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Fluorescent thrombin binding aptamer-tagged nanoparticles for an efficient and reversible control of thrombin activity Claudia Riccardi, Irene Russo Krauss, Domenica Musumeci, Francois Morvan, Albert Meyer, Jean Jacques Vasseur, Luigi Paduano, and Daniela Montesarchio ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b11195 • Publication Date (Web): 29 Aug 2017 Downloaded from http://pubs.acs.org on August 30, 2017

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ACS Applied Materials & Interfaces

FLUORESCENT THROMBIN BINDING APTAMERTAGGED NANOPARTICLES FOR AN EFFICIENT AND REVERSIBLE CONTROL OF THROMBIN ACTIVITY Claudia Riccardi,a,‡ Irene Russo Krauss,a,b,‡ Domenica Musumeci,a,c Francois Morvan,d Albert Meyer,d Jean-Jacques Vasseur,d Luigi Paduano,a,b,* Daniela Montesarchioa,* a

Department of Chemical Sciences, University of Naples Federico II, Via Cintia 21, I-80126,

Napoli, Italy b

CSGI – Consorzio Interuniversitario per lo Sviluppo dei Sistemi a Grande Interfase, Via della

Lastruccia 3, I-50019, Sesto Fiorentino (Fi), Italy c

Institute of Biostructures and Bioimages, CNR, Via Mezzocannone 16, I-80134 Napoli, Italy

d

Institut des Biomolécules Max Mousseron, UMR 5247, Univ. Montpellier, CNRS, ENSCM,

University of Montpellier, place E. Bataillon, 34095 Montpellier Cedex 5, France

*Corresponding authors. E-mail addresses: [email protected] (L.Paduano); [email protected] (D. Montesarchio) ‡

these authors contributed equally to this work.

ABSTRACT Progress in understanding and treatment of thrombotic diseases requires new effective methods for the easy, rapid and reversible control of coagulation processes. In this frame, the use of aptamers, and particularly of the thrombin binding aptamer (TBA), has aroused a strong interest, due to its enormous therapeutic potential, associated with a large number of possible applications in biotechnological and bioanalytical fields. Here, we describe a new TBA analogue (named trismTBA), carrying three different pendant groups: a dansyl residue at the 3’- and a β-cyclodextrin moiety at the 5’-end – providing a host-guest system which exhibits a marked fluorescence enhancement upon TBA G-quadruplex folding – and a biotin tag, allowing the attachment of the aptamer onto biocompatible streptavidin-coated silica nanoparticles (NPs) of 50 nm hydrodynamic diameter (Sicastar®). The use of nanoparticles for the in vivo delivery of TBA, expected to induce per se increased nuclease resistance and improved pharmacokinetic properties of this oligonucleotide, offers as additional advantage the possibility to exploit multivalency effects, due to 1 ACS Paragon Plus Environment


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the presence of multiple copies of TBA on a single scaffold. In addition, the selected fluorescent system allows monitoring both the presence of TBA on the functionalized NPs and its correct folding upon immobilization, also conferring enhanced enzymatic resistance and bioactivity. The anticoagulant activity of the new tris-mTBA, free or conjugated to Sicastar® NPs, was evaluated by dynamic light scattering experiments. Highly effective and reversible inhibition of thrombin activity toward fibrinogen was found for the free tris-mTBA and especially for the tris-mTBA-conjugated NPs, demonstrating great potential for the biomedical control of blood clotting.

Keywords: Thrombin Binding Aptamer, thrombin, fluorescent probe, silica nanoparticles, biotinstreptavidin interaction, multivalency, anticoagulant activity, Dynamic Light Scattering.

1. INTRODUCTION The 15-mer G-rich oligonucleotide TBA (Thrombin Binding Aptamer or TBA15) is the best characterized aptamer of thrombin, a “trypsin-like” serine protease involved in coagulation processes able to convert soluble fibrinogen into insoluble strands of fibrin.1–7 TBA has been proposed as a valuable alternative to classical thrombin inhibitors used in clinic, such as heparin, warfarin and bivalirudin, showing severe side effects or suffering from narrow therapeutic windows.8–10 Upon folding into an antiparallel, chair-like G-quadruplex structure, TBA can strongly and selectively recognize the fibrinogen-binding exosite I of human thrombin,11–14 inhibiting its key function in the coagulation cascade.15 Due to suboptimal dosing profiles during trial stages also due to its intrinsic instability in physiological media, TBA did not progress to advanced clinical trials, being blocked after Phase I studies. Since then, a huge number of TBA analogues have been proposed in the literature, carrying either backbone modifications16–20 or specific conjugations (with streptavidin21 or different kinds of nanoparticles22–32), aimed at increasing its thermal stability, bioactivity and nuclease resistance, thus generally improving its pharmacokinetic profile.33–38 TBA and its variants are very interesting not only for their therapeutic potential, but also for possible applications as biotechnological and bioanalytical tools. Noteworthy is the possibility to label these aptamers with fluorescent probes,27,31,39–43 which in principle, following their turnoff/turn-on fluorescence responses, allow monitoring the target recognition processes, particularly crucial for sensitive thrombin detection. In this work, with the aim of optimizing TBA bioactivity, a new tris-conjugated TBA (here named tris-mTBA, Figure 1) containing an environment-sensitive fluorescent probe has been prepared,

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characterized and then incorporated into 50 nm hydrodynamic diameter silica NPs commercially available as Sicastar®.

O HN

NH H N S

biotin

-

O O P O O-

C O

O

O

O HO

OHOHO

HO O OH

N N

OH O HO

OH O

N N N

dansyl

OH O

β-cyclodextrin

HO

O S NH O

N O

OH O OH O

O 5' 3' O GGT TGG TGT GGT TGG O P O O-

O

N HO

O O P

OH O OH OH

OH OHO

OH OOH

O

O OH

Figure 1. Molecular structure of the tris-mTBA described in this work. The TBA oligonucleotide sequence and the dansyl moiety are highlighted in yellow and green, respectively.

Tris-mTBA has been designed to carry a dansyl group at the 3’-, and a β-cyclodextrin and a biotin moiety at the 5’-end. The host-guest system dansyl/β-cyclodextrin is based on the well-known inclusion properties of cyclodextrins,42,44–50 able to capture the dansyl fluorescent group increasing its fluorescence emission. When TBA is unfolded, the dansyl probe, very sensitive to external environment conditions,51–53 is exposed to bulk water and only basal fluorescence is observed. In turn, when TBA is folded into an antiparallel, unimolecular G-quadruplex structure, the 5’ and 3’ends of the oligonucleotide strand are in close proximity, favouring the encapsulation of the dansyl group into the hydrophobic cyclodextrin cavity. Under these conditions a marked fluorescence enhancement is observed, as demonstrated in our previous work, investigating as a model system the parent bis-modified TBA (bis-mTBA), derivatized with the same host-guest system.42 The dansyl/β-cyclodextrin dyad has been here selected to effectively assess the NP functionalization with TBA and also monitor the correct G-quadruplex folding of the aptamer on the NP surface via simple fluorescence measurements. The conjugation of the NPs with TBA has been realized exploiting the high affinity and specificity of biotin-streptavidin interactions54–56: to this purpose the fluorescent TBA has been derivatized with an additional tag, a biotin residue, to 3 ACS Paragon Plus Environment


