Near-infrared-traceable DNA nano-hydrolase: specific eradication of telomeric G-overhang in vivo

Abstract Telomeric DNA, whose length homeostasis is closely correlated with immortality of cancer cells, is regarded as a molecular clock for cellular lifespan. Regarding the capacity in forming G-quadruplex, G-rich 3′-overhang (G-overhang) has been considered as an attractive anticancer target. However, it is still challenging to precisely target telomeric G-overhang with current ligands because of the polymorphism of G-quadruplexes in cells. Herein, we construct a telomeric G-overhang-specific near-infrared-traceable DNA nano-hydrolase, which is composed of four parts: (i) dexamethasone for targeting cell nuclei; (ii) complementary DNA for hybridizing with G-overhang; (iii) multinuclear Ce(IV) complexes for hydrolyzing G-overhang; and (iv) upconversion nanoparticles for real-time tracking. The multivalent targeted DNA nano-hydrolase can be traced to precisely digest telomeric G-overhang, which contributes to telomeric DNA shortening and thereby causes cell aging and apoptosis. The anticancer treatment is further proved by in vivo studies. In this way, this design provides a telomeric G-overhang-specific eradication strategy based on a non-G-quadruplex targeting manner.


Near-Infrared-Traceable DNA Nano-Hydrolase: Specific Eradication of Telomeric G-Overhang in vivo
Yuhuan Sun, Chuanqi Zhao, Tingting Cui, Hongshuang Qin, Jingsheng Niu, Jinsong Ren and Xiaogang Qu* * Corresponding author. Email: xqu@ciac.ac.cn Methods Figure S1. Material characterization of UCNPs and UCNPs@PDA. Figure S2. 1H-NMR and mass spectrogram of NTA derivative and PEG-Dex. Figure S3. Material characterization of UCeCD. Figure S4. The ability of UCeCD to capture single strand DNA with UV-Vis spectra determination. Figure S5. Nuclease-like activity of UCe based on BNPP substrate. Figure S6. Denaturing PAGE experiments for observation of DNA cleavage. Figure S7. The stability of UCeCD with time course. Figure S8. Double immunofluorescence staining assays to test the DNA damage in MCF-7 cells. Figure S9. the photothermal effect of 980 nm excitation on cell viability, senescence and apoptosis. Figure S10. Photographs of the H22 tumor-bearing mice before or after treatments. Figure S11-S14. The side-effect evaluation of UCeCD in vivo. Table S1 ICP-MS and elemental analysis results of UCeCD. Table S2. DNA sequences used in this study.

Synthesis of (1S)-N-(5-amino-1-carboxypentyl)iminodiacetic acid (NTA derivative) (4)
The synthetic route of NTA derivative was realized in two steps: (i) N ε -benzyloxycarbonyl-L-lysine (8.4 g) was dissolved in NaOH solution (2 M, 45 mL), and the solution was added dropwise with stirring to a cooled solution (0 °C) of bromoacetic acid (8.34 g) in NaOH solution (2 M, 30 mL) for 2 h. The mixture was stirred overnight at 25 °C and then heating for 2 h at 70 °C, and subsequently 1 M HCl (90 mL) was added to the solution. After the mixture had been cooled, the precipitate was filtered off and dried to afford a crude white powder. This was purified by further dissolution in 1 M NaOH (100 mL) and precipitation with 1 M HCl (100 mL) to give the pure (1S)-N-(5-carbobenzyloxyamino-1-carboxypentyl)iminodiacetic acid.
(ii) (1S)-N-(5-carbobenzyloxyamino-1-carboxypentyl)iminodiacetic acid (6 g) was dissolved in methanol (95 mL)/H2O (5 mL) solution, and 10% Pd/C catalyst (0.6 g) was added. The reaction was keeping stirred in H2 at 25 °C and 760 mmHg overnight. Afterwards, the catalyst was filtered off and rinsed with H2O (50 mL), and the solvents were removed from the filtrate to give a white paste which crystallized in pentane. The product was re-dissolved in H2O (20 mL), and then ethanol (15 mL) was added until the solution became cloudy; after heating to give a limpid solution, the mixture was allowed to stand at -20 °C with seeds. The white crystals were filtered off and dried to afford (1S)-N-(5-amino-1-carboxypentyl)iminodiacetic acid, the structure of which was confirmed by the 1H-NMR (D2O) spectra in the supporting information.

