Exploring the conformational behaviour and

International Journal of Biological Macromolecules 118 (2018) 1384–1399

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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac

Exploring the conformational behaviour and aggregation properties of lipid-conjugated AS1411 aptamers Claudia Riccardi a, Domenica Musumeci a,b, Irene Russo Krauss a,c, Marialuisa Piccolo d, Carlo Irace d, Luigi Paduano a,c, Daniela Montesarchio a,e,⁎ a

Department of Chemical Sciences, University of Naples Federico II, Via Cintia 21, I-80126 Napoli, Italy Institute of Biostructures and Bioimages, CNR, Via Mezzocannone 16, I-80134 Napoli, Italy CSGI – Consorzio Interuniversitario per lo Sviluppo dei Sistemi a Grande Interfase, Via della Lastruccia 3, I-50019 Sesto Fiorentino (Fi), Italy d Department of Pharmacy, School of Medicine and Surgery, University of Napoli Federico II, Via D. Montesano 49, 80131 Napoli, Italy e Institute for Endocrinology and Oncology “Gaetano Salvatore”, CNR, Via Pansini 5, 80131 Napoli, Italy b c

a r t i c l e

i n f o

Article history: Received 28 May 2018 Received in revised form 23 June 2018 Accepted 26 June 2018 Available online 4 July 2018 Keywords: AS1411 G-quadruplex Lipid conjugates Conformational behaviour Biophysical characterization Aggregation properties

a b s t r a c t AS1411 is a nucleolin-binding aptamer which attracted great interest as active targeting ligand for the selective delivery of therapeutic agents to tumour cells. In this work we selected three AS1411 derivatives 5′-conjugated with lipophilic tails and studied their properties in view of their application in liposomial formulations and/or lipid coated-nanoparticles for targeted therapies. The conformational behaviour of these AS1411 analogs has been investigated in comparison with the unmodified aptamer by CD, UV, PAGE, SEC-HPLC, DLS and thioflavin T (ThT) fluorescence assays to get insight in their secondary structure and aggregation properties. This study has been performed in pseudo-physiological buffers mimicking the extra- and intracellular environments, and at different concentrations in the μM range, paying special attention to the effects of the lipophilic tail on the overall aptamer conformation. The 5′-lipidated AS1411 derivatives proved to fold into stable, parallel unimolecular G-quadruplex structures, forming large aggregates, mainly micelles, at conc. N10 μM. Preliminary bioscreenings on selected cancer cells showed that these derivatives are less cytotoxic than AS1411, but maintain a similar biological behaviour. This study demonstrated that lipophilic tails dramatically favour the formation of AS1411 aggregates, however not impairing the formation and thermal stability of its peculiar G4 motifs. © 2018 Elsevier B.V. All rights reserved.

1. Introduction AS1411 is a 26-mer G-quadruplex-forming oligodeoxyribonucleotide targeting nucleolin, a multifunctional protein involved in cell survival, growth and proliferation, overexpressed on the outer membrane of most cancer cells, regardless of tissue origin. This aptamer entered several Phase I/Phase II clinical trials which demonstrated its efficacy as a first-in-class anticancer agent with very good overall tolerability [1]. If the anticancer efficacy of AS1411 per se is not particularly high compared with other known antitumourals, extremely interesting is its unique ability to selectively target cancer cells, ensuring the effective delivery of other drugs or imaging agents used in combination therapies. Thus far, AS1411 has been exploited as an active targeting ligand in the construction of a variety of nanosystems (nanoparticles [2–6], carbon dots [7], dendrimers [8, 9], liposomes [10], micelles [11–13],

⁎ Corresponding author at: Department of Chemical Sciences, University of Naples Federico II, Via Cintia 21, I-80126 Napoli, Italy. E-mail address: [email protected] (D. Montesarchio).

https://doi.org/10.1016/j.ijbiomac.2018.06.137 0141-8130/© 2018 Elsevier B.V. All rights reserved.

DNA tetrahedrons [14, 15] etc.) and AS1411-linked conjugates [16–18] for anticancer applications, always with high efficacy. Even though much attention has been paid to AS1411, also in consideration of recent findings on its potent anti-HIV activity [19, 20], its mechanism of action in vivo has not been fully elucidated yet. Also its structural features, and particularly its bioactive conformation, are still largely unknown (for a recent review covering the state-of-the-art knowledge on uses and mechanisms of AS1411, see P. J. Bates et al. [21]). Indeed, some discrepancies on the preferred AS1411 conformation - described as a parallel, in some cases [22–27], and as an antiparallel Gquadruplex (G4) structure, in others [28] - have been reported. Particularly, only one study has been carried out to get detailed structural information on this aptamer, proving to be highly polymorphic, folded into multiple, essentially mono- but also bimolecular G-quadruplex structures [23]. More recently, the spectroscopic properties and thermal stability of AS1411 in the presence of different metal ions (K+ and Pb2+) and in molecular crowding conditions have been investigated [29], showing that AS1411 folds mainly into parallel G4 structures in the presence of both metal ions as well as of PEG.

C. Riccardi et al. / International Journal of Biological Macromolecules 118 (2018) 1384–1399

To reduce the conformational polymorphism of AS1411, Phan et al. [30] have then analysed several related oligonucleotides with single nucleotide substitution, identifying a new AS1411-derived sequence, named AT11, able to form a single major G-quadruplex conformation maintaining similar anti-proliferative activity as AS1411. The NMR study revealed that AT11 adopts a four-layer G-quadruplex structure comprising of two propeller-type parallel-stranded subunits connected through a central linker. In addition to studying AS1411-related oligonucleotide sequences, further strategies to restrict the conformational space of this aptamer include the insertion of modified nucleosides within its sequence and/ or the conjugation with suitable reporter groups providing special physico-chemical features. Even though the relationships between conformational preferences and biological activity of AS1411 have not been clarified, interesting biological results have been obtained following the first approach [31–33]. Less explored in this perspective is the chemical conjugation strategy [16–18]. With the aim of exploiting AS1411 as an active targeting agent for multifunctional nanosystems in anticancer strategies, we have here investigated a set of AS1411 derivatives (Fig. 1) conjugated with different lipophilic tails at their 5′-end, designed to ensure their subsequent incorporation in liposomial formulations or to decorate lipid coatednanoparticles. In detail, we have selected an AS1411 derivative 5′-conjugated with a stearyl residue, i.e. a saturated 18-carbon alkyl chain, and other two conjugates, here named 5′-cholesteryl-TEG- and 5′cholesteryl-C6-AS1411, carrying a cholesterol residue linked to the oligonucleotide through a tetraethylene glycol or a 6-carbon atoms alkyl linker, respectively. In particular, 5′-stearyl-AS1411 has a lipophilic tail of the same length as 1-stearoyl-2-hydroxy-sn-glycero-3phosphocholine (18LPC), recently used to build the coating of superparamagnetic nanoparticles (SPIONs) with a core/double shell architecture, developed by our research group [34, 35]. An 18 carbon atoms tail proved to be very effective in stabilizing ad hoc designed nucleolipids within liposomes or other lipophilic nanosystems by hydrophobic interactions [36–39]. Exploiting the same approach, also cholesterol residues ensured stable incorporation of nucleolipidic anticancer agents in liposomal nanosystems [40, 41] or into 18LPCfunctionalized SPIONs [35].

