Dynamically Long-Term Imaging of Cellular RNA by Fluorescent

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Dynamically long-term imaging of cellular RNA by fluorescent carbon dots with surface isoquinoline moieties and amines Yunying Cheng, Chun Mei Li, Rui Zhu Mu, Yuan Fang Li, Tian Tian Xing, Bin Bin Chen, and Cheng Zhi Huang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02301 • Publication Date (Web): 31 Aug 2018 Downloaded from http://pubs.acs.org on September 1, 2018

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Analytical Chemistry

Dynamically long-term imaging of cellular RNA by fluorescent carbon dots with surface isoquinoline moieties and amines Yunying Cheng†, Chunmei Li*,‡, Ruizhu Mu†, Yuanfang Li†, Tiantian Xing†, Binbin Chen‡, Chengzhi Huang*,†,‡ †

Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, P. R. China. ‡ College of Pharmaceutical Science, Southwest University, Chongqing 400716, P. R. China. ABSTRACT: Cellular RNA dynamics are closely associated with a vast range of physiological processes that are mostly longlasting. To uncover the association between RNA dynamics and these processes, fluorescent RNA probes with high specificity, photostability and biocompatibility are compulsory. Herein, a series of fluorescent carbon dots (CDs) have been prepared by onepot hydrothermal treatment of o-, m-, or p-phenylenediamines with triethylenetetramine. Only CDs derived from the meta precursor (m-CDs) with excellent photostability and biocompatibility can specifically bind to cellular RNA, allowing successfully long-term (up to 3 days) monitoring of RNA dynamics during cell apoptosis, mitosis, and proliferation. This RNA affinity can be attributed to the isoquinoline moieties and amines on the surface of m-CDs, which can bind to RNA through π-π stacking and electrostatic bonding, respectively. The cellular internalization of m-CDs is time-, temperature-, ATP-, caveolar and microtubule dependent. Additionally, investigations on the in vivo behavior of m-CD suggest that they can be efficiently and rapidly excreted from the zebrafish larvae body after 48 h. Our results provide a powerful tool for clarifying complex relationships between RNA dynamics and basic biological processes, disease development or drug interactions.

INTRODUCTION Ribonucleic acids (RNA) are responsible for a wide range of functions in biological systems spanning from transcriptional regulation to essential catalytic roles.1,2 For instance, the dynamics of ribosomal RNAs (rRNA) that assemble in the nucleolus are related to varieties of physiological activities, including cell mitosis, proliferation, stress response and some diseases.3 Spatial-temporal dynamic changes in rRNA synthesis may correlate with the most important molecular alterations in cancer cells and are helpful to evaluate prognosis in tumor.4 One strategy to elucidate the complex roles of RNA dynamics in cellular functions is real-time monitoring of their temporal-spatial distribution, which can be visualized by targeted imaging with fluorescent probes. Various methods have been developed to image RNA, including fluorescence-labelled beacons,5 fluorescence in situ hybridization,6 gold nanoparticle-labelled oligonucleotides probes,7-10 DNAzyme motor,11 and organic small molecules (OSMs).12-14 Despite these progresses in investigations of cellular RNA dynamics, challenges, such as specificity, targeting ability, and long-term imaging capability,15 still remain for easy photobleaching and high cytotoxicity of these imaging probes.16 These challenges, especially the requirements for long-term imaging under continuous irradiation, are critical in studying the dynamics of RNA in cellular processes, including cell apoptosis, mitosis, proliferation, and stress response, which are very slow that may last for several hours or days.17 Fluorescent carbon dots (CDs) have emerged as new outstanding alternatives for fluorescent OSMs and semiconductor quantum dots (QDs), because of their intrinsic advantages.18

