A Carbonized Fluorescent Nucleolus Probe Discloses RNA Reduction in the Process of Mitophagy

Abstract

Open AccessCCS ChemistryRESEARCH ARTICLE5 Aug 2022A Carbonized Fluorescent Nucleolus Probe Discloses RNA Reduction in the Process of Mitophagy Hua Liu, Xin Geng, Xin Wang, Lin Wei, Zhaohui Li, Shen Lin and Lehui Xiao Hua Liu College of Chemistry, Zhengzhou University, Zhengzhou 450001 State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, College of Chemistry, Nankai University, Tianjin 300071 , Xin Geng College of Chemistry, Zhengzhou University, Zhengzhou 450001 , Xin Wang State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, College of Chemistry, Nankai University, Tianjin 300071 , Lin Wei College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081 , Zhaohui Li College of Chemistry, Zhengzhou University, Zhengzhou 450001 , Shen Lin State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, College of Chemistry, Nankai University, Tianjin 300071 and Lehui Xiao *Corresponding author: E-mail Address: [email protected] State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, College of Chemistry, Nankai University, Tianjin 300071 https://doi.org/10.31635/ccschem.021.202101371 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Mitophagy is a complicated process of cell metabolism that exhibits dynamic spatiotemporal coordination between multiple organelles. Despite its relevance to nucleoli, visualizing the distribution of nucleolar RNA in the process of mitophagy remains a great challenge because of the difficulty in specifically labeling RNA. Herein, we demonstrate a robust carbonized fluorescent probe with folic acid (FA) and m-phenylenediamine (m-PD) as the precursors, which displays superior RNA-anchoring ability and fast cellular permeability to tag nucleolar RNA in living cells. More importantly, this probe possesses spontaneous blinking behavior in physiological conditions, which is suitable for nanoscopic imaging of subtle changes in the distribution of nucleolar RNA. Overall, this work presents a new way for in situ observation of nanoscopic behavior of nucleolar RNA in living cells, providing a powerful approach for further exploration of cellular metabolism in mitophagy. Download figure Download PowerPoint Introduction Mitochondria are membrane-bound organelles that play important roles in supplying energy for cellular metabolic processes.1,2 The reactive oxygen species (ROS) accompanying the energy generating process can induce oxidative damage of mitochondria.3 To maintain a healthy population of mitochondria, the mitophagy process, a specialized form of autophagy, is required to selectively eliminate damaged mitochondria. Typically, damaged mitochondria are engulfed by autophagosomes and digested in lysosomes, which is an important cellular mechanism to sustain cellular metabolism. To further understand this process, various fluorescent probes targeting lysosome-related organelles have been designed to visualize the spatiotemporal coordination among various organelles.4–9 Nucleoli, consisting of ribosomal DNA (rDNA), ribosomal RNA (rRNA), and proteins, are important and highly dynamic subnuclear structures inside the cell that manipulate the synthesis of lysosome-related hydrolases and respond to the cellular environment.10 Despite the significant roles of nucleoli in the mitophagy process, little attention is paid to the dynamic change of nucleoli. Inhibition of the mammalian target of rapamycin (mTOR) signaling can promote mitophagy and diminish nucleolar size and function.11 Upon nutrient starvation, mTOR signaling is inhibited and nucleolar stress triggers several response pathways to maintain cell homeostasis. For instance, the nucleolus is depopulated of proteins in response to cellular stress, driving its reorganization.12 Therefore, having a good understanding of the crosstalk between the reorganization of the nucleolus and mitophagy is significant. However, it is still challenging to reveal the dynamic variation of nucleoli during the mitophagy process because of the targeting difficulty and compact structure of nucleoli. rRNA is synthesized and processed in the nucleoli. The compartmentalization, segregation, aggregation, and cleavage of nucleolar RNA are critical indicators for cell transcription, division, and apoptosis. One of the strategies to elucidate the dynamic behavior of nucleoli in the mitophagy process is to visualize nucleolar RNA by RNA-targeted fluorescent probes. However, monitoring their spatiotemporal distribution is difficult because of the imperfection of current fluorescent probes in differentiating between DNA and RNA, crossing cellular barriers, and being biocompatible.13 Recently, carbonized fluorescent probes, such as carbon dots (CDs), have emerged as an important class of fluorescent probes and been widely applied in live cell imaging due to their excellent biocompatibility, facile