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Received 00th January 20xx, Accepted 00th January 20xx
Jianbin Chen,‡a Jikui Wu‡ab and Yunhan Hong*a
DOI: 10.1039/x0xx00000x www.rsc.org/
A non-invasive fluorescent probe, morpholino molecular beacon (MO-MB), was designed for RNA visualization in vivo. Featuring negligible toxicity, stability, and high target specificity in living embryos, MO-MB is superior to conventional probes and has the potential for specific RNA visualization in basic biological and clinical research. The visualization of specific RNA in vivo is of vital significance in biological and clinical research by providing detailed spatiotemporal information of gene expression as well as cell differentiation.1 A variety of fluorescent probes have been developed for imaging RNA in fixed and live cells, 2,3 but due to the low biocompatibility and stability of these probes in living organisms, efficient probes have been lacking for in vivo RNA visualization. The intracellular environment in living organisms is more delicate and rigorous than that in the cultured cells for fluorescent probes. Therefore, ideal fluorescent probes for in vivo application should exhibit the following features: 1) negligible toxicity, the probe should not affect the viability and the normal function of the organisms; 2) stability, the probe should be resistant to degradation by enzymes and be immune to nonspecific binding proteins; 3) target specificity, the probe should be capable of distinguishing its target RNA from nontarget RNA in the complex in vivo environment. Molecular beacons (MBs) have the potential to meet these requirements. MBs are hairpin-structured oligonucleotide probes duallabelled with a reporter fluorophore at one end and a quencher at the other end. In the absence of RNA target, fluorescence of the fluorophore is quenched. Hybridization with their RNA target opens the hairpin and produces fluorescent signals. Although DNA molecular beacon (DNA-MB) has been used for RNA visualization in living cells,4 it is prone
a. Department
of Biological Sciences, National University of Singapore, Singapore 117543, Singapore. E-mail: [email protected] b. College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China. † Electronic supplementary information (ESI) available: Detailed experimental procedures and results. See DOI: 10.1039/x0xx00000x ‡ These authors contributed equally to this work
to be degraded by endogenous nuclease and disrupted by nonspecific binding of DNA/RNA binding proteins, thereby resulting in high background fluorescence and false positive signals. The stability of MBs has been significantly improved by chemically modifying the oligonucleotide backbone or replacing them with artificial nucleic acids, including negatively charged phosphorothioate,5 2'-O-methyl RNA bases,6 peptide nucleic acids,7 locked nucleic acids,8 and serinol nucleic acids.9 However, for in vivo application, these DNA-MB analogues encountered some limitations, such as poor aqueous solubility, toxicity, self-aggregation, slow hybridization rate, and unintended binding to proteins. Therefore, a safe, stable, specific fluorescent probe for visualizing RNA in vivo is demanded. Morpholino oligonucleotides (MOs) are morpholino ringbased oligonucleotide analogues with non-ionic phosphorodiamidate intersubunit linkages (Fig. 1A). This
Figure 1. (A) The structure of morpholino. (B) The stem-loop conformation of the designed share-stem morpholino molecular beacon (MO-MB). (C) The melt curves of MO-MB in the absence (dash line) and presence (solid line) of perfect matched RNA target. (D) Emission spectra of MO-MB before (dash line) and after (solid line) the addition of perfectly matched RNA target. (E) Normalized hybridization kinetics of MO-MB with perfect matched (PM, ○) and singlebase mismatched (1MM, △) RNA target. Conditions: 500 nM MO-MB, 5-fold excess of RNA targets, 10 mM phosphate buffer (pH=7.0, 100 mM NaCl, 3 mM MgCl2), (D) 25 ℃, (E) 28 ℃.
