular transfection is nonspecific and the conjugation between the Au NPs and the oligonucleotides is time-consuming.7 Recently, Tan’s group proposed an approach to combine mRNA detection and gene therapy using molecular beacon micelle flares (MBMFs). MBMFs were prepared by self-assembly of diacyllipid molecularbeacon conjugates and a fluorescence enhancement was observed upon target mRNA binding.8 It is a great step forward, but development of novel fluorescence probes which are easy to prepare, capable of self-delivery and highly sensitive and selective for mRNA optical imaging are still driving researchers. DNA stabilized silver nanoclusters (Ag NCs), which possess good fluorescence properties, excellent photostability, subnanometer size, as well as low cytotoxicity, have become an important alternative to quantum dots (QDs) and other fluorophores for the development of biolabels and molecular sensors.9 Ag NC fluorescence properties are highly dependent on the DNA sequence and are sensitive to oligonucleotide surroundings, which give them their potential for gene diagnosis. Based on this principle, a combination of a functional motif of DNA (such as an aptamer) with a DNA template for fluorescent DNA/Ag NCs has been used for cell-type-specific imaging. We synthesized aptamer-functionalized Ag NCs by designing a DNA scaffold composed of AS1411 aptamer, poly(cytosine) and a –TTTTT– loop connecter. Nuclei of MCF-7 human breast cancer cells were specifically stained with red color.10 Aptamer-functionalized Ag NCs with green color emissions have also been developed to target tumor cells in another study.11 Recently, we proposed aptamer-functionalized Ag NCs for the specific delivery of siRNA into a target cell and for simultaneous and noninvasive imaging. This showed promising potential for DNA templated Ag NCs as the fluorescence probe in cell imaging.12 Utilizing the Ag NCs’ fluorescent sensitivity to oligonucleotide surroundings, Yang et al. designed a DNA sequence containing a specific complementary sequence to the target microRNA and a 12 nucleotide scaffold to produce red emitting Ag NCs. They found the red fluorescence of the DNA/Ag NCs probe is diminished in the
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Fig. 1 Scheme illustrating the aptamer-functionalized Ag NC-mediated in vitro DNA detection and intracellular mRNA imaging. (1) Fluorescent Ag NCs were first synthesized through the reduction of Ag+ by NaBH4 with our designed DNA scaffold. (2) Then the ‘‘light-up’’ and ‘‘spectrum-shift’’ response to the hybridization with target DNA in vitro was observed. Ag NCs were further utilized for intracellular mRNA imaging with the help of aptamer internalization.
presence of target microRNA and developed a DNA/Ag NC-based homogeneous assay for microRNA detection.13 However, the ‘‘turn-off’’ fluorescence signal and the lack of involvement of an internalization agent made it unsuitable for intracellular RNA imaging. Herein, we designed a multifunctionals DNA scaffold for the synthesis of Ag NCs to realize intracellular tumor-related mRNA imaging. As shown in Fig. 1, this DNA scaffold contained three functional parts: Sgc8c aptamer as the specific internalization part, fluorescent Ag NCs nucleation sequence, and complementary sequence (cDNA) which could hybridize with target DNA or RNA to bring changes on the fluorescent properties of Ag NCs. We chose TK1 mRNA as the model target to test this hypothesis. TK1 is associated with cell division and is proposed to be a marker for tumor growth.14 To develop assays for intracellular mRNA imaging, usually a synthetic complementary DNA is first used in vitro as the target to test the feasibility of the design.5,6,8,15 Thus, hybridization studies were first performed with the perfectly matched DNA targets for the complementary sequence to TK1 mRNA in our study. Based on our previous studies,10,12 two DNA scaffolds named NC-Sgc8c-L5T-cTK1 and NC-Sgc8c-L5T-cTK1-2 were designed initially (the detailed sequences can be found in the ESI†). The difference between the two sequences is three extra nucleotides in NC-Sgc8c-L5T-cTK1-2, which is used to hybridize with the target. NC-Sgc8c-L5T-cTK1 and NC-Sgc8c-L5T-cTK1-2 were used as the template to produce Ag NCs. Fluorescence enhancement of both NC-Sgc8c-L5T-cTK1-templated Ag NCs and NC-Sgc8cL5T-cTK1-2-templated Ag NCs was observed in the presence of the DNA target. The enhancement was higher with NC-Sgc8c-L5TcTK1-2-templated Ag NCs (Fig. S1, ESI†). However, considering the relatively low level of TK1 mRNA expression in living cells, such a fluorescence response was not sensitive enough for their detection and imaging. So we modified the DNA scaffold to stabilize the Ag NCs. The new DNA scaffold was named ‘‘cTK1-NC-Sgc8c-L5T’’. The new probe responded with an unexpected 8.9-fold increase of fluorescence signals in the presence of 3 nmol DNA target (Fig. 2). More interestingly, after hybridization with the DNA target, the excitation wavelength of the cTK1-NC-Sgc8c-L5T stabilized Ag NCs shifted from 555 nm to 480 nm and the emission wavelength shifted from 620 nm to 595 nm accordingly. In the absence of DNA target, no fluorescence emission of the cTK1-NC-Sgc8c-L5T
7108 | Chem. Commun., 2014, 50, 7107--7110
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Fig. 2 Fluorescence emission spectra of cTK1-NC-Sgc8c-L5T stabilized Ag NCs in the absence and presence of DNA target (A) and the corresponding bar graph (B). Excitation wavelengths were 480 nm and 555 nm.
