Hadi Hijazi

Frédéric Leroy

Guillaume Monier

Gabin Grégoire

Evelyne Gil

Agnès Trassoudaine

Vladimir G Dubrovskii

Dominique Castelluci

Nebile Isik Goktas

Ray R Lapierre

Yamina André

Christine Robert-Goumet

Dynamics of Au Droplet Formation on SiO2/Si(111) Surface

Au droplets are used as a catalyst for the growth of nanowires on Si(111) substrate via the vapor-liquid-solid (VLS) mechanism. The dewetting of a Au thin film is the most common method to obtain these droplets. The control of this step is crucial to adjust the density and the diameter of the nanowires during VLS growth. When the Si(111) substrate is covered with a silicon dioxide layer, the kinetics of Au droplet formation is strongly modified. The dependence of the diameter and spatial distribution of the droplets on the surface have been studied by scanning electron microscopy with respect to the thickness of the silicon dioxide layer, the thickness of the Au film and the temperature of the substrate during deposition and post-deposition annealing. In-situ low energy electron microscopy and low energy electron diffraction revealed the dynamics of the Au droplet formation after annealing. The Au droplets are shown to catalyze the decomposition of silicon dioxide at high temperature (> 650-700 °C) and form a wetting layer of Au-(Ö3´Ö3)-Si(111). Consequently, the droplets absorb silicon atoms from the substrate, migrate perpendicular to the atomic steps and grow by the Smoluchowski ripening process.

1-INTRODUCTION

Au-assisted growth of nanowires (NWs) based on different materials such as ZnO, GaAs, GaN, InGaAs, Si, Ge and InAs have been performed using many growth techniques: molecular beam epitaxy (MBE), metalorganic vapor phase epitaxy (MOVPE) and hydride vapor phase epitaxy (HVPE). [START_REF] Sharma | Synthesis of Thin Silicon Nanowires Using Gold-Catalyzed Chemical Vapor Deposition[END_REF][START_REF] Orvatinia | Effect of Catalyst Layer on Morphology and Optical Properties of Zinc-Oxide Nanostructures Fabricated by Carbothermal Evaporation Method[END_REF][START_REF] Joyce | Unexpected Benefits of Rapid Growth Rate for III-V Nanowires[END_REF] The Au droplets act as a mediator between the growth precursors and the solid NW phase. In VPE methods, the Au droplets have also a catalytic role; i.e., they assist in the decomposition of the precursors on their surface. The Au droplets subsequently collect and accumulate atoms, which are deposited as a solid phase at the substrate-droplet interface once saturation is reached inside the droplet. Depending on the temperature, different growth mechanisms can occur: the vapor-liquid-solid (VLS) process where the catalyst metallic droplet is liquid during NW growth, [START_REF] Gil | Record Pure Zincblende Phase in GaAs Nanowires Down to 5 nm in Radius[END_REF][START_REF] Ramdani | Fast Growth Synthesis of GaAs Nanowires with Exceptional Length[END_REF] and the vapor-solid-solid (VSS) process where the growth occurs via solid metallic catalyst droplets. [START_REF] Lekhal | Catalyst-Assisted Hydride Vapor Phase Epitaxy of GaN Nanowires: Exceptional Length and Constant Rod-like Shape Capability[END_REF] In both cases, the diameters and the position of the resulting NWs on the surface are pre-defined by the size and distribution of the Au catalysts. Thus, the control of the morphology and the spatial distribution of the Au droplets on the substrate are crucial.