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Morpholino molecular beacon for specific RNA visualization in vivo
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backbone render several advantages of MOs over other oligonucleotide analogues such as high aqueous solubility, good biological compatibility, high affinities toward RNA and low propensity toward self-aggregation.10 Hence, we reason that MOs might be an attractive candidate to design novel fluorescent probes for RNA imaging in vivo. For this purpose, we developed a shared-stem morpholino molecular beacon (MO-MB). A "random" sequence (25 bp), which does not have any exact match in the medaka genome or transcripts, was chosen to design a model beacon. This MO-MB was composed totally of morpholino and synthesized with an 11-mer loop and 7-mer stem, where FAM was the 3'-end reporter and DABCYL was the 5'-end quencher (Fig. 1B, Fig. S1, ESI†). The purified MO-MB was confirmed by MALDI-TOF mass spectroscope and UV-Vis absorption spectra (Fig. S2, Fig. S3, ESI†). To ascertain whether our designed MO-MB possessed the desired characteristics of conventional molecular beacons, we first investigated the thermodynamic behaviour of MO-MB by monitoring the fluorescence change in the absence and presence of RNA target over a temperature profile. In the absence of RNA target, thermal denaturation measurements of MO-MB showed sigmoidal melting curves, which indicated cooperative base pairing of the MO-MO homoduplex in the
Journal Name stem (Fig. 1C, dash line).11 In the presence of RNA as View target, Article Online DOI: 10.1039/C5CC07124K the temperature slowly raised, MO-MB exhibited a characteristic three-state behaviour which was similar to that of conventional MBs, demonstrating that MO-MB had the conformational constraint of stem-loop structure (Fig. 1C, solid line).12 Further support was provided by the exploration of hybridization properties and sequence discrimination ability of MO-MB using fluorescent measurements. Without RNA target, MO-MB showed very weak fluorescence (Fig. 1D, dash line) and was therefore predominantly in the closed state. Once hybridized with the perfect matched (PM) RNA target, MO-MB underwent a conformation change and opened its stem-loop structure, thereby resulting in more than 12-fold increase of fluorescence intensity (Fig.1D, solid line). As expected, MO-MB discriminated correctly between PM RNA target and singlebase mismatched (1MM) RNA target in kinetic profiles (Fig. 1E, Fig. S4, ESI†). Furthermore, the hybridization kinetics of MOMB was very fast, which was attributed to the high affinity of MO toward RNA. Together, these results suggested that the proposed MO-MB held similar properties to that of DNA-MB and might be used as a fluorescent probe for RNA monitoring in living organisms. To examine the feasibility of MO-MB in vivo, we chose
Figure 2. (A) Images of wild-type medaka adults and embryo. Embryo was imaged under dark field, FAM and Texas Red channels, showing the transparency and essentially free of autofluorescence of the medaka embryo. (B) Biocompatibility of MO-MB and DNA-MB. MO-MB and DNA-MB of 100μM were injected to living embryos at 1 hpf. Morphological change of embryos at 6 and 24 hpf were shown. (C) In vivo toxicity of MO-MB. Morphological distribution of the wild-type embryos (3 dph) and the embryos injected with 100 μM or 250 μM MO-MB at 1 dpf, 5 dpf, 3 dph were depicted. (D) Representative images from the in vivo toxicity assay. Random samples from each group was shown at different time point with the abnormal embyros pointed out. ab, abnormal.