stabilized Ag NCs was observed with excitation at 480 nm (Fig. 2). The possible mechanism is described in the ESI.† This would benefit a low background signal from nonspecific binding. Furthermore, cTK1-NC-Sgc8c-L5T retained the same secondary structure as the Sgc8c aptamer, which is essential for its specific binding to PTK7 protein (shown in Fig. S5, ESI†).16 To detect the target DNA, the concentration of cTK1-NCSgc8c-L5T stabilized Ag NCs was optimized using 200 nM target DNA. With an increase in the concentration of cTK1-NC-Sgc8cL5T stabilized Ag NCs, the fluorescence emissions at both 595 nm and 620 nm were enhanced (Fig. S6, ESI†). Considering the intracellular imaging, 500 nM cTK1-NC-Sgc8c-L5T stabilized Ag NCs was chosen for the detection of DNA target (detailed explanation is shown in ESI†). Fig. 3 shows that the fluorescence intensity of the nanoprobe exhibits dose-dependent increases in response to target DNA concentrations from 0 to 2 mM with a wide dynamic range (0 to 200 nM). It suggests that the signals of fluorescence enhancement were generated from hybridization of the DNA scaffold with the target DNA. The detection limit was 10 nM (3 pmol) based on a signal to background ratio of 3. Next, the specificity of this nanoprobe was investigated using other DNA sequences. Synthetic survivin, K-ras, HER-2/neu, C-myc, GalNAc-related DNA sequences as well as single base mutated target DNA were used under the same experimental conditions as those for TK1 mRNA. The fluorescence response of mutated cTK1NC-Sgc8c-L5T stabilized Ag NCs to the target DNA was also tested. The fluorescence emissions at 595 nm of cTK1-NC-Sgc8c-L5T stabilized Ag NCs were low even with 80 times higher concentrations
Fig. 3 Fluorescence response of cTK1-NC-Sgc8c-L5T stabilized Ag NCs to target DNA with concentrations ranging from 0 to 2 mM (A). (B) Intensity of fluorescence (emissions at 595 nm) increased with the concentration of target DNA and reached a plateau in the presence of 1 mM target DNA. (C) The linear dynamic range was from 0 to 200 nM.