These advantages, particularly the features of low cytotoxicity and photobleaching, enable promising applications for fluorescent CDs in a variety of fields, especially in biomedical imaging.19-22 Despite of these substantial advantages, CDs still suffer from a significant challenge, namely precise control of selectivity in complex biological systems.23 To achieve target ability, most of CDs generally have to be modified with functional moieties,24 resulting in more complicated experimental operation. Hence, the one-pot preparation of CDs with high photostability, excellent biocompatibility, specificity and generality for long-term imaging cellular RNA is desired. According to our incomplete survey, RNA-selective dyes exhibit a common feature of having aromatic rings, nitrogencontaining heterocycles and amines (Table S1).12-14 As significant precursors for producing heterocycles and polymers, phenylenediamines have been frequently used to prepare CDs which have good photoluminescence properties and cellular imaging capability.25-27 Inspired by the above-mentioned points, herein, we have prepared three types of CDs (o-CDs, m-CDs and p-CDs) by the reactions of triethylenetetramine (TETA) and the respective phenylamines (ophenylenediamine (o-PD), m-phenylenediamine (m-PD), and p-phenylenediamine (p-PD) via a one-pot hydrothermal process. Only CDs derived from the meta precursor (m-CDs) which have isoquinoline-like groups and amines, can leverage π-π stacking and electrostatic bonding for selective targeting imaging of RNA in biological systems (Scheme 1). The mCDs have been used for real-time monitoring of cellular RNA dynamics during apoptosis, mitosis and proliferation for several hours and up to 3 days, demonstrating their long-term imaging applicability. The m-CDs conclusively exhibit great

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promise in cell biology and medical imaging involving longterm changes of cellular RNA dynamics, thus providing novel insights into complex relationships between RNA dynamics and cell processes.

followed by incuba-tion at 37 °C in 5% CO2 for 3 h. The cells were then stained with either m-CDs (50 µg/mL) or SYTO RNA Select (5 µM) in PBS solution. After washing with PBS, the fluorescence images were obtained using equal exposure times. To improve picture quality, the brightness and contrast were adjusted using Image-Pro plus software ipwin32. Long-term monitoring RNA dynamics during cell mitosis, proliferation and apoptosis. For m-CDs, HEp-2 cells were incubated with medium containing m-CDs (50 µg/mL), and then placed on the microscope platform with a live-cell incubation chamber. Fluorescence images were acquired every 20 min lasting for 8 h to monitor the dynamic change of cellular RNA during mitosis. For cell proliferation, the HEp-2 cells were acquired every 24 h lasting for 3 days on the GFP475 channel. For apoptosis, HEp-2 cells were separately treated with doxorubicin (DOX, 10 µg/mL) and actinomycin D (Act D, 0.05 µg/mL) after incubation with m-CDs for 2 h or SYTO RNA Select (5 µM) for 30 min. Then the dishes were placed on the microscope platform with a live-cell incubation chamber. Fluorescence images of cells stained with m-CDs on the GFP475 channel were acquired every 30 min lasting for 5 h (for Act D) or every 2 h lasting for 24 h (for DOX). The images of cells incubated with SYTO RNA Select were obtained every 30 min lasting for 5 h (for Act D), but every 1 h lasting for 12 h (for DOX) because of high cytotoxicity of SYTO RNA Select. The cells stained by m-CDs exposure to ultraviolet (UV) were irradiated with the laser of confocal microscopy (power intensity: 400 µJ/cm2) and simultaneously imaged every 30 min lasting for 3 h. Because of high cytotoxicity and photo-bleaching, the cells treated with SYTO RNA Select were irradiated and imaged every 10 min lasting for 30 min.

Scheme 1. Reaction route and cellular RNA imaging ability of mCDs.