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As a part of our ongoing research on anticancer multifunctional nanosystems, the conformational behaviour of these AS1411 derivatives has been here investigated in pseudo-physiological solutions in comparison with unmodified AS1411. For this characterization, different biophysical techniques have been used, i.e. CD, CD-melting, UVmelting, gel electrophoresis, size exclusion chromatography, dynamic light scattering (DLS) analyses and thioflavin T (ThT) fluorescence assays, to get an insight into the preferred conformations and thermal stability of these AS1411 derivatives. This study has been carried out at different oligonucleotide concentrations and in two different buffered solutions, i.e. the PBS buffer, containing 147 mM Na+, and the 10 mM KH2PO4, 70 mM KCl, 0.2 mM EDTA solution, containing 80 mM K+ ions, respectively mimicking the extra- and intracellular media. Then, preliminary in vitro bioscreens have been carried out to get information on the overall effects of the lipid tail on the antiproliferative properties of AS1411. 2. Materials and methods 2.1. General methods All the reagents and solvents were of the highest commercially available quality and were used as received. AS1411 (5′GGTGGTGGTGGTTG TGGTGGTGGTGG3′), 5′-stearyl-AS1411, 5′-cholesteryl-TEG-AS1411 and 5′-cholesteryl-C6-AS1411 oligonucleotides were purchased from Biomers (Germany). The identity and purity of the oligomers were proved by MALDI-TOF mass spectrometry and high performance liquid chromatography (HPLC) provided by the commercial supplier. Ethidium bromide and thioflavin T were purchased from Sigma Aldrich; 5× Green GoTaq Flexi Buffer was from Fisher Scientific. 2.2. Preparation of the oligonucleotide samples Lyophilized oligonucleotides were dissolved in a known volume of Milli-Q water and their concentrations were determined by UV spectroscopy on a JASCO V-550 spectrophotometer equipped with a Peltier Thermostat JASCO ETC-505T, in 1 cm path length couvette measuring the absorbance at 260 nm (90 °C) using the molar extinction coefficient

AS1411:’ 5’-GGT GGT GGT GGT TGT GGT GGT GGT GG-3’

5’-cholesteryl-C6-AS1411

5’-stearyl-AS1411

5’-cholesteryl-TEG-AS1411 Fig. 1. AS1411 oligonucleotide sequence and schematic representation of the lipid tails of the AS1411 derivatives investigated in this study.

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of 281,700 cm−1 M−1, calculated for the unstacked oligonucleotide. The spectra were recorded in the range 220–380 nm with a medium response, a scanning speed of 100 nm/min and a 2.0 nm bandwidth, and corrected by subtraction of the background scan with the buffer. Then the oligonucleotides stock solutions (574, 459, 425 and 234 μM for AS1411, 5′-stearyl-AS1411, 5′-cholesteryl-TEG-AS1411 and 5′cholesteryl-C6-AS1411, respectively) were diluted in the selected K+ or Na+ buffer and annealed by heating each system for 5 min at 90 °C and then leaving it to slowly cool to room temperature overnight. Annealed samples were then kept at 4 °C until use. 2.3. Spectroscopic characterization of AS1411 and its derivatives 2.3.1. CD experiments CD spectra and CD-monitored melting curves were recorded on a Jasco J-715 spectropolarimeter equipped with a Peltier-type temperature control system (model PTC-348WI), using a quartz couvette with a path length of 1 cm (3 mL internal volume, Hellma). CD parameters for spectra recording were the following: spectral window 220–320 nm, data pitch 1 nm, band width 2 nm, response 4 s, scanning speed 100 nm/min, 3 accumulations. The oligonucleotide sequences were characterized at 0.5, 2.5, 5.0 and 10 μM concentrations in K+ (10 mM KH2PO4, 70 mM KCl, 0.2 mM EDTA, pH = 7.0) and Na+ (PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH = 7.4) phosphate buffer solutions, taking a suitable initial aliquot from the stock solution in H2O. Thermal denaturation-renaturation curves were recorded following the CD signal at 263 nm vs. the temperature (heating/cooling rate of 1.0 °C/min) in a range of temperature slightly different depending on saline conditions (15–90 °C for the K+ buffer and 15–80 °C for the Na+ buffer). Each experiment was performed in duplicate. The Tm values were estimated as the maxima of the plots of the first derivatives of the melting/annealing curves and the error associated with the Tm determination was ±1 °C. 2.3.2. UV–vis absorption experiments UV-melting curves were obtained on a Cary 5000 UV–Vis-NIR spectrophotometer equipped with a temperature controller system, using 1 cm path length couvette (1 mL internal volume, Hellma). UVmelting experiments were performed analogously to the CD-melting experiments (heating/cooling rate of 1.0 °C/min, temperature range of 15–90 °C and 15–80 °C respectively for the analyses in K+ and Na+ buffer solutions). The characterization of the oligonucleotide sequences was performed at 0.5, 2.5, 5.0 and 10 μM concentrations in the K+ and Na+ buffers, taking a suitable initial aliquot from the stock solution in H2O. Each experiment was performed in duplicate. The Tm values were calculated as the maxima of the plots of the first derivatives of the melting curves (associated error: ±1 °C). 2.3.3. Gel electrophoresis Slowly annealed samples of AS1411, 5′-stearyl-AS1411, 5′cholesteryl-TEG-AS1411 and 5′-cholesteryl-C6-AS1411 in 40% of dye (5× Green GoTaq Flexi Buffer) were loaded on 7% acrylamide gels in TBE (Tris-Borate-EDTA, 0.5×) buffer at the different concentration (5, 10 and 50 μM) and saline conditions analysed (in the K+ and Na+ buffers). The samples were then run at 100 V at room temperature for 75 min, stained with ethidium bromide and finally visualized with a UV transilluminator (BioRad ChemiDoc XRS). In the case of 5′cholesteryl-TEG-AS1411 and 5′-cholesteryl-C6-AS1411, additional runs of 2 h 30 min and 3 h 15 min were performed to better investigate the retarded bands. 2.3.4. Size exclusion chromatography SEC-HPLC analyses were performed using an Agilent HPLC system, equipped with a UV/vis detector and a Yarra 3 μm column (300 × 4.60 mm; flow rate 0.5 mL min−1, Phenomenex). Elution was monitored at λ = 254 nm. The mobile phases used consisted of the K+ and

Na+ buffers. All the oligonucleotides tested were injected from stock solutions of the samples annealed at 5, 10 and 50 μM concentrations. 2.3.5. Dynamic light scattering DLS measurements on AS1411 and its lipid derivatives were carried out with a home-made instrument composed of a Photocor compact goniometer, a SMD 6000 Laser Quantum 50 mW light source operating at 533 nm, a photomultiplier (PMT-120-OP/B) and a correlator (Flex0201D) from Correlator.com. The experiments were carried out at room temperature at a scattering angle θ of 90°. The scattered intensity correlation function was analysed using a regularization algorithm. The diffusion coefficient of each population was calculated as the z-average of the diffusion coefficients of the corresponding distributions. In the case of diluted samples, the Stokes–Einstein equation was used to evaluate the hydrodynamic radius, RH, of the oligonucleotides and their aggregates from their translation diffusion coefficient, D. DLS profiles were also normalized by using the Precision Deconvolve program to identify the most abundant species in solution, regardless of their size. 2.3.6. Thioflavin T (ThT) fluorescence assays The ThT fluorescence assays were carried out on a FluoroMax-4 spectrofluorometer (Horiba Scientific) equipped with a temperaturecontroller system (LFI-3751). A 165 μM stock solution of thioflavin T was prepared in double distilled water using the molar extinction coefficient of 36.000 M−1 cm−1 at 412 nm and then filtered with 0.2 μm Millipore filters according to a previously reported procedure [42]. Fluorescence emission spectra were recorded at 25 °C from 450 to 600 nm in a 1 cm path-length quartz cell exciting at 420 nm and maintaining the excitation and emission slits at 3 nm. Slowly annealed samples of AS1411, 5′-stearyl-AS1411 and 5′cholesteryl-TEG-AS1411 dissolved in the K+ buffer (10 mM KH2PO4, 70 mM KCl, 0.2 mM EDTA, pH = 7.0) at different concentrations (0.5, 1, 2, 3, 5 and 10 μM) were incubated in the dark with 1 μM ThT for 30 min and then analysed. All the measurements were performed in triplicate. The results were plotted as the fluorescence intensity enhancement (FI/FI0) of ThT, i.e. the ratio between the ThT fluorescence in the presence of the oligonucleotide (FI) and the background fluorescence of ThT alone (FI0) at 490 nm after subtraction of the buffer fluorescence, as previously described. [43] The fluorescence intensity at 490 nm was also plotted vs. the concentration of the oligonucleotides in order to calculate the scaling factor a using the Eq. (1): a ¼ Y−Y 0 =Y max −Y 0 ;

ð1Þ

where Y0 is the initial F490 value (i.e. in the absence of the oligonucleotide), and Ymax is the highest fluorescence value obtained (i.e. at the highest concentration tested). The data were then fitted, according to previous reports [44], with the Hill equation (Eq. (2)) in order to estimate the apparent dissociation constant (KD) at 25 °C: n