Preparation of PDA-Coated UCNPs (5)
The as-prepared UCNPs (20 mg) was re-dispersed in cyclohexane (2 mL). For synthesizing PDA-coated UCNPs with nano-sized PDA shell (named as UCNPs@PDA), the mixture of triton X-100 (3.6 mL), hexanol (3.6 mL), cyclohexane (14 mL), and water (680 µL) were stirred to form a homogeneous solution. Then, cyclohexane solution of UCNPs was added and being ultrasound for 30 min. After being stirred for another 30 min, dopamine hydrochloride aqueous solution (50 µL, 25 wt %) was injected into the above reaction mixture at a rate of 1.5 µL min -1 . Afterward, ammonium hydroxide (75 µL, 28 wt % in water) was added into mixture. After being stirred for 6 h, the nanoparticles were precipitated by adding ethanol, collected by centrifugation (10,000 rpm for 10 min) and washed with ethanol and water. Finally, the UCNPs@PDA were re-dispersed in water and dried by vacuum evaporation.

Preparation of UCe
The as-prepared UCNPs@PDA was reacted with NTA in Tris buffer (pH 8.5) under vigorous stirring for 12 h. Then, NTA-modified UCNPs@PDA (named as UCNPs@NTA) were obtained by centrifugation (10000 rpm, three times), and washed with water. Finally, the UCNPs@NTA nanoparticles were stirred with freshly prepared Ce(NH4)2(NO3)6 solution (in acetonitrile) for 4 h and the supernatant solution was discarded. The final nanoparticles (UCe) were purified by rinsing with water for three times.

Preparation of UCeCD
UCe (10 mg/mL) was dispersed into tris buffer (10 mM, containing 50 mM Na + , pH 8.5). Then PEG-Dex (1 mg) was added with stirring for 6 h. Centrifugation, the precipitation obtained is re-dissolved into tris buffer (containing 50 mM Na + , pH 6.5). Subsequently, C-DNA-amino (5 OD) was added to the mixture. The reaction lasted four hours at room temperature, followed by 48 hours at 4 o C. Finally, centrifugation and wash with water for three times. The final product is dissolved in water and stored at 4 o C.
Immunofluorescence detection MCF-7 cells were digested into single cells with trypsin. Then the single cell suspension (1×10 4 cells) was transferred to a coverslip pre-coated with poly-L-lysine. After fixation with 4% paraformaldehyde for 30 min at room temperature, the cells were permeabilized with 0.5% Triton X-100 for 5 min, blocked with 5% bovine serum albumin at room temperature for 30 min and probed overnight with primary antibodies against γ-H2AX, and TRF1 at 4 º C. After rinsing three times with PBS for 5 min each, cells were incubated with a fluorochrome-conjugated secondary antibody, diluted in antibody dilution buffer, for 30 min at room temperature in the dark. Labeled cells were then rinsed with PBS and analyzed under a LSCM (Nikon Eclipse Ni-E, Japan).

Quantification of telomere length by using quantitative real-time polymerase chain reaction (qRT-PCR)
Genomic DNA was extracted from each cell sample, and relative telomere length was measured using a qRT-PCR method described (6). The technique measured the factor by which the sample differed from a reference DNA sample in its ratio of telomere repeat copy number to single copy gene copy number. This ratio was proportional to the average telomere length. A revised primer set (telo-F: 5-CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT-3 and telo-R: 5-GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT-3) was used for telomere amplification and acidic ribosomal phosphoprotein P0 (RPLP0) gene primers (36B4F: 5-ACTGGTCTAGGACCCGAGAAG-3; 36B4R: 5-TCAATGGTGCCTCTGGAGATT-3) used as a single-copy gene reference. Each 20 μL PCR reaction included 10 μL Syber Green PCR Master Mix (Brilliant III Ultra-Fast QPCR) 100 ng genomic DNA, and primers at final concentrations: 200 nM for each telomere primer or 300 nM for each of the 36B4 primers. The amplification was performed in an Agilent MX3000P Light cycler system using the following conditions: 95 °C for 1 min, followed by 40 cycles of 95 °C for 15 s, 58 °C for 15 s, and 72 °C for 40 s. The MxPro software was used for calculation of the Ct values and the relative telomere length. The relative telomere/single copy gene ratio (T/S value) was calculated using the formula T/S ≈ 2 ΔCt , where ΔCt = Ct (36B4) -Ct (Tel).