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of control DNA sequences than target DNA (Fig. S8, ESI†). These results confirmed the excellent selectivity of the nanoprobe. We wonder whether this kind of DNA scaffold could be used to detect other tumor-related mRNA by simply changing the complementary sequence part. We chose another tumor-related mRNA, C-myc mRNA as the target sequence. C-myc is a potent activator of tumorigenesis, and it is deregulated in a range of cancers.17 A DNA scaffold called ‘‘C-myc-NC-Sgc8c-L5T’’ was designed and the same in vitro hybridization test was performed with C-myc related DNA target as that for TK1 mRNA. As shown in Fig. S9 (ESI†), a similar phenomenon was observed and a dynamic range from 5 to 500 nM was achieved, which indicated that our design is applicable to other synthetic DNA targets. Simultaneous detection and imaging of multiple tumor-related mRNA by this simple nanoprobe could be achieved using different Ag NCs nucleation sequences for multicolor fluorescent Ag NCs. The one-pot synthesis of aptamer-functionalized Ag NCs with multiplexed color emissions is under way in our group. Having established the signaling ability of cTK1-NC-Sgc8c-L5T templated Ag NCs with synthetic DNA targets, their application to image intracellular mRNA was then investigated. The cytotoxicity of exogenous materials in living organisms is a major concern for developing imaging probes.18 To employ the nanoprobe for TK1 mRNA intracellular imaging, the biocompatibility of the nanoprobe must be evaluated. We performed a MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in human cervical carcinoma HeLa cells and human lung fibroblast HLF-1 cells to evaluate the cytotoxicity. Both HeLa and HLF-1 cells were exposed to cTK1-NC-Sgc8c-L5T stabilized Ag NCs ranging from 5 nM to 1 mM (Fig. S10, ESI†). No significant cellular toxicity of cTK1-NC-Sgc8c-L5T stabilized Ag NCs was observed for HLF-1 cells even under a concentration of 1 mM. But cytotoxicity was observed in HeLa cells exposed to 1 mM nanoprobe. Considering our previous study,12 without the complementary segment to TK1 mRNA, no obvious cytotoxicity was observed in HeLa and NIH-3T3 cells even incubated with 1 mM NC-Sgc8c-L5T stabilized Ag NCs. The toxicity from cTK1-NC-Sgc8c-L5T stabilized Ag NCs to HeLa cells might come from the silence of target gene after a DNA–RNA hybrid was formed with the target mRNA, leading to the suppression of cancer cell growth.8,19 Finally, intracellular imaging of TK1 mRNA was performed by the incubation of HeLa cells expressing PTK 7 protein with 500 nM cTK1-NC-Sgc8c-L5T stabilized Ag NCs. NIH-3T3 cells deficient in PTK 7 protein as well as mutated cTK1-NC-Sgc8c-L5T stabilized Ag NCs were used as controls (Fig. 4). No fluorescence emission was observed when excited at 543 nm (red color, channel 2), but an obvious fluorescence signal was displayed with excitation at 488 nm (purple color, channel 1), indicating that the fluorescence was from the hybridization between TK1 mRNA and its complementary sequence of cTK1-NC-Sgc8c-L5T. In addition, no fluorescence was observed in NIH-3T3 cells and HeLa cells incubated with mutated cTK1-NC-Sgc8c-L5T stabilized Ag NCs with both excitation at 543 nm or 488 nm, showing its good cell-specificity. The absence of fluorescence signal in NIH-3T3 cells might come from the unable internalization of nanoprobe. Thus, our proposed cTK1-NC-Sgc8c-L5T stabilized Ag NCs could produce fluorescence
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Fig. 4 Confocal laser scanning microscopy images of HeLa human cervical carcinoma cells and NIH-3T3 mouse fibroblast cells incubated with cTK1NC-Sgc8c-L5T stabilized Ag NCs (1, 2) and mutated cTK1-NC-Sgc8c-L5T stabilized Ag NCs (3). Channel 1: excited at 488 nm. Channel 2: excited at 543 nm. DAPI was excited with UV. Scale bar, 20 mm.
response only in the presence of both PTK7 protein and target mRNA, leading to high specificity. It should be noted that a cell line which expresses PTK-7 protein with lower level of TK1 mRNA expression is more suitable as a control in the intracellular imaging study. But unfortunately, at present we have not found such a cell line. To further quantify the sensing ability of cTK1-NCSgc8c-L5T stabilized Ag NCs to intracellular mRNA, a flow cytometry assay was also performed and the result indicated the good behavior of our designed nanoprobe in intracellular mRNA sensing (Fig. S12, ESI†). In summary, we have designed a novel multi-functional DNA scaffold for the preparation of fluorescent Ag NCs. It consists of a cell-specific internalization aptamer, Sgc8c, fluorescent Ag NC nucleation sequence and complementary sequence to the target DNA or mRNA. Such DNA stabilized Ag NCs could be internalized into tumor cells specifically, and more importantly, it presented a switchable fluorescence excitation and emission wavelength to target tumor-related mRNA. This design allows high signal-to-background (S/B) ratio and benefits intracellular target detection, especially for relatively low expressed mRNA. Additionally, such design can be expanded to other synthetic DNA targets. Using multicolor aptamer-functionalized Ag NCs, the detection and imaging of multiple tumor-related mRNAs in living cells might be realized by this simple one-pot synthetic approach. However, it should be mentioned that at present, our proposed probe could not be used to detect TK1 mRNA in cellular extracts. Because the fluorescence of cTK1-NC-Sgc8c-L5T stabilized Ag NCs was quenched strongly by the cellular lysis solution (Fig. S13 in ESI†). Thus, improvement of the sensitivity and photostability of Ag NCs are the important issues we have to solve in the future. Additionally, real-time monitoring needs high cellular stability of the probe. Modified bases are a good way to increase cellular stability of the foreign DNA probe. The influence from fluorescence sensitivity of DNA-templated Ag NCs to the DNA sequence, base number, secondary structure, and the environment requires systematic study in the future.