EXPERIMENTAL SECTION Materials. TETA, o-PD, m-PD, and p-PDs were purchased from Aladdin Reagent Co., Ltd (Shanghai, China). Durapore PVDF membrane filters were obtained from Millipore™ with nominal pore size of 0.22 µm. RNA from baker’s yeast, 5-(NEthyl-N-isopropyl) Amiloride (EIPA), sodium azide (SA), 2deoxy-d-glucose (2-DG), filipin, chlorpromazine, nocodazole, and Ribonuclease A (RNase A) were acquired from SigmaAldrich (UK). SYTO RNA-Select and Cholera Toxin Subunit B (594) were commercially available from Invitrogen (USA). Deoxyribonuclease I (DNase I) and cordycepin were purchased from Sangon Biotech Co., Ltd (Shanghai, China). Tubulin-tracker red and Hoechst33342 were obtained from Beyotime (Nantong, China). All other reagents used were of analytical grade. Preparation of the CDs. TETA (0.2 mL) and m-PD (o-PD or p-PD) (100 mg) were dissolved in double distilled water (4.8 mL), followed by heating hydrothermally at 220 °C for 24 h in Teflon-equipped stainless-steel autoclave. After the reaction, the reactors were cooled to room temperature naturally. The brown resultant solutions were separated by silica-gel column chromatography (300~400 mesh) with a mixture of methylene chloride and methanol as eluents. After removing solvents and further drying under vacuum, three kinds of purified CDs (o-CDs, m-CDs and p-CDs) were finally obtained. The reaction yield of m-CDs is about 20%. DNase and RNase digestion tests. HEp-2 cells were first fixed in pre-chilled pure methanol for 1 min. The cell membrane was then permeabilized with 1% Triton X-100 for 2 min at ambient temperature. After rinsing with PBS twice, DNase (50 µg/mL), DNase-free RNase (100 µg/mL) and PBS solution(as control experiment) were added to separate samples,

RESULTS AND DISCUSSIONS Development of cellular RNA-specific CDs. The asprepared o-CDs, m-CDs and p-CDs exhibited maximum emission at 480, 510 and 520 nm when excited at 360, 360 and 370 nm (Figure S1, Figure 1a, and b), with absolute quantum yield (QY) of 4%, 6% and 7%, respectively. The transmission electron microscopy (TEM) images showed that m-CDs are monodispersed with a diameter distribution of 2.75 ± 1.08 nm (Figure 1c), which is in agreement with hydrodynamic diameter determined by dynamic light scattering (DLS) (Figure S2). It also indicated that m-CDs comprised of 5–9 layers, corresponding to the heights ranging from 1.5 to 2.9 nm in AFM (Figure 1c, Figure S3). The lattice spacings of 0.21 nm and 0.32 nm are attributed to the lattice fringes of graphene (100 facet) with in-plane sp2-hybridized carbon atoms and the spacing of graphitic carbon (002 facet) with interfacial sp3hybridized carbon atoms, respectively. The o-CDs and p-CDs also displayed uniform nanoparticles with average sizes of about 1.9 nm and 2.1 nm, respectively (Figure S4). These three types of CDs were separately used to stain HEp-2 cells (Figure 1a). The m-CDs are distributed throughout the cytoplasm, but mainly concentrated in the nucleoli, while o-CDs and p-CDs are docked into the perinuclear region and failed to target RNA in the nucleoli. A comparison of the distribution in living cells shows an perfect correspondence between m-CDs and commercial RNA-specific dye, SYTO RNA Select (Figure S5), suggesting that m-CDs could be potentially served as optical probes for cellular RNA imaging.


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Analytical Chemistry CDs at various concentrations have been recorded (Figure S7). The results suggest that the m-CDs preferably bind to RNA in solution. Other biomolecules and ions, including protein, amino acids, glucose, ATP, K+ and HCO3¯, did not show any interfering effects. Accordingly, these observations clarify that the m-CDs can selectively bind to RNA. To investigate the general applicability of m-CDs for RNA imaging, three types of human cell lines, including cancer cells (A549 and HEp-2) and normal cells (NHBE), animal tissue (cells of paraffin-embedded tilapia testes) and plant cells (onion cells) were separately incubated with the m-CDs. As shown in Figure 2b, all fluorescence images showed significant green emission from m-CDs in the nucleoli, while only weak green fluorescence emission was visible in the cytoplasm. It is known that rRNA (from nucleoli), tRNA (from nuclei) and mRNA (from nuclei), respectively accounting for almost 80%, 15% and 5% of the total RNA in the eukaryotic cell, are eventually transferred to the cytoplasm. Several researches on preparing CDs with nucleolus-targeting capability for their rRNA affinity have been previously reported.31-33 Compared to these CDs, imaging of RNA by using m-CDs could provide more dynamic information of mRNA and tRNA. Furthermore, the m-CDs show better selectivity through DNase and RNase digest experiments. Similarly, a clear distinction between the signals of m-CDs in the nucleoli and the nuclei indicates that m-CDs perform better than SYTO RNA Select for imaging nucleolar RNA in living cells (Figure S5).