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allow its rapid and efficient binding to streptavidin-coated nanoparticles. Notably, the streptavidinbiotin complex is stable in a broad range of pH, ionic strength and temperature,57,58 and can thus ensure a very stable TBA decoration on the nanoparticles. Among the commercially available silica NPs, Sicastar® with streptavidin coating and hydrodynamic diameter of 50 nm have been selected. According to the literature, NPs of this size seem to be the best compromise between optimization of the in vivo NP biodistribution and preservation of the biotin-streptavidin interactions. As far as the NP pharmacokinetic profile is concerned, it is generally accepted that very small NPs (hydrodynamic radius < 5 nm) are too rapidly excreted from the body through extravasation or renal clearance,59,60 while NPs with size in the µm range can easily accumulate in the body, especially in liver, spleen and bone marrow with severe toxic effects.61,62 On the contrary, NPs with diameter of 50-100 nm range, though showing a different biodistribution and cellular uptake behaviour critically depending on the surface charge and administration route,63 in most cases are trapped by mechanical filtration operated by sinusoids in the spleen and then rapidly removed from the bloodstream by the reticuloendothelial system (RES) cells.64 Concerning the preservation of the biotin-streptavidin interactions on the silica NP surface, a typical stability trend has been observed as a function of the NP size: on increasing their hydrodynamic diameter from 50 to 200 nm, the affinity (in terms of Kd) between biotin and streptavidin is progressively reduced, as reported by Piletska and Piletsky.65 This effect could be due to steric constraints or diffusion limitations, negatively affecting the ligand-protein interactions, leading to conclude that NPs suitable for bioanalytical applications should not exceed the 50-100 nm size. Then, as for the kind of NP core here used, we specifically selected silica NPs, rather than metal or metal oxide-based platforms, for their low cytotoxicity and high biocompatibility, as well as for the additional advantage of not causing fluorescence quenching or general interference with TBA properties.66–70 Once synthesized and purified, the new modified aptamer tris-mTBA has been investigated by means of UV, CD and fluorescence spectroscopy to get information on its conformational behaviour under different solution conditions. In addition, its resistance to nucleases has been analyzed in comparison with the unmodified TBA and a biotinylated TBA, here prepared as control and indicated as biotin-TBA. The inhibitory activity of tris-mTBA towards human α-thrombin has been determined by means of dynamic light scattering experiments, following the conversion of fibrinogen into fibrin promoted by the protein, and compared with biotin-TBA and unmodified TBA.


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Then, tris-mTBA has been immobilized onto Sicastar® nanoparticles, obtaining the corresponding tris-mTBA/Sicastar® NPs system. The functionalized NPs have been characterized using a combined approach including dynamic and static light scattering (DLS and SLS), gel electrophoresis and fluorescence spectroscopy analyses. Their inhibitory activity has been evaluated by DLS and compared to that of the free aptamers and unfunctionalized NPs. Control experiments with the oligonucleotide sequence complementary to TBA (cTBA), carried out on the tris-mTBA both in free form and conjugated to the silica nanoparticles, allowed verifying that – exploiting the higher thermodynamic stability of the duplex structure vs. the corresponding G-quadruplex + single strand system – the anticoagulant activity of these TBA-functionalized NPs, as well as of the trismTBA, can be reversed and blood clotting restored, by simply using cTBA as an antidote.

2. RESULTS AND DISCUSSION 2.1 Synthesis of tris-mTBA In our synthetic procedure, tris-mTBA 8 was prepared in three main steps, as described in Scheme 1. First, dansyl azide derivative 271 was immobilized via a Cu(I)-catalyzed azide-alkyne cycloaddition protocol (CuAAC)72,73 on alkyne-functionalized solid support 174 using the CuSO4/sodium ascorbate catalytic system under microwave irradiation75 for 1 h at 60 °C. Then, the resulting solid support 3 was used for the solid phase assembly of the TBA oligonucleotide sequence d(GGTTGGTGTGGTTGG) on a DNA synthesizer using standard phosphoramidite chemistry. Once the desired sequence was assembled, two additional couplings were performed: the first one with the DMTr-THME propargyl phosphoramidite 4,74 introducing an alkyne moiety in the oligonucleotide chain, and the second one with the commercially available biotin phosphoramidite 5. After final DMTr removal followed by standard ammonia deprotection of the functionalized solid support, the 5’-biotin, 3'-dansyl TBA 6 was released in solution, purified by reverse-phase HPLC and then characterized by MALDI-TOF MS (see Figures S1 and S2 in Supporting Information). Finally, β-cyclodextrin, in the form of mono-azide derivative 7,76 was coupled in solution with the alkyne-containing oligonucleotide 6 through Cu(I)-promoted cycloaddition for 1.5 h at room temperature, yielding the target tris-mTBA 8. The desired oligomer was purified by gel filtration and characterized by MALDI-TOF MS (see Figures S3 and S4 in Supporting Information). Successively, 8 was converted into the corresponding potassium salt by treatment with a 1 M KCl aq. solution and then desalted using size exclusion chromatography.



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a)

N

O

= DANS

O

DMTrO

2 O

HN

O

O

(2 eq)

O S O

O

DMTrO

N

N3

CuSO4 (0.4 eq) Sodium ascorbate (2 eq) DANS N H CH3OH/H2O (48:52, v/v) 1 h, 60 °C

1 (1 eq)

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N N

3

b) SPOS with: (i) dG-CE* and T-CE amidites (ii) O

(iii)

O

DMTrN

O HN

c)

NH H N

S

O O P O O

O

O P

4

NH

NH4OH

O

O O P O O

CN

O

H N

S

N(iPr)2 O P CN O

5

O GG TT GG TGT GG TT GG O P O O

O

N3

N(iPr)2

DMTrO

N3

N

DANS

d)

52 % N N

N H

CuSO4 (5 eq) Sodium ascorbate (25 eq) H2O/CH3OH/DMSO (75:22:3, v/v/v) 1.5 h, RT

β

7

O

O

6 (HPLC purification)

=

OH

(3.5 eq) HN

NH H N

S O

O O P O O

O O P O O

O GG TT GG TGT GG TT GG O P O O

OH O

O

8 CPG solid support

N N

DMTr 4,4′-Dimethoxytriphenylmethyl

β

SPOS Solid Phase Oligonucleotide Synthesis G*

N

N

N-isobutyryl dG

Scheme 1. Synthetic scheme for the preparation of tris-mTBA 8.


DANS

N H

N N

97 %

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2.2 Spectroscopic properties of tris-mTBA in solution The conformational behaviour in solution of tris-mTBA 8 was investigated combining UV, CD and fluorescence data. The spectra collected with the different techniques were obtained from a phosphate buffered solution containing 80 mM K+ ions (10 mM KH2PO4, 70 mM KCl, 0.2 mM EDTA, pH 7.0), in analogy with our previous study performed on bis-mTBA,42 used as a model to evaluate the host-guest system dansyl/β-cyclodextrin on this G-quadruplex forming oligonucleotide. The CD spectrum of tris-mTBA at 10 °C shows two positive bands with maxima centred at 295 and 246 nm and a negative band with minimum at 266 nm (Figure 2a). These spectral features are consistent with those reported for the unmodified TBA under the same solution conditions, forming a chair-like antiparallel G-quadruplex structure;16,77–79 thus confirming that the conjugation with the three sterically hindered reporter groups (i.e., dansyl, β-cyclodextrin and biotin) does not impair the G-quadruplex structuring of TBA nor affects the topology of its folding.

a)

b)

23

20

295

10

20

CD[mdeg]

CD[mdeg]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


246

Tm = 39 °C ± 1 °C

10

0

266 -10 220

0 240

260

280

300

320

10

20

40

60

80 85

Temperature [°C]

Wavelength [nm]