Several methods are used to control the Au droplet deposition. The spin coating of a colloidal solution containing Au nanoparticles and the dewetting of ultra-thin metallic films are the most common methods. The latter approach has been an important topic in material science and technology. [START_REF] Kwon | Comparison of the Agglomeration Behavior of Au and Cu Films Sputter Deposited on Silicon Dioxide[END_REF][START_REF] Ruffino | Towards a Laser Fluence Dependent Nanostructuring of Thin Au Films on Si by Nanosecond Laser Irradiation[END_REF][START_REF] Ruffino | Controlled Dewetting as Fabrication and Patterning Strategy for Metal Nanostructures[END_REF][START_REF] Thompson | Solid-State Dewetting of Thin Films[END_REF][START_REF] Henley | Pulsed-Laser-Induced Nanoscale Island Formation in Thin Metal-on-Oxide Films[END_REF][START_REF] Ruffino | Normal and Abnormal Grain Growth in Nanostructured Gold Film[END_REF] The high surface-to-volume ratio of these films may produce a high driving force for dewetting, which tends to agglomerate matter to reduce the total surface/interface energy of the system. [START_REF] De Gennes | Wetting: Statics and Dynamics[END_REF][START_REF] Bonn | Wetting and Spreading[END_REF] Consequently, the size distribution of the droplets is determined by the kinetics of mass transfer that depends on many parameters such as the temperature, the film thickness and the substrate chemical nature, orientation and roughness. Solid state dewetting and ripening have been mostly studied and described in non-reactive systems, [START_REF] Leroy | Catalytically Enhanced Thermal Decomposition of Chemically Grown Silicon Oxide Layers on Si(001)[END_REF] whereas chemical reactions between the film and the substrate may occur and lead to complex behaviors such as triple line pinning at reactive fronts [START_REF] Leroy | Catalytically Enhanced Thermal Decomposition of Chemically Grown Silicon Oxide Layers on Si(001)[END_REF] or modification of wetting properties by alloying phenomena [START_REF] Ressel | Wetting of Si Surfaces by Au-Si Liquid Alloys[END_REF] . In previous work, [START_REF] Hijazi | Influence of Silicon on the Nucleation Rate of GaAs Nanowires on Silicon Substrates[END_REF] we demonstrated that GaAs NW growth via the VLS-HVPE mechanism mediated by Au droplets on bare Si(111) substrate was ineffective whereas a high density of structures nucleate and grow on oxidized Si(111) substrate. We assumed that, in addition to its role of forming pinning sites for catalyst droplets, [START_REF] Colombo | Ga-Assisted Catalyst-Free Growth Mechanism of GaAs Nanowires by Molecular Beam Epitaxy[END_REF][START_REF] Fontcuberta I Morral | Nucleation Mechanism of Gallium-Assisted Molecular Beam Epitaxy Growth of Gallium Arsenide Nanowires[END_REF][START_REF] Wagner | Vapor-Liquid-Solid Mechanism of Single Crystal Growth[END_REF] the presence of the oxide layer acts as a barrier against the absorption of silicon atoms from the substrate at high growth temperatures. It is the aim of the present work to unravel more precisely the role of this oxide layer on the droplet dynamics at the surface, and to determine the most appropriate conditions of Au droplets formation for subsequent NW growth with VPE methods.

We have investigated the agglomeration dynamics of an ultra-thin Au film on Si(111) substrates when covered by a silicon dioxide (SiO2) layer. The density and size distribution of the Au droplets at the surface were characterized with respect to the thickness of the Au film and oxide layer, temperature of deposition, and post-deposition annealing. Real time in-situ measurements by low energy electron microscopy have revealed the interplay between the decomposition of the SiO2 layer favored by the Au droplets and the droplet growth via static and dynamic coalescence.

The phenomena of Au droplet pinning and Si substrate dissolution on the surface are discussed.

The improved understanding of the Au dewetting mechanisms provided in this paper is not only useful for NW growth, it can be of interest for a wide range of applications. [START_REF] Ruffino | Nanoscale Voltage Tunable Tunnel Rectifier by Gold Nanostructures Embedded in SiO2[END_REF][START_REF] Temple | Optical Properties of Gold and Aluminium Nanoparticles for Silicon Solar Cell Applications[END_REF] 2-EXPERIMENTAL Three types of sample were used in this study, all fabricated using (111) n-doped silicon wafers (1 Ω.cm) but with different oxide layers: (1) a native oxide of thickness ~ 1.2 nm and a surface roughness of 0.55 nm RMS, (2) a 5 nm-thick thermal oxide of surface roughness ~ 0.35 nm RMS, grown by dry oxidation method after a chemical etching of the native oxide (by dipping in 1% HF solution for 3 minutes) and (3) a 500 nm-thick thermal oxide of surface roughness less than 0.3 nm as estimated by the supplier (SIL'TRONIX). The native oxide thickness was estimated by Xray photoelectron spectroscopy (XPS).

