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Functional Titanium Carbide MXenes-Loaded Entropy-Driven RNA Explorer for Long Noncoding RNA PCA3 Imaging in Live Cells Song Wang, Wenlu Song, Shaohua Wei, Shu Zeng, Sihui Yang, Chunyang Lei, Yan Huang, Zhou Nie, and Shouzhuo Yao Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b02040 • Publication Date (Web): 30 May 2019 Downloaded from http://pubs.acs.org on May 31, 2019

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Analytical Chemistry

Functional Titanium Carbide MXenes-Loaded Entropy-Driven RNA Explorer for Long Noncoding RNA PCA3 Imaging in Live Cells

Song Wang, Wenlu Song, Shaohua Wei, Shu Zeng, Sihui Yang, Chunyang Lei*, Yan Huang, Zhou Nie*, and Shouzhuo Yao

State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan Provincial Key Laboratory of Biomacromolecular Chemical Biology, Hunan University, Changsha 410082, P.R. China.

*to whom corresponding should be addressed. Tel: +86-731-88821626; Fax: +86-731-88821848 Email: [email protected]; [email protected].

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ABSTRACT: The visualization of the long noncoding RNA of prostate cancer gene 3 (lncRNA PCA3), a specific biomarker for androgen receptor-positive prostate cancer, in living cells not only directly reflects the gene expression and localization, but also offers a better insight into its roles in the pathological processes. Here, we loaded an entropy-driven RNA explorer (EDRE) on the TAT peptide-functionalized titanium carbide MXenes (Ti3C2–TAT) for the imaging of nuclear lncRNA PCA3 in live cells. The EDRE was condensed on the Ti3C2–TAT (Ti3C2–TAT@EDRE) by electrostatic interaction. Ti3C2–TAT@EDRE enables to enter cells and release TAT peptides and EDRE in the cytoplasm by the glutathione (GSH)-triggered cleavage of the double bonds in Ti3C2–TAT. The released EDRE is delivered into the nucleus by the nucleustargeted guidance of TAT peptides, and initiated by the target lncRNA PCA3, subsequently leading to the continuous accumulation of fluorescence signals. As a consequence, fluorescence analysis of lncRNA PCA3 at low-picomolar concentrations in vitro, as well as sensitive live cell imaging of lncRNA PCA3 in the nuclear of androgen receptor-positive LNCaP prostate cancer cells was achieved, providing a versatile strategy for the monitoring of nucleic acid biomarkers in the nucleus of living cells.

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INTRODUCTION Prostate cancer (PCa), a global disease that seriously endangers men's health, has been considered to be the second leading cause of cancer death in developed countries.1,2 And acquiring the PCa-associated information by quantitative analysis of the related biomarkers is crucial for monitoring the PCa progression and early treatment.3-5 The serum prostate-specific antigen (PSA) test is sensitive for PCa screening, but its specificity for PCa diagnosis remains controversial.6 With the significant advances in molecular medicine, increasing evidences have revealed that the long non-coding RNA (lncRNA) transcribed from prostate cancer antigen 3 (PCA3) gene could be a promising biomarker of PCa.7,8 LncRNA PCA3 is extremely specific for the diagnosis of PCa, because it is significantly over-expressed only in androgen receptor (AR)-positive PCa tumors, while is rarely expressed in adjacent nonneoplastic tissue, benign prostatic hyperplasia tissue, and other human cancerous tumors.9-12 Currently, some traditional techniques have been adopted for the detection of LncRNA PCA3, but most of the methods (e.g. Northern blot13 and RT-PCR14) are limited to fixed cells, cell lysates or cell-free samples. Moreover, live cell imaging of LncRNA PCA3 enables us to gain better insight into the cellular processes involving LncRNA PCA3.15 However, the development of imaging probe for the monitoring of LncRNA PCA3 still exists two major challenges, including 1) the poor target accessibility derived from its location in cell nucleus,16,17 and 2) its low-abundance expression level.18 The two-dimensional (2D) nanomaterials including graphene oxide,19,20 transition