preparation, and high cell permeability.14–20 Additionally, CDs have abundant precursors available making them advantageous in designing fluorescent probes with specific targeting ability.21 To achieve RNA-specific targeting capability, an ingenious solution is to synthesize CDs with RNA affinity materials. It has been reported that folic acid (FA) can bind with RNA by interacting with its major groove and terminal loop region.22,23 Moreover, m-phenylenediamine (m-PD) has been widely employed as a precursor in the preparation of CDs.24–26 Therefore, in this work, FA and m-PD were used as precursors to prepare RNA-CDs with RNA-anchoring ability and fluorescent emission properties. Because of the tiny size and highly compressed structure of nucleoli, some subtle but important changes may be missed when using conventional fluorescence microscopy due to the diffraction resolution barrier. Advances in super-resolution microscopy exceed the diffraction resolution barrier and can be employed to observe unresolvable details, providing novel insights into microcosmos and a chance to visualize the subtle changes in the higher-order organization of RNA.27,28 Single-molecule localization microscopy (SMLM) based on the accurate localization of the point spreading function (PSF) from an individual fluorophore has received much attention because it can achieve nanoscopic images without further modification of the microscope.29–32 To get a nanoscopic image with SMLM, the primary task is to satisfy the requirement of the blinking property. Some approaches regulate the blinking behavior of fluorescent probes by, for instance, using imaging buffer devoid of oxygen but containing additives such as thiols, ascorbic acid, and oxygen scavenger.33 However, the addition of additives may change the physiological environment of the cell, disturbing cellular metabolism. Thus, developing fluorescent probes with spontaneous blinking behaviors is important for nanoscopic imaging. In this work, FA and m-PD were used as the precursors to prepare RNA-CDs. As expected, the prepared RNA-CDs exhibit excellent RNA-anchoring ability and display yellow-green fluorescence. Because of the high cell permeability, RNA-CDs accumulate in nucleoli, enabling the imaging of nucleoli in live cells. Interestingly, the prepared RNA-CDs exhibit remarkable blinking property without any additives. They were employed to visualize the dynamic changes of nucleolar morphology and the distribution of RNA in the mitophagy process induced by nutrient starvation (Schemes 1a,b). We found that mitophagy has a significant effect on the nucleolar morphology and nucleolar RNA distribution. The RNA network in a single nucleolus was gradually destroyed in mitophagy. This work provides deep insight into the relation between the dynamic changes of nucleolar RNA and mitophagy, which is also helpful to comprehend other nucleolus-related processes in pathological situations. Scheme 1 | (a) Synthesis of RNA-CDs and their nucleolar RNA imaging capability. (b) In situ tracking of nucleolar RNA with nanoscopic imaging in the mitophagy process. Download figure Download PowerPoint Experimental Methods Fabrication of RNA-CDs RNA-CDs are prepared by one-step hydrothermal synthesis with FA and m-PD as the precursors. Specifically, FA aqueous solution (2.0 mg/mL) and m-PD aqueous solution (5.2 mg/mL) were mixed in equal volumes. After sonication for 10 min, the mixture was transferred to the reaction kettle and reacted at 200 °C for 12 h. After being cooled in the air, the mixture was centrifuged at 10,000 rpm for 10 min and the supernatant was collected. Subsequently, the supernatant was further purified by silica gel column chromatography with a mixture of ethyl acetate and methanol (1:1, v/v) as the eluent. Finally, the prepared RNA-CDs were dissolved in deionized water to a final concentration of 5.0 mg/mL. The quantum yield of the prepared RNA-CDs in water was calculated to be 21.8% by using rhodamine 6G as the reference. Cytotoxicity assay The cytotoxicity assay of RNA-CDs was performed based on a standard methylthiazolyldiphenyl-tetrazolium bromide (MTT) test. In brief, HepG2 cells were first seeded in 96-well plates with a density of 3 × 104 cells per well and cultured for 16 h. Afterwards, the cells were incubated with RNA-CDs (final concentration of 0, 0.1, 0.5, 1, 5, and 10 μg/mL) for 24 h at 37 °C. Then 20 μL of MTT solution (5 mg/mL) was added to each well and incubated with cells for another 4 h. Finally, supernatants were discarded and sediments were dissolved in 150 μL of dimethyl sulfoxide (DMSO). The optical density at 490 nm was measured by a microplate reader. The relative cell viability was determined by the equation: cell viability = (A – Ablank)/(Acontrol – Ablank), where A is the absorbance of the experimental group (cells and RNA-CDs), Ablank is the absorbance of the blank group (without cells and RNA-CDs), and