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We acknowledge the support by National Natural Science Foundation of China (21305120, 21121091), Natural Science Foundation of Jiangsu Province, China (BK20130211), Natural Science Fund for Colleges and Universities in Jiangsu Province (13KJB150036) and State Key Laboratory of Analytical Chemistry for Life Science (SKLACLS1203). This research was also supported by National Basic Research Program of China (2011CB933502).
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Notes and references 1 D. Hanahan and R. A. Weinberg, Cell, 2000, 100, 57; A. Teraoka, K. Murakoshi, K. Fukamauchi, A. Z. Suzuki, S. Watanabe and T. Furuta, Chem. Commun., 2014, 50, 664. 2 H. Schwarzenbach, D. S. B. Hoon and K. Pantel, Nat. Rev. Cancer, 2011, 11, 426. 3 Z. Dvorak, J.-M. Pascussi and M. Modriansky, Biomed. Pap., 2003, 147, 131; W. J. Kang, Y. L. Cho, J. R. Chae, J. D. Lee, K.-J. Choi and S. Kim, Biomaterials, 2011, 32, 1915. 4 Z. H. Wang, K. Zhang, K. L. Wooley and J.-S. Taylor, Org. Biomol. Chem., 2013, 11, 3159. 5 D. S. Seferos, D. A. Giljohann, H. D. Hill, A. E. Prigodich and C. A. Mirkin, J. Am. Chem. Soc., 2007, 129, 15477. 6 A. E. Prigodich, P. S. Randeria, W. E. Briley, N. J. Kim, W. L. Daniel, D. A. Giljohann and C. A. Mirkin, Anal. Chem., 2012, 84, 2062.
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ChemComm 7 D. S. Seferos, D. A. Giljohann, H. D. Hill, A. E. Prigodich and C. A. Mirkin, J. Am. Chem. Soc., 2007, 129, 15477. 8 T. Chen, C. S. Wu, E. Jimenez, Z. Zhu, J. G. Dajac, M. X. You, D. Han, X. B. Zhang and W. H. Tan, Angew. Chem., Int. Ed., 2013, 52, 2012. 9 N. Souza, Nat. Methods, 2007, 4, 540. 10 J. J. Li, X. Q. Zhong, F. F. Cheng, J.-R. Zhang, L.-P. Jiang and J.-J. Zhu, Anal. Chem., 2012, 84, 4140. 11 J. J. Yin, X. X. He, K. M. Wang, Z. H. Qing, X. Wu, H. Shi and X. H. Yang, Nanoscale, 2012, 4, 110. 12 J. J. Li, W. J. Wang, D. F. Sun, J. N. Chen, P.-H. Zhang, J.-R. Zhang, Q. H. Min and J.-J. Zhu, Chem. Sci., 2013, 4, 3514. 13 S. W. Yang and T. Vosch, Anal. Chem., 2011, 83, 6935; P. Shah, A. Rorvig-Lund, S. B. Chaabane, P. W. Thulstrup, H. G. Kjaergaard, E. Fron, J. Hofkens, S. W. Yang and T. Vosch, ACS Nano, 2012, 6, 8803. 14 P. Broet, S. Romain, A. Daver, G. Ricolleau, V. Quillien, A. Rallet, B. Asselain, P. M. Martin and F. Spyratos, J. Clin. Oncol., 2001, 19, 2778. 15 N. Li, C. Y. Chang, W. Pan and B. Tang, Angew. Chem. Int. Ed., 2012, 51, 7426. 16 Z. Y. Xiao, D. H. Shangguan, Z. H. Cao, X. H. Fang and W. H. Tan, Chem. – Eur. J., 2008, 14, 1769. 17 N. Meyer and L. Z. Penn, Nat. Rev. Cancer, 2008, 8, 976. 18 Z. Liu, M. Winters, M. Holodniy and H. J. Dai, Angew. Chem., Int. Ed., 2007, 46, 2023. 19 M. Elsabahy, A. Nazarali and M. Foldvari, Curr. Drug Delivery, 2011, 8, 235.