Figure 2. CD spectrum registered at 10 °C (a) and CD melting profile recorded at 295 nm (temperature scan rate = 1 °C/min) (b) of a 5 µM tris-mTBA solution in 10 mM KH2PO4, 70 mM KCl, 0.2 mM EDTA (pH 7.0). The CD melting profile, monitoring at 295 nm, provided a melting temperature of 39 °C in the selected buffer (Figure 2b). No hysteresis emerged on comparing the heating and cooling profiles, indicating that, under the used conditions (scan rate of 1 °C/min), the system is at equilibrium. The CD spectrum of tris-mTBA was also recorded in PBS, the buffer solution here used for the successive functionalization of Sicastar® NPs (see below), containing a lower K+ concentration with respect to the previously used buffer (4.5 vs. 80 mM), and a high content of Na+ ions (157 mM). Also under these conditions, tris-mTBA maintains the same CD profile as in the 80 mM K+-buffer (Figure S5), with the positive CD band having the maximum at 295 nm, diagnostic of an antiparallel G-quadruplex structure. In PBS tris-mTBA has a lower structuration degree than in the 7 ACS Paragon Plus Environment


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phosphate buffer containing only K+ as cation, as expected considering that K+ can stabilize Gquadruplex structures better than Na+.80–82 In Figure S5, the CD spectrum of tris-mTBA in pure water is also shown for comparison, proving that in the absence of significant concentrations of stabilizing cations this aptamer is very poorly structured. Next, the UV spectrum of tris-mTBA, registered at 5 µM in the 10 mM KH2PO4, 70 mM KCl, 0.2 mM EDTA (pH 7.0) buffer solution, revealed the characteristic absorption of the TBA sequence, with the double-hump band between 230 and 300 nm (Figure S6a). As determined from comparison with the unmodified TBA under the same concentration and solution conditions, the contribution to the 260 nm absorbance of the dansyl, β-cyclodextrin and biotin moieties can be considered negligible in comparison with that of the oligonucleotide. From the UV-melting curve recorded at 295 nm,83,84 a Tm of 39 °C was derived (Figure S6b), in perfect accordance with the Tm obtained from the CD melting experiments registered at the same wavelength. Even in this case, no hysteresis was observed on comparing heating and cooling (data not shown), indicating fully reversible processes. The thermal denaturation of tris-mTBA was also analyzed by monitoring the changes of the fluorescence emission of the dansyl group, upon excitation at 327 nm, as a function of the temperature on an annealed aptamer solution. On increasing the temperature, a gradual, marked decrease in the fluorescence intensity, as well as an emission red shift from 534 to 551 nm, were observed (Figure 3, panel a and b). These fluorescence features clearly indicate that the dansyl group experiences dramatically different environments upon temperature-induced unfolding. These findings are consistent with the dansyl residue being encapsulated inside the β-cyclodextrin ring in the folded state (low temperature) and pointing outside, exposed to the aqueous solution, in the unfolded state (high temperature), as already observed in the case of bis-mTBA.42 The fluorescence-monitored melting curve was obtained reporting the wavelength at the fluorescence emission maximum as a function of the temperature (Figure 3b).42 The obtained Tm value (40 °C) is, within the experimental error, in good accordance with the results obtained from CD and UV data, indicating that the dansyl group is thrown out from the cyclodextrin cavity in concomitance with the unfolding of the whole G-quadruplex structure. Also in this case, the heating and cooling profiles were essentially superimposable (data not shown).


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a)

b) 180000

548

534

Increasing temperature

160000 140000 120000

546 544 λ max (nm)

Fluorescence intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


100000 80000 60000

542 540

Tm = 40 °C ± 1 °C

538 536

40000

551

534

20000 0 450

500

550

532

600

20

30

Emission wavelength (nm)

40

50

60

70

80

Temperature (°C)

Figure 3. a) Overlapped emission fluorescence spectra (λex = 327 nm) of tris-mTBA, recorded at 2 µM concentration in a 10 mM KH2PO4, 70 mM KCl, 0.2 mM EDTA (pH 7.0) buffer solution, on increasing the temperature from 20 to 80 °C; b) fluorescence melting profile obtained reporting the wavelength at the fluorescence emission maximum as a function of temperature (range 20-80 °C). In summary, these results show that tris-mTBA qualitatively behaves as bis-mTBA42 and the unmodified TBA.16,78,79 As expected, the presence of three bulky conjugating groups affects the apparent Tm of tris-mTBA, which is ca. 10 °C lower than that of TBA and of bis-mTBA (39 vs. 49 °C), showing a lower thermal stability in the explored conditions. However, these data also confirm that, under pseudo-physiological conditions, tris-mTBA is mainly present as a G4 structure. It can be also speculated that the G4 structuring of tris-mTBA is further enhanced when conjugated to the NPs, if other interfering surface processes do not intervene. In fact, once the biotin residue of trismTBA is captured by the streptavidin coating of the NPs, any competition between the dansyl and biotin for cyclodextrin recognition - which is however possible in solution since the binding constants for their complexes with β-cyclodextrin are in the same order of magnitude (Ka ~102 M−1)85,86 - is prevented, thus not disturbing the correct G4 folding.

2.3 CD experiments with cTBA sequence Rapid reversibility of binding between inhibitors and coagulant factors is essential in drug treatments. In fact, in case of clinical complications, such as haemorrhage, physicians need rapidly and safely reversing coagulation inhibition. The 15-mer d(CCAACCACACCAACC), here indicated as cTBA, complementary to the TBA sequence and able to hybridize it forming a duplex structure, can profitably modulate the activity of TBA, reversing its anticoagulation activity.87,88 Thus TBA and cTBA can be considered a very effective drug/antidote pair for thrombin.89 9 ACS Paragon Plus Environment


In this frame, the ability of tris-mTBA to form a duplex structure with cTBA was investigated by CD and compared with the unmodified TBA. The CD experiments were carried out using a tandem cell, a quartz cuvette composed of two distinct compartments, which allows recording the sum and mix spectra of two components (i.e. the spectrum obtained by maintaining the cTBA and TBA as separate systems, and the one obtained after mixing the two solutions).

a)

4

b)

tris-mTBA / cTBA sum mix

4

TBA15 / cTBA

2

CD[mdeg]

2

CD[mdeg]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0

sum mix

0

Tandem cell

Tandem cell

-2

-2

-3

-3 220

240

260

280

300

320

220

240

Wavelength [nm]

260

280

300

320

Wavelength [nm]

Figure 4. CD spectra registered as sum (red lines) and mix (blue lines) of tris-mTBA/cTBA (a) and unmodified TBA/cTBA (b) systems in a tandem cell. Each oligonucleotide was analyzed at 2.5 µM in PBS solution.

In both cases the sum (red lines) and mix (blue lines) spectra were not superimposable, denoting the rapid interaction of the strands (Figure 4). Thus, for tris-mTBA the formation of a duplex structure upon cTBA addition efficiently competes with the G-quadruplex, in analogy with the unmodified TBA behaviour. After mixing the solutions and stabilization of the CD signal, a denaturation curve was recorded at 268 nm for each duplex (Figure S7), showing similar Tm values for the two systems (62 and 60 °C for tris-mTBA/cTBA and TBA/cTBA, respectively). An end-stacking stabilizing effect produced by the dansyl moiety could account for the slightly higher Tm of the duplex structure containing tris-mTBA. The CD melting curves of the duplexes were almost superimposable to the corresponding annealing profiles, not evidencing hysteresis phenomena. These data clearly indicate that the residues inserted at the ends of tris-mTBA do not perturb the ability of the oligonucleotide to recognize its complementary strand and form with it a stable duplex structure under pseudo-physiological conditions, necessary pre-requisite for using tris-mTBA/cTBA as an anticoagulant/antidote pair.