F 490 ¼ a½ThT = K D þ ½ThT

n

þ y0

ð2Þ

where n is the Hill coefficient. 2.4. Biological in vitro evaluation 2.4.1. Cell cultures Human breast adenocarcinoma cells (MCF-7), human colorectal cancer cells (HCT-116), and malignant melanoma cells (A-375) were grown in Dulbecco's modified Eagle's medium (DMEM, Invitrogen, Paisley, UK) containing high glucose content (4.5 g/L). Media were supplemented with 10% fetal bovine serum (FBS, Cambrex, Verviers, Belgium), L-glutamine (2 mM, Sigma, Milan, Italy), penicillin (100 units/mL, Sigma) and streptomycin (100 μg/mL, Sigma), according to ATCC


recommendations. All cells were cultured in a humidified 5% carbon dioxide atmosphere at 37 °C. 2.4.2. In vitro bioscreens The bioactivity of AS1411 and of its derivatives (5′-stearyl-AS1411, 5′-cholesteryl-C6-AS1411 and 5′-cholesteryl-TEG-AS1411) was investigated by the estimation of a “cell survival index”, arising from the combination of cell viability evaluation with cell counting. More specifically, the cell survival index is calculated as the arithmetic mean between the percentage values derived from the MTT assay and the automated cell count, thus providing a more accurate parameter of the concrete number of cells that survive after a preclinical in vitro study. Cells were inoculated in 96-microwell culture plates at a density of 104 cells/well, and allowed growing for 24 h. The medium was then replaced with fresh medium and the cells were treated for further 48 and 72 h with a range of concentrations (1 → 25 μM) of each sample. Cell viability was evaluated using the MTT assay, which measures the level of mitochondrial dehydrogenase activity using the yellow 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, Sigma) as substrate [45]. The assay is based on the redox ability of living mitochondria to convert dissolved MTT into insoluble purple formazan. Briefly, after the treatments, the medium was removed and the cells were incubated with 20 μL/well of a MTT solution (5 mg/mL) for 1 h in a humidified 5% CO2 incubator at 37 °C. The incubation was stopped by removing the MTT solution and then adding 100 μL/well of DMSO to solubilize the obtained formazan. Finally, the absorbance was monitored at 550 nm using a microplate reader (iMark microplate reader, Bio-Rad, Milan, Italy). Cell number was determined by TC20 automated cell counter (Bio-Rad, Milan, Italy), providing an accurate and reproducible total count of cells and a live/dead ratio in one step by a specific dye (trypan blue) exclusion assay [46]. Bio-Rad's TC20 automated cell counter uses disposable slides, TC20 trypan blue dye (0.4% trypan blue dye w/v in 0.81% sodium chloride and 0.06% potassium phosphate dibasic solution) and a CCD camera to count cells based on the analyses of captured images. Once the loaded slide is inserted into the slide port, the TC20 automatically focuses on the cells, detects the presence of trypan blue dye and provides the count. When the cells are damaged or dead, trypan blue is internalized into the cell, allowing living cells to be counted. Operationally, after treatments in 96-microwell culture plates, the medium was removed and the cells were collected; 10 μL of cell suspension, mixed with 0.4% trypan blue solution at 1:1 ratio, were then loaded into the chambers of disposable slides. The results are expressed in terms of total cell count (number of cells per mL). If trypan blue is detected, the instrument also accounts for the dilution and shows live cell count and percent viability. Total counts and live/dead ratio from random samples for each cell line were subjected to comparisons with manual hemocytometers in control experiments. The calculation of the concentration required to inhibit the net increase in the cell number and viability by 50% (IC50) is based on plots of data (n = 4 for each experiment) and repeated three times (total n = 12). IC50 values were obtained by means of a dose-response curve by nonlinear regression using a curve fitting program, GraphPad Prism 5.0, and are expressed as mean values ± SEM (n = 12) of three independent experiments.

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indicated as Na+ buffer), respectively mimicking the intracellular and extracellular media, in order to evaluate the effect of these different saline conditions on the structuring ability of the aptamer. In addition, we tested four different concentrations (10, 5.0, 2.5 and 0.5 μM) in order to investigate the effect of concentration on the aptamer conformation and stability. The CD spectra at r.t. of AS1411 at the analysed concentrations, in both K+ and Na+ buffer solutions, showed a broad negative band with a minimum at ca. 241 nm, and two positive bands with maxima centred at 263 nm, the highest, and at ca. 295 nm, the weakest one (Fig. 2). In the Na+ buffer the band at ca. 295 nm was more evident and, overall, the aptamer exhibited a lower degree of structuration than in the K+ buffer, as expected considering that the latter cation can better stabilize G4 structures than Na+, in accordance with literature studies [47–52]. These spectral features are consistent with predominantly parallel G4 structures [53–59] and in agreement with the CD spectrum of AS1411 reported in previous works [23, 60, 61], with only a low fraction of an antiparallel G4 conformation, evidenced by the weak band at ca. 295 nm. The CD melting experiments, monitored at 263 nm in the K+ buffer, provided a nice sigmoidal behaviour with apparent Tm values of 66 °C at 10 μM, 68 °C at 5.0 and 2.5 μM and 65 °C at 0.5 μM concentration (Fig. 3), indicating the formation of quite stable G4 structures, also in accordance with previously reported data [23]. The heating and cooling profiles were essentially superimposable (as a representative example, the 10 μM sample is reported in Fig. 3a), indicating that, under the experimental conditions used (heating/cooling rate: 1 °C/min), the related denaturation/renaturation processes were reversible. Lower Tm values (37–38 °C) were observed in the CD melting curves recorded in the Na+ buffer (Fig. 4), as previously reported [23]. For these systems, only limited hysteresis emerged on comparing the heating and cooling profiles, as shown for the 10 μM sample, here reported as a representative example in Fig. 4a. From the UV-melting experiments monitored at 295 nm [58, 62, 63], apparent Tm values of 64–66 °C in the K+ buffer (Fig. 5) and of 38–39 °C in the Na+ buffer (Fig. 6) were obtained at the tested concentrations (10, 5.0, 2.5, 0.5 μM), thus confirming the formation of stable G4 structures. Also in the UV-melting profiles, both in K+ and Na+ buffer solution, no significant hysteresis was observed on comparing the heating and cooling processes (data not shown). In summary, a very good agreement between the apparent Tm values determined by UV- and CD-melting experiments, within the experimental error, was always found. Under the studied conditions, AS1411 forms essentially parallel G4 structures, with only minor amounts of antiparallel G4 conformations, more stable in the K+ than in the Na+ buffer. These experiments showed that the thermal stability of the G4 structures of this aptamer was mainly dependent on the saline conditions and essentially independent from the concentration, thus indicating the sole presence of unimolecular G-quadruplex structures, at least in the low μM concentration range analysed. 3.2. Spectroscopic properties and solution behaviour of lipid AS1411 derivatives

3. Results and discussion 3.1. Spectroscopic properties and solution behaviour of AS1411 Having clear evidence of the highly polymorphic nature of AS1411 at mM concentrations [23], we here examined the conformational behaviour of this oligonucleotide in μM range solutions combining CD, CDmelting and UV-melting data. The spectra and melting/annealing profiles were recorded in two different phosphate buffered solutions containing a high content of K+ (10 mM KH2PO4, 70 mM KCl, 0.2 mM EDTA, pH 7.0, here indicated as K+ buffer) or of Na+ ions (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4, here

All the here examined lipid AS1411 derivatives were analysed under the same conditions used to investigate AS1411, aiming at assessing if, and to what extent, the presence of the lipophilic tail influences the conformational behaviour of the G4-forming aptamer in solution. Thus, UV and CD experiments were performed at different concentrations (10, 5.0, 2.5 and 0.5 μM) and in both Na+ and K+ buffers, so to evaluate their macroscopic features in systems mimicking the extra- and intracellular environment. The CD spectra at r.t. of 5′-stearyl-AS1411, 5′-cholesteryl-TEGAS1411 and 5′-cholesteryl-C6-AS1411 showed an intense positive band centred at 263 nm and a weak negative band at ca. 241 nm

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Fig. 2. CD spectra of AS1411 at 10 μM (a), 5.0 μM (b), 2.5 μM (c) and 0.5 μM (d) concentration registered in the K+ buffer (red line) and Na+ buffer (blue line) solutions.