Figure 1. Development of o-CDs, m-CDs and p-CDs for targeting imaging of cellular RNA. a) Preparation routes of oCDs, m-CDs and p-CDs by mixing TETA with the respective phenylenediamine isomers (i.e., o-PD, m-PD and p-PD), corresponding photographs of solutions in daylight and under 365 nm UV irradiation, and fluorescence images of HEp-2 cells separately stained with o-CDs, m-CDs and p-CDs. Scale bar, 10 µm. All images are representative of replicate experiments (n=5). b) UV-vis absorption and fluorescence spectra of the m-CDs. c) TEM and high resolution TEM images of m-CDs. Inset is the size distribution of m-CDs. d, e) XPS and FT-IR spectra of m-CDs.

Specificity and generality of m-CDs for imaging cellular RNA. To verify the RNA-selectivity of m-CDs in the cells, ribonuclease (RNase) and deoxyribonuclease (DNase), which can specifically hydrolyse RNA and DNA, respectively, were used to treat HEp-2 cells. A dramatic decrease in the fluorescence intensity of m-CDs occurred in the cytoplasm and nucleolus following exposure to RNase, but no obvious change was observed upon the treatment with DNase, strongly indicating that m-CDs reliably bind RNA in living cells (Figure 2a). The most interesting observation is that the decrease in fluorescence intensity of m-CD is more pronounced than that of SYTO RNA Select after RNase treatment. Additionally, the RNA selectivity of m-CDs in solution was also clarified by dark field imaging and resonance light scattering (RLS) technique which are suitable for studying interaction between the molecules for high sensitivity, rapid procedure and simple instrumentation.28 The m-CDs showed different RLS responses after adding different biomolecules and ions (Figure S6). The addition of RNA induced strong enhancement of RSL signal, which is caused by aggregation of m-CDs and RNA. The formation of aggregates was confirmed by dark-field imaging. DNA also slightly affected RSL intensity of m-CDs. The similar phenomenon has been also reported in other RNA-specific dyes, such as MPI and StyrylTO.29,30 In order to further investigate difference in the affinity of m-CDs between RNA and DNA, RLS titration curves of m-

Figure 2. Specificity and generality of m-CDs for imaging cellular RNA. a) DNase and RNase digestion experiments. Hoechst 33342, a DNA-specific dye; SYTO RNA Select, a RNA-specific dye. Scale bar, 20 µm. b) Counterstaining of mCDs with Hoechst33342 in living cells. Scale bar, 20 µm. c) Imaging of cellular RNA in the paraffin-embedded sections of tilapia with m-CDs. Scale bar, 200 µm. d) Imaging of RNA in onion cells with m-CDs. Scale bar, 100 µm. All images are representative of replicate experiments (n=5). Moreover, the m-CDs also can specifically image RNA in animal and plant tissue (Figure 2c, d). Bright green fluorescence was clearly visible in the nucleoli of cells in paraffin-



embedded animal tissues and onion cells after incubation with m-CDs. Both RNase digestion in paraffin-embedded animal tissues and treatment of paraffin-embedded onion cells with cordyceptin (an inhibitor of RNA synthesis) resulted in faint fluorescence, further indicating the RNA specificity of m-CDs. Cellular RNA targeting mechanism of m-CDs. The FT-IR and XPS spectra demonstrated the existence of pyridinic nitrogen and N-H bond in m-CDs (Figure 1d, e, and Figure S8), in agreement with the results of 1H and 13C NMR spectra (Figure S9 and S10). In high resolution mass spectrum (HRMS) of m-CDs, the fragments at m/z 173.10688 and m/z 187.12288 were assigned to the molecular formulas of C11H12N2 and C12H14N2 and identified as N-(isoquinolin-1-ylmethyl) ethanamine and 1-(isoquinolin-1-yl)-N-methylmethanamine, respectively (Figure 3a, Table S2), indicating the existence of both amines and isoquinoline-like rings.