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medaka fish embryos as the model. The developing embryo provides a stringent environment with its high sensitivity to toxics and its dramatic change in gene expression as well as cell morphology during the developmental process. Medaka is widely used as an excellent vertebrate model in basic and applied research such as stem cell biology and sex development.13 The fish is small, easy to maintain and produces eggs every day. More importantly, its embryos are transparent and essentially free of auto-fluorescence (Fig. 2A), which facilitates direct, real-time visualization of the fluorescent probes in vivo. For the feasibility examination of MO-MB in living embryos, we first tested whether MO-MB affected the proper embryonic development and normal cell behaviour of medaka. DNA-MB of the same sequence was used for a comparison. We injected 100 μM MBs into 1-cell stage medaka embryos and followed the development of the embryos during the first 24 h after fertilization. The embryos injected with DNA-MB displayed sever developmental delay/defect and significant cell death, demonstrating that DNA-MB was toxic to the developing embryos. By contrast, the MO-MB-injected embryos developed normally without any obvious differences with respect to wild-type embryos, indicating the good biocompatibility of MO-MB (Fig. 2B). We then quantitatively assessed the potential toxicity of MO-MB to the early development of medaka by morphological analysis. We categorized the MO-MB-injected embryos into three groups (normal, abnormal, and dead) on the basis of changes in some morphological features at 1 day post fertilization (dpf), 5 dpf, and 3 days post-hatching (dph) (Fig. 2D, Fig. S5, Table S2, ESI†). As shown in Fig. 2C, the embryos after MO-MB microinjection at 100 μM displayed a survival rate of 84.7%, which was slightly lower than 89.6% for the wild-type embryos. Even at a significantly higher concentration of 250 μM, a high survival rate (84.5%) was maintained by the MO-MB-injected embryos, demonstrating the negligible toxicity or adverse effect of MOMB in vivo. Next, we evaluated the in vitro stability of MO-MB by two types of experiments: enzymatic digestion (DNase I, RNase H) and nucleic acid binding proteins interaction (single-stranded DNA binding protein, SSB). DNA-MB was degraded rapidly by DNase I (Fig. S6A, ESI†), and RNase H also digested the target RNA that was bound by DNA-MB (Fig. S6B, ESI†), evidenced by the significant changes in fluorescence intensity. In contrast, MO-MB showed the remarkable stability under the identical conditions, even after incubation for 24 h. In addition, in the presence of SSB, a fluorescence increase was observed for DNA-MB, whereas MO-MB showed no fluorescence increase, indicating that MO-MB can efficiently avoid signal interference from nucleic acid binding protein (Fig. S7, ESI†).The in vitro results encouraged us to further examine the stability of MOMB in developing embryos. The mixture of MBs and Texas Red Dextran, which serves as a reference dye, was comicroinjected into 1-cell stage medaka embryos. The dynamic images of the MBs-injected embryos were recorded by fluorescence microscopy at different time ranging from 15 to 120 min (Fig. 3A). The MO-MB-injected embryo possessed a
significant lower background than the embryo View injected into Article Online 10.1039/C5CC07124K the same concentration of DNA-MB. WeDOI: further analysed the images in a pixel-by-pixel manner and quantitatively compared the fluorescence change using the ratio of the intensity of FAM to that of Texas Red. As shown in Fig. 3B (blue line), the fluorescence intensity of the DNA-MB-injected embryo continued to increase and reached a plateau within 45min after injection. The signal increase probably resulted from continuous degradation by nucleases as well as open of the stem-loop structure by protein binding in living embryo. Contrarily, the signal for the MO-MB-injected embryo showed no change over a period of 120 min. After 120 min, fluorescence intensity of the DNA-MB-injected embryo gradually decreased, and disappeared at 24 hpf, implying that the injected DNA-MB was completely digested (Fig. S8, ESI†), whereas it was difficult for MO-MB-injected embryo to
Figure 3. (A) Stability of MO-MB and DNA-MB in vivo. Mixtures containing 5 mg/mL Texas Red and 100 μM MBs were microinjected into 1-cell stage embryos. Embryos were imaged under FAM channel and Texas Red channel (inset left corner) from 15 min to 120 min after injection. (B) In vivo stability evaluated by the ratio of FAM/Texas Red fluorescence intensity (blue line). Fluorescence intensity of each image in (A) was also shown. The intensity from the dash square frame in each channel was sampled and averaged respectively for the calculation of FAM/Texas Red ratio. (C) The mass spectrometry spectra of standard MO-MB and the lysate of the MO-MB injected embryo (24 h post injection).