(Figs. S1–S3), characteristic of parallel G4 conformations, in both K+ and Na+ buffers. Interestingly, the weak positive band at 295 nm found in the CD spectra of unmodified AS1411 was not evident for the lipidated aptamers in both buffers, indicating the apparent absence of antiparallel G4 conformations and the formation of uniquely parallel G4 structures. Also in these cases, K+ ions showed higher structuring ability than Na+ in the G4 formation. In the K+ buffer the CD melting profiles, monitored at 263 nm, always provided nice sigmoidal curves, with apparent Tm values of 59 °C for the 5′-stearyl-AS1411 at all the tested concentrations (Fig. S4),

and values in the range of 62–65 °C and 63–65 °C for 5′-cholesterylTEG-AS1411 (Fig. S5) and 5′-cholesteryl-C6-AS1411 (Fig. S6), respectively. Only limited hysteresis was observed on comparing the melting and cooling profiles, suggesting essentially reversible folding/unfolding processes at the heating/cooling rate used (1 °C/min). Remarkably, the apparent Tm values obtained in the K+ buffer for 5′stearyl-AS1411 were lower than those observed for unmodified AS1411 (59 °C, ΔTm = −7 °C), but however indicative of stable G4 structures. In contrast, in the case of the CD analysis performed in the Na+ buffer, a more complex behaviour was observed for the lipidic derivatives of AS1411. Indeed, the CD melting profiles of 5′-stearyl-AS1411

Fig. 3. CD-melting profiles of AS1411 in the K+ buffer at 10 μM (a), 5.0 μM (b), 2.5 μM (c) and 0.5 μM (d) concentration, recorded at 263 nm (heating/cooling rate: 1 °C/min). In panel (a) the CD annealing profile of AS1411 at 10 μM concentration is also reported.


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Fig. 4. CD-melting profiles of AS1411 in the Na+ buffer at 10 μM (a), 5.0 μM (b), 2.5 μM (c) and 0.5 μM (d) concentration, recorded at 263 nm (heating/cooling rate: 1 °C/min). In panel (a) the CD annealing profile of AS1411 at 10 μM concentration is also reported.

in the Na+ buffer showed essentially sigmoidal curves with apparent Tm values of 40, 39 and 36 °C, respectively at 5.0, 2.5 and 0.5 μM concentration (Fig. S7), with a small hysteresis (3 °C) only at 5.0 μM concentration (Fig. S7a). Then, at 10 μM a special behaviour was found: the melting profile did not show a sigmoidal shape, expected for a unique, cooperative transition, but a curve with multiple transitions and not complete G4 denaturation even at 80 °C (Fig. S8). The analysis of the CD spectra on varying the temperature showed a weak shoulder at 285 nm at temperatures higher than 75 °C (light blue and green dashed lines in Fig. S8b). In addition, on increasing the temperature, a small shift in the positive band at 263 nm was also observed, overall indicating that

not a single transition but multiple events occurred on increasing the temperature, plausibly related to unfolding of different G4 species. As in the case of 5′-stearyl-AS1411, 5′-cholesteryl-TEG-AS1411 showed a concentration-dependent behaviour in the Na+ buffer. In particular, at the lowest concentrations investigated (i.e. 5.0, 2.5 and 0.5 μM), a behaviour similar to 5′-stearyl-AS1411 at 10 μM concentration was found (Fig. S9). Then, on the 10 μM sample in the Na+ buffer, the CD melting gave another special behaviour (Fig. S10). Initially, the CD signal slightly decreased up to 40 °C, with the positive maximum stable at 263 nm. At temperatures higher than 45 °C, the CD signal increased up to 70 °C and then again slightly decreased up to 80 °C, still however

Fig. 5. UV-melting profiles of AS1411 in the K+ buffer at 10 μM (a), 5 μM (b), 2.5 μM (c) and 0.5 μM (d) concentration, recorded at 295 nm (heating/cooling rate: 1 °C/min).

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Fig. 6. UV-melting profiles of AS1411 in the Na+ buffer at 10 μM (a), 5 μM (b), 2.5 μM (c) and 0.5 μM (d) concentration, recorded at 295 nm (heating/cooling rate: 1 °C/min).

maintaining a high intensity at this temperature. During the heating process, a shift of the positive band maximum from 263 to 262 nm was observed in the 40–50 °C temperature range, and again moved back to 263 nm starting from 65 °C. Clearly, multiple events occurred, with the main transition in the 40–70 °C range having an apparent Tm of 57 °C. This particular CD melting profile could be attributed to different events, related to aggregation processes in solution and scattering phenomena in suspension. One possible explanation could be that at ca. 57 °C the aggregates were dispersed, with the suspension concomitantly becoming more transparent and producing the observed changes at 263 nm. Concerning 5′-cholesteryl-C6-AS1411 analysed in the Na+ buffer, the CD melting profiles showed at all the tested concentrations nonsigmoidal melting curves (Fig. S11). In contrast, the UV-melting profiles, monitored at 295 nm, both in K+ and Na+ buffers provided for all the investigated systems nice sigmoidal curves, with no significant hysteresis observed in most cases on comparing the heating and cooling processes, indicative of equilibrium processes (data not shown). In particular, the analysis of 5′stearyl-AS1411 showed an apparent Tm of 59 °C in the K+ (Fig. S12) and of 39–40 °C in the Na+ buffer (Fig. S13) at all the tested concentrations, in accordance with CD results. UV-melting curves of 5′cholesteryl-TEG-AS1411 and 5′-cholesteryl-C6-AS1411 provided apparent Tm values respectively in the range 62–65 (Fig. S14) and 62–67

°C (Fig. S15) in the K+ buffer. In turn, UV-melting analysis in the Na+ buffer showed apparent Tm values in the range 44–45 and 41–45 °C respectively for 5′-cholesteryl-TEG-AS1411 (Fig. S16) and 5′-cholesterylC6-AS1411 (Fig. S17), indicating a higher thermal stability with ΔTm of +4–+6 °C with respect to unmodified AS1411. In summary, the apparent Tm values determined by UV- and CDmelting experiments were generally in good agreement among them and with those obtained for the unmodified AS1411, except in few cases (Table 1). Taken together these results indicated a high polymorphism of the AS1411 derivatives, however with different features with respect to unmodified AS1411. In all cases the lipid AS1411 derivatives formed multiple species, all adopting parallel G4 conformations with similar thermal stability, essentially dependent on cation effects and roughly independent on concentration. Peculiar CD-melting curves were observed in the Na+ buffer, mainly at the highest concentrations investigated. In Table 1 an overview of the apparent Tm values derived by UV and CD-melting experiments performed on all the analysed oligonucleotides is reported. To better elucidate the conformational behaviour of these G4 structures, further investigations using gel electrophoresis, size exclusion chromatography, dynamic light scattering (DLS) analyses and ThT fluorescence assays have been carried out.

Table 1 Melting temperature values obtained by UV- and CD-melting experiments for AS1411 and the here investigated lipid analogs (n.d. = not determined). K+ buffer AS1411

Na+ buffer 5′-stearyl-AS1411

5′-chol-TEG-AS1411

5′-chol-C6-AS1411

AS1411

5′-stearyl-AS1411

5′-chol-TEG-AS1411

5′-chol-C6-AS1411

CD Tm (°C) ± 1 10 μM 66 5 μM 68 2.5 μM 68 0.5 μM 65

59 59 59 59

62 64 65 63

64 63 64 65

37 38 37 38

n.d. 40 39 36

57 n.d. n.d. n.d.

n.d. n.d. n.d. n.d.