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metal,21,22 and black phosphorus,23,24 have attracted extensive research interest for the development of biosensing platforms over the past decade due to their unique physicochemical properties. Recently, the MXene family that contains a large number of 2D transition metal carbides/carbonitrides,25,26 has been developed with superior conductivity, high surface area and strong and broad absorption, holding great promise applications in the field of energy storage,27,28 water purification,29,30 biomedicine,31-32 and biosensor.33-36 For instance, on the account of the superior fluorescence quenching and delivery capacities of titanium carbide (Ti3C2) MXenes, our group developed a nanoprobe for the simultaneous mapping of transmembrane glycoprotein mucin 1 and cytoplasmic microRNA-21, exhibiting the great potential of MXenes in the profiling of RNA biomarkers in live cells.35 Meanwhile, the DNA-based amplifiers, a kind of nucleic acid molecular machines, have shown excellent signal amplification ability in the clinical diagnosis of low-abundance biomarkers. Especially, the DNA-based nonenzymatic and isothermal signal amplification techniques that can operate in an autonomous and reconfigurable manner, have been demonstrated as powerful toolkits in the live cell imaging of cytoplasmic RNA, such as messenger RNA and micorRNA.37-41 Taken together, we sought to fabricate a fluorescent nanoprobe for the imaging of nuclear LncRNA PCA3 in living cells by integrating the Ti3C2 MXenes with the DNA-based nonenzymatic and isothermal amplifiers. Aimed at the objective, we herein deployed functionalized Ti3C2 MXenes as the nanocarrier for the nuclear-targeted delivery of an entropy-driven RNA explorer (EDRE) into live cells. As illustrated in Scheme 1A, the surface decoration of 4


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poly(acrylic acid) (PAA) highly stabilizes Ti3C2 MXenes, as well as provides linkage sites for cystamine by amidation reaction. Then, the nucleus-targeted TAT peptides were covalently assembled on the surface of amino-functionalized Ti3C2 MXenes, resulting in the formation of Ti3C2 MXenes-based nanocarrier (denoted as Ti3C2–TAT). The negatively charged DNA components of the EDRE were loaded on the positively charged surface of Ti3C2-TAT by electrostatic interaction (Scheme 1B). When incubated with cells, the complexes (Ti3C2–TAT@EDRE) enter into the cytoplasm, in which the high concentrations of glutathione (GSH) break the disulfide bonds in Ti3C2TAT by thiol-disulfide exchange, leading to the efficient release of TAT peptides and EDRE. The released EDRE is delivered into nucleus by the TAT peptide-mediated target delivery. The nuclear LncRNA PCA3 in PCa tumor cells triggers the entropydriven cascaded strand-displacement reaction between the linear DNA components of EDRE, continuously releasing and accumulating the fluorophore-labeled DNA probes. Benefiting from the effective signal amplification and low circuit leakage of the entropy-driven amplification strategy,38,39 this integrated nanoprobe has achieved sensitive and accurate fluorescence imaging of the low-abundance lncRNA PCA3 in live AR-positive PCa tumor cells. EXPERIMENTAL SECTION Chemicals. Ti3AlC2 was obtained from 11 Technology Co., Ltd (Jilin, China). Cystamine (Cys), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), Sodium hydroxide, N-hydroxysuccinimide (NHS), and poly(acrylic acid) (PAA,

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MW = 3400) were purchased from Sigma Aldrich (St. Louis, MO, USA). LNCaP (ARpositive), PC3 and RWPE-1 prostate cancer cell lines, and A549 lung cancer cell line were obtained from the cell bank of the central laboratory at Xiangya hospital (Changsha, China). All chemical reagents were of analytical grade and used without further purification. All solutions were prepared with ultrapure water (18.25 MΩ·cm) from the Millipore system. All the oligonucleotides (sequences shown in Table S1 and S2) and TAT peptide (NH2-YGRKKRRQRRR-COOH) were synthesized and purified by Sangon (Shanghai, China). Synthesis of Ti3C2–TAT. The Ti3C2 MXenes were prepared based on a previously reported method.25 In brief, 1.5 g of LiF was slowly added into 20 mL of HCl (9 M), and the solution was stirred with a magnetic stirrer for 5 min until LiF was completely dissolved. Next, 2.0 g of Ti3AlC2 powder was added within 10 min and the whole reaction system was held at 35 ℃ with stirring for 24 h. Then, the black solution was washed several times with deionized water under 3500 rpm rotating speed until the final pH of the solution reached 5.0–6.0. Delaminated Ti3C2Tx solution was obtained by shaking for 30 min and then sonicating under N2 for another 1h, followed by centrifuging at 5000 rpm for 30 min and the supernatant was collected for later use. For Ti3C2–PAA preparing, 6 mL of PAA-3400 was added into 40 mL of Ti3C2 MXenes solution and under N2 atmosphere sonicated for 5 h. The large-size masses were removed by centrifugation at 5000 rpm for 5 min while the unbound PAA–3400 was removed by dialyzing (retained molecular weight, 10 kDa) for 24 h. The obtained