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monitoring the fluorescence change of the cells due to rapid cell division in the embryos. However, we still detected MOMB in the embryonic cells at 6 hpf (Fig. S9, ESI†) and 24 hpf (Fig. 3C) using LC-MS, further confirming the long-term stability of MO-MB in live embryos. A critical issue in RNA imaging in vivo is target specificity. For hybridization-based probes, stringency (i.e. salt) was required for precise control during probe/target pairing because too high or low stringency can generate false negatives or positives signal. Considering the low salt environment in live embryos, we compared melting temperature (Tm) of MO-MB/RNA and DNA-MB/RNA duplex as a function of salt concentration. Interestingly, in contrast to DNA-MB, MO-MB can effectively hybridize to PM RNA targets in low salt concentrations and Tm values of MO-MB/RNA duplex were independent on the ionic strength of the medium (Fig. S10, ESI†), suggesting that the hybridization function of MO-MB was not interfered by the high stringency condition in live embryos. This finding enabled us to further investigate hybridization potentials of MO-MB in developing embryos. MO-MB was injected to the two 1-cell stage embryos. 3 h after injected, one of the cells of the two embryos at the stage 6 was injected with 10 μM PM RNA target or 1MM RNA target, respectively. As shown in Fig. 4, a single-base mismatch prevented the hybridization of MO-MB resulting in a fluorescent signal indistinguishable from the background fluorescence. By comparison, MO-MB quickly yielded a strong response to the PM RNA targets with signal increase of around 2-fold, indicating high specificity of MO-MB for targeting RNA in developing embryos. In conclusion, we have developed a novel, non-invasive fluorescent probe, morpholino molecular beacon, for RNA visualization in live embryos. MO-MB combines the advantages of MB with the superior properties of morpholino. This probe fulfilled the key features of ideal fluorescent probes for in vivo imaging, namely good biocompatibility, negligible toxicity, long-term stability, and high target specificity. Combined signal amplification with further optimization of MO-MB, this probe should find broad applications regarding RNA visualization in living organisms. Further work will make use of MO-MB to monitor RNA in the highly dynamic processes of embryonic development or tumour development for a
Journal Name better understanding of cell migration and differentiation in View Article Online DOI: 10.1039/C5CC07124K these processes. This work is supported by National Research Foundation of Singapore (NRF-CRP7-2010-03).
Notes and references 1 2
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P. Pantazis, W. Supatto, Nat. Rev. Mol. Cell Biol. 2014, 15, 327- 339; b) S. Pitchiaya, L. A. Heinicke, T. C. Custer, N. G. Walter, Chem. Rev. 2014, 114, 3224-3265. a) A. M. Femino, F. S. Fay, K. Fogarty, R. H. Singer, Science 1998, 280, 585-590; b) A. Raj, P. van den Bogaard, S. A. Rifkin, A. van Oudenaarden, S. Tyagi, Nat methods, 2008, 5, 877-879; c) E. Lubeck, L. Cai, Nat methods, 2012, 9, 743-748. a) S. Tyagi, Nat. Methods 2009, 6, 331- 338; b) N. Li, C. Y. Chang, W. Pan, Angew. Chem. Int. Ed. 2012, 51, 7426-7430; Angew. Chem. 2012, 124, 7544-7548; c) F. Höelmann, I. Gaspar, S. Loibl, E. A. Ermilov, B. Roder, J Wengel, A. Ephrussi, O. Seitz, Angew. Chem. Int. Ed. 2014, 53, 11370-11375; Angew. Chem. 2014, 126, 11553-11558; d) S.Sato, M. Watanabe, Y. Katsuda, A. Murata, D. O. Wang, M. Uesugi, Angew. Chem. Int. Ed. 2015, 54, 1855-1858; Angew. Chem. 2015, 127, 1875-1878. a) D. L. Sokol, X. L. Zhang, P. Lu, A. M. Gewirtz, Proc. Natl. Acad. Sci. USA 1998, 95, 11538-11543; b) D. P. Bratu, B. J. Cha, M. M. Mhlanga, F. R. Kramer, S. Tyagi, Proc. Natl. Acad. Sci. USA 2003, 100, 13308-13313; c) P. Santangelo, N. Nitin, L. LaConte, A. Woolums, Gang Bao, J.Virol 2006, 80, 682-688. a) V. Vijayanathan, T.Thomas, L.H. Sigal, T.J. Thomas, Antisense Nucleic Acid Drug Dev, 2002, 12, 225–233; b) R. Shah, W. S. El-Deiry, Cancer Biol Ther, 2004, 3, 871-875. D.P. Bratu, B.J. Cha, M.M. Mhlanga, F.R. Kramer, S. Tyagi, Proc. Natl. Acad. Sci. USA 2003, 100, 13308-13313. a) C.W Xi, M. Balberg, S. A. Boppart, L. Raskin, Appl. Environ. Microbiol. 