Claudia Riccardi

Albert Meyer

Jean-Jacques Vasseur

Irene Russo Krauss

Luigi Paduano

François Morvan
email: francois.morvan@umontpellier.fr

Daniela Montesarchio
email: daniela.montesarchio@unina.it

Fine-tuning the properties of the Thrombin Binding Aptamer through cyclization: effect of the 5′-3′ connecting linker on the aptamer stability and anticoagulant activity

Keywords: thrombin binding aptamer, G-quadruplex, biophysical characterization, thrombin, anticoagulant activity

teaching and research institutions in France or abroad, or from public or private research centers.

Fine-tuning the properties of the thrombin binding aptamer through cyclization: Effect of the 5'-3' connecting linker on the aptamer stability and anticoagulant activity

INTRODUCTION

Among the biologically active aptamers, the Thrombin Binding Aptamer (TBA, 5′ GGTTGGTGTGGTTGG 3′ ) -adopting a stable chair-like, antiparallel G-quadruplex (G4) structure and able to strongly inhibit fibrin clot formation by its binding to thrombin -has attracted huge attention as effective thrombin activity modulator, representing one of the most extensively studied systems in the development of thrombin-targeting agents. [START_REF] Aviñó | Thrombin binding aptamer, more than a simple aptamer: chemically modified derivatives and biomedical applications[END_REF][START_REF] Musumeci | Polyvalent nucleic acid aptamers and modulation of their activity: a focus on the thrombin binding aptamer[END_REF][START_REF] Zavyalova | G-quadruplex aptamers to human thrombin versus other direct thrombin inhibitors: the focus on mechanism of action and drug efficiency as anticoagulants[END_REF][START_REF] Platella | G-quadruplex-based aptamers against protein targets in therapy and diagnostics[END_REF] Albeit clinical trials to validate its anticoagulant efficacy have been halted after Phase I studies for its suboptimal dosing profiles, [START_REF] Schwienhorst | Direct thrombin inhibitors -a survey of recent developments[END_REF] TBA is still actively investigated for its potential applications in therapeutics [START_REF] Platella | G-quadruplex-based aptamers against protein targets in therapy and diagnostics[END_REF] and diagnostics, [START_REF] Musumeci | Fluorescence sensing using DNA aptamers in cancer research and clinical diagnostics[END_REF] mainly in cardiovascular diseases, and a large number of its variants have been described in the literature. In the past decades, both serendipity and targeted rational design have contributed to discover chemically modified TBA analogues endowed with markedly improved physico-chemical and biological properties. Among the approaches thus far explored to obtain modified TBA variants with promising pharmacokinetic profiles, either backbone modifications [START_REF] Smirnov | Effect of Loop Sequence and Size on DNA Aptamer Stability[END_REF][START_REF] Sacca | The effect of chemical modifications on the thermal stability of different G-quadruplex-forming oligonucleotides[END_REF][START_REF] Martino | A new modified thrombin binding aptamer containing a 5′-5′ inversion of polarity site[END_REF][START_REF] Peng | G-quadruplex induced stabilization by 2′-deoxy-2′-fluoro-darabinonucleic acids (2′F-ANA)[END_REF][START_REF] Nagatoishi | Loop residues of thrombin-binding DNA aptamer impact G-quadruplex stability and thrombin binding[END_REF][START_REF] Pasternak | Improved thrombin binding aptamer by incorporation of a single unlocked nucleic acid monomer[END_REF][START_REF] Borbone | Investigating the role of T7 and T12 residues on the biological properties of thrombinbinding aptamer: enhancement of anticoagulant activity by a single nucleobase modification[END_REF][START_REF] De Tito | Fluorescence enhancement upon Gquadruplex folding: synthesis, structure, and biophysical characterization of a dansyl/cyclodextrin-tagged thrombin binding aptamer[END_REF][START_REF] Virgilio | Site-specific replacement of the thymine methyl group by fluorine in thrombin binding aptamer significantly improves structural stability and https://doi[END_REF][START_REF] Scuotto | Site specific replacements of a single loop nucleoside with a dibenzyl linker may switch the activity of TBA from anticoagulant to antiproliferative[END_REF][START_REF] Esposito | Backbone modified TBA analogues endowed with antiproliferative activity[END_REF][START_REF] Kotkowiak | Thermodynamic, anticoagulant, and antiproliferative properties of thrombin binding aptamer containing novel UNA derivative[END_REF][START_REF] Chai | TBA loop mapping with 3′-inverted-deoxythymidine for fine-tuning of the binding affinity for α-thrombin[END_REF] or incorporation onto suitable nanoplatforms -including magnetic, [START_REF] Yigit | MRI detection of thrombin with aptamer functionalized superparamagnetic iron oxide nanoparticles[END_REF][START_REF] Musumeci | G-quadruplex-forming oligonucleotide conjugated to magnetic nanoparticles: synthesis, characterization, and enzymatic stability assays[END_REF][START_REF] Yu | Bare magnetic nanoparticles as fluorescence quenchers for detection of thrombin[END_REF] gold, [START_REF] Shiang | Aptamer-conjugated nanoparticles efficiently control the activity of thrombin[END_REF][START_REF] Shiang | Gold nanoparticles presenting hybridized self-assembled aptamers that exhibit enhanced inhibition of thrombin[END_REF][START_REF] Hsu | 103379 control of thrombin activity by multivalent nanoparticles[END_REF][START_REF] Hsu | Highly flexible and stable aptamer-caged nanoparticles for control of thrombin activity[END_REF][START_REF] Huang | Gold nanoparticles modified with self-assembled hybrid monolayer of triblock aptamers as a photoreversible anticoagulant[END_REF] silica-based nanoparticles [START_REF] Gao | Selective recognition of co-assembled thrombin aptamer and docetaxel on mesoporous silica nanoparticles against tumor cell proliferation[END_REF][START_REF] Babu | Highly sensitive optical biosensor for thrombin based on structure switching aptamer-luminescent silica nanoparticles[END_REF][START_REF] Yue | A convenient sandwich assay of thrombin in biological media using nanoparticle-enhanced fluorescence polarization[END_REF][START_REF] Riccardi | Fluorescent thrombin binding aptamer-tagged nanoparticles for an efficient and reversible control of thrombin activity[END_REF] and graphene [START_REF] Lai | Selfassembled, bivalent aptamers on graphene oxide as an efficient anticoagulant[END_REF][START_REF] Lin | Supramolecular Aptamers on Graphene Oxide for Efficient Inhibition of Thrombin Activity[END_REF] -have been successfully exploited.

As a general strategy to improve the overall properties of anticoagulant aptamers and obtain TBA analogues better performing in vitro and in vivo, we have recently explored the cyclization, extending an approach similar to that already adopted for peptides [START_REF] Riahifard | Design, synthesis, and evaluation of amphiphilic cyclic and linear peptides composed of hydrophobic and positively-charged amino acids as antibacterial agents[END_REF] and peptidomimetics, [START_REF] Qvit | Peptidomimetic therapeutics: scientific approaches and opportunities[END_REF] as well as peptide nucleic acids (PNA) [START_REF] Moggio | Solid-phase synthesis of cyclic PNA and PNA-DNA chimeras[END_REF] and glycomimetics [START_REF] Di Fabio | Cyclic phosphate-linked oligosaccharides: synthesis and conformational behavior of novel cyclic oligosaccharide analogues[END_REF][START_REF] Coppola | Novel amphiphilic cyclic oligosaccharides: synthesis and self-aggregation properties[END_REF], but only limitedly investigated for oligonucleotides. [START_REF] Napoli | PEGsupported synthesis of cyclic oligodeoxyribonucleotides[END_REF][START_REF] Lietard | New strategies for cyclization and bicyclization of oligonucleotides by click chemistry assisted by microwaves[END_REF] In this frame, valuable multivalent circular analogues of antithrombotic oligonucleotides have been also studied. [START_REF] Di Giusto | Construction, stability, and activity of multivalent circular anticoagulant aptamers[END_REF] As a proof-of-concept, we have previously synthesized a cyclic TBA analogue obtained by covalently connecting the 3′-and 5′-ends of this G-rich oligonucleotide with a 20 atom long linker, ensuring approximately the same length of a trinucleotide (Figure 1). [START_REF] Riccardi | Stability is not everything: the case of the cyclization of the thrombin binding aptamer[END_REF] cyclic TBA TBA vs. The biophysical characterization of this first cyclic TBA prototype, that we named cycTBA, highlighted the noteworthy advantages obtained upon cyclization of the aptamer: indeed, a remarkable increase in the thermal stability of its G-quadruplex structure (ΔT m of ca. + 18 °C in both the analysed K + -and Na + -rich buffer solutions) and a dramatically higher resistance to nuclease degradation (ca. 180-fold higher half-life in PBS) were observed, denoting a much more compact and tightly assembled structure compared to unmodified TBA. [START_REF] Riccardi | Stability is not everything: the case of the cyclization of the thrombin binding aptamer[END_REF] This cyclic TBA analogue was obtained by covalently linking the 3′-and 5′-ends of the TBA sequence (Figure 1), which in the folded antiparallel G4 structure point in the same direction and are far away from the TT loops recognized by the protein. [START_REF] Krauss | Thrombinaptamer recognition: a revealed ambiguity[END_REF][START_REF] Krauss | Highresolution structures of two complexes between thrombin and thrombin-binding aptamer shed light on the role of cations in the aptamer inhibitory activity[END_REF][START_REF] Troisi | Several structural motifs cooperate in determining the highly effective anti-thrombin activity of NU172 aptamer[END_REF] Thus, the presence of a circularizing linker in principle should not impair the recognition and interaction of the aptamer with the target protein.