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Ti3C2–PAA was stored at 4℃ in refrigerator. Furthermore, the prepared Ti3C2–PAA solution was reacted with 1 mg mL-1 EDC and 1 mg mL-1 NHS for 1.0 h, and then 10 mg of cysteine was added and reacted for 24 h. Finally, the mixture solution was filtered by ultrafiltration (MWCO of 3 kDa, Millipore) to remove the unreacted Cys, EDC, and NHS to obtain the aminofunctionalized Ti3C2 (Ti3C2–NH2).50,51 Subsequently, 20 mL of Ti3C2–NH2 solution was mixed with 2.0 mg of EDC, 2.0 mg of NHS and stirred for 1 h. Then TAT peptide with a final concentration of 1.0 μM was added into the mixture solution and further reacted for 24 h, Ti3C2-TAT was extracted by centrifugation at 12000 rpm and re-dispersed in water for further usage. In Vitro Fluorescence Measurements. All fluorescence measurements were performed on the F-4500 fluorescence spectrophotometer (Hitachi, Japan). For the detection of the targets in buffer solution, different concentrations of PCA3-mimic DNA were added into 200 µL of Tris–HCl solution (25 mM Tris, 5 mM MgCl2 and pH 8.0) containing 100 nM EDRE. The fluorescence spectra of the mixture were collected from 500 to 600 nm with the excitation wavelength of 488 nm in a 200 µL quartz cuvette. All experiments were repeated for at least three times. For the selectivity tests, other types of control RNAs, including M1, M3 (one and three base mismatches, respectively), let-7a, TK1, C-myc and miR-21 RNA were incubated with the EDRE at room temperature for 1.0 h and the fluorescence signals were measured under the same experimental conditions. 7



Fluorescence Imaging in Live Cells. LNCaP, PC3, RWPE-1 and A549 cells grown in RPMI-1640 medium with 10% FBS and 1% penicillin-streptomycin with 5% CO2 at 37 °C. All cells were plated on a 35–mm confocal laser culture dish for 24 h. 300 nM EDRE and 10 μg mL-1 Ti3C2-TAT were mixed for 10 min before adding into 200 µL 1640 medium and then incubated with cells at 37 °C for the appropriate time. After washing with PBS three times, confocal fluorescence imaging studies were performed on confocal laser scanning microscope. RESULTS AND DISCUSSION Synthesis and Characterization of the Ti3C2–TAT. The Ti3C2 MXenes were prepared by one-step etching of Ti3AlC2 MAX-phase powder.42 The synthesis of the Ti3C2 MXenes with the typical flakelike morphology from the original MAX phase was first confirmed by the results of the transmission electron microscopy (TEM) images (Figure 1A). And the exfoliation and synthesis of Ti3C2 MXenes from the MAX phase were validated by the disappearance of the most intense diffraction peak of Ti3AlC2 (2θ ≈ 38°) with a shift of (002) peak at the low-angle area in the X-ray diffraction (XRD) patterns (Figure S1).43 The chemical composition of the as-prepared Ti3C2 MXenes was examined by X-ray photoelectron spectroscopy (XPS). And the mains peaks in the XPS spectra indicated the existence of Ti, C, O and F elements (Figure 1B). Moreover, the peaks of Ti−C (455.2 eV) and Ti−O/F (458.3 eV) were observed in the high resolution Ti 2p spectrum without the Ti-Al peak at 452 eV (Figure S2), confirming the typical composition of Ti3C2 nanosheets and the removal of the Al layers.44 The