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As featured in:
A “light-up” and “spectrum-shift” response of aptamer-functionalized silver nanoclusters for intracellular mRNA imaging A novel multifunctional DNA scaffold for the one-pot synthesis of fluorescent silver nanoclusters (Ag NCs) was designed. The obtained DNA/Ag NCs presented a “light-up” and “spectrum-shift” response to target DNA in vitro and could further image the tumor-related mRNA in living cells. See Kai Xu, Jun-Jie Zhu et al., Chem. Commun., 2014, 50, 7107.
www.rsc.org/chemcomm Registered charity number: 207890
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Published on 21 March 2014. Downloaded by Purdue University on 31/08/2014 19:19:29.
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Cite this: Chem. Commun., 2014, 50, 7107 Received 8th January 2014, Accepted 21st March 2014
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A ‘‘light-up’’ and ‘‘spectrum-shift’’ response of aptamer-functionalized silver nanoclusters for intracellular mRNA imaging† Jingjing Li,ab Jia You,b Yinping Zhuang,b Cuiping Han,ab Junfeng Hu,b Aming Wang,b Kai Xu*ab and Jun-Jie Zhu*c
DOI: 10.1039/c4cc00160e www.rsc.org/chemcomm
We have designed a novel multifunctional DNA scaffold for the synthesis of fluorescent silver nanoclusters (Ag NCs) using a onepot approach. The obtained DNA/Ag NCs presented a ‘‘light-up’’ and ‘‘spectrum-shift’’ response to target DNA in vitro and could further image the tumor-related mRNA in living cells.
Estimation of abnormalities in gene expression in living cells is an important way to identify cancer at the cellular level at an early stage.1 Tumor-related messenger RNA (mRNA) has been widely used as a specific marker to identify cancer cells.2 There are various techniques available for mRNA detection, for example, the northern blot (NB) technique, ribonuclease protection assays (RPA), the reverse-transcription quantitative polymerase chain reaction (RT-qPCR) and optical imaging. Because of its simplicity and visualization, optical imaging has been an area of great interest.3 The difference between the signal and background of this technique is critical for accurate target detection, especially for targets with a relatively low expression in living cells. In order to reduce the background signal, probes that produce fluorescence only in the presence of target mRNA have been designed based on fluorescence resonance energy transfer (FRET).4 Mirkin’s group developed nanoflares to generate an intracellular fluorescence signal proportional to the relative quantity of a specific intracellular RNA. In their study, Au NPs were functionalized with thiolated oligonucleotides complementary to target mRNA. The hybridization between thiolated oligonucleotides and a short cyanine (Cy 5) dye-terminated reporter sequence resulted in a weak fluorescence emission due to the proximity of the Au NP surface. In the presence of target mRNA, the reporter sequence was displaced and released from the Au NP, resulting in an increased fluorescence signal. Au NPs served as both a
Department of Radiology, Affiliated Hospital of Xuzhou Medical College, Xuzhou 221006, China. E-mail: [email protected] b School of Medical Imaging, Xuzhou Medical College, Xuzhou 221004, China c State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China. E-mail: [email protected] † Electronic supplementary information (ESI) available: Experimental details and additional figures and tables. See DOI: 10.1039/c4cc00160e
This journal is © The Royal Society of Chemistry 2014
the fluorescence quencher and the cellular transfection agent.5 They further designed multiplexed nano-flares for simultaneous detection of two distinct mRNA targets inside living cells.6 However, such cellular transfection is nonspecific and the conjugation between the Au NPs and the oligonucleotides is time-consuming.7 Recently, Tan’s group proposed an approach to combine mRNA detection and gene therapy using molecular beacon micelle flares (MBMFs). MBMFs were prepared by self-assembly of diacyllipid molecularbeacon conjugates and a fluorescence enhancement was observed upon target mRNA binding.8 It is a great step forward, but development of novel fluorescence probes which are easy to prepare, capable of self-delivery and highly sensitive and selective for mRNA optical imaging are still driving researchers. DNA stabilized silver nanoclusters (Ag NCs), which possess good fluorescence properties, excellent photostability, subnanometer size, as well as low cytotoxicity, have become an important alternative to quantum dots (QDs) and other fluorophores for the development