Anyhow, its anticoagulant activity -determined by dynamic light scattering (DLS) analysis in phosphate buffer solutions (PBS) of fibrinogen in the presence of human -thrombin -was ca. 50 % that of unmodified TBA, also correlated with a ca. halved binding affinity to thrombin. [START_REF] Riccardi | Stability is not everything: the case of the cyclization of the thrombin binding aptamer[END_REF] These data can be rationalized considering that some mutual interactions between the aptamer and the protein occur which are definitively responsible for TBA bioactivity, as suggested also by other research groups. [START_REF] Martino | A new modified thrombin binding aptamer containing a 5′-5′ inversion of polarity site[END_REF][START_REF] Troisi | Several structural motifs cooperate in determining the highly effective anti-thrombin activity of NU172 aptamer[END_REF] In this frame, the increased rigidity of the G4 structure of TBA produced by cyclization, if over a certain limit, could somehow reduce its affinity for the protein, and thus its bioactivity.

However, the overall results obtained on the first cyclic TBA prototype -clearly demonstrating the favourable impact of cyclization on the biophysical features of TBA -proved that there is significant room for improvement, particularly by finely tuning the design of the connecting linker. This structural component can indeed be optimized to realize the best compromise between preorganizing/stabilizing the TBA G4 structure, necessary for thrombin recognition, and maintaining some structural flexibility degrees, so to achieve an improved target affinity and bioactivity.

Stimulated by our previous study, we here report the design, synthesis and characterization of a small library of novel cyclic TBA analogues, indicated as cycTBAs I-IV (Table 1), differing for the linker connecting the TBA ends. In particular, these novel analogues exhibit flexible linkers with increased length with respect to the previously reported cycTBA, which should provide a still compact, stable G4 core, however allowing higher structural flexibility compared to their precursor.

In particular, cycTBA I and II were obtained using a chemical cyclization strategy based on the oxime ligation method, [START_REF] Edupuganti | Head-to-Tail oxime cyclization of oligodeoxynucleotides for the efficient synthesis of circular DNA analogues[END_REF][START_REF] Meyer | Phthalimide-oxy derivatives for 3′-or 5′conjugation of oligonucleotides byoOxime ligation and circularization of DNA by "bisor tris-Click" oxime ligation[END_REF] while cycTBA III and IV were circularized using a Cu(I)-assisted azide-alkyne cycloaddition (CuAAC) protocol, [START_REF] Pourceau | Azide solid support for 3′-conjugation of oligonucleotides and their circularization by click chemistry[END_REF] also adopted to obtain our first prototype, https://doi.org/10.1016/j.bioorg.2019.103379 cycTBA. [START_REF] Riccardi | Stability is not everything: the case of the cyclization of the thrombin binding aptamer[END_REF] More in detail, cycTBA I was prepared also to evaluate whether the oxime ligation approach was more efficient, in terms of cyclization yields, than the alternative, previously explored CuAAC. Therefore, cycTBA I was designed with a linker only slightly longer than the one used in cycTBA (22-vs. 20-atom length). In turn, in cycTBA II one triethylene glycol unit -a flexible linker typically used in bioconjugation strategies -was inserted at each end, providing a 30-atom long linker. Then, cycTBA III and IV were prepared using the CuAAC protocol, analogously to cycTBA, [START_REF] Riccardi | Stability is not everything: the case of the cyclization of the thrombin binding aptamer[END_REF] introducing one or two tetraethylene glycol units, thus producing connecting linkers 34-and 48-atom long, respectively (Table 1).

The exploited synthetic strategies allowed obtaining circularizing linkers varying not only in length but also featuring marked differences in their chemical structure.

With this second generation of cyclic TBAs, we intended to improve the potency of TBA in inhibiting fibrin clot formation, aiming at obtaining suitable derivatives concomitantly featuring increased thermal stability and nuclease resistance. Then, as additional goal, we intended to evaluate the cyclization approach from a synthetic point of view, probing the chemical scope of the different cyclization reactions, with special attention to the yield optimization, as well as from a functional point of view, analysing the impact of the length and chemical nature of the linker on the TBA performance. In this frame, our studies were also aimed at identifying the structural elements more effective for TBA cyclization in order to extend this strategy to other G4-based aptamers of great biological interest, involved in clotting disorders but also in other pathologies, such as cancer.

Table 1. Molecular structure and length of the linkers connecting the 3′-and 5′-ends of TBA, providing the new cycTBAs I-IV, here investigated. In the Table, the previously studied cycTBA [START_REF] Riccardi | Stability is not everything: the case of the cyclization of the thrombin binding aptamer[END_REF] is also reported for comparison. In addition, the anticoagulant activity of these cyclic oligonucleotides compared to that of unmodified TBA was determined by dynamic light scattering (DLS) measurements in PBS (Na +rich buffer), which mimes the blood salt composition and is indeed relevant from a biological point of view.

EXPERIMENTAL SECTION

Preparation of the oligonucleotide samples.

Purified and lyophilized TBA and cyclic TBAs oligonucleotides (here named as cycTBA I, cycTBA II, cycTBA III and cycTBA IV, Table 1) were dissolved in a defined volume of Milli-Q water. Their concentrations were determined by UV measurements on a JASCO V-530 UV-vis spectrophotometer equipped with a Peltier Thermostat JASCO ETC-505T, using a 1 cm path length cuvette (1 mL internal volume, Hellma), recording the absorbance at 260 nm and 90 °C. The molar extinction coefficient of 158.480 cm -1 M -1 was used for both TBA and the cyclic TBAs (assuming negligible for the latter ones the contribution of the linkers at 260 nm), as calculated for the unstacked oligonucleotides. The UV spectra were recorded in the range 200-320 nm with a medium response, a scanning speed of 100 nm/min and a 2.0 nm bandwidth with the appropriate baseline subtracted. Taking a suitable aliquot from the initial stock solutions in H 2 O, all the investigated oligonucleotides were then diluted in the selected K + -(10 mM KH 2 PO 4 /K 2 HPO 4 , 70 mM KCl, 0.2 mM EDTA, pH = 6.9) or Na + -rich (PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM NaH 2 PO 4 /Na 2 HPO 4 , 1.8 mM KH 2 PO 4 /K 2 HPO 4 pH = 7.0) phosphate buffer solutions. Thereafter, the samples were annealed by heating the solutions for 5 min at 95 °C and then allowed slowly cooling to r.t. overnight. The annealed samples were eventually kept at 4 °C until their subsequent use.

UV Thermal Difference Spectra.

The UV spectra measurements were performed on a JASCO V-530 UV-vis spectrophotometer equipped with a Peltier Thermostat JASCO ETC-505T, using 1 cm path length cuvette (1 mL internal volume, Hellma). cycTBAs I-IV were dissolved in the selected buffer so to obtain 2 μM solutions and annealed. The absorbance spectra were recorded at 15 and 90 °C in the range 200-320 nm using a scanning speed of 100 nm/min with the appropriate baseline subtracted. The thermal difference spectra (TDS) were obtained by subtracting the UV spectrum recorded at a temperature below the T m (15 °C), at which the aptamer is fully structured, from the one obtained at a temperature above the T m (90 °C), where the oligonucleotide G4 structure is fully denatured. [START_REF] Mergny | Thermal difference spectra: a specific signature for nucleic acid structures[END_REF][START_REF] Mergny | UV Melting of G-Quadruplexes[END_REF][START_REF] Malgowska | How to study Gquadruplex structures[END_REF] Each experiment was performed in triplicate.

Circular dichroism (CD) spectroscopy.

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 cuvette 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. cycTBAs I-IV were analysed at 2 μM concentration in the selected buffers. Thermal denaturation-renaturation curves were recorded following the CD signal at 295 nm vs. the temperature (scan rate 1.0 °C/min) and recording spectra from 220 to 320 nm in 5 °C steps, in the 20 -90 °C temperature range for both the K + -and Na + -rich buffer solutions. Each experiment was performed in triplicate. The molar ellipticity [θ] (deg cm 2 dmol -1 ) was calculated from the equation [θ] = θ obs /10 × l × C, where θ obs is the observed ellipticity (mdeg), C is the oligonucleotide molar concentration, and l is the optical path length of the cell (cm). Data were also converted into folded fraction (α) calculated as: α = [θ(T) -θ U )/(θ F -θ U )], where θ(T) is the molar ellipticity at 295 nm at each temperature, while θ F and θ U are the molar ellipticity at 295 nm for the folded (T = 20 °C) and unfolded (T = 90 °C) oligonucleotide, respectively. The T m values were estimated as the maxima of the first derivative plots of the melting/annealing curves and the error associated with the T m determination was ± 1 °C. The ΔT m , values were calculated by subtracting the measured T m of unmodified TBA from that observed for each cyclic TBA analogue. The CD melting curves showed sigmoidal profiles and were also modelled by a two-state transition, using a theoretical equation for an intramolecular association, according to the van't Hoff analysis. [START_REF] Mergny | UV Melting of G-Quadruplexes[END_REF][START_REF] Marky | Calculating thermodynamic data for transitions of any molecularity from equilibrium melting curves[END_REF] Thermodynamic data were obtained using the following equations:

θ u (T)= θ u 0 + m u *T θ l (T)= θ l 0 + m l *T α = [θ(T) -θ l (T)]/[θ u (T) -θ l (T)] θ(T) = α* [(θ u 0 + m u *T) -θ l 0 + m l *T] + (θ l 0 + m l *T) α = K a /(1 + K a ) K a = exp(ΔH v.H /RT)*[(T/T m ) -1]
where:

θ u and θ l are linear equations describing the upper and lower baselines, respectively, θ u 0 and θ l 0 are fitted parameters for the intercepts of the upper and lower baseline, while m u and m l are the respective slopes. θ(T) is the dependent variable and is the experimentally determined molar ellipticity (θ) at each temperature (T). Ka is the equilibrium constant for the unstructured → structured oligonucleotide transition for an intramolecular process and α is the folded fraction.