Au film deposition and post-deposition annealing were performed in ultra-high vacuum (UHV) with a base pressure of ~10 -7 Pa. Au (purity of 99.9998%) was deposited by a Knudsen cell at a rate of about 0.02 nm•min -1 . The deposition rate was determined by several Au deposition/XPS measurement cycles as shown in Ref. [23].

Scanning electron microscopy (SEM) was used to characterize the samples after growth or annealing, while the dynamics of the Au droplets during annealing was studied by in-situ low energy electron microscopy (LEEM) coupled with low energy electron diffraction (LEED). The SEM images were taken in a Zeiss Supra 55VP field emission scanning microscope with in-lens detection mode at a working distance of 5.5 mm and an incident energy of 3 kV. Ex-situ non-contact AFM (PSIA-XE 100) was used to characterize the sample topography. In-situ measurements (LEEM and LEED) were performed in an UHV chamber equipped with an Elmitec LEEM III microscope. [START_REF] Cheynis | Combining Low-Energy Electron Microscopy and Scanning Probe Microscopy Techniques for Surface Science: Development of a Novel Sample-Holder[END_REF] The observations were carried out in bright field mode, with electron beam energy of 3 eV. Sequential images were recorded at a rate of 1 Hz. This allowed for a good compromise between image quality and real-time acquisition. Diffraction patterns of the surface were taken by LEED (electron beam energy of 15 eV).

3-RESULTS AND DISCUSSION

Many experimental parameters may influence the size and spatial distribution of Au droplets on the surface. In the following, we discuss the effects of (i) oxide thickness and substrate temperature, (ii) Au film thickness and (iii) annealing time.

To examine the effect of the temperature on the spatial distribution of Au droplets, the equivalent of 1 nm (4 monolayers (ML)) of Au was deposited at room temperature (RT), and at 600 °C on oxidized surfaces (native oxide, 5 nm and 500 nm thermal oxides). For the samples processed at 600 °C, the substrate temperature was cooled down to RT directly after Au deposition. SEM images showing the spatial distributions of Au morphology are given in Figure 1. After deposition at RT, the density and size distribution of Au droplets on all samples are very similar (Figure 1a-c) regardless of the oxide thickness. On the other hand, the deposition at 600 °C results in different behavior for the three samples. The density and the size distribution decrease considerably when increasing the SiO2 thickness (Figure 1d-f). This is likely related to an enhanced diffusion of Au droplets on thicker SiO2 layers where the roughness, and therefore the developed surface and the morphological defects, are considerably reduced. Thus, Au atoms on thick SiO2 surfaces at high temperature can diffuse at larger distances before forming larger clusters. The high diffusivity of Au at 600°C on 500 nm-thick thermal oxide prevents the formation of nanometer-size Au droplets that are desirable for NW growth (NW growth temperature by HVPE is above 600 °C). Thus, we have chosen Si(111) substrates with native oxide and 5 nmthick thermal oxide to study the effect of the Au film thickness (from 0.2 to 2 nm) on the droplet formation kinetics. The Au films were deposited at RT, and subsequently annealed at 600 °C for 10 minutes in UHV. The SEM images of Figure 2 indicate an increase of the droplet size and decrease of the droplet density with Au film thickness (hAu). For hAu= 2 nm deposited on native SiO2, SEM images (Figure 2d) reveal a bimodal droplet size distribution. The largest droplets appear with irregular shapes surrounded by depleted zones. These droplets also have a darker contrast with respect to the smallest ones that may be attributed to a change of chemical composition. From these measurements, we attribute the irregular droplet formation to the local decomposition of the oxide layer, allowing the Au droplets to spread onto the Si(111) surface and absorb silicon to reach the liquidus composition (~30% Si at 600 °C [START_REF] Gerlach | Gold Silicon Phase Diagram[END_REF] ). This will be studied in more detail in the second part of this paper. No irregular droplets were observed on the 5 nm-thick thermal oxide, suggesting that the oxide layer was probably not entirely decomposed (as expected, a thicker SiO2 layer requires more time to be decomposed).