2003, 69, 5673-5678; b)Y. Kam, A. Rubinstein, A. Nissan, D. Halle, E. Yavin, Mol. Pharmaceutics 2012, 9, 685693. a) L. Wang, C.Y J.Yang, C. D. Medley, S. A. Benner, W.H Tan, J. Am. Chem. Soc. 2005, 127, 15664-15665; b) I.E. Catrina , S.A. Marras , D.P. Bratu, ACS Chem Biol. 2012, 7, 1586-1595. K. Murayama, Y. Kamiya, H. Kashida, H. Asanuma, ChemBioChem 2015, 16, 1298-1301. a) D. R. Corey, J.M. Abrams, Genome Biol 2001, 2, 1015.11015.3; b) S. Karkare, D. Bhatnagar, Appl Microbiol Biotechnol 2006, 71, 575-586. X. H. Ouyang, I. A. Shestopalov, S. Sinha, G.H. Zheng, C. L. W. Pitt, W. H. Li, A. J. Olson, J. K. Chen, J. Am. Chem. Soc. 2009, 131, 13255-13269. G .Bonnet, S. Tyagi, A. Libchaber, F.R. Kramer, Proc. Natl. Acad. Sci. USA 1999, 96, 6171-6176. a) M.S. Yi, N. Hong, Y.H. Hong, Science 2009, 326, 430-433;b) J. Wittbrodt, A. Shima, M. Schartl, Nat Rev Genet 2002, 3, 5364.
Figure 4. Target specificity of MO-MB in vivo. MO-MB of 200 μM was injected into 1-cell stage embryos, followed by the single-cell injection of 10 μM perfectly matched (PM) or single-base mismatched (1MM) RNA targets mixed with 5 mg/mL Texas Red, respectively, 3 h after MO-MB injection.
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This article can be cited before page numbers have been issued, to do this please use: J. Chen, J. Wu and Y. Hong, Chem. Commun., 2016, DOI: 10.1039/C5CC07124K.
This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains.
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Received 00th January 20xx, Accepted 00th January 20xx
Jianbin Chen,‡a Jikui Wu‡ab and Yunhan Hong*a
DOI: 10.1039/x0xx00000x www.rsc.org/
A non-invasive fluorescent probe, morpholino molecular beacon (MO-MB), was designed for RNA visualization in vivo. Featuring negligible toxicity, stability, and high target specificity in living embryos, MO-MB is superior to conventional probes and has the potential for specific RNA visualization in basic biological and clinical research. The visualization of specific RNA in vivo is of vital significance in biological and clinical research by providing detailed spatiotemporal information of gene expression as well as cell differentiation.1 A variety of fluorescent probes have been developed for imaging RNA in fixed and live cells, 2,3 but due to the low biocompatibility and stability of these probes in living organisms, efficient probes have been lacking for in vivo RNA visualization. The intracellular environment in living organisms is more delicate and rigorous than that in the cultured cells for fluorescent probes. Therefore, ideal fluorescent probes for in vivo application should exhibit the following features: 1) negligible toxicity, the probe should not affect the viability and the normal function of the organisms; 2) stability, the probe should be resistant to degradation by enzymes and be immune to nonspecific binding proteins; 3) target specificity, the probe should be capable of distinguishing its target RNA from nontarget RNA in the complex in vivo environment. Molecular beacons (MBs) have the potential to meet these requirements. MBs are hairpin-structured oligonucleotide probes duallabelled with a reporter fluorophore at one end and a quencher at the other end. In the absence of RNA target, fluorescence of the fluorophore is quenched. Hybridization with their RNA target opens the hairpin and produces fluorescent signals. Although DNA molecular beacon (DNA-MB) has been used for RNA visualization in living cells,4 it is prone
a. Department
of Biological Sciences, National University of Singapore, Singapore 117543, Singapore. E-mail: [email protected] b. College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China. † Electronic supplementary information (ESI) available: Detailed experimental procedures and results. See DOI: 10.1039/x0xx00000x ‡ These authors contributed equally to this work
to be degraded by endogenous nuclease and disrupted by nonspecific binding of DNA/RNA binding proteins, thereby resulting in high background fluorescence and false positive signals. The stability of MBs has been significantly improved by chemically modifying the oligonucleotide backbone or replacing them with artificial nucleic acids, including negatively charged phosphorothioate,5 2'-O-methyl RNA bases,6 peptide nucleic acids,7 locked nucleic acids,8 and serinol nucleic acids.9 However, for in vivo application, these DNA-MB analogues encountered some limitations, such as poor aqueous solubility, toxicity, self-aggregation, slow hybridization rate, and unintended binding to proteins. Therefore, a safe, stable, specific fluorescent probe for visualizing RNA in vivo is demanded. Morpholino oligonucleotides (MOs) are morpholino ringbased oligonucleotide analogues with non-ionic phosphorodiamidate intersubunit linkages (Fig. 1A). This