RESULTS AND DISCUSSION

Synthetic procedures for the preparation of cyclic TBA variants

Four different cyclic TBA analogues -exhibiting linkers of different length between the 3′ and 5′

terminal 2′-deoxyguanosines of the TBA sequence (Table 1) -were successfully synthesized and characterized. The cyclic TBAs indicated as cycTBA I and cycTBA II, with a 22-and 30-atom linker, were respectively prepared by using oxime [START_REF] Edupuganti | Head-to-Tail oxime cyclization of oligodeoxynucleotides for the efficient synthesis of circular DNA analogues[END_REF] and bis-oxime [START_REF] Meyer | Phthalimide-oxy derivatives for 3′-or 5′conjugation of oligonucleotides byoOxime ligation and circularization of DNA by "bisor tris-Click" oxime ligation[END_REF] ligation procedure; while cycTBA III and cycTBA IV, with a 34-and 48-atom linker, respectively, were obtained by

CuAAC. [START_REF] Riccardi | Stability is not everything: the case of the cyclization of the thrombin binding aptamer[END_REF][START_REF] Pourceau | Azide solid support for 3′-conjugation of oligonucleotides and their circularization by click chemistry[END_REF] cycTBA I was synthesized according to the strategy developed by Dumy et al. [START_REF] Edupuganti | Head-to-Tail oxime cyclization of oligodeoxynucleotides for the efficient synthesis of circular DNA analogues[END_REF], exploiting the oxime ligation between an oxyamine linker introduced at the 5′-end and an aldehyde function Thus, starting from the commercially available glycerol-derivatized solid support 5, the TBA oligonucleotide sequence was elongated and 4 was coupled at its 5′-end using standard phosphoramidite chemistry. After ammonia treatment on 6, the 3′-glycerol-derivatized oligonucleotide 7 was dissolved in a KCl aq. solution to allow its G-quadruplex folding, and the glycerol moiety at the 3′-end was oxidized to the aldehyde derivative 8. After removal of the obtained formaldehyde by evaporation, the residue was dissolved in ammonium acetate buffer to deprotect the 5′-aminooxy function and allow its coupling with the 3′-aldehyde to form the target cyclic TBA by oxime ligation. The resulting cycTBA I was then purified by HPLC and characterized by MALDI-TOF MS analysis (Figure S3). Scheme 2. Synthetic scheme for the preparation of cycTBA I by oxime ligation.

For the synthesis of cycTBA II, a bis-oxime ligation [START_REF] Meyer | Phthalimide-oxy derivatives for 3′-or 5′conjugation of oligonucleotides byoOxime ligation and circularization of DNA by "bisor tris-Click" oxime ligation[END_REF] protocol (Scheme 3) was applied exploiting a strategy recently reported by some of us. Starting from solid support 9, [49] the TBA sequence was elongated, using tBu-phenoxyacetyl (tBu-Pac) protection for the 2′-deoxyguanosine building blocks, and then compound 4 was coupled. The resulting support-bound oligonucleotide 10 was treated first with ammonia solution in methanol, to remove the 2-cyanoethyl and tBu-Pac groups, and then with methoxyamine HCl in ammonium acetate buffer, to obtain its detachment from the solid support and the MMTr removal, affording the linear 3′,5′-bis aminooxy-TBA 11.

After size exclusion chromatography, linear oligonucleotide 11 was circularized by addition of https://doi.org/10.1016/j.bioorg.2019.103379

terephthalaldehyde and KCl to give cycTBA II, then purified by HPLC and characterized by MALDI-TOF MS analysis (Figure S4). Scheme 3. Synthetic scheme for the preparation of cycTBA II by bis-oxime ligation.

As concerning the synthesis of cycTBAs III and IV, respectively exhibiting a linker of 34 and 48 atoms between the two 3′ and 5′-terminal 2′-deoxyguanosines (Table 1), CuAAC protocols were exploited, analogously to the previously developed cycTBA precursor. [START_REF] Riccardi | Stability is not everything: the case of the cyclization of the thrombin binding aptamer[END_REF] In detail, these cyclic TBA analogues were synthesized starting from the azido-functionalised solid support 12 [START_REF] Pourceau | Azide solid support for 3′-conjugation of oligonucleotides and their circularization by click chemistry[END_REF] (Scheme 4). In the case of cycTBA III, a tetraethyleneglycol (EG 4 ) spacer was introduced before the oligonucleotide sequence elongation -using standard phosphoramidite chemistry -and then a pentynyl group [START_REF] Pourceau | Azide solid support for 3′-conjugation of oligonucleotides and their circularization by click chemistry[END_REF] was inserted at the 5′-end by reaction with phosphoramidite 13. In the case of cycTBA IV, an additional EG 4 spacer was introduced after the oligonucleotide assembly, followed by coupling with the pentynyl phosphoramidite 13 (Scheme 4).

Then, support-bound oligonucleotides 14 and 15 were fully deprotected and released in solution by ammonia treatment, affording the linear oligonucleotides 16 and 17. Lastly, these were circularized by CuAAC, using CuSO 4 and sodium ascorbate in a K + -containing solution, introduced to promote the oligonucleotide G-quadruplex folding and thus favour the spatial proximity of their 5′-and 3′ends (Scheme 4). Resulting cycTBAs III and IV were purified by HPLC and then characterized by MALDI-TOF MS analysis (Figures S5 andS6).

https://doi.org/10.1016/j.bioorg.2019.103379

As previously observed, [START_REF] Riccardi | Stability is not everything: the case of the cyclization of the thrombin binding aptamer[END_REF] in all cases the precursor linear oligonucleotides exhibited different retention times vs. their cyclic congeners by analytical RP-HPLC analysis, nicely confirming the effective cyclization (Figures S7-S10). Scheme 4. Synthetic scheme for the preparation of cycTBAs III and IV.

Thermodynamic and spectroscopic features of cyclic TBA variants

UV spectra and TDS

The spectroscopic properties of cycTBAs I-IV were investigated by means of UV and CD spectroscopies in two different buffer solutions, containing a high content of K + or Na + ions, respectively. It is indeed well known that buffer composition, and particularly ion concentration, can determine dramatic changes in the biophysical properties [START_REF] Krauss | Highresolution structures of two complexes between thrombin and thrombin-binding aptamer shed light on the role of cations in the aptamer inhibitory activity[END_REF][START_REF] Hardin | Cation-dependent transition between the quadruplex and Watson-Crick hairpin forms of d(CGCG 3 GCG)[END_REF][START_REF] Hud | The selectivity for K + versus Na + in DNA quadruplexes is dominated by relative free energies of hydration: a thermodynamic analysis by 1 H NMR[END_REF][START_REF] Poniková | A circular dichroism study of the stability of guanine quadruplexes of thrombin DNA aptamers at presence of K + and Na + ions[END_REF][START_REF] Zavyalova | Cation coordination alters the conformation of a thrombin-binding G-quadruplex DNA aptamer that affects inhibition of thrombin[END_REF] as well as in the anticoagulant activity of TBA, due to conformational changes involving key protein-aptamer interactions. [START_REF] Krauss | Highresolution structures of two complexes between thrombin and thrombin-binding aptamer shed light on the role of cations in the aptamer inhibitory activity[END_REF] For all the cyclic TBAs here investigated, UV thermal difference spectra (TDS) at 2 μM concentration were obtained recording spectra at low and high temperatures. Differential spectrarepresenting the spectral difference between the unfolded and the folded oligonucleotide -have a typical and unique shape for G-quadruplexes and can be used as a simple and rapid method to identify the presence of these structures in solution. [START_REF] Mergny | Thermal difference spectra: a specific signature for nucleic acid structures[END_REF][START_REF] Mergny | UV Melting of G-Quadruplexes[END_REF][START_REF] Malgowska | How to study Gquadruplex structures[END_REF] In Figures S11 andS12, the spectra at 15 and 90 °C (panels a and c) as well as the obtained TDS profiles (panels b and d) were reported for cycTBAs I and II as representative examples.

https://doi.org/10.1016/j.bioorg.2019.103379

All the UV spectra at 15 °C, in both saline solutions, revealed the characteristic absorption of TBA, [START_REF] Riccardi | Stability is not everything: the case of the cyclization of the thrombin binding aptamer[END_REF] with the double-hump band between 230 and 300 nm (Figures S11 andS12). The obtained TDS profiles, in both K + -and Na + -rich solutions, showed for all the cyclic TBA analogues here investigated a positive band at 273 nm and a negative band at 295 nm, similarly to unmodified TBA, [START_REF] Riccardi | Stability is not everything: the case of the cyclization of the thrombin binding aptamer[END_REF] providing the typical "fingerprint" of G-quadruplex structures (Figures S11 andS12).[50-52]

CD spectroscopy

In order to determine the effects of the circularizing linker on the conformational properties and thermal stability of the TBA G4 structure, CD spectra and CD thermal denaturation measurements were recorded for all the studied cyclic TBA analogues. The CD spectra at 15 °C of cycTBAs I-IV, at 2 µM concentration in both K + -and Na + -rich buffer solutions, are reported in Figure 2 in comparison with unmodified TBA, analysed under the same experimental conditions. These data nicely showed that all the novel analogues in both saline conditions folded into structures displaying a CD profile very similar to that of TBA, with only tiny differences in the profile or intensity of the bands (Figure 2).