UV Tm (°C) ± 1 10 μM 66 5 μM 66 2.5 μM 66 0.5 μM 64

59 59 59 59

65 64 62 63

64 67 66 64

39 38 38 39

40 40 40 39

44 45 44 44

43 41 44 45


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3.3. Gel electrophoresis analysis Gel electrophoresis analysis was used to further characterize AS1411 and its lipid derivatives, and particularly define their molecularity under the studied conditions. For these studies, we tested different concentrations of the oligonucleotides (5, 10 and 50 μM) in both the K+ and Na+ buffer, using the 26-mer d5′(TTAGGG)4TT3′ taken from the human telomere (tel26) as reference oligonucleotide, known to adopt unimolecular G4 structures in solution [64, 65]. In Figs. 7, 8 and S18 the photographs of 7% polyacrylamide gels, run under native conditions, are reported. In comparison with tel26, which on a polyacrylamide gel migrated as a single band, AS1411 in the same conditions showed two very close bands at 10 μM concentration (lanes 3 and 7) or three, poorly resolved bands at 50 μM concentration (lanes 4 and 8), in both the K+ and Na+ buffers (Fig. 7a). These bands showed a gel mobility similar to tel26 indicating for these systems, carrying the same number of negative charges, similar overall size. In contrast, in the case of 5′-stearyl-AS1411 (Fig. 7b), bands with a marked difference in electrophoretic mobility were observed: a welldefined band, with similar but not identical electrophoretic mobility as tel26 (at low concentrations), was accompanied by a retarded band, attributable to large aggregates. This retarded band, not evident at 5 μM and very poorly detectable at 10 μM (lanes 3 and 7), became clearly visible at 50 μM (lanes 4 and 8). In the case of 5′-cholesteryl-TEG-AS1411 the band with the same electrophoretic mobility as tel26 was observed only in the K+ buffer along with more retarded bands, not well distinguishable, on top of the gel (Fig. 8). In the presence of Na+ ions, only the retarded bands were clearly visible. In order to better investigate these low-migrating bands, the gel was run also for longer times, i.e. 2.5 and 3.3 h, respectively (Fig. 8, right side). Also in these conditions, the bands migrated poorly, indicating the presence of very large species which, in contrast to what observed for 5′-stearyl-AS1411, were more abundant in K+ than in Na+ ions. On analysing 5′-cholesteryl-C6-AS1411, the gel showed at all the tested conditions (different concentrations and saline solutions) the

Fig. 8. 7% polyacrylamide gel electrophoresis under native conditions of the 5′-cholesterylTEG-AS1411 run at 100 V at r.t. for 1 h 15 min. Lane 1: tel26 (5 μM); Lane 2: 5′-cholesterylTEG-AS1411 (5 μM); Lane 3: 5′-cholesteryl-TEG-AS1411 (10 μM); Lane 4: 5′-cholesterylTEG-AS1411 (50 μM), in the Na+ buffer; Lane 5: tel26 (5 μM); Lane 6: 5′-cholesteryl-TEGAS1411 (5 μM); Lane 7: 5′-cholesteryl-TEG-AS1411 (10 μM); Lane 8: 5′-cholesteryl-TEGAS1411 (50 μM), in the K+ buffer; On the right, the magnification of the gel run at 100 V at r.t. for 2.5 h (top) and 3.3 h (bottom) is shown.

concomitant presence of a band with the same mobility as tel26 and of several retarded bands (Fig. S18, left side). Even in this case, the gel was run also for longer times (2.5 and 3.3 h), and, in an attempt to separate the retarded bands, a conspicuous number of different species was evidenced (Fig. S18, right side). Taking into account that gel electrophoresis is a low resolution technique, these results provided a clear evidence that AS1411 and all the here studied derivatives are present in solution as multiple species, which for the lipid derivatives include aggregates, formed in a cationand concentration-dependent manner. The bands with gel mobility similar to tel26 are attributable to unimolecular structures, while the retarded species account for very large aggregates. Bimolecular species, well characterized for AS1411 [23], seem to be absent in all the lipidated systems, while, at high concentrations, large aggregates are the prevailing species, whose presence can explain their complex CD melting profiles. Notably, the formation of stable self-assembled aggregates has been recently reported also for other lipid-conjugated G4-forming oligonucleotides [66, 67]. 3.4. Size exclusion chromatography analysis

Fig. 7. 7% polyacrylamide gel electrophoresis under native conditions of the analysed AS1411 (a) and 5′-stearyl-AS1411 (b) run at 100 V at r.t. for 1 h 15 min. (a) Lane 1: tel26 (5 μM); Lane 2: AS1411 (5 μM); Lane 3: AS1411 (10 μM); Lane 4: AS1411 (50 μM), in the Na+ buffer; Lane 5: tel26 (5 μM); Lane 6: AS1411 (5 μM); Lane 7: AS1411 (10 μM); Lane 8: AS1411 (50 μM), in the K+ buffer; (b) Lane 1: tel26 (5 μM); Lane 2: 5′stearyl-AS1411 (5 μM); Lane 3: 5′-stearyl-AS1411 (10 μM); Lane 4: 5′-stearyl-AS1411 (50 μM), in the Na+ buffer; Lane 5: tel26 (5 μM); Lane 6: 5′-stearyl-AS1411 (5 μM); Lane 7: 5′-stearyl-AS1411 (10 μM); Lane 8: 5′-stearyl-AS1411 (50 μM), in the K+ buffer.

In order to further characterize AS1411 and its lipid derivatives and particularly get a deeper insight into the number of species formed in solution, the studied samples were also investigated via size exclusion chromatography (SEC-HPLC). For these experiments, the oligonucleotide derivatives were analysed at the same concentrations used in the gel electrophoresis assays (5, 10 and 50 μM) in the K+ and Na+ buffers, using tel26 as the reference oligonucleotide. The 26-mer tel26 is well known to fold in solution in different G4 conformations, critically depending on the cation composition of the solution. Indeed, in the presence of sodium, one main species consisting in an antiparallel “basket” G4 structure is observed; on the contrary, in potassium solutions, two types of antiparallel “hybrid” G4 structures are found [64, 65]. In Fig. S19 the HPLC profile of a slowly annealed tel26 solution is shown. In the Na+ buffer (Fig. S19a), only one peak (tR = 6.18 min) was present, while in the K+ buffer (Fig. S19b), in addition to the

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main peak (tR = 6.62 min), another species (tR = 8.61 min, corresponding to 12.6% in area) was detected. These results are consistent with the known G4 structures adopted by tel26 in these solutions. In the case of AS1411, a similar trend was found (Fig. 9). In the Na+ buffer (Fig. 9a), one main peak was observed. At 10 and 50 μM, also another peak with shorter elution times was apparent, accounting for an area of 3.2 and 3.6%, respectively. In the K+ buffer (Fig. 9b), a main peak with tR = 5.90 min was always present, accompanied by another peak with longer elution times (tR = 7.62 min), whose area decreased on increasing the concentration (8.1 and 6.5% for 5 and 10 μM, respectively) and completely disappeared at 50 μM. In addition, two shoulders under the main peak were detected (tR = 5.68 and 5.41 min), accounting for an area of 13.1% at 5 and 10 μM, and of 22.3% at 50 μM. Taken together, these data showed that this oligonucleotide is essentially present in solution as a monomer under the studied conditions, with different conformational features as a function of the buffer composition. For 5′-stearyl-AS1411, several peaks were present even at low concentrations, in both the Na+ and K+ buffer. In the Na+ buffer (Fig. 10a), the 5 μM sample showed a predominant peak at 6.23 min and other three peaks at 3.97, 7.74 and 8.70 min, accounting for 10.6, 19.5 and 4.4% in area, respectively. At 10 μM concentration, the peaks with tR = 7.74 and 8.70 min decreased in intensity (8.1 and 1.2%, respectively) finally disappearing at 50 μM. In turn, the peak at 3.97 min increased from 10.6% at 5 μM to 25.1% at 10 μM, becoming the predominant one (77.7%) at 50 μM. In the chromatogram obtained with the K+ buffer as the mobile phase (Fig. 10b), four peaks were found at 4.02, 6.00, 6.23 and 7.71 min for the 5 μM sample, which then varied in % with the concentration. In particular, similarly to the data obtained in the Na+ buffer, on increasing the concentration from 5 to 10 μM, the peak at tR 7.71 min decreased from 14.7 to 7.5%, finally disappearing at 50 μM, whereas the peak at ca. 4 min increased from 10.5 to 13.9% and became the prevailing one (53.2%) at 50 μM. Species with so short elution times, i.e. excluded volumes of the HPLC column, should be very large aggregates, plausibly corresponding to the retarded bands observed in the PAGE experiments. Thus, for 5′stearyl-AS1411, the peak at ca. 6 min, attributable to monomeric structures, decreased in intensity on increasing the concentration in favour of other species with shorter elution times, attributable to large aggregates. In a representative experiment, the two main peaks from the 50