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Ti3C2 MXenes dispersed in aqueous solution showed the representative absorption peak at 788 nm with a molar extinction coefficient of 28.8 L g–1 cm–1 (Figure S3).33 Next, the surface of Ti3C2 MXenes were coated by carboxy-rich PAA on the basis of the strong coordination between the carboxyl group and titanium atom.45,46 The surface decoration of PAA not just improves the dispersibility of Ti3C2 MXenes, but also provides numerous carboxyl groups for the further functionalization.35 Afterward, cystamine (Cys) and the nucleus-targeted TAT peptide were stepwise coupled on the surface of Ti3C2–PAA via EDC/NHS coupling to construct the TAT peptidefunctionalized Ti3C2 MXenes (Ti3C2–TAT).47-49 The as-prepared Ti3C2–TAT showed the same excellent dispersibility as Ti3C2–PAA in biological solutions including PBS and RPMI-1640 culture, which is much better than that of bare Ti3C2 MXenes (Figure S4). Then the modifications were characterized by atomic force microscope (AFM), dynamic light scattering (DLS) and Fourier transform infrared spectroscopy (FTIR). After the stepwise modifications of PAA and TAT peptides, the topological thickness of Ti3C2 MXenes increased form 1.1 nm to 2.1 nm and 3.4 nm, respectively (Figure 1C). And the surface coating of PAA resulted in an increase in the Zeta potential of Ti3C2 MXenes from –13.6 to –21.9 mV, while the further modification of TAT peptides caused a remarkable surface charge reversal with the Zeta potential of +25.7 mV (Figure 1D), which could be attributed to the plenty basic amino acid residues in the coupled TAT peptides. Moreover, the appearance of the new absorption peaks at 1196 cm–1 (C−O stretching vibration), 1435 cm–1 (COO−stretching vibration), and 1731 cm–1 (C=O vibrations) in FTIR spectra suggested the modification of PAA (Figure 1E, curve 9



2). Analogously, the new peaks at 1556 cm–1 (−NH2 bending vibrations) and 1650 cm–1 (−CO−NH− stretching vibration) indicated the covalent coupling of Cys and TAT peptides, respectively (Figure 1E).50 Therefore, these results confirm the successful preparation of TAT-functionalized Ti3C2 MXenes (Ti3C2–TAT). Fluorescence Response of EDRE Toward lncRNA PCA3 in Vitro. To identify the potential target sequence of lncRNA PCA3, we performed sequence analysis of the transcript of PCA3 gene by Oligo7 software. A specific region of the lncRNA PCA3 between positions +108 and +146 was screened (Figure 2a). The secondary structure of this region was simulated and confirmed as a single chain state (Figure S5), which is conducive for the subsequent hybridization reaction. And a 22-mer truncated sequence (+125 to +146) was used as the target sequence of lncRNA PCA3 for the better design of the DNA-based amplifier.38,39 An EDRE composed of a signal reporter (SR) and fuel unit, was designed for the sensitive detection of lncRNA PCA3 (Figure 2B, and detailed in Figure S6). And the SR consists of three well-designed single stranded DNA (P1, P2 and P3), in which P1 and P3 were labeled by a fluorophore (5’FAM) and quencher (3’-BHQ1) at the terminus, respectively. P3 can hybridize with P1 and P2, leading to the efficient fluorescence quenching of FAM in P1 through FRET. In the presence of PCA3, the EDRE is effective initiated and runs continuously. The hybridization between PCA3 and the toehold 1 in SR triggers a strand-displacement reaction between PCA3 and P2, and forms the P1/P3/PCA3 hybrid duplex with the generation of toehold 2. Then the fuel strand P4 hybridizes with the new generated toehold 2, and subsequently initiates the cascaded strand-displacement reaction, 10