In detail, for three of the here investigated cyclic TBAs (i.e. I, III and IV, orange, light blue and magenta lines, respectively), the recorded spectra are essentially superimposable to that of unmodified TBA showing two positive bands, with maxima at about 294-295 and 246-247 nm and one negative band with minima at 268-269 nm. These spectroscopic properties are fully consistent with an antiparallel G4 structure in which anti and syn guanosines alternate along the strands. [START_REF] Dapić | Biophysical and biological properties of quadruplex oligodeoxyribonucleotides[END_REF][START_REF] Paramasivan | Circular dichroism of quadruplex DNAs: applications to structure, cation effects and ligand binding[END_REF][START_REF] Kypr | Circular dichroism and conformational polymorphism of DNA[END_REF][START_REF] Masiero | A nonempirical chromophoric interpretation of CD spectra of DNA G-quadruplex structures[END_REF][START_REF] Karsisiotis | Topological characterization of nucleic acid G-Quadruplexes by UV absorption and circular dichroism[END_REF][START_REF] Vorlíčková | Circular dichroism and guanine quadruplexes[END_REF] Only in the case of cycTBA II, little changes in the CD spectra were observed (maxima and minima at 293 and 265 nm, in both saline conditions, together with the other maximum at 245/246 nm, respectively in K + /Na + buffer solutions) corresponding to a slight shift in the minimum and in the maximum at low wavelengths (Figure 2), as already observed for the previously developed cycTBA. [START_REF] Riccardi | Stability is not everything: the case of the cyclization of the thrombin binding aptamer[END_REF] Similar differences in the CD profiles of TBA analogues are not unprecedented [START_REF] Virgilio | Site-specific replacement of the thymine methyl group by fluorine in thrombin binding aptamer significantly improves structural stability and https://doi[END_REF] suggesting slight conformational differences in the G4 arrangement in solution. The collected CD data confirmed the ability of cycTBAs I-IV to form a chair-like antiparallel Gquadruplex structure, clearly demonstrating that the cyclization did not impair the G4 structuring ability of TBA nor affected its folding topology, as also previously evidenced in the case of cycTBA. [START_REF] Riccardi | Stability is not everything: the case of the cyclization of the thrombin binding aptamer[END_REF] .

CD thermal denaturation/renaturation measurements

Differences in the behaviour of the cyclic TBA analogues are clearly evident by comparing their CD melting profiles, obtained monitoring the CD signal at 295 nm on varying the temperature. For these experiments, cycTBAs I-IV were analysed at 2 µM concentration in both K + -and Na + -rich buffer solutions in the 20 -90 °C range, in comparison with unmodified TBA. In Figure 3 2), indicating the formation of very stable G4 structures. Conversely, cycTBA IV (Figures 3a andS16a) showed a similar thermal stability as unmodified TBA, with the same apparent T m values within the experimental error (Table 2).

In the Na + -rich buffer (Figure 3b) a similar trend was found, with cycTBAs I and II forming the most stable G4 structures. Notably, the apparent T m values for these aptamers were respectively of showing very similar T m values (Table 2).

In all cases, a nice sigmoidal behaviour was found (Figures 3 andS13-S16), with no significant hysteresis comparing the heating and cooling profiles, thereby indicating that, under the experimental conditions used (heating rate of 1 °C/min), the related denaturation/renaturation processes were reversible (Figures S13-S16). CD spectra, acquired every 5 °C during each melting and cooling experiment (Figures S17-S20), showed a marked reduction of the CD signals at high temperatures, overall indicating that all the cyclic TBAs behaved as unmodified TBA in both saline phosphate buffers from a qualitative point of view. Notably, all the cyclic analogues were completely denaturated at 90 °C and, after the heating/cooling processes, full recovery of the initial spectral features was always observed (Figures S17-S20).

Na + -rich buffer K + -rich buffer

In the case of cycTBAs I-IV, all the thermodynamic parameter values are quite similar to those found for unmodified TBA; [START_REF] Martino | A new modified thrombin binding aptamer containing a 5′-5′ inversion of polarity site[END_REF][START_REF] Virgilio | Site-specific replacement of the thymine methyl group by fluorine in thrombin binding aptamer significantly improves structural stability and https://doi[END_REF][START_REF] Virno | A novel thrombin binding aptamer containing a G-LNA residue[END_REF] and the enthalpy change values are consistent with the destructuring of two G-tetrads. [START_REF] Petraccone | Effect of a modified thymine on the structure and stability of [d(TGGGT)] 4 quadruplex[END_REF][START_REF] Petraccone | Stability and structure of telomeric DNA sequences forming quadruplexes containing four G-tetrads with different topological arrangements[END_REF][START_REF] Olsen | Unfolding of G-quadruplexes: energetic, and ion and water contributions of G-quartet stacking[END_REF][START_REF] Petraccone | 103379 Structure and stability of higher-order human telomeric quadruplexes[END_REF] The whole set of thermodynamic analysis data clearly indicated that, compared to unmodified TBA, cycTBA I and II were the most stable analogues in both buffer solutions; cycTBA III exhibited a higher thermal stability in the K + -rich buffer but not in the Na + -rich one, while cycTBA IV had the same stability as unmodified TBA in both saline conditions. Where observed (i.e. cycTBAs I-III in K + -rich buffer and cycTBAs I and II in the Na + -containing solution) the increased energetic stability of these G4 structures is mainly due to a favourable decrease of the unfolding entropy change, in line with the expected structural preorganization of these cyclic aptamers, which balances the slight unfavourable enthalpy decrease. [START_REF] Riccardi | Stability is not everything: the case of the cyclization of the thrombin binding aptamer[END_REF] Taken together, the CD-monitored thermal denaturation data and the related thermodynamic analysis revealed this trend of G4 stability: cycTBA II > cycTBA I > cycTBA III > cycTBA IV ≈ TBA in the K + -rich buffer, and cycTBA II > cycTBA I > cycTBA III ≈ cycTBA IV ≈ TBA in the Na + -rich buffer solution. Particularly, a noteworthy stabilizing effect on the G-quadruplex structure (ΔT m = + 17) was observed for cycTBA II in both saline conditions, behaving very similarly to the previously described cycTBA. [ ) for the unfolding process of TBA [START_REF] Riccardi | Stability is not everything: the case of the cyclization of the thrombin binding aptamer[END_REF] and cycTBAs I-IV as determined by CD measurements at 295 nm in both saline phosphate buffer solutions at 2 µM concentration. ΔT m is calculated by subtracting the measured T m of unmodified TBA from that observed for each cyclic TBA analogue. ΔG v.H was calculated at 298 K. Errors on thermodynamic parameters are within ± 10 %, while the error associated with the T m determination is ± 1 °C.

Non-denaturing polyacrylamide gel electrophoresis

In order to further confirm that the cyclic TBA analogues were able to preserve the monomolecular G-quadruplex structure typical of TBA, not forming additional superstructures, a non-denaturing PAGE analysis was carried out. In Figure 4, a representative example of a 15 % polyacrylamide gel -in which cycTBAs I-IV were compared with unmodified TBA in both the selected buffer solutions -is reported. Electrophoretic analysis in both saline conditions showed that all the cyclic TBAs migrated as a single band on the gel, with a slightly increased electrophoretic mobility with respect to unmodified TBA.

Taking into account that the gel mobility of the G4 structures mostly affected by their conformation and compactness and that the charge/mass ratio is very similar for all the here investigated oligonucleotides, PAGE results suggested a more compact structure for all the cyclic aptamers, facilitating their movement through the gel, similarly to what already observed for the previously described cycTBA. [START_REF] Riccardi | Stability is not everything: the case of the cyclization of the thrombin binding aptamer[END_REF] This increased mobility is a direct consequence of the cyclic structure of these TBA analogues: in fact, we have previously demonstrated [START_REF] Riccardi | Stability is not everything: the case of the cyclization of the thrombin binding aptamer[END_REF] that the migration on the gel of the linear precursor of cycTBA was the same as unmodified TBA, not being affected by the presence of the 3' and 5'-appendages..

Remarkably, no additional retarded bands, attributable to large aggregates or higher order G4 structures, were found, thus indicating the exclusive formation of monomolecular G4 structures, analogously to the case of unmodified TBA.