Figure 1. (A) The structure of morpholino. (B) The stem-loop conformation of the designed share-stem morpholino molecular beacon (MO-MB). (C) The melt curves of MO-MB in the absence (dash line) and presence (solid line) of perfect matched RNA target. (D) Emission spectra of MO-MB before (dash line) and after (solid line) the addition of perfectly matched RNA target. (E) Normalized hybridization kinetics of MO-MB with perfect matched (PM, ○) and singlebase mismatched (1MM, △) RNA target. Conditions: 500 nM MO-MB, 5-fold excess of RNA targets, 10 mM phosphate buffer (pH=7.0, 100 mM NaCl, 3 mM MgCl2), (D) 25 ℃, (E) 28 ℃.
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backbone render several advantages of MOs over other oligonucleotide analogues such as high aqueous solubility, good biological compatibility, high affinities toward RNA and low propensity toward self-aggregation.10 Hence, we reason that MOs might be an attractive candidate to design novel fluorescent probes for RNA imaging in vivo. For this purpose, we developed a shared-stem morpholino molecular beacon (MO-MB). A "random" sequence (25 bp), which does not have any exact match in the medaka genome or transcripts, was chosen to design a model beacon. This MO-MB was composed totally of morpholino and synthesized with an 11-mer loop and 7-mer stem, where FAM was the 3'-end reporter and DABCYL was the 5'-end quencher (Fig. 1B, Fig. S1, ESI†). The purified MO-MB was confirmed by MALDI-TOF mass spectroscope and UV-Vis absorption spectra (Fig. S2, Fig. S3, ESI†). To ascertain whether our designed MO-MB possessed the desired characteristics of conventional molecular beacons, we first investigated the thermodynamic behaviour of MO-MB by monitoring the fluorescence change in the absence and presence of RNA target over a temperature profile. In the absence of RNA target, thermal denaturation measurements of MO-MB showed sigmoidal melting curves, which indicated cooperative base pairing of the MO-MO homoduplex in the
Journal Name stem (Fig. 1C, dash line).11 In the presence of RNA as View target, Article Online DOI: 10.1039/C5CC07124K the temperature slowly raised, MO-MB exhibited a characteristic three-state behaviour which was similar to that of conventional MBs, demonstrating that MO-MB had the conformational constraint of stem-loop structure (Fig. 1C, solid line).12 Further support was provided by the exploration of hybridization properties and sequence discrimination ability of MO-MB using fluorescent measurements. Without RNA target, MO-MB showed very weak fluorescence (Fig. 1D, dash line) and was therefore predominantly in the closed state. Once hybridized with the perfect matched (PM) RNA target, MO-MB underwent a conformation change and opened its stem-loop structure, thereby resulting in more than 12-fold increase of fluorescence intensity (Fig.1D, solid line). As expected, MO-MB discriminated correctly between PM RNA target and singlebase mismatched (1MM) RNA target in kinetic profiles (Fig. 1E, Fig. S4, ESI†). Furthermore, the hybridization kinetics of MOMB was very fast, which was attributed to the high affinity of MO toward RNA. Together, these results suggested that the proposed MO-MB held similar properties to that of DNA-MB and might be used as a fluorescent probe for RNA monitoring in living organisms. To examine the feasibility of MO-MB in vivo, we chose
Figure 2. (A) Images of wild-type medaka adults and embryo. Embryo was imaged under dark field, FAM and Texas Red channels, showing the transparency and essentially free of autofluorescence of the medaka embryo. (B) Biocompatibility of MO-MB and DNA-MB. MO-MB and DNA-MB of 100μM were injected to living embryos at 1 hpf. Morphological change of embryos at 6 and 24 hpf were shown. (C) In vivo toxicity of MO-MB. Morphological distribution of the wild-type embryos (3 dph) and the embryos injected with 100 μM or 250 μM MO-MB at 1 dpf, 5 dpf, 3 dph were depicted. (D) Representative images from the in vivo toxicity assay. Random samples from each group was shown at different time point with the abnormal embyros pointed out. ab, abnormal.