Senescence-Associated β-Galactosidase Assay
MCF-7 Cells treated with nanoparticles (72 h) were washed twice in PBS, fixed in 4% formaldehyde for 30 min at room temperature, washed again in PBS, and stained by use of an X-gal solution (1 mg/mL, pH 6) for 16 h at 37°C as described in the reported method (7). Cells were viewed with an OLYMPUS BX-51 light microscope and photographed. More than 5 different fields of glass slip were randomly chosen, and over 1000 cells in the chosen fields were counted to calculate the percentage of senescent cells.

Annexin V/propidium iodide (PI) staining
The MCF-7 cells were treated with nanoparticles for 72 h. Cells were harvested and digested into single cells. After rinsing twice with PBS, 1×10 6 cells were re-suspended in 400 μL staining buffer (Annexin V-FLUOS staining Kit, BestBio, China) by mixing 5 μL of Annexin-V-FITC and incubated for 15 min at 4 °C in the dark. Then 10 μL PI was added into the mixture and continued to incubate for 5 min. Samples were analyzed immediately by flow cytometer.

TEM imaging of cellular internalization of nanoparticles MCF-7 cells (cultured in a 6-well tissue culture plate) after 4 hours incubation with
UCeCD (50 µg/mL) were fixed for 1 hour in 2.5% glutaraldehyde, post fixed in 1% osmium tetroxide. Cells were then dehydrated in a graded ethanol series and embedded in epoxy resin (Epon 812, SPI). Thin sections of the embedded cell monolayer were cut with an ultramicrotome (UC7, Leica) equipped with a diamond knife (Ultra 45 o Diatome). And the sections were respectively stained with 2% uranium acetate in saturated ethanol solution and lead citrate. Images were acquired using a HITACHI HT7700 TEM.

Cell Toxicity Assays
Cell viability was measured using the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma-Aldrich) MTT assays. MCF-7 cells and HEK293T cells were cultured in DMEM (Gibco BRL) medium supplemented with 5% FBS, in a 5% CO2 humidified environment at 37 °C. For the MTT assay, cells were plated at a density of 10 4 cells per well on 96-well plates, followed by introduction of nanoparticles 24 h later. After 72 h, the cells were treated with 10 μL MTT (5 mg/mL in PBS) for 4 h at 37 °C and then were lysed in DMSO for 10 min at room temperature in the dark. Absorbance values of formazan were determined using an Ultraspec 2000 (Pharmacia Biotech) at 660 nm (reference wavelength) and 490 nm.

Animal models
Healthy female Kunming mice (20-25 g) were purchased from the Laboratory Animal Center of Jilin University (Changchun, China), and handling procedures were according to the guidelines of the Regional Ethics Committee for Animal Experiments. Hepatoma 22 (H22) tumor-bearing mice were selected as the animal model to assess the antitumor effect. H22 cells were harvested from the peritonea cavity of mice 5-7 days after inoculation. Then, the cells of 2 × 10 5 cells were suspended in saline (about 50 μL) and subcutaneously injected into the right armpit region of mice.
For studying the therapy efficiency via intravenous injection, when the tumor volumes were about 100 mm 3 , tumor-bearing mice were divided into five groups (n = 6 mice/group) randomly for different formulations: (1) saline alone; (2) UCe; (3) UCeC; (4) UCeD; and (5) UCeCD. The nanoparticles solution (500 μL, 0.5 mg mL -1 ) was intravenously injected into mice. The tumor dimensions (length and width) and body weight were measured every other day after the treatment. The mice were sacrificed after 2 weeks post-treatment, the tumors were collected, and photos were taken. For histology, the tumor tissues in each group were harvested from mice after the treatment. The tumor tissues were dissected to make paraffin sections for further hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining assays.