Size exclusion chromatography analysis

Oligonucleotides can adopt a variety of structures depending on the experimental conditions, and the knowledge of their functional conformations in solution is critical to gain insight into their biological role and mechanism. In this context, SE-HPLC recently emerged as a powerful technique to detect the polymorphism of G4 species displaying various structures and/or molecularity and definitively assess the prevailing secondary structures formed by oligonucleotides. [START_REF] Dailey | Resolution and characterization of the structural polymorphism of a single quadruplex-forming sequence[END_REF][START_REF] Largy | Shape matters: size-exclusion HPLC for the study of nucleic acid structural polymorphism[END_REF][START_REF] Zavyalova | Putative mechanisms underlying high inhibitory activities of bimodular DNA aptamers to thrombin[END_REF] Thus, its use can be particularly useful to study the conformational behaviour and decipher the polymorphism of G-quadruplexes, in parallel with electrophoretic methods. [START_REF] Riccardi | Exploring the conformational behaviour and aggregation properties of lipidconjugated AS1411 aptamers[END_REF][START_REF] Moccia | Insights into the G-rich VEGF-binding aptamer V7t1: when two G-quadruplexes are better than one![END_REF] The analysis of cycTBAs I-IV at 2 μM concentration by SE-HPLC analysis showed in all cases only one peak, with retention times of ca. 9 min in both buffer solutions (Figure S21), similar to those found for unmodified TBA, as well as for the previously described cycTBA. [START_REF] Riccardi | Stability is not everything: the case of the cyclization of the thrombin binding aptamer[END_REF] These data further supported the native PAGE results, confirming the formation of a unique G4 conformation, comparable to that of unmodified TBA.

Only for cycTBAs III and IV, a small shoulder under the main peak was detected in both saline conditions, suggesting the possible presence of very low amounts of unstructured cycTBAs or G4 structures with different conformations under the tested conditions. This could be attributed to the presence of long and flexible connecting linkers, which however did not affect the overall conformation and thermal stability of these aptamers, showing in all cases a behaviour very similar to unmodified TBA, as revealed by spectroscopic investigations.

Nucleases stability assay

The cyclization approach, linking the 5′-and 3′-extremities of the TBA sequence and thus preventing exonuclease attacks, is expected to markedly improve the nuclease resistance of the oligonucleotide, as already demonstrated for the previously reported cycTBA. [START_REF] Riccardi | Stability is not everything: the case of the cyclization of the thrombin binding aptamer[END_REF] Thus, cycTBAs I-IV were tested in their resistance to nuclease degradation in a pseudo-physiological environment, in order to evaluate the protective effects of cyclization against enzymatic digestion, an event which is typically limiting in vivo applications.

cycTBAs I-IV -previously annealed and dissolved in PBS -were treated with fetal bovine serum (FBS), using a solution mimicking the extracellular environment in which the aptamer should exert its activity. Thus, in parallel experiments, cycTBAs I-IV were incubated in 80 % FBS (v/v) at 37 °C; at fixed times, aliquots of these mixtures were collected and analysed by HPLC on a Nucleogen https://doi.org/10.1016/j.bioorg.2019.103379

SAX column until disappearance of the peak corresponding to the intact oligonucleotide. The cycTBAs area under their corresponding peak, at each collected time, was then calculated and normalized with respect to the initial one. Percentages of the remaining area of cycTBAs I-IV are reported in Figure 5, in comparison with unmodified TBA data. Then, fitting of the experimental points allowed the half-life values of the aptamers (t 1/2 ). Fitting curves are represented in Figure 5 for an overall view of the nuclease digestion results, but also separately reported for each system in Figure S22.

Overall results clearly indicated that all the cyclic TBAs here investigated had enhanced half-lives with respect to unmodified TBA, used as control (Figure S22). [START_REF] Riccardi | Stability is not everything: the case of the cyclization of the thrombin binding aptamer[END_REF] The nuclease stability was found to be not dependent on the specific nature of the linker inserted: in fact, all the derivatives exhibited similar properties, with a closer behaviour between cycTBA I and II, on one side, and between cycTBA III and IV, on the other.

In particular, cycTBA III and IV showed a t 1/2 of 2.6 h, i.e. 4.9-fold higher than unmodified TBA, which under our experimental assay had a half-life of 0.53 h. [START_REF] Riccardi | Stability is not everything: the case of the cyclization of the thrombin binding aptamer[END_REF] The serum nuclease stability of cycTBA II was found to be enhanced over TBA by a factor of ca.

6.6 (t 1/2 = 3.6 h) while cycTBA I proved to be the most stable derivative, showing a t 1/2 of 4.3 h, i.e.

ca. 8.1-fold higher than TBA. The general trend of serum nuclease resistance was therefore:

cycTBA I > cycTBA II > cycTBA III ≈ cycTBA IV >> TBA.

These data comprehensively supported the initial idea that end-capped G4 structures -as those obtained by cyclization of the 5′ and 3′-ends -are indeed less accessible to the nuclease attack than the G4 structure formed by unmodified TBA.

Coagulation experiments

The ability of cycTBAs I-IV to inhibit the thrombin-catalysed conversion of fibrinogen into fibrin was investigated by means of light scattering experiments and compared with that of unmodified TBA. The analysis of the normalized autocorrelation functions gives a direct information on the size of the species in the system, so we analysed the autocorrelation functions obtained for the different systems upon 5 min monitoring from the beginning of the experiment (Figure S23). The two limit cases are represented by the autocorrelation functions of the sample only containing fibrinogen -which had a well-defined sigmoidal shape and whose position did not change with time -and that of the sample containing fibrinogen and thrombin but no anticoagulant agent, which, already after 5 min, was sensibly shifted towards long delay times and did not reach zero even at very long times, clearly indicating the formation of very big, barely soluble aggregates. Samples containing anticoagulant aptamers, either cycTBAs I-IV or native TBA, showed an intermediate behaviour between these two limit cases. In particular, the autocorrelation function of the sample containing cycTBA I was close to that of the sample only containing thrombin, but in the long run reached zero, indicating the presence in solution of still soluble, smaller species. The curves of the TBA-, cycTBA III-or cycTBA IV-containing samples were very close to each other, suggesting the presence of even smaller species than those present in the case of cycTBA I. Finally, the autocorrelation curve found for the cycTBA II-containing sample was the closest to the fibrinogen autocorrelation curve, indicating a sensibly retarded coagulation process.

Overall, the analysis of the autocorrelation functions clearly proved that the TBA cyclization overall affected the thrombin recognition/inhibition properties of the here tested aptamers and, at the same time, highlighted different inhibiting ability of cycTBAs I-IV. Same results were obtained when the scattered intensity was monitored (Figure 6a). Indeed, a rapid increase of the scattered intensity with time, indicating the progression of fibrin formation, was found in the samples only containing fibrinogen and thrombin, while when cycTBAs I-IV or unmodified TBA were added, the scattered intensity increased much more slowly than in the untreated samples, evidencing the net inhibition of thrombin coagulation activity (Figure 6a). The extent of the scattered intensity increase depended on the used aptamer, indicating again that cycTBAs I-IV displayed different anticoagulant properties. Thus, to quantitatively assess the anti-thrombin activity of cycTBAs I-IV and to compare it with that of unmodified TBA, we calculated the anticoagulant efficacy of each aptamer as the ratio between the coagulation rates, defined as the slope of the line fitting the initial increase of the normalized scattering intensity plotted as a function of time, in the exclusive presence of thrombin and in the presence of both thrombin and each tested aptamer. The results, reported in Figure 6b, notably showed cycTBA II as a more effective anticoagulant agent than TBA, while cycTBA III and cycTBA IV behaved similarly to the first developed cycTBA [START_REF] Riccardi | Stability is not everything: the case of the cyclization of the thrombin binding aptamer[END_REF] proving to be slightly less potent than TBA. Interestingly, cycTBA I was the less potent aptamer of the investigated series. b) Coagulation efficacy (defined as the ratio between the coagulation rate in the exclusive presence of thrombin and in the presence of both thrombin and each aptamer) of cycTBAs I-IV with respect to unmodified TBA and cycTBA.

CONCLUSIONS

Among the known aptamers able to fold into stable G-quadruplex structures, TBA is probably the most studied system, object of wide investigations for its intriguing therapeutic and diagnostic potential, and also used as effective tool to validate sensing strategies. [START_REF] Musumeci | Fluorescence sensing using DNA aptamers in cancer research and clinical diagnostics[END_REF] In a recent communication, [START_REF] Riccardi | Stability is not everything: the case of the cyclization of the thrombin binding aptamer[END_REF] we anticipated that the TBA properties could be markedly improved by covalently linking the extremities of its sequence. Indeed, as a proof-of-concept we prepared a first TBA analogue circularized via a 20 atom-long linker, which exhibited a remarkable serum resistance and a notably increased thermal stability of its G-quadruplex core. Unfortunately, its binding affinity to thrombin and anticoagulant activity were sensibly reduced compared to native TBA. [START_REF] Riccardi | Stability is not everything: the case of the cyclization of the thrombin binding aptamer[END_REF] Thus, stimulated by the favourable impact of cyclization on the aptamer biophysical properties, in the present work we prepared a small library of novel cyclic TBA derivatives containing different flexible circularizing linkers of variable length, ranging from 22 to 48 atoms. Our aim was to obtain a good compromise between increasing the compactness of the aptamer G-quadruplex core, necessary to improve the oligonucleotide resistance to nucleases, and maintaining some structural flexibility, somehow crucial for thrombin recognition.

Four novel derivatives were thus prepared: in particular, cycTBAs I and II were circularized by using the oxime ligation method, while cycTBAs III and IV were obtained using a Cu(I)-catalysed azide-alkyne cycloaddition approach. With this series of novel cyclic TBAs, we have therefore tested and compared two different cyclization procedures, both providing in high yields and few synthetic steps the desired cyclic aptamers, characterized by connecting linkers with different chemical properties and peculiar structural elements.