μM sample, at ca. 4 and ca. 6 min, respectively, were separately collected and then re-injected on the column; interestingly, in both cases each isolated peak gave a mixture containing both of them, indicating that these systems are in a rapid, dynamic equilibrium (data not shown). In the case of 5′-cholesteryl-TEG-AS1411, already at low concentrations, the predominant peak had a retention time of ca. 4 min in both saline conditions tested (Fig. S20). In the Na+ buffer (Fig. S20a) two peaks were evident for the 5 μM sample, a bigger (tR = 3.96 min) and a smaller one (tR = 6.14 min), the latter one gradually decreasing on increasing the concentration (representing 24.8, 15.8 and 5.1% of the whole system at 5, 10 and 50 μM, respectively). In the K+ buffer, in addition to the dominant peak (tR = 4.53 min), also other two peaks (tR = 6.77 and 8.77 min) were observed at 5 μM concentration, representing the 5.5 and 13.7% in area, respectively (Fig. S20b). At higher concentrations, the peak with tR = 6.77 min kept an almost constant intensity, while the one at 8.77 min sensibly decreased (9.9 and 2.5% for the 10 and 50 μM sample, respectively). Concomitantly, for both the 10 and 50 μM samples, minor peaks with tR = 7.24 min and 7.55 min, respectively, were observed. Also for 5′-cholesteryl-C6-AS1411, already at low concentrations and in both saline conditions tested, the peak at ca. 4 min retention time was clearly predominant (Fig. S21). In the Na+ buffer (Fig. S21a), also a weaker peak (tR = 6.13 min) was detected, with an almost constant area at the lowest concentrations investigated (8.4%), but decreasing at 50 μM concentration (5.9%). In the K+ buffer (Fig. S21b), at 5 μM concentration three peaks with retention times of 4.45, 6.65 and 8.67 min were detected. The peak with tR = 4.45 min dramatically increased in intensity (from 35.2 to 94.1%) on going from 5 to 50 μM. The other two peaks decreased in intensity on increasing the concentration from 5 to 50 μM, as dramatically evident for the peak with tR = 8.67 min, going from 56.0% to 2.0%. In turn, the peak with tR = 6.65 min representing the 8.9% in area at 5 μM, showed then a reduced value (3.9%) at both 10 and 50 μM concentration. In summary, SEC-HPLC analysis confirmed the formation of multiple species in solution for the lipid AS1411 derivatives, as also indicated by gel assays under native conditions. These different species include monomolecular systems, prevailing at 5 μM, as well as large aggregates, whose presence expectedly increases on increasing the sample concentration. Interestingly, the propensity to form large aggregates is higher in the Na+ than in the K+ buffer for all the investigated lipid derivatives. In the latter solution, at low concentrations 5′-stearyl-AS1411 is mainly

Fig. 9. Size exclusion chromatography HPLC analysis of AS1411 in the Na+ (a) and K+ (b) buffer at 5, 10 and 50 μM concentration (green, yellow and purple lines, respectively).


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Fig. 10. Size exclusion chromatography HPLC analysis of 5′-stearyl-AS1411 in the Na+ (a) and K+ (b) buffer at 5, 10 and 50 μM concentration (green, yellow and purple lines, respectively).

present as a monomolecular species, whereas the aptamers with a cholesteryl tail tend to form large aggregates at all the tested conditions. 3.5. DLS experiments AS1411 and its lipid derivatives were then investigated by dynamic light scattering (DLS) measurements, to better elucidate the nature and size of the large species observed in gel assays and SEC-HPLC analysis. In these experiments we focused on the systems dissolved in the K+ buffer, with higher stabilizing effects on the G4 structures and higher biological relevance for our studies. Due to the inherent sensitivity characteristics of the technique, the investigated solutions were in the range 50–300 μM, i.e. had concentrations ca. one order of magnitude higher than those studied with spectroscopic, chromatographic and electrophoretic techniques. 3.5.1. AS1411 DLS profiles of AS1411 at 200 and 300 μM concentration showed the presence of multiple species in solution. Particularly, as depicted in Fig. 11a for the 300 μM sample, here reported as a representative example, distributions with hydrodynamic radius (RH) centered at 2, 15, 80 and 200 nm were observed at both tested concentrations. When normalized by the number of scattering objects, only one main population,

i.e. the smallest one, was found in both cases (Fig. 11b). This population can be reasonably associated to the monomolecular G-quadruplex structures of AS1411 also observed by CD, UV, gel electrophoresis and SEC-HPLC analyses, thus confirmed to be the largely prevailing ones also in very concentrated samples. DLS analysis of AS1411 at lower concentrations was not possible due to the very poor scattering intensity of the species in solution. In the case of AS1411 lipid derivatives, a more complex scenario was observed. 3.5.2. 5′-stearyl-AS1411 Several populations were present in the DLS profile of 5′-stearylAS1411 (Fig. 12a) but with different size distribution with respect to unmodified AS1411. Indeed, at 200 μM concentration one population was centered at ca. 5 nm, accompanied by a large population centered around 20–30 nm, and others with RH values of 120 and 400 nm. When the profiles were normalized by the number of scattering objects, only the 15 nm population – and the 5 nm population to a lesser extent proved to be significant in solution (Fig. 12b). While the latter species could be associated to the monomeric 5′-stearyl-AS1411 (the stearyl tail is ca. 2 nm in length if in extended conformation) [68], the prevailing ones could be attributed to aggregated forms in solution, as also suggested by the gel electrophoresis and SEC-HPLC analysis results, and

Fig. 11. DLS profiles of AS1411 in K+ buffer at θ = 90°. (a) Intensity weighed hydrodynamic radius distribution at 300 μM concentration; (b) particle number weighed hydrodynamic radius distribution at different AS1411 concentrations.

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Fig. 12. DLS profiles of 5′-stearyl-AS1411 in K+ buffer at θ = 90°. (a) Intensity weighed hydrodynamic radius distribution at 200 μM concentration; (b) particle number weighed hydrodynamic radius distribution at different 5′-stearyl-AS1411 concentrations.

particularly to micelles, as also observed for other lipid-conjugated Gquadruplex-forming oligonucleotides [66, 67]. Conversely, from the normalized DLS profiles of more diluted samples (100 and 50 μM), only the population with a RH of 15 nm could be observed, indicating this species as the most stable in solution (Fig. 12b). It is worth noting that monomolecular structures of this aptamer could not be detected under these conditions because of the concomitant small size and low concentration. 3.5.3. 5′-cholesteryl-TEG-AS1411 In the case of 5′-cholesteryl-TEG-AS1411 the concentration range analysed was 50–300 μM. At the highest concentration tested, three main populations were observed, respectively centered at RH 13, 30 and 300 nm (Fig. S22a). When the DLS profiles were normalized by the number of scattering objects, only the smallest population was evident in solution (Fig. S22b). The absence of populations with RH lower than 13 nm - even in the normalized profiles - indicated that the monomeric form was virtually absent, or present at concentrations far below the detection limits. In the DLS profiles of more diluted samples (200, 100 and 50 μM concentration), multiple species were detected in solution but also in these cases normalization of the data indicated that the only significant population was the 13 nm and smaller populations were not appreciably present in solution. These data confirm that the cholesteryl-TEG tail confers the aptamer a strong tendency to aggregation. 3.5.4. 5′-cholesteryl-C6-AS1411 Also in the case of 5′-cholesteryl-C6-AS1411 the investigated concentration range was 50–300 μM. The DLS profile at 300 μM conc. was quite similar to that of 5′-cholesteryl-TEG-AS1411 (Fig. S23a), with populations centered at RH 13, 40 and ca. 300 nm. In the normalized profiles (Fig. S23b) only the 13 nm distribution was observed. Once again, small species (with RH ca. 4–5 nm or however lower than 10 nm), attributable to monomolecular structures, were not detected. This effect was clearly due to the presence of the cholesteryl moiety, conferring also in this case a strong tendency to form large aggregates. In the DLS profiles of 5′cholesteryl-C6-AS1411 at lower concentrations, differences with respect to 5′-cholesteryl-TEG-AS1411 were apparent, showing effects due to the nature of the linker (i.e., TEG or C6) attached to the cholestryl tail. Indeed the population at 40 nm was predominant at both 100 and 200 μM, while at 50 μM both 13 and 40 nm populations were observed (Fig. S23b). Taken together, the DLS results confirmed the high polymorphism of AS1411 and revealed also for the unmodified aptamer the tendency to form oligomeric species at high concentrations. As far as the lipidated AS1411 derivatives are concerned, all of them displayed a marked tendency to form aggregates in solution. Remarkably, this tendency was