(1H-indol-3-yl)vinyl) quinaldine (E36), (E)-2-(2,4,6trimethoxystyryl) quinaldine (E144), (E)-4-(2-(6-methoxy quinaldine -2-yl)vinyl)-N,N-dimethylaniline (F22) and StyrylTO (Table S1), implying that these structural features are critical for the specific binding and targeting of cellular RNA. In addition, FT-IR spectra of o-CDs and p-CDs displayed analogous characteristics to that of m-CDs (Figure 1e, Figure S11). However, no corresponding signal of isoquinoline-like ring was observed in the XPS, 1H and 13C NMR spectra of oCDs and p-CDs (Figure S8 and S12). The results suggested the absence of isoquinoline-like rings in the surface of o-CDs and p-CDs which are non-specific for RNA, illustrating the importance of isoquinoline-like rings for targeting RNA. The 1H NMR spectra of the m-CDs measured in the presence of RNA showed that the binding of RNA caused the chemical shifts of m-CDs in the range of 7.5-8.5 ppm to move slightly upfield with a shift of 0.1-0.2 ppm (Figure 3c), which presumably results from the π−π interactions between isoquinoline parts of m-CDs and the nucleobases of RNA in the major groove.14,38 The zeta-potential measurement indicated m-CDs possess positive charge of 22.4 ± 1.2 mV. A further evidence for the direct link between the isoquinoline moieties and amines of m-CDs and RNA targeting was provided by endowing non-target CDs (called PTDA-CDs) 39 with RNA target ability via modification it with (E)-4-(2-(isoquinolin-1yl)vinyl)aniline (IVA) (Figure 3d, Figure S13-15). These results demonstrate that the RNA affinity of m-CDs is originated from the surface isoquinoline-like rings and amines. Long term monitoring of cellular RNA dynamics in realtime with m-CDs. Since different cellular processes exhibit different RNA profiles,40 long-term monitoring of RNA dynamics can provide novel insights into understanding complex cellular mechanisms and the cellular state. Therefore, the photostability and the cytotoxicity of m-CDs were studied to evaluate the suitability for in situ long-term imaging of RNA in living cells. As shown in the Figure S16, the m-CDs possess good photostabilities under continuous irradiation or in solutions with strong acidity, high salt and oxidization. Time-lapse imaging over a period of 120 min under continuous irradiation with a mercury lamp in the focal plane revealed that m-CDs, as compared to SYTO RNA Select, exhibited a remarkable photostability (Figure 4a). Besides, the low cytotoxicity of mCDs was demonstrated by CCK-8 cell viability assay in HEp2 cells (Figure S17).The excellent biocompatibility and photostability of m-CDs, along with their RNA specificity and generality, enable real-time monitoring of the RNA dynamics over a long term in cellular processes. First, we investigated the RNA dynamics in the presence of apoptosis inducers in HEp-2 cells. UV, for example, damages DNA structure, whereas doxorubicin (DOX) and actinomycin D (Act D) inhibit the synthesis of total RNA and rRNA, respectively (Figure 4b, c).41 Under UV irradiation, no significant change of fluorescence emissions was observed in the cells over 5 h, even though cell damage occurred. However, treatment of cells with DOX, which can cease DNA replication and total RNA transcription, resulted in a decrease of fluorescence signals in both the cytoplasm and nucleoli after 24 h. After incubating with Act D, an RNA Pol I-targeted anticancer drug, the fluorescence signals decreased significantly over time in the nucleoli, while barely changed in the cytoplasm, illustrating that only rRNA synthesis was inhibited,