Then, we investigated the structural and biophysical properties of these cyclic TBA analogues by using a combined approach including spectroscopic, electrophoretic as well as chromatographic techniques. UV and CD spectroscopic data confirmed that cycTBAs I-IV adopted antiparallel, chair-like G-quadruplex conformations, essentially similar to that of their natural counterpart. The thermal stability of these structures was strictly solution-dependent. In fact, the melting temperatures of cycTBAs I-IV were in all cases higher than that of unmodified TBA (ΔT m ranging from +1 to +17 °C) in a K + -rich buffer solution, while, in the case of a Na + -rich medium, only cycTBAs I and II proved to be more stable than unmodified TBA, with ΔT m of +10 and +17 °C, respectively. In all the studied buffer solutions, cycTBA II showed the most favourable thermodynamic properties, indicating that its 30 atom-long circularizing linker ensured the highest stabilization of the G4 structure in the investigated series.

Most importantly, the serum resistance of cycTBAs I-IV proved to be always increased with respect to TBA. Indeed, cycTBAs I and II showed a 8.1-and 6.6-fold enhancement in stability to nuclease digestion, with t 1/2 of 4.3 and 3.5 h, respectively, while cycTBAs III and IV displayed a 4.9-fold increased stability, with t 1/2 values slightly lower than 3 h.

Finally, light scattering experiments showed cycTBA II as the most potent anticoagulant agent, with a higher thrombin clotting inhibitory activity than unmodified TBA and the other cycTBAs.

Thus, the obtained results demonstrated an effective dependence of the chemical properties and anticoagulant activity of the synthesized cyclic analogues of TBA on varying the length and nature of the circularizing linker. In particular, among all the designed derivatives, the best candidate proved to be cycTBA II. In this compound the linker of 30 atoms length seems to nicely combine, on one hand, a marked stabilization of the G-quadruplex core and an increased nuclease resistance, and, on the other hand, an improved anticoagulant activity with respect to both unmodified TBA and the previously developed cyclic analogue. Interestingly, the lipophilic benzene ring in the connecting linker could also play a role in the observed enhanced stability by establishing additional stacking interactions with the G-tetrads, finally promoting the G4 structuring and subsequent recognition/inhibition of the protein.

In contrast, in the case of cycTBA I, carrying a linker very similar in length to our first prototype cycTBA but containing one additional nucleotide, and thus one additional phosphate unit, the higher stability of the G4 core and the enhanced resistance to nuclease digestion was not accompanied by an improved thrombin inhibition activity, in contrast proving to be the worst inhibitor of the series. The case of cycTBA I indicates that, as also observed for cycTBA, [START_REF] Riccardi | Stability is not everything: the case of the cyclization of the thrombin binding aptamer[END_REF] a short linker (20-22 atoms) also including additional rigid structural elements somehow prevented the optimal recognition/interaction with the protein.

In turn, cycTBAs III and IV showed a similar behaviour, with little or no stabilization of the Gquadruplex structure, a higher but not exceptional resistance to enzymatic degradation and a reduced antithrombin activity compared to unmodified TBA. In these cyclic analogues, the linker is probably too long (34-48 atoms), somehow causing steric hindrance and/or too much flexibility to the system. As in the case of cycTBA I, the presence of additional phosphates (respectively one and two for cycTBAs III and IV) could also play a role, impairing G-quadruplex structuring and target recognition.

Taken together, the present data also demonstrated that the anticoagulant efficacy of TBA analogues is controlled by multiple phenomena, in which the aptamer enzymatic stability and structural preorganization of their functional folding are only one side of the coin.

Indeed, our results confirmed that a very high rigidity may be detrimental for an effective thrombin binding and inhibition, as suggested in our previous work, [START_REF] Riccardi | Stability is not everything: the case of the cyclization of the thrombin binding aptamer[END_REF] highlighting that long and bulky modifications can also hamper the thrombin-aptamer interaction due to steric hindrance. Finally, the interaction with the target protein seems to be negatively affected by the insertion of additional phosphate groups in the linker, that likely alter the delicate balance between electrostatic and apolar interactions leading to effective thrombin-aptamer recognition.

The remarkable thermal stability and the general properties of cycTBA II, incorporating a flexible circularizing 30 atom-long linker without additional phosphate groups, suggest that this modification could be successfully extended also to other G-quadruplex-forming aptamers to enhance their properties so to develop new, efficient tools for biomedical, supramolecular chemistry and nanotechnology applications. [START_REF] Platella | G-quadruplex-based aptamers against protein targets in therapy and diagnostics[END_REF]

Figure 1 .

 1 Figure 1. Schematic representation of a cyclic TBA structure -with a linker connecting the 3′-and 5′-ends of the oligonucleotide sequence -compared to the antiparallel G-quadruplex structure formed in solution by unmodified TBA.

(0. 1 M 16 : 17 :

 11617 in CH 3 CN, 60 s coupling time). The capping step was performed with acetic or phenoxyacetic (Pac) anhydride using commercially available solutions (Cap A: acetic anhydride:pyridine:THF 10:10:80 v/v/v and Cap B: 10 % N-methylimidazole in THF) for 10 s or Cap A: Pac anhydride:pyridine:THF 10:10:80 v/v/v and Cap B: 10 % N-methylimidazole in THF) for 60 s. The oxidation step was performed with a standard, diluted iodine solution (0.1 M I 2 , THF:pyridine:water 90:5:5, v/v/v) for 15 s. Cyclization by oxime ligation (cycTBA I). Solid-supported oligonucleotide 6 was introduced into a sealed vial and treated with concentrated aqueous ammonia (2 mL) for 5 h at 55 °C. After evaporation, the resulting linear oligonucleotide 7 was purified by HPLC (189 nmol). Analytical RP-HPLC retention time: 11.95 min, MALDI-TOF MS: m/z: [M-H] - = calc. 5682.83, found 5683.77. Then 50 mM KCl (400 µL) and 100 mM NaIO 4 (5 µL) were added to a solution of 7 in water (100 µL). After 2 h, formaldehyde was removed by evaporation (speedvac) and 400 µL of 0.4 M NH 4 Ac buffer (pH = 4.2) were added. The resulting cycTBA I was purified by HPLC affording 74 nmol of the target compound (40 % isolated yields). Analytical RP-HPLC retention time: 10.08 min, MALDI-TOF MS: m/z: [M-H] -= calc. 5360.42, found 5360.74. Cyclization by bis-oxime ligation (cycTBA II). Solid-supported oligonucleotide 10 was treated with a 7 M ammonia solution in methanol at r.t. for 3 h. After washings with methanol, the CPG beads were transferred into a vial and treated with a 50 mM CH 3 ONH 2 HCl solution in 0.4 M NH 4 Ac buffer, pH = 4.2 (2 mL) for 2 h at r.t. The linear TBA 11 was purified by size exclusion https://doi.org/10.1016/j.bioorg.2019.103379 chromatography cartridges (NAP-10), recovering 320 nmol of the target compound which were then lyophilized. Analytical RP-HPLC retention time: 11.56 min, MALDI-TOF MS: m/z: [M-2H + NH 4 + ] -= calc. 5196.41, found 5197.78. The crude was dissolved in water (120 µL) and then 50 mM KCl (500 µL), 0.4 M NH 4 Ac buffer pH = 4.2 (3.0 mL) and 360 nmol of terephthalaldehyde (10 mM in methanol, 36 µL) were sequentially added. After 3 h at r.t., the crude was purified by size exclusion chromatography cartridges (NAP-25) affording cycTBA II in 58 % overall yields (186 nmol). Analytical RP-HPLC retention time: 13.42 and 13.75 min for E/Z isomers,[47] MALDI-TOF MS: m/z: [M-H] - = calc. 5277.49, found 5278.83. Cyclization by CuAAC (cycTBAs III and IV). Oligonucleotide-functionalised CPG beads 16 and 17 were introduced into a sealed vial and treated with concentrated aqueous ammonia (2 mL) overnight at 40 °C. The supernatant was withdrawn and evaporated. The linear modified TBAs 16 and 17 were dissolved in water and then analyzed by UV, HPLC and MALDI-TOF MS. The amount of crude 16 and 17, determined by UV measurements at 260 nm, was 0.93 and 0.95 µmol, respectively. Analytical RP-HPLC retention time: 16.69 min, MALDI-TOF MS: m/z: [M-H] - = calc. 5434.64, found 5433.21. Analytical RP-HPLC retention time: 16.89 min, MALDI-TOF MS: m/z: [M-H] - = calc. 5690.83, found 5691.92. Protocol for TBA cyclization. Freshly prepared, 100 mM aq. solutions of CuSO 4 (5 eq, 5 μmol, 50 μL) and 500 mM sodium ascorbate (10 eq, 10 μmol, 40 μL), were added to a solution of linear TBA 16 (0.93 µmol) or 17 (0.95 µmol) dissolved in 1 mL of H 2 O and KCl (50 mM, 4 mL). The vials containing the resulting mixture were sealed and stirred at r.t. After 1 h, the mixtures were purified by size exclusion chromatography (SEC) on a NAP-25 column (GE-Healthcare), giving crude compounds cycTBAs III and IV, which were frozen and lyophilized. The crudes were then purified by HPLC affording cycTBA III (255 nmol, 27 % overall yields, analytical RP-HPLC retention time: 12.66 min, MALDI-TOF MS: m/z: [M-H] -= calc. 5434.64, found 5435.67) and cycTBA IV (350 nmol, 37 % overall yields, analytical RP-HPLC retention time: 13.14 min, MALDI-TOF MS: m/z: [M-H] -= calc. 5690.83, found 5690.92) which were frozen and lyophilized.