higher for the cholesteryl-modified aptamers, for which no population attributable to monomeric species was observed, than for 5′-stearylAS1411. Moreover, different aggregates formed, depending on the nature of the lipophilic tail. Indeed 5′-stearyl-AS1411 and 5′-cholesterylTEG-AS1411 predominantly gave small, probably micellar aggregates with RH in the range 13–15 nm, whereas 5′-cholesteryl-C6-AS1411 also arranged in different, larger aggregates, e.g. elongated micelles, with ca. 40 nm RH. Therefore the DLS analysis fully corroborated the results of gel electrophoresis (presence of highly retarded bands) and SEC-HPLC analysis (presence of peaks with very short retention time). Although these techniques investigated different concentration ranges due to their different intrinsic sensitivity, the overall results demonstrated that these oligonucleotides can form large aggregates in a concentrationdependent manner. This aggregation propensity is qualitatively less pronounced in 5′-stearyl-AS1411, showing in all cases a behaviour more similar to that of unmodified AS1411, and progressively more marked in 5′-cholesteryl-TEG-AS1411 and 5′-cholesteryl-C6-AS1411, in the order. Thus, not only the nature of the lipid (stearyl vs. cholesteryl), but also the kind of linker (TEG vs. C6) contributes to the aggregation processes of the lipidated aptamer. 3.6. Thioflavin T (ThT) fluorescence assays Thioflavin T (ThT), a commercially available cationic benzothiazole dye, is typically exploited as a sensitive sensor to evaluate the amyloid fibrils formation and aggregation [69–73]. Recently, it has been also used as a fluorescence light-up probe for the detection of Gquadruplex structures [43, 74–77]. Here we used the ThT fluorescence assay to analyse the interaction between ThT and AS1411 and its derivatives on the ThT fluorescence, to further investigate the structure and aggregation of these oligonucleotides in solution. In these experiments ThT was tested on AS1411 and, in parallel, on 5′-stearyl-AS1411 and 5′-cholesteryl-TEG-AS1411, i.e. the lipid derivatives which, according to the DLS analysis, gave more homogeneous aggregates under the studied conditions (Figs. 13 and S24, S25). In all cases the dye was added to the slowly annealed oligonucleotide solutions to obtain a final 1 μM concentration and the fluorescence signal was recorded after 30 min incubation time exciting at 420 nm. Particularly, six different concentrations (0.5, 1, 2, 3, 5 and 10 μM) of the selected oligonucleotides in the K+ buffer were investigated adding a proper volume of ThT from a stock solution prepared as described in a reported procedure [42]. The fluorescence emission spectra of ThT alone and after incubation with increasing concentrations of AS1411, 5′-stearyl-AS1411 and 5′cholesteryl-TEG-AS1411, reported in Fig. 13a, S24a and S25a, respectively, showed a 25- to 35-fold enhancement of the band at ca.


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Fig. 13. a) Fluorescence emission spectra of 1 μM ThT alone (violet line) and in the presence of increasing AS1411 concentrations (0.5, 1, 2, 3, 5, and 10 μM: magenta, yellow, light blue, red, blue and green lines, respectively); b) Bar graph representation of ThT (1 μM) fluorescence enhancement at 490 nm in the presence of increasing AS1411 concentrations. Error bars correspond to S.D., calculated on three different measurements.

490 nm at the 1:1 probe/aptamer ratio, which further increased on increasing the oligonucotide concentration. These data clearly confirmed the ability of ThT to interact with all the investigated oligonucleotides. The obtained results were also plotted as a function of the fluorescence intensity enhancement (FI/FI0) of ThT, i.e. the ratio between the ThT fluorescence in the presence of the oligonucleotide (FI) and the background fluorescence of ThT alone (FI0) at 490 nm, after subtraction of the buffer [43]. The obtained FI/FI0 ratios, depicted in Fig. 13b, S24b and S25b, respectively for AS1411, 5′-stearyl-AS1411 and 5′cholesteryl-TEG-AS1411, showed in all cases, starting from 1 μM oligonucleotide concentration (i.e. ThT/oligonucleotide 1:1 in mol), FI/FI0 values N 20, which is a characteristic hallmark of G-quadruplex structures [43]. A non-linear increase of the ThT fluorescence emission at 490 nm with the concentration was observed for all the investigated systems, as previously observed for other G4 sequences [74]. Particularly, lipid-conjugated AS1411 derivatives were able to induce a stronger fluorescence enhancement compared to unmodified AS1411, reaching at the highest concentration here investigated (10 μM) FI/FI0 values of ca. 100 and 120 (respectively for 5′-cholesteryl-TEG-AS1411 and 5′-stearyl-AS1411 derivatives) compared to 70 observed for unmodified AS1411. Notably FI/FI0 values are dependent on the G4 topology, reflecting the different binding modes preferred by ThT vs. different G4 conformations [43]. However, since the selected oligonucleotides form in all cases parallel G4 structures, the higher ThT fluorescence enhancement observed with the lipid AS1411 derivatives with respect to the unmodified aptamer can be associated to the interaction of the dye with the lipid aggregates, which produce a strongly a polar environment very efficiently potentiating the ThT fluorescence properties, as in the case of amyloid fibrils [69–73]. These experiments allowed confirming the presence of G-quadruplex structures in all the studied systems, also further evidencing that the AS1411 lipid derivatives formed stable aggregates. These experimental results were also fitted with the Hill equation (see Experimental Section) in order to estimate the apparent dissociation constant (KD) at 25 °C, according to a previous work [44]. The fitted curves are reported in Fig. S26, while the obtained KD values and Hill constants (n) are summarized in Table 2. The KD values were in all cases in the 2–3 μM range, with no marked difference between modified and unmodified AS1411. Concerning the n values, although the Hill constant does not directly reflect the number of ThT

molecules bound to a selected G-quadruplex structure [44], the data for 5′-cholesteryl-TEG-AS1411 and 5′-stearyl-AS1411 were higher than for AS1411, suggesting a different binding stoichiometry. This difference essentially depends on the different fluorescence as well as binding extent as a function of the oligonucleotide structure, [44] and could be here attributed to the presence of the lipid tails promoting the formation of large aggregates, clearly allowing multiple binding modes for ThT. 3.7. In vitro biological evaluation Following conformational behaviour and molecularity investigations, we set up targeted in vitro bioscreens in order to evaluate the bioactivity of the here investigated lipid-conjugated AS1411 aptamers on neoplastic cells in terms of antiproliferative effects. In fact, besides displaying an active targeting activity for the selective delivery of nanoparticles, oligonucleotides, and small molecules to cancer cells, several studies demonstrated significant antiproliferative effects for AS1411 in both preclinical and clinical trials. In this context, there is evidence that nucleolin plays a central role also in direct anticancer effects, since AS1411 is able to inhibit multiple cancer-associated functions of this protein [21]. Therefore, via in vitro bioscreens we tested the anticancer activity of the AS1411 derivatives, monitoring the effects due to the presence of different lipophilic tails. To carry out this study in the perspective of preclinical trials, human colon carcinoma cells (HCT-116), breast adenocarcinoma cells (MCF-7), and malignant melanoma cells (A-375) were tested varying the incubation times (time course experiments up to 72 h) and the concentration, explored in the range 1 → 25 μM (Fig. 14). The obtained results were substantially in line with recent reports on the antiproliferative properties of AS1411 in vitro, showing its effects on human cancer cells of different

Table 2 Dissociation constants (KD) at 25 °C and Hill constants (n) for the ThT–oligonculeotide binding. Sequence

KD (μM)

n

AS1411 5′-stearyl-AS1411 5′-cholesteryl-TEG-AS1411

2.1 ± 0.3 2.9 ± 0.5 2.3 ± 0.3

1.3 ± 0.2 1.5 ± 0.2 1.5 ± 0.1

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Fig. 14. Concentration-effect curves reported as “cell survival index” (evaluated by the MTT assay and live/dead cell ratio analysis) of MCF-7, HCT-116, and A-375 cell lines after 48 and 72 h of incubations with the indicated concentrations (from 1 to 25 μM) of AS1411 aptamer and its lipid derivatives (5′-stearyl-AS1411, 5′-cholesteryl-C6-AS1411 and 5′-cholesteryl-TEGAS1411), as indicated in the legend. Cell survival indexes are plotted in line graphs as averages ± SEM values of three independent experiments (n = 12). ⁎p b 0.001 vs. control cells (untreated cultures).