of biolabels and molecular sensors.9 Ag NC fluorescence properties are highly dependent on the DNA sequence and are sensitive to oligonucleotide surroundings, which give them their potential for gene diagnosis. Based on this principle, a combination of a functional motif of DNA (such as an aptamer) with a DNA template for fluorescent DNA/Ag NCs has been used for cell-type-specific imaging. We synthesized aptamer-functionalized Ag NCs by designing a DNA scaffold composed of AS1411 aptamer, poly(cytosine) and a –TTTTT– loop connecter. Nuclei of MCF-7 human breast cancer cells were specifically stained with red color.10 Aptamer-functionalized Ag NCs with green color emissions have also been developed to target tumor cells in another study.11 Recently, we proposed aptamer-functionalized Ag NCs for the specific delivery of siRNA into a target cell and for simultaneous and noninvasive imaging. This showed promising potential for DNA templated Ag NCs as the fluorescence probe in cell imaging.12 Utilizing the Ag NCs’ fluorescent sensitivity to oligonucleotide surroundings, Yang et al. designed a DNA sequence containing a specific complementary sequence to the target microRNA and a 12 nucleotide scaffold to produce red emitting Ag NCs. They found the red fluorescence of the DNA/Ag NCs probe is diminished in the
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Fig. 1 Scheme illustrating the aptamer-functionalized Ag NC-mediated in vitro DNA detection and intracellular mRNA imaging. (1) Fluorescent Ag NCs were first synthesized through the reduction of Ag+ by NaBH4 with our designed DNA scaffold. (2) Then the ‘‘light-up’’ and ‘‘spectrum-shift’’ response to the hybridization with target DNA in vitro was observed. Ag NCs were further utilized for intracellular mRNA imaging with the help of aptamer internalization.
presence of target microRNA and developed a DNA/Ag NC-based homogeneous assay for microRNA detection.13 However, the ‘‘turn-off’’ fluorescence signal and the lack of involvement of an internalization agent made it unsuitable for intracellular RNA imaging. Herein, we designed a multifunctionals DNA scaffold for the synthesis of Ag NCs to realize intracellular tumor-related mRNA imaging. As shown in Fig. 1, this DNA scaffold contained three functional parts: Sgc8c aptamer as the specific internalization part, fluorescent Ag NCs nucleation sequence, and complementary sequence (cDNA) which could hybridize with target DNA or RNA to bring changes on the fluorescent properties of Ag NCs. We chose TK1 mRNA as the model target to test this hypothesis. TK1 is associated with cell division and is proposed to be a marker for tumor growth.14 To develop assays for intracellular mRNA imaging, usually a synthetic complementary DNA is first used in vitro as the target to test the feasibility of the design.5,6,8,15 Thus, hybridization studies were first performed with the perfectly matched DNA targets for the complementary sequence to TK1 mRNA in our study. Based on our previous studies,10,12 two DNA scaffolds named NC-Sgc8c-L5T-cTK1 and NC-Sgc8c-L5T-cTK1-2 were designed initially (the detailed sequences can be found in the ESI†). The difference between the two sequences is three extra nucleotides in NC-Sgc8c-L5T-cTK1-2, which is used to hybridize with the target. NC-Sgc8c-L5T-cTK1 and NC-Sgc8c-L5T-cTK1-2 were used as the template to produce Ag NCs. Fluorescence enhancement of both NC-Sgc8c-L5T-cTK1-templated Ag NCs and NC-Sgc8cL5T-cTK1-2-templated Ag NCs was observed in the presence of the DNA target. The enhancement was higher with NC-Sgc8c-L5TcTK1-2-templated Ag NCs (Fig. S1, ESI†). However, considering the relatively low level of TK1 mRNA expression in living cells, such a fluorescence response was not sensitive enough for their detection and imaging. So we modified the DNA scaffold to stabilize the Ag NCs. The new DNA scaffold was named ‘‘cTK1-NC-Sgc8c-L5T’’. The new probe responded with an unexpected 8.9-fold increase of fluorescence signals in the presence of 3 nmol DNA target (Fig. 2). More interestingly, after hybridization with the DNA target, the excitation wavelength of the cTK1-NC-Sgc8c-L5T stabilized Ag NCs shifted from 555 nm to 480 nm and the emission wavelength shifted from 620 nm to 595 nm accordingly. In the absence of DNA target, no fluorescence emission of the cTK1-NC-Sgc8c-L5T
7108 | Chem. Commun., 2014, 50, 7107--7110
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Fig. 2 Fluorescence emission spectra of cTK1-NC-Sgc8c-L5T stabilized Ag NCs in the absence and presence of DNA target (A) and the corresponding bar graph (B). Excitation wavelengths were 480 nm and 555 nm.