2. 9 .

 9 Anticoagulant activity by light scattering measurements. Evaluation of the anticoagulant activity of cycTBAs I-IV and unmodified TBA, used as control, was performed by means of light scattering (LS) experiments analysing the normalized autocorrelation functions, which give information on the size of the species in the system at a certain time, and monitoring the increase of the light scattered intensity upon conversion of fibrinogen to fibrin catalysed by thrombin.[31] A 1.2 μM solution of fibrinogen in PBS was placed in a LS cuvette and left to equilibrate in the instrument for 20 min. Then, thrombin was added up to a final concentration of 5 nM and the light scattered intensity was registered every 20 s for at least 2 h. Experiments with the different aptamers were performed by adding each oligonucleotide, previously annealed in PBS, to the fibrinogen solutions before addition of thrombin at a concentration providing a thrombin:oligonucleotide molar ratio of 1:5. All the experiments were performed at least in triplicate. By plotting the normalized scattered intensity nI https://doi.org/10.1016/j.bioorg.2019.103379 nI = (I-I 0 )/I 0where I is the scattered intensity at time t and I 0 the scattered intensity at t=0, as a function of time and by linearly fitting the initial increase of nI upon the lag time, it is possible to obtain an estimation of the coagulation rate from the slopes of the fitted lines. The thrombin inhibiting properties of the different oligonucleotides were then compared by calculating the anticoagulant efficacy as the ratio between the coagulation rate in the exclusive presence of thrombin and in the concomitant presence of thrombin and aptamers.[START_REF] Troisi | Several structural motifs cooperate in determining the highly effective anti-thrombin activity of NU172 aptamer[END_REF]

aminooxytriethyleneglycol phosphoramidite 4 starting from phthalimidotriethyleneglycol 1 [ 47 ]Scheme 1 .

 1471 Scheme 1. Synthesis of MMTr-aminooxytriethyleneglycol phosphoramidite 4.

Figure 2 .

 2 Figure 2. Overlapped CD spectra of cyclic TBAs I-IV (orange, green, light blue and magenta lines, respectively), recorded at 15 °C in both the selected K + -(a) and Na + -rich (b) buffer solutions, in comparison with unmodified TBA (black line). The investigated oligonucleotides were analyzed at 2 µM concentration.

  the overlapped CD-melting profiles of cycTBAs I-IV are reported in terms of folded fraction as a function of the temperature, while in Figures S13-S16 the overlapped CD-melting and CDannealing profiles for each compound are shown.In the K + -rich buffer (Figure3a), all the cyclic TBA analogues -except cycTBA IV -revealed a noteworthy increased thermal stability compared to unmodified TBA, although exhibiting different behaviours. Indeed, cycTBAs I, II and III (Figures3a and S13-15a) showed apparent T m values respectively of ca. 63, 68 and 61 °C (with ΔT m of + 12, + 17 and + 10 compared to unmodified TBA, Table

Figure 3 .

 3 Figure 3.Overlapped CD-melting profiles (recorded at 295 nm, with a heating rate of 1 °C/min) reporting the fraction of folded cycTBAs I-IV (orange, green, light blue and magenta lines, respectively) as a function of the temperature, in both the selected K + -(a) and Na + -rich (b) buffers, in comparison to unmodified TBA (black line). All the investigated oligonucleotides were analysed at 2 µM concentration.

Figure 4 .

 4 Figure 4. 15 % polyacrylamide gel electrophoresis under native conditions of TBA and the cycTBAs samples at 20 μM concentration in the selected K + -(lanes 1-5) and Na + -rich (lanes 6-10) buffer solutions, run at 100 V at r.t. for 75 min in TBE 0.5X buffer; lane 1: TBA; lane 2: cycTBA I; lane 3: cycTBA II; lane 4: cycTBA III; lane 5: cycTBA IV; lane 6: TBA; lane 7: cycTBA I; lane 8: cycTBA II ; lane 9: cycTBA III; lane 10: cycTBA IV.

Figure 5 .

 5 Figure 5. Enzymatic resistance experiments on TBA and cycTBAs I-IV incubated in 80 % fetal bovine serum (FBS) as monitored by SAX-HPLC analysis up to 26 h. Chromatographic peak area is expressed as percentage of the each cyclic TBA remaining area (see legend for detail) normalized to the initial one for all the analysed time points (8 and 11 total time points collected in 1 day, respectively for TBA and cycTBAs I-IV). Data are reported as mean values ± SD (error bars) for multiple determinations. Obtained values were also fitted with an equation for first order kinetics, allowing the determination of the half-life of each aptamer (t 1/2 ).

Figure 6

 6 Figure 6. a) Coagulation curves of fibrinogen in the presence of thrombin and different anticoagulant agents (TBA; cycTBA; cycTBAs I-IV, see inset for details) at thrombin:aptamer 1:5 molar ratio.

Name Linker connecting sequence (5'-3') Linker connecting length cycTBA 20 atoms cycTBA I 22 atoms cycTBA II 30 atoms cycTBA III 34 atoms cycTBA IV

  By using several biophysical techniques, the influence of the different inserted linkers on the conformational behaviour, thermodynamic stability, nuclease resistance and anticoagulant activity of cycTBAs I-IV was here analysed.In particular, the novel cyclic analogues were studied, in comparison with the unmodified TBA, by UV, CD, CD-melting, gel electrophoresis and SE-HPLC techniques. In order to evaluate the effects of different saline conditions on the structuring ability of these aptamers, all these investigations were carried out in two different phosphate buffer solutions, containing a high content of K + (10 mM KH 2 PO 4 /K 2 HPO 4 , 70 mM KCl, 0.2 mM EDTA, pH 6.9, here indicated as K + -rich buffer) or of Na + (i.e. PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM NaH 2 PO 4 /Na 2 HPO 4 , 1.8 mM KH 2 PO 4 /K 2 HPO 4 , pH 7.0, here indicated as Na + -rich buffer), respectively mimicking the intra-and extracellular environment.

Monomethoxytritylaminooxytriethyleneglycol 2-cyanoethyl N,N-diisopropyl-phosphoramidite 4. Compound 3 (

	https://doi.org/10.1016/j.bioorg.2019.103379
	Monomethoxytritylaminooxytriethyleneglycol 3. Hydrazine hydrate (437 µL, 9.0 mmol) was Synthesis of the unmodified TBA. TBA was synthesized according to reported protocols[42] by
	added to a solution of phthalimide triethyleneglycol 1[47] (970 mg, 3.28 mmol) in pyridine (14 using commercially available CPG solid support and base-protected 2-cyanoethyl, N,N-
	mL). The reaction, left under stirring, produced a white precipitate already after 5 min. After 1 h, diisopropylphosphoramidite monomers and exploiting standard phosphoramidite chemistry.
	this precipitate was removed by filtration and the solution was then co-evaporated three times with Analytical RP-HPLC retention time: 9.81 min, MALDI-TOF MS: m/z: [M-H] -: for
	anhydrous pyridine. Crude compound 2 was dissolved in anhydrous pyridine (26 mL) at 0 °C, and C 150 H 186 N 57 O 94 P 14 calc.: 4725.04; found: 4725.70.[42]
	then MMTrCl (1.g, 3.2 mmol) was added in three aliquots over 30 min. The solution was stirred at Synthesis of cyclic TBA derivatives. The TBA sequence was elongated from commercially
	r.t. for 2.5 h and then methanol (1 mL) was added. After 5 min the solution was diluted with CH 2 Cl 2 available glycerol-derivatized solid support 5, levulinoyl-derivatized solid support 9[49] or azide-
	(50 mL) and washed with saturated NaHCO 3 aqueous solution (50 mL) after extraction with CH 2 Cl 2 derivatized support 12[48] on an ABI 394 DNA synthesizer, with a 1 μmol scale cycle, according to
	(2 x 50 mL). The organic layers were then dried over anhydrous Na 2 SO 4 and taken to dryness. The standard phosphoramidite chemistry protocols. The detritylation step was performed for 65 s using
	crude was purified by flash chromatography on a silica gel column eluted with a gradient from 0 to 3 % TCA in CH 2 Cl 2 . For the coupling step: benzylmercaptotetrazole (0.3 M in anhydrous CH 3 CN)
	50 % of ethyl acetate in cyclohexane containing 1 % TEA, which afforded compound 3 in 67 % was used as the activator with commercially available thymidine or N 2 -iBu or N 2 -tBuPac-2′-
	isolated yields (980 mg). deoxyguanosine-2-cyanoethyl, N,N-diisopropylphosphoramidite (0.075 M in CH 3 CN, 30 s coupling
	1 H NMR (CDCl 3 , 400 MHz, Figure S1a): δ 2.31 (1H, OH), 3.41-3.58 (10H, CH 2 OH, O-CH 2 -CH 2 -time), MMTrN-EG 3 phosphoramidite 4 (0.1 M in CH 3 CN, 60 s coupling time), DMTr-EG 4 -
	O), 3.71-3.76 (5H, CH 3 O, NO-CH 2 ), 6.72-6.75 (2H, Ar), 6.51 (1H, NH), 7.12-7.25 (12H, Ar). phosphoramidite (0.1 M in CH 3 CN, 60 s coupling time) or pent-4-yn-1-yl phosphoramidite 13[48]
	13 C MR (CDCl 3 , 100 MHz, Figure S1b): δ 55.2, 61.8, 69.7, 70.4, 70.5, 72.5, 73.2, 73.5, 113.0,
	126.8, 127.6, 129.1, 130.4, 136.6, 144.7, 158.3.
	HRMS (ESI/Q-TOF): [M + H] + calc. for C 26 H 32 NO 5 438.2280, found 438.2276.
	650 mg, 1.5 mmol) was coevaporated twice with anhydrous acetonitrile and then
	dissolved in anhydrous CH 2 Cl 2 (10 mL). Anhydrous diisopropylethylamine (DIEA, 325 µL, 1.8
	mmol) and 2-cyanoethyl-N,N-diisopropylphosphoramidochloridite (330 µL, 1.5 mmol) were then
	added to the compound 3 solution under argon atmosphere. After 30 min under stirring, water (0.5
	mL) was added and the organic phase was washed with saturated aqueous NaHCO 3 (70 mL) and
	extracted with CH 2 Cl 2 (2 x 50 mL). The organic layer was then dried over anhydrous Na 2 SO 4 and
	taken to dryness. The crude was purified by silica gel chromatography (cyclohexane/AcOEt/ with 4
	% TEA) from 1:0 to 7:3 to afford pure phosphoramidite 4 in 87 % overall yields (823 mg).
	1 H NMR (CDCl 3 , 300 MHz, Figure S2a): δ 1.11-1.16 (12H, NiPr 2 ), 2.54-2.59 (2H, CH 2 CN), 3.44-
	MALDI-TOF MS studies were performed on a Shimadzu Assurance equipped with a 337 nm 3.80 (19H, CH 2 OP, O-CH 2 -CH 2 -O, N-O-CH 2 , CH 3 O, N-CH), 6.50 (1H, NH), 6.75-6.78 (2H, Ar),
	nitrogen laser. Spectra were recorded in negative ion mode, using 2,4,6-trihydroxyacetophenone 7.16-7.28 (12H, Ar).
	(THAP) with 10 % ammonium citrate as a matrix in H 2 O/CH 3 CN, 1:1, v/v. Aqueous sample 13 C NMR (CDCl 3 , 100 MHz, Figure S2b): δ 19.2, 23.5, 23.6, 23.7, 42.1, 54.2, 57.5, 61.5, 68.7,
	solutions were mixed with the matrix as 1:1 or 1:5 v/v ratio and 1 µL of the solution was then 69.4, 69.6, 70.1, 72.1, 72.5, 111.9, 116.7, 125.7, 126.6, 128.1, 129.3, 135.6, 143.7, 157.3.
	deposited on the stainless steel plate for drying. 31 P NMR (CDCl 3 , 121 MHz, Figure S2c): δ 148.6.
	2.2. Synthetic procedures HRMS (ESI/Q-TOF): [M + H] + = calc. for C 35 H 49 N 3 O 6 P 638.3359, found 638.3351.

2.1. General methods.

All the reagents and solvents were of the highest commercially available quality and were used as received. Acrylamide, GelRed Nucleic Acid Stain, and Tris-Borate-EDTA (TBE) 10X were purchased from VWR. Ammonium persulfate (APS), tetramethylethylenediamine (TEMED), Fetal Bovine Serum (FBS) were purchased from Sigma Aldrich.

The solid support 12 and the pentynyl phosphoramidite 13

[START_REF] Pourceau | Azide solid support for 3′-conjugation of oligonucleotides and their circularization by click chemistry[END_REF]

were synthesized according to reported protocols. Tetraethylene glycol phosphoramidite is commercially available from ChemGenes.

RP-HPLC analyses were performed at 60 °C on a Macherey Nagel Nucleodur 100-3 C 18 ec column (length: 75 mm, ID: 4.6 mm), using a linear gradient of B (0 to 30 %) in A for 20 min at 1 mL min -1 . Solution A: 1 % CH 3 CN/0.05 M TEAA, solution B: 80 % CH 3 CN/0.05 M TEAA.

  Phenomenex). The elution was monitored at λ = 254 nm with 0.35 mL min -1 flow rate. The mobile phases consisted of the K + -(10 mM KH 2 PO 4 /K 2 HPO 4 , 70 mM KCl, 0.2 mM EDTA, pH = 6.9) or Na + -rich (PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM NaH 2 PO 4 /Na 2 HPO 4 , 1.8 mM KH 2 PO 4 /K 2 HPO 4 , pH = 7.0) buffer solutions. cycTBAs I-IV were investigated at 2 µM concentration in the selected buffer solution and slowly annealed before the HPLC analysis. The error associated with the retention time (t R ) determination is within ±5 %.IV in biological media was analysed by incubating the oligonucleotides -previously annealed in PBS buffer -at 10 µM concentration in 80 % of fetal bovine serum (FBS) at 37 °C. Then, at fixed times, 20 µL of the samples were collected, inactivated at 95 °C for 3 min and finally stored at -20 °C until their analysis. These aliquots were then diluted to 100 µL with solution A (20 mM KH 2 PO 4

	Na + -rich buffer solutions were loaded on 15 % polyacrylamide gels in TBE (Tris-Borate-EDTA,
	0.5X) as running buffer. All the samples were supplemented with 5 % glycerol just before loading
	and then run, under native conditions, at 100 V at r.t. for 75 min. Gels were stained with a GelRed

Using these equations, T m and ΔH v.H change values of the unfolding processes were calculated and the reported results provided the best fit of the experimental melting data. The ΔS v.H values were calculated by equation ΔS v.H = ΔH v.H /T m . and free energy change values (at 298 K) by the equation ΔG v.H = ΔH v.H -TΔS v.H . All the thermodynamic parameters are expressed in kJ/mol with the corresponding errors within ± 10 %. 2.6. Non-denaturing polyacrylamide gel electrophoresis (PAGE). Slowly annealed samples of unmodified TBA and cycTBAs I-IV dissolved at 20 μM concentration in both the selected K + -and solution (supplemented with NaCl 0.1 M) for 20 min and finally visualized with a UV transilluminator (BioRad ChemiDoc XRS). Each experiment was performed in triplicate. 2.7. Size exclusion chromatography. SE-HPLC analyses were performed using an Agilent HPLC system, equipped with a UV/vis detector, on a Yarra 3 µm analytical column (300 x 4.60 mm; https://doi.org/10.1016/j.bioorg.2019.103379 2.8. Enzymatic stability assays by Nucleogen-SAX HPLC analysis. The stability of cycTBAs Iaq. solution, pH 6.9, containing 20 % (v/v) CH 3 CN), filtered on 0.2 µm filter and finally analysed by HPLC. For these experiments, a Nucleogen DEAE 60-7 column (Macherey-Nagel, 7 µm, 125 x 4 mm) with a flow rate of 0.8 mL min -1 , detection at 254 nm and a linear gradient of B (0 to 100 %) in A in 30 min was used, until disappearance of the peak attributed to the intact oligonucleotide. Solution A: 20 mM KH 2 PO 4 /K 2 HPO 4 aq. solution, pH 6.9, containing 20 % (v/v) CH 3 CN; solution B: 1 M KCl, 20 mM KH 2 PO 4 /K 2 HPO 4 aq. solution, pH 7.0, containing 20 % (v/v) CH 3 CN. Each experiment was performed at least in triplicate. The oligonucleotide area under the peak, at each collected time, was then calculated and normalized with respect to the initial one. Percentages of the remaining area are reported as mean values ± SD for multiple determinations. Half-life times of each aptamer (t 1/2 ) was obtained by fitting the values with an equation for kinetics of one order.

  ://doi.org/10.1016/j.bioorg.2019.103379 52 and 59 °C, with a ΔT m of + 10 and 17 °C vs. unmodified TBA (Table 2, Figures 3b, S13b and S14b). However, in these saline conditions mimicking the extracellular environment, cycTBAs III and IV (Figures 3b, S15b and S16b) exhibited negligible differences compared to unmodified TBA,

	TBA
	cycTBA I
	cycTBA II
	cycTBA III
	cycTBA IV

https

Table 2 .

 2 Apparent T m values (estimated from the maxima of the first derivative plots) and thermodynamic parameters (ΔH v.H , ΔS v.H and ΔG v.H

		42]
				K + -rich buffer
		CD	ΔT m	ΔH v.H	ΔS v.H	ΔG v.H
		T m (°C) ± 1	(°C)	(kJ mol -1 )	(kJ mol -1 K -1 )	(kJ mol -1 )
	TBA	51	-	152	0.47	12
	cycTBA I	63	+ 12	144	0.43	16
	cycTBA II	68	+ 17	141	0.41	19
	cycTBA III	61	+ 10	143	0.43	15
	cycTBA IV	52	+ 1	130	0.40	11
				Na + -rich buffer
		CD	ΔT m	ΔH v.H	ΔS v.H	ΔG v.H
		T m (°C) ± 1	(°C)	(kJ mol -1 )	(kJ mol -1 K -1 )	(kJ mol -1 )
	TBA	42	-	147	0.47	7
	cycTBA I	52	+ 10	144	0.44	13
	cycTBA II	59	+ 17	132	0.40	13
	cycTBA III	42	0	127	0.41	8
	cycTBA IV	42	0	148	0.47	8

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.