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2.4 Enzymatic stability assays In order to investigate the effects of the 5’- and 3’-end modifications on the enzymatic stability in serum of the TBA sequence, tris-mTBA has been analyzed in comparison with both the unmodified TBA and a non-fluorescent, biotinylated TBA analogue, here named biotin-TBA (Figure S8). The latter oligonucleotide, carrying a biotin and a THME-propargyl group at the 5’- and 3’-ends, respectively, has been here prepared (Figures S9 and S10) as a control to investigate the possible role of the dansyl and β-cyclodextrin moieties in influencing the coagulation properties of TBA (see below). The stability in biological environment of the unmodified and modified TBA oligonucleotides has been tested under pseudo-physiological conditions by incubating the oligomers - previously annealed in PBS buffer - at 10 µM concentration in 79 % fetal bovine serum at 37 °C. Then, at fixed times, aliquots of these mixtures were collected and analyzed by HPLC on a Nucleogen column until disappearance of the peak corresponding to the pure oligonucleotide.90 The unmodified TBA was completely degraded in ca. 20 min (Figure S11a). In turn, both the modified TBAs, i.e. biotin-TBA and tris-mTBA, under the same conditions, completely disappeared in ca. 5 h (Figures S11b and S11c). The higher resistance to enzymatic degradation of these TBA analogues with respect to the unmodified TBA was clearly due to a protective effect of the conjugating groups, as expected for oligonucleotides modified at both their ends, and is highly desirable for potential in vivo studies.

2.5 Preparation of the tris-mTBA-functionalized Sicastar® nanoparticles Once having fully characterized the behaviour of tris-mTBA in solution, this aptamer was conjugated to Sicastar® NPs as schematically represented in Figure 5. The functionalization of the streptavidin-coated silica nanoparticles with tris-mTBA is based on the high affinity of streptavidin/biotin specific recognition (Kd ~10-14 M).56



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SiO2

tris-mTBA Sicastar®

= Biotin moiety

streptavidin/biotin specific recognition

SiO2

= Streptavidin moiety

tris-mTBA/Sicastar® Figure 5. Schematic representation of the Sicastar® functionalization with the tris-mTBA. Thus, by simply mixing in an eppendorf tube the Sicastar® NPs and tris-mTBA solutions both in PBS buffer (using a biotin/streptavidin 1:1 ratio, see section 2.6 for optimization of the functionalization protocol) and leaving the mixture under gentle stirring for 1 h, the conjugated trismTBA/Sicastar® NPs were obtained. The functionalized NPs were then analyzed using different techniques. 2.6 Dynamic and Static Light Scattering analysis of functionalized Sicastar® nanoparticles In order to characterize the tris-mTBA-functionalized Sicastar® NPs, dynamic light scattering analyses were carried out on the nanoparticles before and after the functionalization with the aptamer. The streptavidin-coated Sicastar® NPs – analyzed as received from the commercial supplier - showed a well-defined, single population, with hydrodynamic radius of ca. 25 nm, accounting for more than 95 % of the scattering intensity, accompanied by a very small population of higher size. Analysis of the tris-mTBA-functionalized NPs samples, obtained using different functionalization protocols, in all cases indicated the presence of one main population, with hydrodynamic radius similar to that of unfunctionalized Sicastar® NPs, which was unique only if specific functionalization conditions were adopted. In detail, DLS curves of the functionalized NPs obtained using high biotin/streptavidin ratios or long incubation times (#1-3, Figure S12) showed, in addition to the main population, also detectable amounts of larger species, likely aggregates. On 12 ACS Paragon Plus Environment

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the contrary, when the functionalization was performed mixing Sicastar® NPs and tris-mTBA so to have a 1:1 or 1:0.9 streptavidin/biotin ratio, with short incubation times (functionalization #4 and #5, Figure S12), only a single monodisperse NP population was obtained. As expected, due to the negligible size of the aptamer with respect to the streptavidin-coated silica NPs, no effect on the overall dimensions of the Sicastar® system was detected after functionalization with tris-mTBA, as determined by DLS. Similar results were obtained with Sicastar® NPs functionalized with the control aptamer biotin-TBA (data non shown). Static light scattering analysis and Zimm plot elaboration were also exploited to characterize the tris-mTBA/Sicastar® NPs system, providing the molecular weight of the NPs before and after functionalization. Notably, the tris-mTBA/Sicastar® NPs showed a molecular weight of the same order of magnitude as unfunctionalized NPs (5.4*106 vs. 5.3*106 Da before and after functionalization, respectively). As in the case of the overall size determined by DLS, also in terms of molecular weight the contribution of tris-mTBA, upon conjugation to the Sicastar® NPs, was negligible: indeed, the expected increase in molecular weight after the functionalization fell within the uncertainty of the method, allowing a molecular weight evaluation with an error of ca. ±10 %. However, a quite unexpected and remarkable finding was the different slope of the Zimm plots relative to the NPs before and after the functionalization (Figure S13). In the former case a positive slope was observed, indicating energetically favoured NP system-solvent interactions, while the latter system showed a negative slope, indicating the interactions among the NPs as more favourable than the NPs-solvent ones, and thus an intrinsic tendency of the tris-mTBA/Sicastar® NPs to self-aggregate over time. Moving from this result, DLS was further employed to check the stability over time of Sicastar® NPs before and after the tris-mTBA functionalization. Remarkably, no detectable change in the DLS profiles of unfunctionalized Sicastar® NPs in PBS solution was observed over a period of several months. In contrast, in the case of tris-mTBA/Sicastar® NPs, DLS analysis showed the appearance of new, even if very small, populations of bigger aggregates starting from ca. 4 weeks from the functionalization procedure, in close agreement with the results of the Zimm plot analysis (Figure 6). However, despite their inherent propensity to aggregate with time, these data prove that the functionalized tris-mTBA/Sicastar® NPs can be considered stable in pseudo-physiological solutions for at least one month from their preparation, which is a time range well beyond that needed for practical biomedical applications.



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Figure 6. Overlapped DLS profiles of the tris-mTBA/Sicastar® NPs (functionalization conditions #5 in Figure S12) in PBS solution, registered at t=0, 1 week, 2 weeks, 3 weeks or 4 weeks after their preparation.

2.7 Gel electrophoresis analysis Gel electrophoresis analysis was used to further characterize the modified aptamer and the Sicastar® NPs in solution. In Figure S14, the photograph of a representative example of a 1 % agarose gel, run under native conditions, is reported. A detectable difference in electrophoretic mobility was observed for the unmodified TBA and tris-mTBA, both previously annealed in PBS, proving that the three bulky, pendant groups here introduced on tris-mTBA somehow affected the migration ability of TBA. Interestingly, upon UV visualization, the tris-mTBA band showed a different colour with respect to the unmodified TBA, probably conferred by the dansyl group (cfr. lanes 3 and 4 in Figure S14, respectively). As far as the nanoparticles are concerned, both the unfunctionalized and tris-mTBA/Sicastar® NPs loaded on the gel (lanes 1 and 2, respectively) showed retarded bands with respect to the free aptamers in solution. They revealed the same mobility under the used 14 ACS Paragon Plus Environment

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conditions, clear evidence that the TBA functionalization of the Sicastar® NPs did not alter their overall size and shape, as expected considering the relative weight of the streptavidin-coated NPs and of tris-mTBA and in accordance with the DLS and SLS analyses. Remarkably, the functionalized NPs loaded on the gel (lane 2) did not show detectable bands in correspondence of the free tris-mTBA. However taking into account that gel electrophoresis is a low resolution technique, these results provided a good indication that, within the detection limit of the method, the functionalized NPs did not contain unbound tris-mTBA.

2.8 Fluorescence spectroscopy analysis of free and NP-conjugated tris-mTBA Fluorescence measurements allowed monitoring the fluorescence emission of the dansyl group. Since this fluorescent probe is extremely sensitive to its environment, the persistence of substantial fluorescence emission, with the same maximum as observed in the G4-structured tris-mTBA solution before the NPs functionalization, can provide evidence of complete encapsulation of the dansyl residue within the cyclodextrin cavity even in the NP-bound aptamer. As demonstrated in solution studies, this event is strictly connected with the correct G4-structuring of tris-mTBA and can thus provide information on the effective conformation adopted by the aptamer on the NP surface after the functionalization procedure. In Figure 7 overlapped fluorescence spectra of trismTBA and tris-mTBA/Sicastar® NPs in PBS solution at the same aptamer concentration are reported, after subtracting the contributions of the buffer and of the unfunctionalized Sicastar® NPs. These spectra are qualitatively similar, with a fluorescence difference of less than 20 %. The perfect coincidence of the fluorescence maxima proved that, after immobilization onto Sicastar®, trismTBA maintained its original fluorescence features, thus indicating that the peculiar G-quadruplex conformation of tris-mTBA, essential for thrombin recognition, is overall preserved on the functionalized Sicastar® NPs.



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Figure 7. Overlapped fluorescence spectra of tris-mTBA and tris-mTBA/Sicastar® NPs (black and red curves, respectively) in PBS solution, with the emission maximum marked for each spectrum.

2.9 Anticoagulant activity The anticoagulant activities of tris-mTBA and tris-mTBA/Sicastar® NPs were evaluated following the conversion of fibrinogen in fibrin promoted by thrombin by means of dynamic light scattering experiments, and compared with those of unmodified TBA and biotin-TBA. A rapid increase of the scattered intensity with time indicated the progression of fibrin formation in the samples only containing fibrinogen and thrombin (Figure 8).25 The addition of the aptamers, TBA, biotin-TBA or tris-mTBA, caused a marked decrease of the scattered intensity with respect to the untreated samples, evidencing the inhibition of thrombin coagulation activity. A quantitative analysis of the anticoagulant activity of the investigated systems was obtained by fitting the starting linear increase of the normalized scattered intensity vs. time functions and comparing the slopes of the resulting curves with that obtained for the system only containing thrombin (Figure S15 and Table 1).


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Figure 8. a) Coagulation curves of fibrinogen in the presence of thrombin and different anticoagulant agents (TBA; biotin-TBA; tris-mTBA and tris-mTBA/Sicastar® NPs, see inset for details). b) Normalized autocorrelation function at different times and in the presence of different anticoagulant agents (TBA; biotin-TBA; tris-mTBA and tris-mTBA/Sicastar® NPs) and in the presence of unfunctionalized Sicastar® NPs (see inset for details)

Table 1. Coagulation rates for the different studied systems. s-1 Thrombin

0.25±0.03

Thrombin:TBA (1:5)

0.027±0.04

Thrombin:biotin-TBA (1:5)

0.024±0.06

Thrombin:tris-mTBA (1:5)

0.0026±0.0003

Remarkably, biotin-TBA behaved as unmodified TBA in terms of inhibition of thrombin coagulation activity, whereas tris-mTBA was ca. 10 times more effective than TBA and biotin-TBA under the same experimental conditions, showing that the dansyl/cyclodextrin dyad inserted on trismTBA strongly enhanced the aptamer bioactivity. With the aim of assessing if the pendant groups of tris-mTBA were able to inhibit the thrombin activity per se, the coagulation experiments with tris-mTBA were performed also in the presence of its complementary sequence cTBA. In this case the coagulation curve was almost superimposable to that obtained with thrombin alone (data not shown), suggesting that the pendant groups have an effect only when tris-mTBA is correctly folded in a G-quadruplex structure. Coagulation experiments were performed at different aptamer:thrombin ratios and the observed anticoagulant effects were found to be concentration-dependent for all the investigated aptamers, 17 ACS Paragon Plus Environment


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TBA, biotin-TBA and tris-mTBA (see Figure S16). Even in this respect the differences between biotin-TBA and unmodified TBA were negligible. Valuably, no increase of the scattered intensity over time was detected when the fibrinogen/thrombin system was treated with the tris-mTBA/Sicastar® NPs, suggesting that the coagulation process was completely blocked by the functionalized NPs. Indeed, at the end of the coagulation experiments performed with the thrombin alone or even in the presence of the TBAs in solution, the coagulation process could be visualized at the naked eye following the rapid conversion of the tested solution into a gel in the DLS couvette. On the contrary, in the presence of tris-mTBA/Sicastar® NPs, no gel formation was observed, proving that these NPs are much more efficient anticoagulants than any free TBA. Control experiments on the fibrinogen/thrombin system performed in the presence of unfunctionalized Sicastar® NPs showed an almost sudden increase of the light scattered intensity, which did not change further with time, in concomitance with the coagulation of fibrinogen. To get a further insight into this process, we also analyzed the autocorrelation functions obtained for the different systems at different times. Indeed, the analysis of the normalized autocorrelation function gave a direct information on the size of the species in the system, presenting a different experimental view of the coagulation process. As shown in Figure 8b, in the presence of thrombin, a shift of the curves with respect to pure fibrinogen (fibr t=0) occurred at increasing times due to the formation of fibrin. Remarkably, the autocorrelation curves at the end of the coagulation experiment (t=tf) were different depending on the anticoagulant agent. When no anticoagulant was present, the curve did not reach zero even at very long times, clearly indicating the formation of very big aggregates. In the presence of TBA and biotin-TBA at ca. 2 h from the beginning of the experiment, the position of the curve and the lack of a flat region at long times pointed towards the formation of large species. On the contrary, in the case of tris-mTBA, the position of the curve and the flat region at long times indicated that only soluble, smaller species were present. Finally, in the presence of tris-mTBA/Sicastar® NPs no appreciable shift of the autocorrelation curve was observed with time. Interestingly, in the control system containing unfunctionalized Sicastar® NPs, the initial curve was intermediate between that of the system containing fibrinogen and tris-mTBA/Sicastar® NPs, and that of coagulated fibrinogen, suggesting the presence of some aggregated species obtained upon interaction between the NPs and fibrinogen. However, at the end of the experiment, the normalized autocorrelation curve was fully superimposable to that of coagulated fibrinogen. Indeed, the initial conversion of fibrinogen, likely deposited on the NP surface, into coagulated fibrinogen, did not result into a significant change of intensity, as we would expect in the case of the full conversion of smaller but more abundant species (fibrinogen-NPs) into larger, less abundant ones (coagulated fibrinogen). So, although 18 ACS Paragon Plus Environment

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monitoring the light scattered intensity over time is a good method to follow the coagulation of fibrinogen, nonetheless in some cases it fails to reveal what actually happens in the examined system, and only the concomitant analysis of the autocorrelation function can give a clear picture of the system. Finally, we verified if the anticoagulant activity of tris-mTBA could be controlled by addition of the antidote of TBA (i.e., cTBA) even when the aptamer was bound on the NP surface. A 10-fold excess of cTBA was added to the tris-mTBA/Sicastar® NPs and the scattered intensity of the resulting mixture monitored over time. A significant increase of the scattered intensity was detected ca. 30 min after the cTBA addition (Figure 9a) and formation of a consistent gel was observed in the couvette at the end of the experiment. We also compared the normalized autocorrelation function with that of the other systems containing Sicastar® NPs (Figure 9b). We found that, upon addition of the antidote, at the end of the measurements, the curve was superimposable to that of the control experiment with no anticoagulant agent. Altogether, these results indicate that the antidote is effectively able to fully reverse the tris-mTBA anticoagulant activity even when the aptamer is immobilized on the NP surface.

Figure 9. Effect of the addition of the antidote cTBA on the anticoagulant activity of the trismTBA/Sicastar® NPs. a) Coagulation curves of fibrinogen in the presence of thrombin and different anticoagulant agents (see inset for details). b) Comparison of the normalized autocorrelation function upon addition of the antidote with respect to systems containing unfunctionalized and functionalized Sicastar® (see inset for details).



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3. CONCLUSIONS Nanostructures based on inorganic, organic or hybrid nanoparticles have stimulated a strong interest as new potential tools for the in vivo delivery of drugs and/or imaging agents. Embedding into a nanostructure typically confers to the loaded species increased chemical stability, protection from enzymatic degradation and an almost constant chemical environment.91,92 Among inorganic nanoparticles, silica-based nanoparticles are a popular choice for fabricating nanocarriers since they offer several advantages, such as well-defined and easily tunable size, low cytotoxicity, high biocompatibility, easy preparation and functionalization by well-established siloxane chemistry. Moreover, silica NPs are optically silent in visible and near infrared (NIR) regions, do not cause detectable fluorescence quenching if loaded with fluorescent probes and can be easily enriched with therapeutics agents, thus proving to be ideal candidates for the development of new and efficient diagnostic and therapeutic agents. In this work, we have synthesized and characterized in pseudo-physiological solutions a novel fluorescent TBA analogue, named tris-mTBA, designed for the decoration of streptavidin-coated silica nanoparticles. Tris-mTBA is functionalized with a biotin tag, useful for the incorporation onto commercially available streptavidin-coated silica NPs, exploiting the streptavidin-biotin specific recognition, and the host-guest system dansyl/β-cyclodextrin, useful to monitor the NP functionalization and the correct aptamer folding onto the nanoparticles thanks to the environmentsensitive fluorescence of the dansyl probe. Tris-mTBA has been characterized in solution using combined spectroscopic techniques. CD results show that tris-mTBA is able to form an antiparallel G-quadruplex structure, essentially similar to unmodified TBA, and retains the ability to form a duplex structure with its complementary strand (cTBA), which acts as an antidote to reverse the anticoagulation activity of TBA. Fluorescencemelting measurements, combined with UV- and CD-melting experiments, demonstrate that the disruption of the cyclodextrin/dansyl complex is concomitant with the G-quadruplex unfolding. Moreover, coagulation experiments - performed using DLS measurements monitoring the conversion of fibrinogen in fibrin catalyzed by thrombin – have proved that tris-mTBA inhibits the human thrombin activity ca. 10 fold more efficiently than unmodified TBA and biotin-TBA and in a reversible manner. In fact, when tris-mTBA is mixed with its complementary sequence, the anticoagulant activity of thrombin is recovered as a consequence of the denaturation of the Gquadruplex structure of tris-mTBA due to the tris-mTBA/cTBA duplex formation. The Sicastar® NPs have been functionalized with tris-mTBA, optimizing the conjugation protocol, and characterized using DLS and SLS, gel electrophoresis and fluorescence analysis. Then, the anticoagulant activity of the conjugated NPs on human α-thrombin has been investigated by DLS 20 ACS Paragon Plus Environment

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experiments. Remarkably, the tris-mTBA/Sicastar® NPs completely block the thrombin-catalyzed fibrin formation, while the addition of cTBA to the functionalized NP solution is able to revert the thrombin inhibition process, restoring the thrombin coagulation activity. Taken together, these results demonstrate that the incorporation of tris-mTBA onto silica NPs enables highly efficient and reversible inhibition of thrombin activity toward fibrinogen. The peculiarity of our design – which can be easily extended to other biologically active aptamers resides in the derivatization of TBA with the dansyl/cyclodextrin host-guest system. This proved to be an effective tool to monitor, via simple fluorescence measurements, the correct G-quadruplex folding of the aptamer on the NPs, and thus indirectly their intrinsic responsiveness to thrombin. In principle, the structure-sensitive fluorescence of tris-TBA-functionalized NPs can be also exploited in localization studies in vivo, resulting in effective systems for theranostic applications.

4. EXPERIMENTAL SECTION

4.1 Synthesis of tris-mTBA 8 Preparation of dansyl-modified solid support 3. A 100 mM solution of dansyl propyl azide 2 (2 eq, 6 µmol, 60 µL) in CH3OH, a freshly prepared 40 mM aqueous solution of CuSO4 (0.4 eq, 1.2 µmol, 30 µL) and a 100 mM solution of sodium ascorbate in water (2 eq, 6 µmol, 60 µL) were added to the monoalkyne Controlled Pore Glass (CPG) solid support 1 (3 µmol) suspended in 0.8 mL of a CH3OH/water (1:1, v/v) solution. The vial containing the resulting mixture was sealed and placed in a microwave synthesizer (Monowave 300, Anton Paar) for 60 min at 60 °C with a gentle magnetic stirring (400 r/min). Then, the CPG beads were filtered, washed with water (2 mL) and CH3OH (2 mL), and dried. Solid phase synthesis, deprotection and purification of 5'-biotin-THME-propargyl-GGTTGG TGTGGTTGG-3'-{1-[2-methyl-2-(1-dansylpropyl-1H-[1,2,3]triazol-4-ylmethoxymethyl)]propane -1,3-diol} 6. The dansyl-modified solid support 3 was divided into three reaction columns and the TBA15 sequence (1 µmol scale each) was elongated in parallel on an ABI 394 DNA synthesizer according to standard phosphoramidite chemistry protocols. Detritylation was performed for 65 s. Coupling step: commercially available 2’-deoxyribonucleoside phosphoramidites (0.09 M in CH3CN) and DMTr-THME-propargyl 4 (0.1 M in CH3CN) were introduced into the solid support with a 30 s coupling time, while biotin phosphoramidite 5 (0.1 M in CH3CN) with a 180 s coupling time. The capping step was performed for 10 s. Oxidation was performed for 15 s. After the solid phase synthesis was completed, the CPG beads (3 x 1 µmol) were recombined together into a sealed vial and treated with concentrated aqueous ammonia (1.5 mL) overnight at 55 °C. The supernatant 21 ACS Paragon Plus Environment


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was then withdrawn and evaporated. The residue containing 6 was redissolved in water and analyzed by UV, HPLC (see Figure S1 in Supporting Information) and MALDI-TOF MS (see Figure S2 in Supporting Information). In particular, UV measurements at 260 nm revealed 245 OD of oligonucleotide material. From HPLC analysis on a Macherey Nagel Nucleodur 100-3 C18 ec column (length: 75 mm, ID: 4.6 mm) a 52 % yield in compound 6 was estimated (detection at 254 nm): linear gradient of B (10 to 30 %) in A for 20 min (6 tR: 17.45 min). Solution A: 1 % CH3CN/0.05 M TEAA, solution B: 80 % CH3CN/0.05 M TEAA. The crude was then purified by HPLC on a Macherey Nagel Nucleodur 100-7 C18 ec column (length: 125 mm, ID: 8 mm), using a linear gradient from 20 to 25 % of B in A for 20 min, affording 0.72 µmol of 6. MALDI-TOF MS: m/z: [M-H]-: for C197H259N65O111P17S1 calc.: 5905.29; found: 5905.75. Conjugation of 6 with β-cyclodextrin azide in solution. A 57 mM solution of β-cyclodextrin azide 7 (3.5 eq, 2.5 µmol, 45 µL) in H2O/DMSO (4:3, v/v), and freshly prepared aqueous solutions of CuSO4 (5 eq, 3.6 µmol, 90 µL) and sodium ascorbate (25 eq, 18 µmol, 180 µL), were added to 5'biotin THME-propargyl-TBA15-3’-dansyl 6 (1 eq, 0.72 µmol) dissolved in 240 µL of a CH3OH/water (1:1, v/v) solution. The vial containing the resulting mixture was sealed and stirred at room temperature. After 1.5 h, the crude was purified by size exclusion chromatography (SEC) on a NAPTM-10 column (GE Healthcare), giving compound 8 which was frozen and lyophilized. Then, it was redissolved in 500 µL of 1 M KCl solution, and after 1 h the excess of salts were removed by SEC, affording the target tris-TBA 8 in potassium salt form (0.52 µmol, 4.0 mg). HPLC analysis on the Macherey Nagel C18 ec analytical column revealed a 97 % purity at 254 nm (linear gradient: 10 to 30 % B in A for 20 min, 8 tR: 14.6 min). MALDI-TOF MS m/z: [M-H]-: for C239H328N68O145P17S2 calc.: 7064.29; found: 7064.68 (see Figure S3 and S4 in Supporting Information).

4.2 Synthesis of unmodified TBA and cTBA TBA and cTBA were prepared by solid phase synthesis on a CPG Universal Support (25 mg, 1 µmol) using 1 µmol scale and the DMTr-OFF protocol. The oligonucleotides were deprotected and cleaved from the solid support upon treatment with 1.5 mL 30 % ammonium hydroxide for 16 h at 55 °C. After evaporation of the solvent, the resulting crude mixtures were chromatographed on a C18 analytical column (Thermo Hypersil-100, 5 µm, 4.6×250 mm) using a linear gradient of CH3CN in 0.1 M TEAB solution (flow rate: 1 mL/min) and UV detection at 254 nm. TBA: gradient from 8 to 20 % CH3CN in 0.1 M TEAB in 25 min, tR= 14.1 min; cTBA: gradient from 8 % (2 min) to 28 % CH3CN in 0.1 M TEAB in 26 min, tR= 12.5 min. The purified oligonucleotides were lyophilized, then dissolved in 2.5 mL H2O and desalted using prepacked NAP™-25 columns. 22 ACS Paragon Plus Environment

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4.3 Synthesis of biotin-TBA Biotin-TBA was synthesized starting from propargylated solid support 1 on which the TBA oligonucleotide sequence was elongated and finally conjugated with biotin phosphoramidite 5 according to the above reported conditions. After deprotection by standard ammonia treatment, biotin-TBA was purified by HPLC on C18 reverse phase column, using a linear gradient from 0 to 30 % of B in A for 20 min, affording 0.44 µmol of biotin-TBA (solution A: 1 % CH3CN/0.05 M TEAA; solution B: 80 % CH3CN/0.05 M TEAA). Biotin-TBA: tR 13.18 min. MALDI-TOF MS: m/z: [M-H]-: for C197H259N65O111P17S1 calc.: 5350.71; found: 5350.50 (see Figures S9 and S10 in Supporting Information).

4.4 Preparation of oligonucleotide samples Purified and lyophilized oligonucleotides tris-mTBA 8, unmodified TBA, biotin-TBA and cTBA were dissolved in a known volume of Milli-Q water and their concentrations were determined by UV spectroscopy in 1 cm path length cuvette measuring the absorbance at 260 nm (85 °C) using the following molar extinction coefficients: for unmodified TBA, biotin-TBA and tris-mTBA, ε260 = 158480 cm−1 M−1; for cTBA, ε260 = 154660 cm−1 M−1. Then the oligonucleotides from the stock solutions were diluted in the selected buffer, annealed by heating each solution for 5 min at 90 °C and then allowed to slowly cool to room temperature overnight. The annealed samples were then kept at 4° C until use.

4.5 Spectroscopic characterization of tris m-TBA CD experiments. The CD spectra were collected at 10 °C in the range 220-320 nm. The characterization of tris-mTBA was performed at 5 µM concentration taking a suitable initial aliquot from a 335 µM stock solution in H2O. For the CD melting experiments, the CD signal at 295 nm was recorded vs. the temperature in the range 10-85 °C. The CD experiments carried out with the unmodified TBA or tris-mTBA and the corresponding complementary strand (cTBA) were performed in a tandem cell by placing, in one chamber, the TBA sequence, and in the other the complementary strand, each at 2.5 µM concentration in PBS solution. Two CD spectra were registered for each experiment: the first one was recorded as the sum of the separate components, here named as the sum spectrum, and the second one was recorded after mixing the two solutions and stabilization of the signal, here named as mix spectrum, in analogy with previous studies.93,94 After mixing the two solutions, the duplex concentration was reduced by a factor of 2 and the solution path length increased from 0.437 to 0.875 cm. Thermal denaturation-renaturation curves of 23 ACS Paragon Plus Environment


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the resulting duplex were recorded following the CD signal at 268 nm. The error associated with the Tm values determination was ± 1 °C. UV-vis absorption experiments. The UV-vis measurements were registered in the range 220–380 nm using 1 cm path length cuvette. The absorbance vs. temperature profiles were recorded by following the absorbance changes at 295 nm wavelength on increasing the temperature (heating scan rate 1 °C/min). The tris-mTBA solution used for these experiments was 5 µM in a 10 mM KH2PO4, 70 mM KCl, 0.2 mM EDTA (pH 7.0) buffer, prepared diluting the original stock solution of the oligonucleotide in H2O. The error associated with the Tm value determination was ± 1 °C. Fluorescence spectroscopy experiments. The fluorescence emission spectra were recorded from 440 to 640 nm, exciting at 327 nm and maintaining the excitation and emission slits at 10 nm. The oligonucleotide concentration used was 2 µM in a 10 mM KH2PO4, 70 mM KCl, 0.2 mM EDTA (pH 7.0) solution. Fluorescence melting curves were obtained by reporting the wavelength shift of the emission maximum as a function of temperature (from 20 to 80 °C). The error associated with the Tm value determination was ± 1 °C.