The nucleation probability of NWs is considerably reduced by the presence of Si in the Au droplets. [START_REF] Hijazi | Influence of Silicon on the Nucleation Rate of GaAs Nanowires on Silicon Substrates[END_REF] Therefore, the substrates that have decomposed SiO2 layers are not suitable for NW growth. Consequently, we have focused on the native oxide substrate and deposited only 1 nm of Au. This ensures the formation of an adequate density of small pure Au droplets when annealed at 600 °C. Next, sequential characterization of the surface topography was performed for different annealing times. This is given in Figure 3. The SEM images of Figure 3f-j show that the mean droplet size increased with annealing duration. The evolution of the droplet size distributions is indicated in Figure 3a-e. Initially, the as-deposited droplets have a Gaussian diameter distribution (distribution 1) with a narrow full-width-at-half-maximum (FWHM) and a mean diameter of 10 nm (Figure 3f). We have reported previously that as-deposited droplets have an average height of ~2.2 nm. [START_REF] Mahjoub | XPS Combined with MM-EPES Technique for in-situ Study of Ultra Thin Film Deposition: Application to an Au/SiO2/Si Structure[END_REF] At ta=30 min (Figure 3h), the mean size increases slightly and the FWHM becomes larger. This behavior indicates that large stable droplets grow at the expense of smaller ones to decrease the total surface to volume ratio of the droplets. [START_REF] Chakbavebty | Grain Size Distribution in Thin Films-1. Conservative Systems[END_REF][START_REF] Fischmeister | Ostwald Ripening -A Survey[END_REF][START_REF] Ruffino | Self-Organization of Gold Nanoclusters on Hexagonal SiC and SiO2 Surfaces[END_REF] However, at ta=60 min, a change of behavior occurs where large droplets with irregular shapes start to appear, as shown by the distribution 2 in Figure 3d,e. This behavior is similar to the one described earlier for a 2 nm thick Au film at ta=10 min, and was attributed to the oxide layer decomposition. We conclude that the same phenomenon occurs at later times (ta=60 min) if a smaller amount of Au (hAu=1 nm) is deposited. Figure 4 shows the evolution of the droplet density (number of droplets µm -2 ) and the fraction of area covered with droplets (coverage) with annealing time. These quantities are measured using the SEM images of Figure 3f-j. The colors of the points in Figure 4 correspond to those used for the distributions in Figure 3a-e. Clearly, the droplet density decreases with increasing ta due to the coalescence of small droplets. The coverage also decreases with ta indicating that the droplets formed by coalescence reshape to get closer to the equilibrium shape by reducing their footprint area at the surface. Note that a simple evaluation combining the coverage obtained in Figure 4 and XPS measurement shows that the total amount of gold remains stable up to 30 min of annealing, revealing that Au evaporation is unlikely to be efficient. The metal-promoted reactions of silicon depend strongly on temperature. They are complex but widely studied. [START_REF] Dallaporta | Silicon Dioxide Defects Induced by Metal Impurities[END_REF][START_REF] Hiraki | Formation of Silicon Oxide over Gold Layers on Silicon Substrates[END_REF] To further study the kinetics of oxide decomposition and Au droplet formation, in-situ LEEM coupled with LEED was used. Figures 5 (panels a to c) show the time evolution of the surface under annealing at 700 °C. The Au amount deposited is 1 nm, and the substrate is the same as the native oxidized one used above. The time t = 0 s corresponds to the stage where the droplets (dark contrast) become sufficiently large to be distinguished clearly at the surface. During the annealing, the droplets grow and migrate on the surface (as shown in Figure 3), forming behind them bright regions. These regions, characterized by micro-LEED (beam spot diameter 300 nm), have a hexagonal pattern (Figure 5e) that is characteristic of the Au-(Ö3´Ö3)-Si(111) surface reconstruction. [START_REF] Levine | Thin Film Island Growth Kinetics: A Grazing Incidence Small Angle X-Ray Scattering Study of Gold on Glass[END_REF][START_REF] Gerlach | Gold Silicon Phase Diagram[END_REF] The existence of this surface reconstruction shows that the amorphous SiO2 layer is locally completely etched. Note that the same results can be extrapolated to lower temperatures (~ 600 °C), except that much longer annealing time would be required. In addition to the oxide decomposition mediated by Au droplets, a strong decrease of the droplet density occurs at the surface. Figure 5d shows the evolution of the number of Au droplets per unit area with time. The fit shows an exponential decay with a typical time scale t=190±10 s. This decay cannot be attributed to conventional Ostwald ripening (N(t)~t -3/4 ) that is much too slow. [START_REF] Fischmeister | Ostwald Ripening -A Survey[END_REF] Smoluchowski ripening partially explains the droplet density decay. [START_REF] Smoluchowski | Growth of Oriented Au Nanostructures: Role of Oxide at the Interface[END_REF] Indeed, the largest droplets move spontaneously by lateral migration towards the silicon while etching the surrounding silica and grow fast by capturing neighboring droplets (see LEEM in-situ movie in Supporting Information). LEEM real-time measurements also show that after the passage of the migrating droplets, the Au-reconstructed areas appear as perfectly flat since no atomic step edge can be detected. The AFM images in Figure 6a corroborate this result: the surface is flattened by the droplet migration. As shown on bare Si(111) surface, the directed motion of the droplets is driven by the step structure of the underlying Si(111) substrate. [START_REF] Curiotto | Self-Propelled Motion of Au-Si Droplets on Si(111) Mediated by Monoatomic Step Dissolution[END_REF] As the surface below the oxide was initially vicinal, Au droplets that decompose the oxide climb up the staircase to efficiently absorb Si and reach the liquidus composition (~35% Si at 700 °C). This explains the preferential direction to the Au migration and track formation seen in Figure 5a-c. The track profiles (Figure 6c) behind the droplets show that the slope is locally about 0.1±0.02° misaligned with respect to the average plane of the surface. This angle is compatible with the miscut angle of the Si(111) surface and indicates that the Si steps have been dissolved by the Au-Si droplets. However, the main mechanism for the droplet density decrease is the spontaneous decay of the smallest Au droplets.