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resulting in the release of P1 and PCA3. As a consequence, the fluorescence of FAM in P1 is recovered, and PCA3 is regenerated and triggers the next rounds of cycle for signal gain. To validate this design, the DNA mimic of the lncRNA target was used to test the EDRE in vitro. The response of the EDRE toward the DNA mimic of lncRNA PCA3 was first rationally confirmed by the gel shift assay (Figure S7). Then, a low concentration of the target DNA mimic (10 nM) was incubated with the EDRE, a drastic fluorescence enhancement that is 31.7-fold as high as the control, was observed (Figure S8), suggesting the excellent sensitivity of the proposed EDRE. Then fluorescence kinetics of the EDRE probe toward the target DNA was further studied by real-time monitoring the fluorescence emission at 525 nm of the system. The results showed that the EDRE responded rapidly to the target DNA, and the fluorescence response reached saturation in 1.0 h, while the mutations of the target only induced slight responses (Figure S9). In addition, the effect of the concentration of fuel strand on the fluorescence response was also investigated, and a satisfactory signal could be obtained at 100 nM (Figure S10). Next, the EDRE was incubated with various concentrations of target DNA for 1.0 h to obtain the concentration-dependent fluorescence responses signals. As shown in Figure 2C, fluorescence emission of the system increased gradually as the concentration of PCA3 increasing. Figure 2D illustrates the relationship between the fluorescence intensity at 525 nm and the concentration of PCA3, and a linear relationship in the concentration range of 0 − 0.2 nM was obtained with a limit of detection of 2.6 pM (S/N = 3). Furthermore, the existence of the TAT 11



peptides showed a negligible influence on the signal amplification capability of the EDRE probe (Figure S11). Finally, the specificity of the EDRE toward PCA3 was evaluated by testing the potential interferents including one- and three-base mismatched mutations (M1 and M3), microRNAs (let-7a and miRNA-21), and mRNAs (C-myc and TK1), and these interferents could only induced negligible fluorescence signals (Figure 2E). Taken together, the results of the in vitro assay have demonstrated the prominent sensitivity and specificity of the EDRE for the detection of lncRNA PCA3, suggesting its reliability for bioimaging of the target lncRNA. Nucleus-targeted Delivery of Nucleic Acids by Ti3C2–TAT. Prior to the imaging of lncRNA PCA3 in live cells, the nucleus-targeted delivery of nucleic acids by Ti3C2– TAT was investigated using a FAM-labeled ssDNA probe (P5) as the target model. Previous studies revealed that TAT peptide-functionalized nanomaterials with a diameter of less than 50 nm can efficiently be translocated into cell nucleus,49 which means that Ti3C2–TAT cannot directly deliver the nuclei acids into cell nucleus because of their relatively large scales (＞80 nm). However, when the Ti3C2–TAT carriers enter cytoplasm, the high concentration of cytoplasmic GSH will trigger the release of TAT peptides and the loaded nucleic acid cargoes.50,51 Then the released TAT peptides can deliver the nucleic acids into cell nucleus. Owing to the superior and broad-spectrum fluorescence quenching capacity, the fluorescence of FAM in the loaded P5 was efficiently quenched, and thus the GSH-triggered release of P5 can be estimated by monitoring the fluorescence of FAM (Figure S12). As showed in Figure 3A, the fluorescence of FAM was quenched 96.1% after the mixing of P1 with Ti3C2–TAT, 12


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while the addition of GSH could restore 67.4% of the initial fluorescence. In contrast, there was only a negligible release when a control Ti3C2-TAT carrier without the disulfide crosslinking was used under the same conditions (Figure S13). Then the dynamic of the GSH-triggered release was investigated by monitoring the fluorescence of released P5 under different concentrations of GSH at different times. The results in Figure 3B and Figure S14 exhibited a dose- and time-dependent release behavior, and GSH at millimolar concentrations could trigger effective release of the loaded nucleic acids. Since the concentrations of cytoplasmic GSH were reported in the range of 2.0– 10 mM,51,52 it is anticipated that the GSH-triggered release of nucleic acids loaded by Ti3C2–TAT carrier could also perform in live cells. To test this hypothesis, LNCaP cells were incubated with Ti3C2–TAT@P5 and monitored by confocal laser scanning microscope (CLSM). Previous research indicated that nanosheets with lateral size ≤ 200 nm could be internalized into mammalian cells by endocytosis,53-55 and thus Ti3C2– TAT@P5 can enter into cytoplasm. After the incubation with Ti3C2–TAT@P5 for 1.0 h, green fluorescence spots emerged in the cytoplasm of LNCaP cells, indicating that the internalization of Ti3C2–TAT@P5, as well as the release of TAT@P5 have occurred (Figure S15). As the time was prolonged to 4.0 h, intensive green fluorescence that overlaps with the fluoresce of DAPI was observed in the CLSM images (Figure 3C), which indicated that the P5 was effectively released from the nanocarrier and delivered into the nucleus. As a control, there were no visible fluorescence signal in the cytosolic or cell nucleus of the LNCaP cells when the Ti3C2-TAT without disulfide bonds was used as the carriers under the same experimental conditions (Figure S16). These results 13