Acontrol is the absorbance of the control group (cells only). Single-particle fluorescence imaging Single-particle fluorescence imaging was carried out by a Ti-U inverted epi-fluorescence microscope (Nikon, Tokyo, Japan) equipped with a single-mode fiber-coupled semiconductor laser. RNA-CDs were excited with a 473 nm laser (Changchun New Industries Optoelectronics Technology Co., Ltd., Changchun, China). The fluorescent signals were collected by a 100× (numerical aperture (NA) 1.49) total internal reflection fluorescence (TIRF) objective. For the nanoscopic imaging of nucleolar RNA, HepG2 cells were seeded in confocal dishes for 24 h and washed with phosphate buffered saline (PBS) twice. Dulbecco's modified eagle medium containing 10% fetal bovine serum and RNA-CDs (1 ng/mL) was added to the confocal dishes and incubated for 15 min at 37 °C, and then serum-free medium was added to the cells to induce mitophagy. The fluorescent images were recorded at different time points, such as 0, 3, 6, and 9 h. The exposure time was 50 ms and the electron multiplying gain of the electron multiplying charge coupled device (EMCCD) (Andor iXon Ultra 897) was set to 5–60. Image J was used to process the results, and appropriate references were introduced to eliminate the system drift. Results and Discussion Fabrication and characterization of RNA-CDs Developing RNA-targeted fluorescent probes is the cornerstone to observe the spatial distribution of nucleolar RNA. It is reported that RNA-targeted fluorescent probes commonly consist of aromatic rings, nitrogen heterocyclic rings, and amine groups.34,35 Phenylenediamines are commonly used precursors for preparing CDs to provide heterocyclic compounds and polymers. According to previous reports, CDs prepared by m-PD have the potential to target nucleolar RNA.36,37 However, an additional precursor is required to improve the RNA targeting capability. FA is a water-soluble vitamin with pteridine, p-aminobenzoic acid, and glutamate moieties, which is involved in the synthesis of nucleic acids. It was reported that FA can bind with RNA by interacting with its major groove and terminal loop region.22,23 Therefore, FA and m-PD were used as the precursors to prepare RNA-CDs by a one-step hydrothermal method to incorporate both merits of FA and m-PD. To translocate through the nuclear pore, probes of small size are a prerequisite for nucleolar RNA imaging. Transmission electron microscopy (TEM) images show that the prepared RNA-CDs have a homogeneous morphology distribution and a small size dimension with an average diameter of 2.2 ± 0.2 nm (Figures 1a and 1b). The optical properties of prepared RNA-CDs were characterized, and RNA-CDs exhibited a maximum absorption at 445 nm and fluorescence emission at 535 nm (Figures 1c and 1e). Excitation-independent fluorescence emission spectra confirmed the homogeneous size distribution of RNA-CDs. To explore the structure of RNA-CDs, Fourier transform infrared (FTIR) spectra of m-PD, FA, and RNA-CDs were obtained (Figure 1f). Some characteristic signals were observed, such as stretching vibrations from O–H (3442 cm−1), N–H (3337 cm−1), C–H (2922 cm−1), C=O (1682 cm−1), C=C (1632 cm−1), C=N (1601 cm−1), and C–N (1400 cm−1).25 X-ray photoelectron spectroscopy (XPS) analysis was performed to further explore the functional groups on the surface of RNA-CDs. In the XPS spectrum (Figure 1g), three peaks that appeared at 284.8, 399.2, and 531.3 eV were attributed to C 1s, N 1s, and O 1s, respectively. In the N 1s spectrum, the binding energies of 399.2 and 400.4 eV confirmed the presence of pyridinic and pyrrolic N groups, respectively.38 The peaks at 284.8, 286.4, and 288.4 eV from C–C/C=C, C–O/C–N, and C=O groups in the C 1s spectrum and the peaks at 531.3 and 532.7 eV from C=O and C–O groups in the O 1s spectrum implied the presence of carbonyl groups and nitrogen-containing compounds on the surface of RNA-CDs.39 These results are in good agreement with the FTIR spectral measurements. Figure 1 | (a) TEM image of RNA-CDs. (b) Size distribution of RNA-CDs. (c) UV–vis absorption spectra of the RNA-CDs. The inset shows the photographs of RNA-CDs under daylight (left) and 365 nm UV light illumination (right). (d) Zeta potential of RNA-CDs. (e) Fluorescence excitation (λem = 535 nm) and emission spectra (λex = 390, 410, 430, 450, 470, and 480 nm) of the RNA-CDs. (f) FTIR spectra of FA, m-PD, and RNA-CDs. (g) XPS spectra of RNA-CDs. Download figure Download PowerPoint Blinking behaviors of single RNA-CDs One of the solutions to achieve nanoscopic resolution with fluorescence microscopy is SMLM ( Supporting Information Figure S1). Random blinking of individual probes is the foundation of nanoscopic imaging. Although the blinking property of fluorophores can be modulated by adding chemical reagents, probes with spontaneous blinking property are still the best choice for nanoscopic imaging. To ascertain whether RNA-CDs can be employed for nanoscopic imaging based on SMLM, the single particle fluorescent behaviors of RNA-CDs without any additives were investigated. Typical fluorescence intensity trajectories of single RNA-CDs are shown in Supporting Information Figure S2a. Spontaneous blinking behavior was evident. After several switches between fluorescent ("on") and dark ("off") states, RNA-CDs maintained high fluorescence emission under 25 s continuous illumination. However, under the same conditions, the fluorescence of single rhodamine B was quenched after blinking a few times ( Supporting Information Figure S2b). To quantitatively understand the blinking performance of single RNA-CDs, the duty cycle (proportion of time in "on" states), the frequency of "on" time (τon), and the frequency of photon-number of each switching event from single RNA-CDs were statistically analyzed from hundreds of trajectories. Fluorescent probes with long-lived "on" states correlate to simultaneous illumination of adjacent fluorophores. If their PSFs overlap, fluorophores cannot be positioned accurately based on the Gaussian fitting. High duty cycle increases the probability of localizing many probes within a diffraction-limited area simultaneously. A long "on" state also triggers similar inaccuracy. Though the "on" time of probes should be as small as possible in theory, the "on" time far less than exposure time will impair the collection of sufficient photons, which is not conducive for nanoscopic imaging. RNA-CDs with low duty cycle (0.06) and "on" times within the range of 0.1–0.3 s are suitable for nanoscopic imaging ( Supporting Information Figures S2c and S2e). As described in previous studies, the photon-number of single fluorescent probes greatly affects precise localization.29–31 Photon distribution of single RNA-CDs in the "on" state is shown in Supporting Information Figure S3. The photon-number from single RNA-CDs is ∼3000 per switching event, which is sufficient for nanoscopic imaging. Despite the remaining debate of the nanocrystals fluorescence blinking, it is consistent to contribute the fluorescence blinking behavior to charge carrier trapping,40 which can impede charge transport and recombination in nanocrystals. Analysis of the duration in the "on" and "off" states is a powerful method to probe charge trapping dynamics. From Supporting Information Figure S4, the probability density of τoff follows an inverse power law distribution, P(τ)off ∝ τ − α off , with a power law coefficient, αoff = 1.3. However, exponential truncation is observed in the probability density of τon, which is fit to a truncated power law, P ( τ ) on ∝ τ − α on × e ( − τ / τ c ) , with αon = 1.4 and cross-over time (τc) is 3.0 s. τc indicates the start of exponential truncation. The distribution of τoff and τon is rationalized. The truncated power law behavior of τon is interpreted as an increase in charge trapping rate. The blinking behaviors of single RNA-CDs can contribute to energetic diffusion of the corresponding charge trapping states or trap states undergoing a process of activation-deactivation. Evaluation of the RNA targeting ability of RNA-CDs RNA is a negatively charged polymer composed of nitrogenous bases, five-carbon sugars, and phosphate groups. Therefore, the common strategy for RNA binding is to construct probes with positively charged aromatic structures based on hydrogen bond interactions and electrostatic interactions. Herein, zeta potential was measured to estimate the surface functional groups of RNA-CDs. Different than common RNA-targeted fluorescent probes, RNA-CDs exhibit a negative zeta potential (−18.5 mV), which might originate from the –COOH group of RNA-CDs (Figure 1d). Compared with those probes with high positive charges, the prepared RNA-CDs can avoid undesirable interactions with negatively charged intracellular organelles and proteins. The effect of various biomolecules on the fluorescent spectra of RNA-CDs, including histidine (His), glycine (Gly), glucose (Glc), galactose (Gal), adenosine triphosphate (ATP), bovine serum albumin (BSA), double-stranded λ-DNA (dsDNA), single-stranded DNA (ssDNA), and RNA, were explored to assess the RNA-selectivity of RNA-CDs (Figure 2e). Generally, the isoelectric points of His, Gly, and BSA are 7.59, 5.97, and 4.9, respectively. Under the test conditions, the pH value is above the isoelectric points of Gly and BSA; therefore, Gly and BSA release protons and carry negative charges. Interestingly, the fluorescence intensity of RNA-CDs increases instantly by more than 10-fold after binding to RNA. Under the same experimental conditions, the fluorescence intensity of RNA-CDs mixed with other biomolecules exhibited negligible fluctuation even in the presence of dsDNA. In the presence of ssDNA, the fluorescence intensity of RNA-CDs increased 2.7 times, which is also far lower than that in the presence of RNA. Previous studies have demonstrated that FA with DNA and RNA at different The fluorescence of RNA-CDs may be due to the different binding with DNA and RNA. From Figures and it is found that the fluorescence intensity of RNA-CDs increases with the addition of RNA. Upon with the fluorescence intensity of RNA-CDs to the value (Figure Figure | (a) in solution internal (b) Fluorescence emission spectra of RNA-CDs (5 μg/mL) in the presence of various of RNA. (c) The between the relative fluorescence intensity and are the fluorescence at 535 nm in the and presence of RNA, and of RNA. (d) Fluorescence emission spectra of RNA-CDs (5 μg/mL) in different situations. (e) studies of RNA-CDs (5 μg/mL) in the presence of potential including His, Gly, 1 ssDNA, and RNA. (f) fluorescence emission spectra of RNA-CDs in with different Download figure Download PowerPoint As shown in Figure fluorescent probes are from the state to the excited state and The excited states are and to the state through three including and To explore the fluorescence mechanism of RNA-CDs after binding with RNA, the fluorescence spectra of RNA-CDs in solutions with different were by using a mixture of and the solution gradually the fluorescence of RNA-CDs in the mixture also was in the solution ( Supporting Information Figures and the fluorescence excitation spectra of RNA-CDs exhibited an with solution consistent with that of RNA-CDs in RNA solution ( Supporting Information Figures and Thus, of RNA-CDs after binding with RNA should be the of fluorescence The fluorescence emission of RNA-CDs in with different was further Generally, fluorescent probes exhibit in However, the fluorescence of RNA-CDs with a in (Figure The fluorescence emission can be by It is reported that a network can the and energy to the fluorescence targeting of nucleolar RNA by the excellent RNA-selectivity of RNA-CDs in HepG2 cells were with both RNA-CDs and to assess the imaging of RNA-CDs in living cells. Figure shows that the RNA-CDs specifically accumulate in the nucleoli, the potential RNA-anchoring ability of RNA-CDs. The corresponding intensity of the shown in Figures and that RNA-CDs in the nucleoli and not with the DNA by results are also observed with other cell including cells cell and cells cell the nucleolar targeting of RNA-CDs in different cell ( Supporting Information Figure images of HepG2 cells with RNA-CDs at different through the further demonstrate the nucleolar targeting ability ( Supporting Information Figure To whether the RNA-CDs in the nucleolar is from RNA, cells and with solution by RNA-CDs were respectively. HepG2 cells were by for 20 min to maintain their structures and and by containing for After the HepG2 cells were incubated with RNA-CDs for 15 min, RNA-CDs exhibited specific in the nucleolar and not with the DNA consistent with the results from live cells ( Supporting Information Figure 25 solution was employed to specifically RNA by with the HepG2 cells at 37 °C for h. As shown in Figures and a in the fluorescence intensity of RNA-CDs in the nucleoli after that the of RNA-CDs in the nucleolar is based on the RNA-anchoring ability of RNA-CDs. Figure 3 | (a) images of HepG2 cells with RNA-CDs μg/mL) and (b) intensity of the fluorescence images from the in (c) intensity the in (d) images of HepG2 cells with μg/mL) and RNA-CDs μg/mL) after or with (e) intensity the in Download figure Download PowerPoint RNA can provide insights into the cellular under different cellular Therefore, the cytotoxicity of RNA-CDs was to the for in situ imaging of RNA in living cells Supporting Information Figure After HepG2 cells were seeded and cultured for 16 were with RNA-CDs 0.1, 0.5, 1, 5, and 10 μg/mL) for 24 respectively. More than of cells at the concentration of for the imaging an excellent and to image RNA in living cells. and intracellular transport of RNA-CDs The and intracellular transport mechanism of RNA-CDs are to elucidate their possible and potential for After HepG2 cells were seeded in the confocal for 24 RNA-CDs were added into the to the images a of 15 min are shown in Supporting Information Figures and RNA-CDs the cell within min and the fluorescence increase gradually with the increase of A is gradually when the time 10 The cellular of RNA-CDs may be attributed to their It is within RNA-CDs in the nucleoli as shown in Supporting Information Figure To understand the intracellular transport mechanism of RNA-CDs, HepG2 cells were with RNA-CDs at physiological and low for 15 min, respectively. The results that cellular of RNA-CDs was at low which might be by the in of various for at low that is involved in the cellular process. The and processes are the pathways for cells. Therefore, of of and of on the cellular of RNA-CDs were investigated. It can be observed from Figures and that the cellular fluorescence signals of RNA-CDs are inhibited by and further

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