4.6 Enzymatic stability assays The stability of tris-mTBA in biological media was analyzed by incubating the oligonucleotide previously annealed in PBS buffer - at 10 µM concentration in 79 % FBS at 37 °C. Then, at fixed times, 20 µL of the samples were collected and stored at -20 °C. These aliquots were diluted to 100 µL with solution A (20 mM KH2PO4 aq. solution, pH 7.0, containing 20 % (v/v) CH3CN at pH = 7.0), filtered on 0.2 µm filter and finally analyzed by HPLC on a Nucleogen DEAE 60-7 column (Macherey-Nagel, 7 µm, 125 x 4 mm) using a linear gradient of B (0 to 100 %) in A in 30 min (solution A: 20 mM KH2PO4 aq. solution, pH 7.0, containing 20 % (v/v) CH3CN; solution B: 1 M KCl, 20 mM KH2PO4 aq. solution, pH 7.0, containing 20 % (v/v) CH3CN) and 0.8 mL min-1 flow rate (detection at 254 nm), until disappearance of the peak attributed to the intact oligonucleotide. Parallel experiments, using the same experimental conditions, have been carried out with biotinTBA and unmodified TBA. 4.7 Sicastar® functionalization and characterization 750 µL of a 0.4 µM solution of either tris-mTBA or biotin-TBA in PBS were first subjected to annealing, heating the sample at 95 °C for 5 min and then leaving it to slowly cool to room temperature. The formation of the desired G-quadruplex structure of tris-mTBA was confirmed in this buffer solution by CD and fluorescence spectra. Then 250 µL of a Sicastar® NPs suspension in PBS buffer (10 mg/mL) were added to the aptamer reaching a final volume of 1 mL (Sicastar® NPs 24 ACS Paragon Plus Environment

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2.5 mg/mL; tris-mTBA or biotin-TBA 0.3 µM) and the mixtures were gently stirred at room temperature for 1 h. Dynamic Light Scattering analysis. The NP dimensions were determined by means of Dynamic Light Scattering before and after the functionalization with tris-mTBA and biotin-TBA. DLS measurements were performed with a home-made instrument composed of a Photocor compact goniometer, a SMD 6000 Laser Quantum 50 mW light source operating at 5325 Å, a photomultiplier (PMT-120-OP/B) and a correlator (Flex02-01D) from Correlator.com. The experiments were carried out at 25.0 °C and at a scattering angle θ = 90°. The scattered intensity correlation function was analyzed using a regularization algorithm.95 The diffusion coefficient of each population of diffusing particles was calculated as the z-average of the diffusion coefficients of the corresponding distributions. Since diluted solutions were used, the Stokes–Einstein equation could be exploited to determine the hydrodynamic radius, RH, of the nanoparticles from their translation diffusion coefficients, D.95 Static Light Scattering analysis. Light scattering experiments were also employed to evaluate the molecular weight of the NPs before and after the functionalization with tris-mTBA. Indeed the intensity of the scattered light is related to the molecular weight of the scattering object through the Zimm equation.96 In particular, the scattered light is used to define the Rayleigh ratio as:

where “I” indicates the scattered intensity, “n” the refractive index, the subscript “S” indicates the sample, the subscript “0” indicates the solvent, the subscript “R” indicates the reference, toluene, and “θ” is the scattering angle, in our case 90°. “Rθ,R” is the Rayleigh ratio of the reference and is a tabulated value. In the case of scattering objects whose mean radius is less than λ/10, where λ is the wavelength of the incident light, the Rayleigh ratio is related to the molecular weight of the scattered object (Mw) through:

where “c” is the mass concentration, “A2” is the second virial coefficient and “K” is a constant, namely:



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

with “n0” the refractive index of the solvent, “λ” the wavelength of the incident light, “NA” the Avogadro number and

the variation of the refractive index of the sample solution with respect to

the sample concentration. By plotting Kc/Rθ as a function of c, a straight line with intercept equal to 1/MW was obtained, allowing also the determination of the second virial coefficient from its slope. Gel electrophoresis analysis. Non denaturing agarose (1 % w/v) gel electrophoresis was performed loading unmodified TBA, tris-mTBA, Sicastar® NPs and tris-mTBA/Sicastar® NPs on the gel with 3 % of GelRed dye. The samples were then run at 100 V for 30 min at r.t. in Tris-acetate-EDTA (TAE) buffer and visualized under UV light. Fluorescence analysis. Fluorescence spectra of the tris-mTBA/Sicastar® NPs solution were recorded in PBS buffer (0.3 µM oligonucleotide concentration) from 400 to 650 nm, exciting at 340 nm and maintaining the excitation and emission slits at 10 nm. Fluorescence spectra of the free trismTBA in solution under the same conditions were also registered.

4.8 Anticoagulant activity experiments Evaluation of the anticoagulant activity of tris-mTBA and tris-mTBA/Sicastar® NPs with respect to unmodified TBA and biotin-TBA was performed by monitoring the increase of light scattered intensity upon conversion of fibrinogen to fibrin catalyzed by thrombin and analyzing the normalized autocorrelation functions for the different systems at different times of the experiment. A 1.2 µM solution of fibrinogen in PBS was placed in a DLS cuvette and left to equilibrate in the instrument for 20 min. Then thrombin was added so to reach a final concentration of 5 nM and the light scattered intensity was registered every 20 s for at least 1 h. In the case of the experiments in the presence of anticoagulant agents, the TBAs were added to the fibrinogen solution before addition of thrombin so that their final concentration was 10-fold, 5-fold, 2-fold the thrombin concentration (for TBA and biotin-TBA) or 5-fold, 2-fold or equal to the thrombin concentration for tris-mTBA. Each experiment was performed in triplicate. Comparison of the anticoagulant activities was performed by plotting the normalized scattered intensity nI

as a function of time and linearly fitting the initial increase of nI upon the lag time. The slopes of the fitted lines give an estimation of the coagulation rate and of the thrombin inhibiting properties of the different anti-coagulant agents. The dose-effect dependence for the three aptamers, i.e. TBA, biotin-TBA and tris-mTBA, was also evaluated by plotting the ratio between the coagulation rate in 26 ACS Paragon Plus Environment

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the exclusive presence of thrombin and that in the presence of thrombin + aptamers as a function of the aptamer:thrombin molar ratio. In the case of the tris-mTBA/Sicastar® NPs, the nanoparticle concentration was chosen so to have the desired aptamer concentration in solution, knowing the NP concentration and assuming that all the binding sites on the NPs (as provided by the supplier) were functionalized with tris-mTBA. The same concentration of NPs was used for the control experiments with unfunctionalized Sicastar® and biotin-TBA/Sicastar® NPs. In all these experiments, the scattered intensity was monitored for longer times and also in this case each experiment was performed in triplicate.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Materials, apparatus and general methods. HPLC and MALDI characterization of oligonucleotide 6, of biotinTBA and of tris-mTBA. CD and UV spectra of tris-mTBA; UV melting profile of tris-mTBA. CD melting curves for tris-mTBA/cTBA and unmodified TBA/cTBA. Experiments of stability in FBS of tris-mTBA in comparison with biotin-TBA and unmodified TBA. DLS profiles and Zimm plots of Sicastar® NPs and tris-mTBA/Sicastar® NPs. Fibrinogen coagulation rates in the presence of the tested anticoagulants and dose-effect dependence for TBA, biotin-TBA and tris-mTBA.

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work was supported by the Italian Association for Cancer Research (AIRC) (IG2015 n. 17037 to D.M.). F.M. is member of INSERM.

ABBREVIATIONS CD

Circular dichroism

CE

2-cyanoethyl

DMSO

dimethyl sulfoxide

DMTr

4,4′-dimethoxytriphenylmethyl

EDTA

ethylenediaminetetraacetic acid

ec

end-capped 27 ACS Paragon Plus Environment


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HPLC

High Performance Liquid Chromatography

ID

internal diameter

MALDI

Matrix-Assisted Laser Desorption Ionization

MS

Mass Spectrometry

PBS

phosphate-buffered saline

tR

retention time

SEC

Size Exclusion Chromatography

TAE

Tris-acetate-EDTA

TBA

thrombin binding aptamer

TCA

trichloroacetic acid

TEAA

triethylammonium acetate

THF

tetrahydrofuran

THME

1,1,1-tris(hydroxymethyl)ethane

Tm

melting temperature

TOF

time of flight

UV

Ultraviolet Spectroscopy


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