suggested that the designed nanocarrier could not only realize a redox-triggered cargo release, but also promote the carried nucleic acid for efficient nuclear targeting. Therefore, by integrating the endocytosis-mediated internalization with GSH-triggered release, the proposed Ti3C2–TAT carrier was able to efficiently deliver nucleic acid cargos into cell nucleus. Imaging of Nuclear LncRNA PCA3 in Live Cells. Encouraged by the results outlined above, we sought to deliver the EDRE into cell nucleus based on the Ti3C2–TAT nanocarrier, and perform the function of the EDRE for the imaging of nuclear lncRNA PCA3 in live cells. First, the ratio between EDRE probe and Ti3C2–TAT was optimized by the fluorescence titration experiment (Figure S17). And the biocompatibility of the system was investigated by the MTT assay (Figure S18), the Ti3C2–TAT@EDRE showed no significant cytotoxicity to LNCaP, PC3, RWPE-1, and A549 cells at the concentration of 40 μg mL−1. Then, the four cell lines were incubated with Ti3C2TAT@EDRE and imaged by CLSM. As shown in Figure 4A, several green spots were in the nucleus of AR-positive LNCaP cells, while no detectable green fluoresce was observed in the nuclei of PC3, RWPE-1 and A549 cells. And the average numbers of the spots in each LNCaP, PC3, RWPE-1 and A549 cells were calculated to be 8.70, 0.82, 0.32 and 0.14, respectively, which consists well with the expression levels of lncRNA PCA3 in these cell lines quantified by RT-PCR (Figure S19). Moreover, the control experiments revealed that both the Ti3C2–TAT and EDRE are indispensable for the imaging of nuclear lncRNA PCA3 (Figure S20, S21). To confirm that the fluorescence spots were specifically originated from the target lncRNA PCA3-triggered 14


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signal amplification, control EDREs for one- and three-base mutation targets (EDREM1and EDRE-M3), and a random target (EDRE-R) were designed for the imaging of lncRNA PCA3 under the same experimental conditions (Figure S22). And the average numbers of fluorescence spots in each cell were determined to 1.08, 0.22 and 0.02 for EDRE-M1, EDRE-M3 and EDRE-R, respectively. Therefore, with the use of the proposed Ti3C2–TAT@EDRE, imaging of the low-abundance nuclear lncRNA PCA3 in live cells has been achieved. CONCLUTION In summary, we deployed Ti3C2 MXenes as a nanocarrier (Ti3C2–TAT) to efficiently deliver an entropy-driven RNA explorer (EDRE) into living cells for the imaging of nuclear lncRNA PCA3. The Ti3C2–TAT-loaded EDRE can enter the cytoplasm, and release TAT and EDRE by GSH-triggered thiol-disulfide exchange. Then the EDRE can be delivered into cell nucleus by TAT guidance, enabling sensitive and selective imaging of target nuclear lncRNA PCA3 in live cells. The versatility of the Ti3C2– TAT@EDRE probe have been demonstrated in the mapping of lncRNA PCA3 level in different cancer cells, including LNCaP, PC3, RWPE-1 and A549 cells, with high sensitivity and reliability. Therefore, this work not just provides a sensing platform for the sensitive live cell imaging of nuclei acid biomarkers in cell nucleus, but also presents a versatile strategy that deploys Ti3C2 MXenes as a promising nanocarrier for nucleus-targeted delivery, which will stimulate deeper applications of MXenes in biosensing and biomedicine.

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ACKNOWEDGEMENTS This research was financially supported by National Natural Science Foundation of China (Nos. 21575038, 21725503, and 21675044), the Young Top-notch Talent for Ten Thousand Talent Program, and the Fundamental Research Funds for the Central Universities. S. Wang was supported by Hunan Provincial Innovation Foundation For Postgraduate (CX2018B186). Notes The authors declare no competing financial interest. Supporting Information Available Additional supplementary material, including the description of extensive method, oligonucleotide sequences, RT-PCR assay, XPS, UV-vis-NIR and fluorescence spectra, CLSM images and results.