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medaka fish embryos as the model. The developing embryo provides a stringent environment with its high sensitivity to toxics and its dramatic change in gene expression as well as cell morphology during the developmental process. Medaka is widely used as an excellent vertebrate model in basic and applied research such as stem cell biology and sex development.13 The fish is small, easy to maintain and produces eggs every day. More importantly, its embryos are transparent and essentially free of auto-fluorescence (Fig. 2A), which facilitates direct, real-time visualization of the fluorescent probes in vivo. For the feasibility examination of MO-MB in living embryos, we first tested whether MO-MB affected the proper embryonic development and normal cell behaviour of medaka. DNA-MB of the same sequence was used for a comparison. We injected 100 μM MBs into 1-cell stage medaka embryos and followed the development of the embryos during the first 24 h after fertilization. The embryos injected with DNA-MB displayed sever developmental delay/defect and significant cell death, demonstrating that DNA-MB was toxic to the developing embryos. By contrast, the MO-MB-injected embryos developed normally without any obvious differences with respect to wild-type embryos, indicating the good biocompatibility of MO-MB (Fig. 2B). We then quantitatively assessed the potential toxicity of MO-MB to the early development of medaka by morphological analysis. We categorized the MO-MB-injected embryos into three groups (normal, abnormal, and dead) on the basis of changes in some morphological features at 1 day post fertilization (dpf), 5 dpf, and 3 days post-hatching (dph) (Fig. 2D, Fig. S5, Table S2, ESI†). As shown in Fig. 2C, the embryos after MO-MB microinjection at 100 μM displayed a survival rate of 84.7%, which was slightly lower than 89.6% for the wild-type embryos. Even at a significantly higher concentration of 250 μM, a high survival rate (84.5%) was maintained by the MO-MB-injected embryos, demonstrating the negligible toxicity or adverse effect of MOMB in vivo. Next, we evaluated the in vitro stability of MO-MB by two types of experiments: enzymatic digestion (DNase I, RNase H) and nucleic acid binding proteins interaction (single-stranded DNA binding protein, SSB). DNA-MB was degraded rapidly by DNase I (Fig. S6A, ESI†), and RNase H also digested the target RNA that was bound by DNA-MB (Fig. S6B, ESI†), evidenced by the significant changes in fluorescence intensity. In contrast, MO-MB showed the remarkable stability under the identical conditions, even after incubation for 24 h. In addition, in the presence of SSB, a fluorescence increase was observed for DNA-MB, whereas MO-MB showed no fluorescence increase, indicating that MO-MB can efficiently avoid signal interference from nucleic acid binding protein (Fig. S7, ESI†).The in vitro results encouraged us to further examine the stability of MOMB in developing embryos. The mixture of MBs and Texas Red Dextran, which serves as a reference dye, was comicroinjected into 1-cell stage medaka embryos. The dynamic images of the MBs-injected embryos were recorded by fluorescence microscopy at different time ranging from 15 to 120 min (Fig. 3A). The MO-MB-injected embryo possessed a
significant lower background than the embryo View injected into Article Online 10.1039/C5CC07124K the same concentration of DNA-MB. WeDOI: further analysed the images in a pixel-by-pixel manner and quantitatively compared the fluorescence change using the ratio of the intensity of FAM to that of Texas Red. As shown in Fig. 3B (blue line), the fluorescence intensity of the DNA-MB-injected embryo continued to increase and reached a plateau within 45min after injection. The signal increase probably resulted from continuous degradation by nucleases as well as open of the stem-loop structure by protein binding in living embryo. Contrarily, the signal for the MO-MB-injected embryo showed no change over a period of 120 min. After 120 min, fluorescence intensity of the DNA-MB-injected embryo gradually decreased, and disappeared at 24 hpf, implying that the injected DNA-MB was completely digested (Fig. S8, ESI†), whereas it was difficult for MO-MB-injected embryo to