Figure 3. Mechanistic investigation of RNA targeting of mCDs. a) HRMS spectra of m-CDs. b) Possible formation mechanism of the isoquinoline-like rings. c) Partial 1H NMR spectra of m-CDs (100 µg/mL) in presence (black line) or absence (red line) of RNA. RNA (50 µM) from yeast was dissolved in D2O as stock solution. d) Cellular imaging of HEp-2 cells after incubation with the original PTDA-CDs (20 µg/mL) and IVA-modified PTDA-CDs (PTDA-CDs-IVA, 20 µg/mL). The fluorescence images are representative of replicate experiments (n=5). Scale bar, 20 µm. The possible formation mechanism of isoquinoline-like rings is depicted in Figure 3b. Firstly, treatment of m-PD in TETA at 220 °C gave compound 1.34 Next, compound 1 thermally dissociates into phenylenediamine and N1-(2-aminoethyl) -N2-ethylethane-1,2-diamine cation, and the latter attack the phenylenediamine ring preferentially in the electronically and sterically favored ortho position (compound 2) through Hofmann−Martius rearrangement.35 Then compound 2 transform to 3 as depicted in literature and finally form isoquinoline-like moiety.36,37 Interestingly, both amines and quinoline-like rings are also present in other RNA-specific dyes, such as (E)-2-(2-


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Analytical Chemistry which is well consistent with previous work.42 The dynamic changes in the fluorescence intensity of m-CDs are well consistent with the treatment effect of different drugs, proving the ability of m-CDs to monitor cellular RNA dynamics.

rRNA synthesis. With the onset of anaphase, several small fluorescent spots were observed, and became larger and brighter in the nucleolar region at telophase, implying the reassembly of the nucleoli and restoration of rRNA synthesis. That means m-CDs can be simultaneously applied for imaging nucleoli, which are closely linked with physiological stress (such as starvation, hypoxemia and drug exposure) and diseases (such as cancer).43,44 Consequently, m-CDs have the capability to provide novel insight into the cellular state and complex relationships between nucleoli (or rRNA) and diseases. Finally, we investigated whether the m-CDs can image RNA over a long time scale in living cells. HEp-2 cells incubated with m-CDs were imaged over 3 days, during which the cell number increased from 7 to 17 (Figure 4e, f), confirming that m-CDs have excellent biocompatibility and photostability. As a clear contrast, the treatment of RNA SYTO Select not only disrupted the cell proliferation, but also altered the cellular morphology. Cellular internalization, and intracellular transport of m-CDs. The internalization and intracellular transport mechanism of m-CDs are of utmost importance in elucidating their possible hazards and potentials for studying biological behaviour and beneficial use. Therefore, the internalization time of m-CDs, an essential factor in elucidating the efficiency of the cellular internalization,45 was monitored by time-lapse imaging over a period of 120 min (Figure 5a). The m-CDs exhibited high speed (within 5 minutes) and efficiency of nuclear internalization as compared to other CDs,46 which may be attributed to the positive charges and ultra-small size of mCDs.47 This provides an insight for development of new classes of highly effective drug delivery systems. To understand the intracellular transport mechanism of mCDs, a systematic study using confocal fluorescent microscopy, flow cell cytometry and co-localization has been performed in the presence of diverse endocytosis inhibitors (Figure 5b-g). The results showed that cellular internalization of m-CDs was significantly blocked by low temperature (4 °C) and ATP depletion (SA and 2-DG) (Figure 5b). This internalization blocking may be caused by the decrease in activities of various enzymes for ATP production at low temperature and inhibition in the respiratory chain, indicating that energydependent endocytosis is predominant pathway for the internalization of m-CDs. Furthermore, the fluorescence signals of m-CDs were not appreciably influenced by ethylisopropylamiloride (EIPA, a micropinocytosis inhibitor) or chlorpromazine (Chlorp., an inhibitor of clathrinmediated endocytosis), but were significantly inhibited by filipin (caveolin inhibitor) and nocodazole (nocod., an inhibitor of microtubuledependent transport), suggesting the involvement of caveolin and microtubules in the cellular internalization of m-CDs. These results were confirmed by flow cell cytometry analysis and the co-localization of m-CDs and cholera toxin subunit B (a marker of caveolin) or Tubulin-Tracker red (a marker of microtubules) (Figure 5c-g). Accordingly, we presume that the m-CDs are trapped in endocytic vesicles by caveolin-mediated endocytic pathways, subsequently transported to the perinuclear region by microtubule-dependent endosomes translocation. Owing to the existence of amines on the surface, the m-CDs can escape from the endosomes via the proton sponge effect, and associate with the nuclear envelope.48 There are many nuclear pore complexes