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FIGURE CAPTIONS Scheme 1. Schematic illustration of (A) the fabrication of the Ti3C2–TAT nanocarrier, and (B) the Ti3C2–TAT-loaded EDRE for the imaging of nuclear lncRNA PCA3 in live cells.

Figure 1. (A) TEM image, and (B) XPS of the Ti3C2 MXenes. (C) AFM images and height profiles, (D) Zeta potentials, and (E) FT-IR spectra of the Ti3C2 MXenes, Ti3C2–PAA, Ti3C2–NH2, and Ti3C2–TAT.

Figure 2. (A) A specific sequence in lncRNA PCA3 (the PCA3 transcript variant 2, NR_015342.2), and the sequences of the DNA mimics of lncRNA PCA3 target and its mutations. (B) Illustration of the EDRE for PCA3 sensing with catalytic fluorescence signal enhancement. (C) Fluorescence spectra of the EDRE in the presence of different concentration of PCA3. (D) Correlation curve between the fluorescence signal and concentration of PCA3. (E) Fluorescence responses of the EDRE toward several interferences. EDRE, 100 nM; and the concentration of each oligonucleotide is 10 nM.

Figure 3. (A) Fluorescence spectra of probe P5 (100 nM), Ti3C2-TAT@P5, and Ti3C2-TAT@P5 incubated with GSH (5.0 mM). (B) Fluorescence intensity 23



changes at 525 nm of the Ti3C2-TAT@P5 after the incubation with various concentrations of GSH for different times. (C) CLSM images of LNCaP cells after the incubation with Ti3C2-TAT@P5. Scale bar, 20 μm. The graphs on the right show the green and blue fluorescence channel intensities along the lines on the CLSM images.

Figure 4. (A) CLSM images of LNCaP, PC3, RWPE-1 and A549 cells after the incubation with Ti3C2-TAT@EDRE probe for 6.0 h. Scale bar, 20 μm. (B) The distributions of green spots detected in each cell nucleus. The average number of green spots per cell in each cell line was quantified from approximately 50 cells.

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Scheme 1.

Scheme 1. Schematic illustration of (A) the fabrication of the Ti3C2–TAT nanocarrier, and (B) the Ti3C2–TAT-loaded EDRE for the imaging of nuclear lncRNA PCA3 in live cells.

25



Figure 1.

Figure 1. (A) TEM image, and (B) XPS of the Ti3C2 MXenes. (C) AFM images and height profiles, (D) Zeta potentials, and (E) FT-IR spectra of the Ti3C2 MXenes, Ti3C2–PAA, Ti3C2–NH2, and Ti3C2–TAT.

26


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Figure 2.

Figure 2. (A) A specific sequence in lncRNA PCA3 (the PCA3 transcript variant 2, NR_015342.2), and the sequences of the DNA mimics of lncRNA PCA3 target and its mutations. (B) Illustration of the EDRE for PCA3 sensing with catalytic fluorescence signal enhancement. (C) Fluorescence spectra of the EDRE in the presence of different concentration of PCA3. (D) Correlation curve between the fluorescence signal and concentration of PCA3. (E) Fluorescence responses of the EDRE toward several interferences. EDRE, 100 nM; and the concentration of each oligonucleotide is 10 nM.

27



Figure 3.

Figure 3. (A) Fluorescence spectra of probe P5 (100 nM), Ti3C2-TAT@P5, and Ti3C2-TAT@P5 incubated with GSH (5.0 mM). (B) Fluorescence intensity changes at 525 nm of the Ti3C2-TAT@P5 after the incubation with various concentrations of GSH for different times. (C) CLSM images of LNCaP cells after the incubation with Ti3C2-TAT@P5. Scale bar, 20 μm. The graphs on the right show the green and blue fluorescence channel intensities along the lines on the CLSM images.

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Figure 4.

Figure 4. (A) CLSM images of LNCaP, PC3, RWPE-1 and A549 cells after the incubation with Ti3C2-TAT@EDRE probe for 6.0 h. Scale bar, 20 μm. (B) The distributions of green spots detected in each cell nucleus. The average number of green spots per cell in each cell line was quantified from approximately 50 cells.

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