Figure 3. (A) Stability of MO-MB and DNA-MB in vivo. Mixtures containing 5 mg/mL Texas Red and 100 μM MBs were microinjected into 1-cell stage embryos. Embryos were imaged under FAM channel and Texas Red channel (inset left corner) from 15 min to 120 min after injection. (B) In vivo stability evaluated by the ratio of FAM/Texas Red fluorescence intensity (blue line). Fluorescence intensity of each image in (A) was also shown. The intensity from the dash square frame in each channel was sampled and averaged respectively for the calculation of FAM/Texas Red ratio. (C) The mass spectrometry spectra of standard MO-MB and the lysate of the MO-MB injected embryo (24 h post injection).
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monitoring the fluorescence change of the cells due to rapid cell division in the embryos. However, we still detected MOMB in the embryonic cells at 6 hpf (Fig. S9, ESI†) and 24 hpf (Fig. 3C) using LC-MS, further confirming the long-term stability of MO-MB in live embryos. A critical issue in RNA imaging in vivo is target specificity. For hybridization-based probes, stringency (i.e. salt) was required for precise control during probe/target pairing because too high or low stringency can generate false negatives or positives signal. Considering the low salt environment in live embryos, we compared melting temperature (Tm) of MO-MB/RNA and DNA-MB/RNA duplex as a function of salt concentration. Interestingly, in contrast to DNA-MB, MO-MB can effectively hybridize to PM RNA targets in low salt concentrations and Tm values of MO-MB/RNA duplex were independent on the ionic strength of the medium (Fig. S10, ESI†), suggesting that the hybridization function of MO-MB was not interfered by the high stringency condition in live embryos. This finding enabled us to further investigate hybridization potentials of MO-MB in developing embryos. MO-MB was injected to the two 1-cell stage embryos. 3 h after injected, one of the cells of the two embryos at the stage 6 was injected with 10 μM PM RNA target or 1MM RNA target, respectively. As shown in Fig. 4, a single-base mismatch prevented the hybridization of MO-MB resulting in a fluorescent signal indistinguishable from the background fluorescence. By comparison, MO-MB quickly yielded a strong response to the PM RNA targets with signal increase of around 2-fold, indicating high specificity of MO-MB for targeting RNA in developing embryos. In conclusion, we have developed a novel, non-invasive fluorescent probe, morpholino molecular beacon, for RNA visualization in live embryos. MO-MB combines the advantages of MB with the superior properties of morpholino. This probe fulfilled the key features of ideal fluorescent probes for in vivo imaging, namely good biocompatibility, negligible toxicity, long-term stability, and high target specificity. Combined signal amplification with further optimization of MO-MB, this probe should find broad applications regarding RNA visualization in living organisms. Further work will make use of MO-MB to monitor RNA in the highly dynamic processes of embryonic development or tumour development for a
Journal Name better understanding of cell migration and differentiation in View Article Online DOI: 10.1039/C5CC07124K these processes. This work is supported by National Research Foundation of Singapore (NRF-CRP7-2010-03).
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Figure 4. Target specificity of MO-MB in vivo. MO-MB of 200 μM was injected into 1-cell stage embryos, followed by the single-cell injection of 10 μM perfectly matched (PM) or single-base mismatched (1MM) RNA targets mixed with 5 mg/mL Texas Red, respectively, 3 h after MO-MB injection.
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