Figure 4. Long-term in situ imaging of cellular RNA with mCDs. a) Time-lapsed imaging of RNA by m-CDs and SYTO RNA Select (upper) and corresponding fluorescence intensity (lower). b) Time-lapse imaging of RNA in HEp-2 cells after treatment with different apoptosis-inducing strategies. c) Corresponding mean fluorescence intensity of m-CDs in nucleoli (dot) and cytoplasm (triangle) of b. d) Time-lapse fluorescence imaging of RNA in HEp-2 cells during mitosis after incubation with m-CDs. Red boxes indicate the cells during mitosis. e, f) Long time-lapse imaging of RNA in HEp-2 cells using mCDs and SYTO RNA Select in the range of 0-3 days (e) and corresponding cell numbers. (f) All images are representative of replicate experiments (n=5). Scale bar, 20 µm. To further provide proof for monitoring RNA dynamics by m-CDs, the cells exposed to the apoptosis inducers were stained with SYTO RNA Select (Figure S18) during the cell apoptosis. The UV irradiation caused a dramatic decrease in the fluorescence intensity of SYTO RNA Select in both of nucleoli and cytoplasm within 30 min, which is attributed to the high photo-bleaching under the continuous UV irradiation (Figure 4a). Except for UV irradiation, the dynamic changes of RNA in the cells stained with SYTO RNA Select after treatment with other apoptosis inducers (DOX and Act D) are well identical with that of m-CDs, confirming that m-CDs can specifically monitor cellular RNA dynamics. These results also provide the potential of m-CDs for evaluating the therapeutic effect of RNA Pol I-targeted anticancer drugs. Next, we monitored RNA dynamics during cell mitosis for a cell division cycle. Bright fluorescence was observed at interphase, then became distorted and disappeared in nucleoli with a dramatic decrease in the fluorescence signals at prophase (Figure 4d, Movie S1), indicating the repression of



embedded in the nuclear envelope, and small diffusional channels with a diameter of 5−9 nm are located in the nuclear pore complex.49 These small diffusional channels allow passive diffusion of molecules smaller than 5 nm in and out of the nucleus. The m-CDs is small enough (average diameter of 2.75 nm) to cross the nuclear membrane by passive diffusion. Therefore, the escaped m-CDs may ultimately entry into the nuclear through nuclear pore complex.

confirming the high biocompatibility of m-CDs. The majority of m-CDs were accumulated in the lens, pronephros, intestine, and vessel after incubation (Figure S24), which was confirmed by comparing with the previous reports51 and the Zebrafish Information Network (an online biological database of zebrafish). Strong signals were observed in the intestine with relatively weak signals in the pronephros after 24 h (Figure S25), while no significant fluorescence was detected in any organs after 48 h, indicating that m-CDs were all cleared out from the body. Accordingly, we presumably conclude that the clearance of m-CDs was involved in two major pathways after entering the body via swallowing. One pathway is by direct excretion via intestine and cloacal aperture (Figure S24c and e), while the other pathway is that m-CDs were absorbed to the blood, subsequently transported to pronephros which is a functional kidney in early teleosts and responsible for the disposal of metabolic waste products,52 and eventually excreted through cloacal aperture (Figure S24b, d and e). This pathway is consistent with that of other CDs previous reported in mouse.53 These results lay the foundation for exploring the biosafety of m-CDs in biomedical application.