histological origin. Overall, breast cancer cells were the most sensitive to the antiproliferative activity of AS1411 (IC50 of ca. 20 and 10 μM after 48 and 72 h of incubation, respectively), followed by colon cancer cells (IC50 of ca. 30 μM after 72 h of incubation) and melanoma cells (IC50 always N50 μM). These IC50 values, showing a decreasing trend on increasing the incubation time, are typically indicative of a moderate anticancer activity in vitro. Notably, all the investigated AS1411 derivatives showed lower antiproliferative activity than AS1411, especially after 48 h of treatments. However, as evident from the analysis of concentration-effect curves, their in vitro behaviour did not differ much from that of AS1411, always being time- and concentration-

dependent, and reaching in MCF-7 and HCT-116 significant levels of cell growth inhibition. In fact, following 72 h of incubation at the highest tested concentration (25 μM), the lipid AS1411 derivatives were able to interfere with cell proliferation and viability of MCF-7 and HCT-116 cells by about 45% and 25%, respectively. The most bioactive AS1411 analog in the series was 5′-stearyl-AS1411 on MCF-7 cells, showing an IC50 value of about 30 μM after 72 h of incubation. Less evident antiproliferative effects were observed on A-375, both in the case of AS1411 and of its derivatives. It should be also noted that the three lipid AS1411 derivatives always exhibited similar bioactivities in all the tested cell lines. Hence, based on the data presented herein, it can be concluded that


the structural changes introduced in this aptamer interfere with its biological properties, even though without drastically compromising its anticancer activity. In line with the biophysical analyses, outcomes from these preclinical trials would in fact suggest that the recognition motifs of AS1411, responsible for their biological activity, are essentially maintained in these lipid derivatives, a central concern in view of their future nanobiotechnological applications. 4. Conclusions In this work a set of lipid AS1411 derivatives - presenting at their 5′ends stearyl- or cholesteryl-based tails - were selected and investigated by CD, UV, PAGE, SEC-HPLC, DLS and ThT fluorescence assays in order to get information on their conformational behavior and aggregation propensity in comparison with unmodified AS1411. The biophysical characterization was performed in two different buffered solutions and at different concentrations in the μM range to highlight peculiar concentration or salt-dependent effects. Then, preliminary in vitro bioscreens were performed on human cancer cell lines to explore the antiproliferative activity of these oligonucleotides. In accordance with previous reports, CD analysis demonstrated that AS1411 essentially formed parallel G4 structures, with only minor amounts of antiparallel G4 conformations. The thermal stability of the coexisting AS1411 G4 structures proved to be mainly dependent on the saline conditions and essentially independent from the concentration in the studied range, thus showing the sole presence of unimolecular G4 structures. In turn, the lipid AS1411 derivatives exclusively formed parallel G4 structures, giving generally CD- and UV-melting curves with nice sigmoidal behaviour and apparent Tm values quite similar to those obtained for the unmodified AS1411. Only in few cases - particularly in the Na+ buffer, mimicking the extracellular environment in which the G4 structures are less stable – CD-melting curves evidenced multiple transitions, ascribable to different processes. The combined picture of the PAGE and size exclusion HPLC data demonstrated that AS1411 is essentially present in the form of unimolecular species, while its lipid derivatives show concentrationdependent equilibria between monomeric forms and large aggregates as micelles or more complex aggregates, the latter ones predominant in the case of the cholesteryl conjugates. For all the lipid derivatives the presence of aggregates is validated by native gel electrophoresis (multiple, dramatically retarded bands with respect to the monomeric G4 structures observed in native PAGE experiments), SEC-HPLC analyses data (peaks corresponding to species totally excluded from the column) and DLS measurements (species with RH of 15 nm or larger), as well as by thioflavin T fluorescence assays (showing higher ThT fluorescence enhancement in the presence of the lipid AS1411 derivatives vs. unmodified AS1411). Remarkably, these different species formed in all cases parallel G4 structures with similar Tm values, as deduced by CD and UV experiments. It can be concluded that, in spite of the variety of aggregated forms of the lipid-conjugated oligonucleotides, the G-quadruplex motif maintains its integrity and overall stability. This makes us hypothesize that the G-quadruplex core is always well kept and exposed to the solvent, even in complex, highly structured aggregates, and not detectably impaired in its folding by the presence of the attached lipid tail. In vitro experiments on selected human cancer cell lines of different histological origin showed for the lipid derivatives a biological response similar to that of unmodified AS1411, albeit in a general context of reduced anticancer activity. It is plausible that steric hindrance effects in lipid AS1411 derivatives negatively interfere with the recognition and subsequent binding to nucleolin, resulting in an overall decrease of the antiproliferative activity, however maintained in the most sensitive MCF-7 cells. In conclusion, conjugation with lipids critically affects the structuring of AS1411 which, in the concentration-dependent equilibria

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between monomeric and aggregate forms, in all cases forms G4 structures, more stable in the presence of K+ than Na + cations. The nature of the lipid and of the linker exerts a key role in tuning the size and stability of the obtained aggregates (simple micelles vs. elongated, more complex aggregates), as also found with other biomolecules [78, 79]. However, though introducing more complex systems due to the lipiddriven self-aggregation process, from the point of view of the Gquadruplex formation the presence of the lipid drives the AS1411 oligonucleotide in a sensibly simplified conformational space with respect to the unmodified sequence, as also recently observed by others [66, 67]. Indeed, as far as the G4 structure of AS1411 is concerned, the presence of a covalently linked lipid seems to favour exclusively the formation of unimolecular, parallel G4 structures. Then, it is worth mentioning that all the here reported data clearly show that in the low μM concentration range all the lipid derivatives are mainly present in solution as monomeric forms, and aggregated forms are predominant only at concentrations higher than 50 μM. These results provide precious information for future studies aimed at incorporating these lipophilic derivatives into suitable nanosystems, thus producing a variety of finely tunable AS1411-decorated supramolecular architectures for effective nucleic acid-based therapeutics and/ or nanotechnological applications. Studies are in progress to extend lipidation also to other bioactive aptamers as a general strategy for a fine-tuning of their biophysical properties. Abbreviations CD Chol DLS G4 HPLC NPs PAGE PBS SEC TBE ThT Tm tR UV

circular dichroism spectroscopy cholesteryl dynamic light scattering G-quadruplex high performance liquid chromatography nanoparticles polyacrylamide gel electrophoresis phosphate-buffered saline size exclusion chromatography tris-borate-EDTA thioflavin T melting temperature retention time ultraviolet spectroscopy

Author contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding sources This work was supported by the Italian Association for Cancer Research (AIRC) (IG2015 n. 17037 to D.M.). Acknowledgment Dr. Alessandra Picariello is gratefully acknowledged for her valuable contribution in the DLS analyses. Conflict of interest The authors declare no competing financial interests. Appendix A. Supplementary data UV-melting profiles of AS1411 in the K+ and Na+ buffer. CD spectra, CD-melting and UV-melting profiles of 5′-stearyl-AS1411, 5′-

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cholesteryl-TEG-AS1411 and 5′-cholesteryl-C6-AS1411. Native PAGE analysis of 5′-cholesteryl-C6-AS1411. SEC-HPLC analysis of tel26, 5′cholesteryl-TEG-AS1411 and 5′-cholesteryl-C6-AS1411. DLS profiles of 5′-cholesteryl-TEG-AS1411 and 5′-cholesteryl-C6-AS1411. ThT fluorescence assays data on 5′-stearyl-AS1411 and 5′-cholesteryl-TEG-AS1411. Supplementary data to this article can be found online at doi: https:// doi.org/10.1016/j.ijbiomac.2018.06.137.

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Exploring the entire conformational space of ... - Semantic Scholar

Exploring the conformational landscape of menthol ... - Semantic Scholar

Exploring the function and behaviour of Natural ...

Exploring the link between behaviour and health - Bert Arnrich

Exploring the Acquisition and Consumption Behaviour of ... - MIR Labs

exploring the eco-attitudes and buying behaviour of facebook ... - Core

Exploring Consumer Decision Making and Behaviour in the At - FCA

Exploring Consumer Decision Making and Behaviour in the At - FCA

A unique conformational behaviour of glutamine peptides - Scientific ...

Exploring herding investment behaviour on Zagreb ...

Exploring Large Business Tax Strategy Behaviour - Gov.uk

SESAME: Exploring small businesses' behaviour to enhance ...

Exploring Gender Differences in Computer-Related Behaviour: