Quantum Dot Nanobeacons for Single RNA Labeling and Imaging

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Quantum Dot Nanobeacons for Single RNA Labeling and Imaging Yingxin Ma, Guobin Mao, Weiren Huang, Guoqiang Wu, Wen Yin, Xinghu Ji, Zishi Deng, Zhiming Cai, Xian-En Zhang, Zhike He, and Zongqiang Cui J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b04659 • Publication Date (Web): 24 Jul 2019 Downloaded from pubs.acs.org on July 24, 2019

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Journal of the American Chemical Society

Quantum Dot Nanobeacons for Single RNA Labeling and Imaging Yingxin Ma1, 2‡, Guobin Mao3‡, Weiren Huang2‡, Guoqiang Wu2, Wen Yin1, Xinghu Ji3, Zishi Deng2, Zhiming Cai2, Xian-En Zhang4, Zhike He3*, Zongqiang Cui1* 1

State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, P.R. China Key Laboratory of Medical Reprogramming Technology, Shenzhen Second People’s Hospital, The First Affiliated Hospital of Shenzhen University, Shenzhen University School of Medicine, Shenzhen P.R. China; 3 Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Sciences, Wuhan University, Wuhan, Hubei, P.R. China; 4 National Key Laboratory of Biomacromolecules, CAS Center for Biological Macromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, P.R. China 2

ABSTRACT: Detection and imaging RNAs in live cells is highly in demand. Methodology for such a purpose is still a challenge, particularly for single RNA detection and imaging in live cells. In the present study, a type of quantum dot (QD) nanobeacons with controllable valencies was constructed by precisely conjugating the black hole quencher (BHQ1) and phosphorothioate co-modified DNA onto CdTe: Zn2+ QDs via a one-pot hydrothermal method. The nanobeacon with only one conjugated DNA was used to label and detect low-abundance nucleic acids in live cells, and single HIV-1 RNAs were detected and imaged in live HIV-1 integrated cells. Additionally, QD nanobeacon-labeled HIV-1 genomic RNAs were encapsulated in progeny viral particles, which can be used to track the uncoating process of single viruses. The current study provides a platform for nucleic acid labeling and imaging with high sensitivity, being especially meaningful for tracking of individual RNAs in live cells.

INTRODUCTION Detection and imaging of RNAs in live cells is demanded in many biological and biomedical studies. Nevertheless, a methodology is still a challenge, particularly for single RNA detection and imaging in live cells. Traditional techniques, including quantitative real-time polymerase chain reaction and Northern blotting, require complex RNA extraction steps and are not capable of in vivo detection. Recently, fluorescent assays based on nanomaterials showed to be a promising technology to monitor RNA in live cells via fluorescence imaging.1-4 Nanobeacons (NBs) emerged as an attractive probe to support for nucleic acids monitoring in live cells. NBs are expected to improve the sensitivity and specificity of molecular imaging.5-7 A number of fluorescent nanomaterials with different modifications, such as quantum dots (QDs), carbon dots, metal nanocluster, and upconversion nanoparticles, have been tried to employ as fluorophores to construct nanobeacon probes.8-12 Among available fluorescent materials, QDs have been thought as excellent fluorophores, due to their unique optical properties, small enough particle size, and easy modification.13-15 However, the available QD-based nanobeacons (QD-NBs) cannot control over valency. This disadvantage greatly diminishes their sensitivity and specificity for nucleic acid imaging, and thus they are unable to be used for single molecule labeling and imaging.

(BHQ1) can be precisely regulated from 1 to 4 on each QD. The nanobeacon with one conjugated DNA was suitable for labeling and imaging single RNA in live cells. Scheme 1A illustrates the processes for the construction of CdTe: Zn2+ QDs with the conjugation of BHQ1-DNA. Target nucleic acid sequences are expected to hybridize to the stem-loop hairpin DNA and recover the QD fluorescence for ultra-sensitive detection in live HIV-1 integrated cells (Scheme 1B). Selfassembled viruses labeled by the QD-NB can be further used for imaging analysis of the uncoating process of single viral particles, and to accomplish single RNA tracking in the live host cells (Scheme 1C).

In the present study, we develop a series of QD-NBs with controllable QD valency, in which DNA-black hole quencher

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(XPS), and P (2p) peak was attributed to the conjugated BHQ1-DNA (Figure 2E).19 X-ray diffraction (XRD) characterization indicated that the constructed QD-NB-1 was of cubic zinc blend structure, which was unchanged by the introduction of BHQ1-DNA (Figure 2F).20 These results suggested that the developed method is able to successfully provide control over valency to further construct QD-based nanobeacons.

Figure 1. Representative TEM images of DNA-programmed quantum dot complexes. Quantum dot assemblies built using dots with valencies of 1–4. Scale bar: 5 nm.

Scheme 1. A) Schematic diagram of the procedure for preparing QD-NBs. Schematic illustration of QD-NBs for B) HIV-1 genomic RNAs detection in living cell, and C) fluorescence labeling single virus particle.

RESULTS AND DISCUSSION Strict control of QD valency is necessary for effective fluorescence recovery of fluorescence resonance energy transfer (FRET)-based hairpin nanobeacon.16-18 Here, BHQ1 and phosphorothioate co-modified DNA was used as a multifunctional ligand for the preparation of QD-NBs. QD valency was regulated by the number of phosphorothioate on the DNA strand and the concentration of DNA used in the synthesis.19 In order to validate the valencies of QDs, a series of QD complexes were constructed by assembling several structures. As shown in Figure 1, the constructed QD complexes were assembled using similar QDs with different valencies, and visualized under a transmission electron microscope (TEM). QD complexes with 2, 3, 4, and 5 QDs were observed. Thus, a series of QD-based nanobeacons (QD-NB-1, QD-NB-2, QDNB-3, and QD-NB-4) were engineered to have valencies of 14 for nucleic acids labeling and imaging in live cells (Table S1). The morphologies and size of QD-NB-1 were characterized by TEM and dynamic light scattering (DLS). As shown in Figure 2A and 2B, QD-NB-1 was well dispersed and spherical with an average size of approximately 4 nm, similar to DNA-free QDs (Figure S1A and S1B). Due to the presence of conjugated DNA, the DLS diameter of QD-NB-1 was 9.36 nm (Figure 2C), a slightly larger than DNA-free QDs (Figure S1C). QD-NB-1 was demonstrated to have a more negative zeta potential than DNA-free QDs (Figure S1D). In addition, UV-vis absorption spectra was determined to show that BHQ1-DNA was successfully modified to QDs, according to two absorption peaks of QD-NB-1 for DNA at 257 nm and BHQ1 at 530 nm overlapping with QD absorption peak (Figure 2D). The chemical compositions of QD-NB-1 surface were also investigated by X-ray photoelectron spectroscopy

Figure 2. A) TEM imaging of QD-NB-1. Scale bar: 20 nm. Inset: HRTEM image. B) Size distribution of QD-NB-1. C) DLS distribution of QD-NB-1. D) UV-vis absorbance spectrum of QD-NB1 at room temperature. E) XPS of QD-NB-1. F) XRD pattern of QD-NB-1.

Feasibility of QD-NBs for nucleic acids labeling and imaging was investigated by incubation with the targeted DNA sequence and assayed using a spectrophotometer. As shown in Figure 3A, the developed QD-NB-1 exhibited poor green fluorescence (curve b), which is due to the formation of a stemloop. The sequence of the target nucleic acid was able to light up the quenched QDs after an incubation of 30 min. The recovered fluorescence (curve c) was similar to the intensity of


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Journal of the American Chemical Society DNA-free QDs (curve a). The stability of QD-NB-1 was evaluated by treatment with deoxyribonuclease I (DNase I). The emission of QD-NB-1 had no obvious change, while the fluorescence of FAM from traditional molecular beacon (MB) was highly recovered (Figure 3B), which demonstrated that QDNB-1 performed much better stability than commercial MB. The negatively charged surfaces of QDs and resultant high local salt concentrations deactivated enzymatic activity and increased the stability of QD-NB-1.21-23 As illustrated in Figure 3C, the fluorescence intensity dramatically recovered in the presence of the target sequence and exhibited a linear relationship with a nucleic acid concentration in the range of 10 300 nM. The limit of detection (LOD) was 2.06 nM. The sensitivity of QD-NB-1 was performed based on the variation of QD fluorescence associated with the concentration of the target sequence. QD-NB-1 was comparable or even more sensitive than previously published results for nucleic acid detection (Table S2). The selectivity of QD-NB-1 for the target sequence and the mismatched (MT) sequences (Table S3) was shown in Figure 3D. The single-base-mismatched sequence slightly recovered the fluorescence, and other mismatched sequences had no effect on QD-NB-1. These results demonstrate that the constructed QD-NB-1 is an excellent platform for a selective and sensitive assay to target nucleic acids.

Figure 3. A) Fluorescence spectra and photos of DNA-free QD (a), QD-NB-1 before (b) and after (c) incubation with target sequence. B) The stability studies of QD-NB-1 and MB with deoxyribonuclease I (DNase I) treatment (200 U/L). C) Calibration plots for target DNA. Inset: Evolution of QD-NB-1 fluorescence spectra with increasing target sequence concentration. D) Fluorescence spectra of QD-NB-1 incubated with target sequence and mismatched target sequences, respectively.

tions. Within the initial 30 min, just a little fluorescence signal was detected, and the fluorescence slightly started to increase after 60 min. With longer incubation times, the green emission of QD-NB-1 lighted up the cells as expected (Figure 4A). To achieve live imaging of low-abundance nucleic acids, QDNB-1 was incubated with U1 cells activated by TNF-α at different time durations. The expression of target nucleic acids for cells activated with different periods was quantified by using qPCR analysis (Figure S4). The fluorescence intensity dramatically decreased with shorter activation times, due to less expression of target nucleic acids. Fluorescence signals were still able to be visualized when U1 cells were only activated for 6 h, indicating that the constructed QD-NB-1 had excellent sensitivity in live cell detection (Figure 4B). Realtime detection of single RNA was achieved by further reducing the activation time to 1 h and the incubation time to 15 min, and only one fluorescent dot was visualized in the selected U1 cell. Several or more fluorescent dots could be observed in a single cell, which was activated for 2 h or 3 - 4 h (Figure 4C, Movie S1, and Movie S2). For statistical analysis, 300 cells were randomly selected at each activation time to investigate the precise amount of fluorescent dots in each cell (Figure S5). The visualized detection of single RNA was done at a short activation time, and there were too many fluorescent dots in cells to distinguish and count as the increase of activation time. The detection efficiency was evaluated by adding exogenous target sequences into inactive U1 cells, and the fluorescent dots can still be visualized at the concentrations as low as 2.5 zmol/cell. Compared with QD-NB-1, other constructed QD-NBs with valencies of 2 - 4 possessed poorer fluorescence recoveries and lower detection sensitivities with the increase of conjugated DNA on the QD surface. In ultrasensitive detection, low concentrations of target nucleic acids can hybridize to only one or part of the stem-loop hairpin DNA for each nanobeacon with multiple valencies, and the QD fluorescence was slightly recovered, which was difficult for single RNA imaging in live HIV-1 integrated cells. QDNB with mismatched sequence cannot be used to detect or image nucleic acids in live U1 cells (Figure S6). The results indicate that the designed QD-NB-1 is a great biosensor for visual monitoring of single RNA in live cells, and a one to one combination of QD and BHQ1-DNA greatly benefits the recover fluorescence effectively.

To achieve live imaging of target nucleic acids in HIV-1 integrated cells, QD-NBs with different valencies were incubated with U1 cells activated by TNF-α for a period of 48 h and observed with a spinning disk confocal microscope. First, the cytotoxicity of QD-NB-1 was investigated using Cell Counting Kit-8 (CCK-8) assay. No clear cytotoxic effect was observed in the U1, 293T, and TZM-bl cells (Figure S2). The stability of QD-NB-1 was further evaluated by incubation with non-activated U1 cells for different time durations. Nonspecific fluorescence from QD-NB-1 was not observed, however, background signal of traditional MB was obvious (Figure S3). For activated U1 cell imaging, the feasibility of QDNB-1 to visualize target nucleic acids in live HIV-1 integrated cells has been further tested at different incubation time dura-



detected inside the virions (Figure 5B), while no dots were observed in the wild type particle (Figure S10A). The infectivity of HIV-1 virus was quantified by infecting an HIV-reporter cell line (TZM-bl cells). HIV-1-QD has similar infectivity as the wild type particles, and HIV-1-QD-ReAsH possessed approximately 50% of the infectivity of the wild type virions, which shows that the incorporation of QD-NB-1 did not diminish the viral infectivity (Figure 5C).

Figure 4. A) Confocal images of activated U1 cells after incubation with QD-NB-1 (75 nM) for different time. U1 cells were activated by TNF-α (100 U/mL) for 48 h at 37 °C. B) Confocal images of U1 cells activated for 6 - 36 h and incubated with 75 nM of the QD-NB-1 for 120 min at 37 °C. C) Snapshots of single RNA labeled by the QD-NB-1 in live U1 cells, which were activated by TNF-α (100 U/mL) for 1 - 4 h at 37 °C and incubated with 75 nM of the QD-NB-1 for 15 min at 37 °C. Confocal microscopy images collected by DIC channel and green channel with 488-nm laser excitation. Scale bar: 10 µm.

To assess the potential application of QD-NBs, they were packaged into virions, through hybridization to genomic RNAs during the HIV-1 assembly. The QD nanobeaconlabeled progeny virions (HIV-1-QD) were confirmed by immunostain against the capsid protein (p24) or by labeling the viral envelope with a lipophilic membrane dye (DiD). The results in Figure S7 showed that the majority of QDs (approximately 75%) co-localized with p24 or DiD, demonstrating that HIV-1 particles were efficiently labeled with QD-NB-1. Compared with QD-NB-1, other constructed QD-NBs with valencies of 2 - 4 or mismatched sequence were inefficient for labeling HIV-1 genomic RNAs, and cannot be used to visualize single viral particle in live cells (Figure S8). During fluorescence labeling of viral genomic RNA, the nanobeacons with multiple valencies hybridized with more than one HIV-1 genomic RNA were easy to form complex, which was not conductive to viral self-assembly. To obtain dual-labeled HIV1 particles, a tetracysteine (TC) motif was inserted into the capsid protein (p24), and interacted with the ReAsH fluorescent dye. The fluorescence signal of ReAsH co-localized with immunofluorescence (p24) or DiO staining, which suggests that the capsid proteins were efficiently labeled with the ReAsH fluorescent dye (Figure S9). Dual-color virions (HIV1-QD-ReAsH) were prepared by labeling of the genomic RNAs with QD-NB-1 and the capsid with the TC-ReAsH. As shown in Figure 5A, fluorescence colocalization of QD and ReAsH indicated a successful production of dual-fluorescence virus. HIV-1-QD-ReAsH was further characterized by negative staining in TEM, where a dark electron-dense dot was

As a direct test to track the dynamic processes of a single virus, TZM-bl cells were infected with HIV-1-QD-ReAsH virions, and real-time imaging performed for viral uncoating by the use of spinning disk confocal microscopy. It is commonly accepted that the HIV-1 uncoating is an obligatory step for the release of the viral genome during early infection.24, 25 After the transportation of virions into the cytoplasm, the red dot was separated from the green dot, indicating the release of genomic RNAs from the capsid (Figure 5D, Figure S10B, and Movie S3). The dynamic trajectories, velocities, and mean square displacement (MSD) for the two fluorescent dots are given in Figure 5E - 5G. At 60 - 120 min post infection, 27 dissociations were observed by tracking 30,000 individual HIV-1-QD-ReAsH virions. This low proportion of viral uncoating is consistent with the very low productive infection efficiency of HIV-1 stocks, in which approximately 0.1% of HIV-1 particles undergo productive infection.24, 26, 27 The realtime imaging results show that the developed QD-NB-1 can be applied as an excellent fluorescent probe to label viral genomic RNAs and to track the dynamic processes of single particles.

Figure 5. A) Colocalization of QD and ReAsH signals in HIV-1QD-ReAsH virions. Scale bar: 2 µm. B) TEM imaging of HIV-1QD-ReAsH virus. C) The infectivity assay of viral particles. D) Snapshots of the dynamic separation of the RNA-QD and the capsid-ReAsH. Scale bar: 1 µm. E-G) Trajectories (E), mean velocities (F), and MSD plots (G) of the RNA-QD (green) and the capsid-ReAsH (red).

CONCLUSION In the current study, a new type of QD-based nanobeacons with controllable valencies has been developed for labeling and imaging single RNA in live cells. The QD nanobeacons are synthesized by conjugating the BHQ1 and phosphorothioate co-modified DNA onto CdTe: Zn2+ QDs via a one-pot


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Journal of the American Chemical Society hydrothermal method. The QD-based nanobeacons were engineered to have valencies of 1 - 4. QD-based nanobeacon with just one conjugate DNA (QD-NB-1) is proved to be suitable and effective for imaging single HIV-1 RNA in live HIV-1 integrated cells. QD nanobeacon was also used to label HIV-1 genomic RNAs and be encapsulated in progeny viral particles for tracking the uncoating process of single viruses. The imaging and labeling single RNA by the QD-based nanobeacon demonstrates a significant advance in the detection sensitivity of nucleic acid and fluorescence labeling of viral genome. Our developed QD-based nanobeacons with controllable valencies may provide a novel platform for effectively labeling and imaging nucleic acid in live cells, especially for individual RNAs.

ASSOCIATED CONTENT Supporting Information Experimental details and additional data. The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author [email protected], [email protected]

Author Contributions ‡These authors contributed equally.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by Strategic Priority Research Program of Chinese Academy of Sciences (XDB29050201), National Natural Science Foundation of China (21675119, 21705110, 81772737), National Major Science and Technology Projects (2018ZX10301405), Natural Science Foundation of Guangdong (2018B030306046), China Postdoctoral Science Foundation (BX201700161, 2017M622860), Shenzhen Municipal Government of China (JCYJ20170413161749433, JSGG20160301161836370), Sanming Project of Shenzhen Health and Family Planning Commission (SZSM201412018, SZSM201512037), High Level University’s Medical Discipline Construction (2016031638), Shenzhen Health and Family Planning Commission (201606019).

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Quantum Dot Nanobeacons for Single RNA Labeling and Imaging

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