Droplets decay abruptly by edge retraction and this mechanism can be assigned to an unpinning phenomena of the triple-line SiO2-Si-Au(droplet) that initiates the motion. The driving force for unpinning is, however, difficult to assess. At 700°C Au in the Au-Si alloy droplets may slowly evaporate or dissolve into the oxide layer and Si bulk. Moreover, droplets are in competition with the Au-(Ö3´Ö3)-Si(111) surface reconstruction that needs also to consume 1 ML of Au to cover the whole surface. Ex-situ AFM measurements of the droplet morphology allows an estimation of the wetting angle q of the droplets (Figure 6b and Figure 6d). The AFM tips are made of Si with a conic shape (angle <40°) and a typical radius of curvature of 8 nm. Assuming a spherical shape, the wetting angle q is about 155±10°, which is larger than that expected on the Si(111) surface. [START_REF] Ressel | Wetting of Si Surfaces by Au-Si Liquid Alloys[END_REF] This result suggests Au-Si alloy droplets pinned at the triple-line SiO2-Si-Au(droplet) due to Au loss decay in height. This height reduction induces a force for the unpinning of the droplet edge that initiates the decay. Considering these in-situ observations and results obtained by SEM (Figure 2 and3), the mechanisms of Au droplet formation and SiO2 layer decomposition can be summarized as follows:

(i) At 600 °C-700 °C, a thin film of Au agglomerates into droplets that grow. After an incubation time for Au atoms to pass through the SiO 2 layer (depending on temperature and Au film thickness), the decomposition of the oxide layer is initiated. As reported elsewhere, [START_REF] Leroy | Catalytically Enhanced Thermal Decomposition of Chemically Grown Silicon Oxide Layers on Si(001)[END_REF][START_REF] Dallaporta | Silicon Dioxide Defects Induced by Metal Impurities[END_REF] the presence of metal atoms (including Au) at the SiO 2 /Si interface catalyzes the oxide decomposition by lowering the temperature of the reaction (800-900 °C without metal impurities). This process continues until the full decomposition of the oxide layer underneath the droplets.

(ii) After the oxide decomposition, the Au droplets start to absorb Si atoms from the substrate forming a Au-Si alloy that is liquid at T ≥ 370 °C. [START_REF] Hiraki | Formation of Silicon Oxide over Gold Layers on Silicon Substrates[END_REF]36 Therefore, the droplets tend to spread at the surface as wetting is more favorable on Si than on SiO2.

(iii) To reach the liquidus composition, the largest droplets unpin from the initial site and migrate in the upward direction of the steps and absorb Si atoms by consuming the step edge. This process favors dynamic coalescence of the droplets. Meanwhile, the smallest droplets are unstable and decay spontaneously.

4-CONCLUSION

In conclusion, we have presented a study of the agglomeration dynamics of ultra-thin Au films on oxidized Si(111) substrates. When Au is deposited at RT, there is no effect of the oxide thickness on the size distribution and the density of the droplets. Under annealing in the range of 600-700 °C, two behaviors are predominant. On thick thermal oxides, Au diffusivity is strongly enhanced and prevents the formation of nanometer size Au droplets. For thermal SiO2 layers, the decomposition of the oxide is activated by the Au atoms and this process modifies the Au droplet growth. Irregular-shape droplets start to appear after a sufficient annealing time, which is larger for smaller Au thickness deposited. This is attributed to the decomposition of the oxide layer and the absorption of Si atoms from the underlying substrate to form AuSi alloy. This process was studied in-situ by LEEM and LEED experiments supported by AFM measurements. We have shown that large Au droplets decompose the SiO2 layer and unveil the Si(111) substrate while migrating perpendicular to the step edges. Small droplets are pinned and decay spontaneously. This study brings important information for NW growth using vapor phase epitaxy methods. Our results allow the control of the density and the diameters of the Au droplets for subsequent NW growth.

Figure 1 :

 1 Figure 1: SEM images showing the distribution of as-deposited Au after evaporation of 1 nm of Au at RT (upper row) and 600 °C (lower row) on different types of substrate: (a and d) native oxide, (b and e) 5 nm-thick oxide, and (c and f) 500 nm-thick oxide. Scale bar = 100 nm.

Figure 2 :

 2 Figure 2: SEM images showing the distribution of Au droplets after deposition of (a,e) 0.2, (b,f) 0.5, (c,g) 1 and (d,h) 2 nm of Au at RT followed by annealing at 600 °C for 10 minutes under UHV. The upper two rows correspond to samples with native SiO2 and the bottom two rows correspond to samples with 5 nm thick thermal oxide. The white and yellow scale bars represent 200 nm and 50 nm, respectively.

Figure 3 :

 3 Figure 3: Distribution of Au droplets after deposition at RT and annealing at 600 °C of 1 nm of Au on the native oxide substrate for different annealing times ta: (a) 0, (b) 10, (c) 30, (d) 60 and (e) 180 minutes. The black-doted and the red distributions represent respectively distribution 1 and distribution 2 discussed in the text. The right panels are the ex-situ SEM images and the left ones are the size distributions of the droplets. After 60 minutes of annealing, a bimodal distribution appears, indicating a partial decomposition of the SiO2 layer beneath the large droplets and the formation of a Au-Si alloy after absorbing Si atoms from the substrate.