stabilized Ag NCs was observed with excitation at 480 nm (Fig. 2). The possible mechanism is described in the ESI.† This would benefit a low background signal from nonspecific binding. Furthermore, cTK1-NC-Sgc8c-L5T retained the same secondary structure as the Sgc8c aptamer, which is essential for its specific binding to PTK7 protein (shown in Fig. S5, ESI†).16 To detect the target DNA, the concentration of cTK1-NCSgc8c-L5T stabilized Ag NCs was optimized using 200 nM target DNA. With an increase in the concentration of cTK1-NC-Sgc8cL5T stabilized Ag NCs, the fluorescence emissions at both 595 nm and 620 nm were enhanced (Fig. S6, ESI†). Considering the intracellular imaging, 500 nM cTK1-NC-Sgc8c-L5T stabilized Ag NCs was chosen for the detection of DNA target (detailed explanation is shown in ESI†). Fig. 3 shows that the fluorescence intensity of the nanoprobe exhibits dose-dependent increases in response to target DNA concentrations from 0 to 2 mM with a wide dynamic range (0 to 200 nM). It suggests that the signals of fluorescence enhancement were generated from hybridization of the DNA scaffold with the target DNA. The detection limit was 10 nM (3 pmol) based on a signal to background ratio of 3. Next, the specificity of this nanoprobe was investigated using other DNA sequences. Synthetic survivin, K-ras, HER-2/neu, C-myc, GalNAc-related DNA sequences as well as single base mutated target DNA were used under the same experimental conditions as those for TK1 mRNA. The fluorescence response of mutated cTK1NC-Sgc8c-L5T stabilized Ag NCs to the target DNA was also tested. The fluorescence emissions at 595 nm of cTK1-NC-Sgc8c-L5T stabilized Ag NCs were low even with 80 times higher concentrations
Fig. 3 Fluorescence response of cTK1-NC-Sgc8c-L5T stabilized Ag NCs to target DNA with concentrations ranging from 0 to 2 mM (A). (B) Intensity of fluorescence (emissions at 595 nm) increased with the concentration of target DNA and reached a plateau in the presence of 1 mM target DNA. (C) The linear dynamic range was from 0 to 200 nM.
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of control DNA sequences than target DNA (Fig. S8, ESI†). These results confirmed the excellent selectivity of the nanoprobe. We wonder whether this kind of DNA scaffold could be used to detect other tumor-related mRNA by simply changing the complementary sequence part. We chose another tumor-related mRNA, C-myc mRNA as the target sequence. C-myc is a potent activator of tumorigenesis, and it is deregulated in a range of cancers.17 A DNA scaffold called ‘‘C-myc-NC-Sgc8c-L5T’’ was designed and the same in vitro hybridization test was performed with C-myc related DNA target as that for TK1 mRNA. As shown in Fig. S9 (ESI†), a similar phenomenon was observed and a dynamic range from 5 to 500 nM was achieved, which indicated that our design is applicable to other synthetic DNA targets. Simultaneous detection and imaging of multiple tumor-related mRNA by this simple nanoprobe could be achieved using different Ag NCs nucleation sequences for multicolor fluorescent Ag NCs. The one-pot synthesis of aptamer-functionalized Ag NCs with multiplexed color emissions is under way in our group. Having established the signaling ability of cTK1-NC-Sgc8c-L5T templated Ag NCs with synthetic DNA targets, their application to image intracellular mRNA was then investigated. The cytotoxicity of exogenous materials in living organisms is a major concern for developing imaging probes.18 To employ the nanoprobe for TK1 mRNA intracellular imaging, the biocompatibility of the nanoprobe must be evaluated. We performed a MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in human cervical carcinoma HeLa cells and human lung fibroblast HLF-1 cells to evaluate the cytotoxicity. Both HeLa and HLF-1 cells were exposed to cTK1-NC-Sgc8c-L5T stabilized Ag NCs ranging from 5 nM to 1 mM (Fig. S10, ESI†). No significant cellular toxicity of cTK1-NC-Sgc8c-L5T stabilized Ag NCs was observed for HLF-1 cells even under a concentration of 1 mM. But cytotoxicity was observed in HeLa cells exposed to 1 mM nanoprobe. Considering our previous study,12 without the complementary segment to TK1 mRNA, no obvious cytotoxicity was observed in HeLa and NIH-3T3 cells even incubated with 1 mM NC-Sgc8c-L5T stabilized Ag NCs. The toxicity from cTK1-NC-Sgc8c-L5T stabilized Ag NCs to HeLa cells might come from the