CONCLUSIONS Our study highlights the following three attractive feathers: (1) by

exploiting the molecular structure characteristics of the RNAspecific dyes, we have prepared a novel and modification-free fluorescent CDs merely by selecting precusors via a one-pot hydrothermal process. This work demonstrates that, by appropriate selection of the precusors, precise control over the selectivity of CDs is a viable process. (2) The obtained CDs, namely m-CDs, showed excellent specificity, photostability, biocompatibility, allowing for long-term (up to 3 days) imaging cellular RNA in biological systems, which hasn’t been adequately explored in the traditional probes. With the capability of long-term RNA imaging, m-CDs have been successfully applied to real-time monitor RNA dynamics during cell apoptosis, mitosis and proliferation. (3) The RNA targeting mechanism of the m-CDs has been investigated and explicated adequately, demonstraing that these cellular RNA affinities are mainly attributed to strong electrostatic interactions and π-π stacking of the isoquinoline moieties of m-CDs with the major groove of RNA. Thus, as a very smart probe for cellular RNA imaging, the m-CDs are expected to expedite the discovery of complex relationships between RNA dynamics and cellular physiological process (such as disease), and are valuable for further development of visualized screening and pharmacodynamic evaluation of RNA polymerase I-targeted anticancer drug.

Figure 5. Cellular internalization and intracellular transport of m-CDs. a) Time-dependent cellular internalization of m-CDs in the range of 0-120 min. b) Endocytic pathway of m-CDs in the presence of different specific inhibitors. c, d) Colocalization of m-CDs and Cholera Toxin Subnit B or Tubulin Tracer in HEp-2 cells. e, f) Flow cytometry analysis corresponding to b. g) Mean fluorescence calculated from e and f using Flowjo 7.6.2 software. All images are representative of replicate experiments (n=5). Scale bars, 20 µm. Biocompatibility, bio-distribution and metabolic pathway of m-CDs in vivo. Investigations on in vivo behaviours of nanomaterials, including biocompatibility, bio-distribution and metabolic pathway, are also critical to illuminate their possible hazards for human health and ecological environment. As an important model for studying metabolic pathway of nanomaterials, the zebrafish larvae were used to elucidate the biodistribution and metabolic pathway of m-CDs for their transparent body and sensibility to environmental change.50 As depicted in the Figure S19-S23, the m-CDs did not significantly interfere with the viability and development of zebrafish embryos even at high concentration as 1 mg/mL,

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Additional experimental details; characterization of o-CDs, m-CDs and p-CD; fluorescent images of cells stained by SYTO RNA Select; dark field images and resonance light scattering intensity of m-CDs in presence of different substances; investigations on cellular RNA targeting


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mechanism of m-CDs; the photostability and biocompatibility of m-CD; proof of monitoring cellular RNA dynamics by m-CD, Movie for monitoring RNA dynamics by m-CDs during cell mitosis; in vivo biodistribution and metabolic pathway of m-CDs (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]； *E-mail: [email protected], Tel: (+86) 23 68254659, Fax: (+86) 23 68367257.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS We sincerely appreciate Prof. Na Li and Prof. Wenxiong Zhang at Peking University, Prof. Yuanjiang Pan at Zhejiang University for helpful discussions. We also acknowledge Dr. Kathryn R. Williams at the University of Florida for her assistance with manuscript revision. This work was support by the National Natural Science Foundation of China (NSFC, No. 21535006 and No. 21705131).

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Analytical Chemistry

for Table of Contents only

A novel type of fluorescent carbon dots (CDs) with excellent biocompatibility, photostability and specificity has been developed to long-term in situ monitor RNA dynamics in living cells during varies biological processes through surface isoquinoline moieties and amines.


Dynamically Long-Term Imaging of Cellular RNA by Fluorescent

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