Figure 4 :

 4 Figure 4: Density of droplets and surface coverage as function of the annealing time. The colors of the data points correspond to those used for the distributions in Figure 3.

Figure 5 : 2 Figure 6 :

 526 Figure 5: (a)-(c) LEEM images showing the evolution of the Au droplet growth and migration on the surface while decomposing the native SiO2 at 700 °C. The thickness of Au is 1 nm, deposited at RT before annealing. For the complete movie see Supporting Information. (b) Droplet density as a function of time (squares) and exponential fit (red dashed line). (c) LEED pattern of the tracks formed behind the migrating droplet. The surface exhibits a Au-(Ö3´Ö3)-Si(111) reconstruction.

ACKNOWLEDGMENTS

This work has been supported financially by the CPER MMASYF of Region Auvergne-Rhone Alpes that we acknowledge gratefully. It was also funded by the program "Investissements d'avenir" of the French ANR agency, the French government IDEX-SITE initiative 16-µIDEX-0001 (CAP20-25), the European Commission (Auvergne FEDER Funds) and the Region Auvergne in the framework of the LabEx IMobS3 (ANR-10-LABX-16-01) and CPER. HH and VGD gratefully acknowledge the Russian Science Foundation for financial support under the Grant 19-72-30004. Electron microscopy was performed at the Canadian Centre for Electron

Microscopy (also supported by NSERC and other government agencies). We also thank Alain Ranguis for AFM measurements.

AUTHOR INFORMATION

Corresponding Authors

* Hadi Hijazi, ITMO University, Kronverkskiy pr. 49, 197101 St. Petersburg, Russia E-mail: hadi.hijazi@uca.fr, hijazi@itmo.ru Tel. : +33 (0) 6 60 37 32 65

SUPPORTING INFORMATION DESCRIPTION:

In-situ LEEM movie showing the evolution of the Au droplet growth and migration on the surface while decomposing the native SiO2 at 700 °C. The thickness of Au is 1 nm, deposited at RT before annealing. Total time 400 s. Electron energy: 3 eV. Field of view: 4.1 microns.


Mégane Debiais

Prof. Dr Amandine Lelievre

Jean-Jacques Vasseur

Sabine Müller
email: sabine.mueller@uni-greifswald.de

Michael Smietana
email: michael.smietana@umontpellier.fr

Dr ] M Debiais

Prof. Dr J.-J Vasseur

Boronic acid-mediated activity control of split 10-23 DNAzymes

Keywords: Split DNAzyme, RNA cleavage, Boronic Acid, Functional nucleic acid, autoligation

The 10-23 DNAzyme is an artificially developed Mg 2+dependent catalytic oligonucleotide that can cleave a RNA substrate in a sequence-specific fashion. In this study, we designed new split 10-23 DNAzymes made of two non-functional fragments, the first one carrying a boronic acid group at its 5' end, while the other one is composed of a ribonucleotide at its 3' end. We demonstrate herein that the addition of Mg 2+ ions leads to the assembly of the fragments, which in turn induces the formation of a new boronate internucleoside linkage that restores the DNAzyme activity. A systematic evaluation has been performed that identified the most performant system. Our results highlight key features for an efficient control of DNAzymes activity through the formation of boronate linkages.