silence of target gene after a DNA–RNA hybrid was formed with the target mRNA, leading to the suppression of cancer cell growth.8,19 Finally, intracellular imaging of TK1 mRNA was performed by the incubation of HeLa cells expressing PTK 7 protein with 500 nM cTK1-NC-Sgc8c-L5T stabilized Ag NCs. NIH-3T3 cells deficient in PTK 7 protein as well as mutated cTK1-NC-Sgc8c-L5T stabilized Ag NCs were used as controls (Fig. 4). No fluorescence emission was observed when excited at 543 nm (red color, channel 2), but an obvious fluorescence signal was displayed with excitation at 488 nm (purple color, channel 1), indicating that the fluorescence was from the hybridization between TK1 mRNA and its complementary sequence of cTK1-NC-Sgc8c-L5T. In addition, no fluorescence was observed in NIH-3T3 cells and HeLa cells incubated with mutated cTK1-NC-Sgc8c-L5T stabilized Ag NCs with both excitation at 543 nm or 488 nm, showing its good cell-specificity. The absence of fluorescence signal in NIH-3T3 cells might come from the unable internalization of nanoprobe. Thus, our proposed cTK1-NC-Sgc8c-L5T stabilized Ag NCs could produce fluorescence
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Fig. 4 Confocal laser scanning microscopy images of HeLa human cervical carcinoma cells and NIH-3T3 mouse fibroblast cells incubated with cTK1NC-Sgc8c-L5T stabilized Ag NCs (1, 2) and mutated cTK1-NC-Sgc8c-L5T stabilized Ag NCs (3). Channel 1: excited at 488 nm. Channel 2: excited at 543 nm. DAPI was excited with UV. Scale bar, 20 mm.
response only in the presence of both PTK7 protein and target mRNA, leading to high specificity. It should be noted that a cell line which expresses PTK-7 protein with lower level of TK1 mRNA expression is more suitable as a control in the intracellular imaging study. But unfortunately, at present we have not found such a cell line. To further quantify the sensing ability of cTK1-NCSgc8c-L5T stabilized Ag NCs to intracellular mRNA, a flow cytometry assay was also performed and the result indicated the good behavior of our designed nanoprobe in intracellular mRNA sensing (Fig. S12, ESI†). In summary, we have designed a novel multi-functional DNA scaffold for the preparation of fluorescent Ag NCs. It consists of a cell-specific internalization aptamer, Sgc8c, fluorescent Ag NC nucleation sequence and complementary sequence to the target DNA or mRNA. Such DNA stabilized Ag NCs could be internalized into tumor cells specifically, and more importantly, it presented a switchable fluorescence excitation and emission wavelength to target tumor-related mRNA. This design allows high signal-to-background (S/B) ratio and benefits intracellular target detection, especially for relatively low expressed mRNA. Additionally, such design can be expanded to other synthetic DNA targets. Using multicolor aptamer-functionalized Ag NCs, the detection and imaging of multiple tumor-related mRNAs in living cells might be realized by this simple one-pot synthetic approach. However, it should be mentioned that at present, our proposed probe could not be used to detect TK1 mRNA in cellular extracts. Because the fluorescence of cTK1-NC-Sgc8c-L5T stabilized Ag NCs was quenched strongly by the cellular lysis solution (Fig. S13 in ESI†). Thus, improvement of the sensitivity and photostability of Ag NCs are the important issues we have to solve in the future. Additionally, real-time monitoring needs high cellular stability of the probe. Modified bases are a good way to increase cellular stability of the foreign DNA probe. The influence from fluorescence sensitivity of DNA-templated Ag NCs to the DNA sequence, base number, secondary structure, and the environment requires systematic study in the future.
Chem. Commun., 2014, 50, 7107--7110 | 7109
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We acknowledge the support by National Natural Science Foundation of China (21305120, 21121091), Natural Science Foundation of Jiangsu Province, China (BK20130211), Natural Science Fund for Colleges and Universities in Jiangsu Province (13KJB150036) and State Key Laboratory of Analytical Chemistry for Life Science (SKLACLS1203). This research was also supported by National Basic Research Program of China (2011CB933502).
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7110 | Chem. Commun., 2014, 50, 7107--7110
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