The spontaneous and reversible reaction between boronic acids and cis-diols leading to the formation of cyclic boronate esters has been comprehensively investigated [1] and has found widespread use for biomedical, [2] sensing, [3] materials, [4] and supramolecular applications. [5] This very unique reaction is notably characterized by the ability of boronic acids to operate in aqueous media, and by the reactivity of the boron atom, which can be either sp 2 -or sp 3 -hybridized depending on the addition of a Lewis base. [6] In this context, we recently developed reversible DNA-and RNA-templated ligation systems occurring through the reaction of two oligonucleotide fragments, the first one composed of a boronic acid group at its 5' end, while the second one carried a ribonucleotide at its 3' end (Figure 1A). [7] The resulting ligated strand therefore differs from natural DNA by having a boronate internucleoside linkage instead of a phosphodiester. Inspired by these results we decided to evaluate if the formation of boronate esters at key positions of engineered split DNAzymes could be used to control their activity (Figure 1B). Like ribozymes, DNAzymes are capable of supporting a wide variety of reactions. [8] Yet, the most prominent and best studied representatives of the DNAzyme family are RNA cleaving DNAzymes. [9] Composed of a catalytic core and substraterecognizing domains RNA-cleaving DNAzymes catalyze the phosphodiester bond cleavage of their RNA substrate with the aid of a divalent cation. Among them, the 10-23 DNAzyme, which was named after the round and clone number of its respective in vitro selection, has been the most widely studied RNA-cleaving DNAzyme. [10] Its catalytic core is composed of a loop flanked by two complementary substrate-recognition domains. Upon binding the substrate (which is a full oligoribonucleotide or an oligodeoxyribonucleotide with a single ribonucleotide at the cleavage site), the catalytically competent structure is formed in the presence of magnesium ions, followed by cleavage of a specific phosphodiester bond. [11] The 10-23 DNAzyme catalytic core has been well studied through base or phosphate substitutions and deletions. The G9-G14 and C21-G22 regions were demonstrated to be directly involved in forming the catalytic site. [12] Since DNAzymes are easy to synthesize and to modify, many studies have been devoted to improve their stability and catalytic properties. [12d, 13] In this context split-systems, which rely on dividing a DNAzyme into a series of two or more independent fragments, able to assemble in the presence of a specific target, are emerging as novel biosensing tools for the modulation of DNAzyme activity with high spatial and temporal resolution. [14] Although split systems are yet easier to synthesize and carry less negative charges per strand, the design of efficient split systems can be a challenging task. The choice of the split site and the resulting stability and efficiency of the split DNAzymesubstrate complex are key issues that need to be considered, in particular when application of the split system in diagnostics or therapy is planned. Most of the systems rely solely on the target-driven assembly of the fragments, which however, often results in unstable and/or poorly active systems. This drawback can be counterbalanced by the use of fragments modified with chemical functionalities that upon addition of the target will be brought in close proximity to allow covalent end-joining of the fragments. Ideally, this reaction occurs without prior chemical activation. Therefore, fragments design and experimental verification of the activity of the assembled structure are required for the development of split DNAzymes.

To achieve this control, the 10-23 DNAzyme was split in two fragments individually at all thymidine positions, located either in the catalytic loop or in the flanked arm. To monitor the performance of the different systems, the RNA substrate 5'-GGAGAGAGAUGGGUGCG-3' was fluorescently labeled with ATTO680 at its 3' extremity and the cleavage rates (k) of DNAzymes Dz1-Dz6 (Figure 2C), were determined by quantification of the amount of cleaved RNA at eight different time points, analyzed by denaturing polyacrylamide gel electrophoresis (PAGE) on a LICOR DNA sequencer (Figure 2A). Cleavage reactions were performed under single turnover conditions, hence binding of the substrate and release of the cleavage products were not needed to be considered. The first fragment of each DNAzyme was modified at its 3'extremity with a cytosine ribonucleotide, while the second fragment was modified at its 5'-extremity with a boronic acid moiety. Three phosphoramidite derivatives of boronic acid, namely T Bn , Ph Bn and Napht Bn were prepared and incorporated individually at all thymidine positions of the 10-23 DNAzyme (Figure 2B and Figure 2C). The phosphoramidite of T Bn was prepared according to previously reported procedures from our laboratory. [7d, 15] Phosphitylation of commercially available 4hydroxyphenylboronic acid pinacol ester delivered the phosphoramidite derivative of Ph Bn , while the phosphoramidite of Napht Bn was obtained from 6-hydroxynaphthalene-2-boronic acid after protection of the boronic acid as a MIDA ester and phosphitylation (ESI). These derivatives were then incorporated at the 5'-end of the oligonucleotide sequences on an automated DNA synthesizer using conventional phosphoramidite chemistry.

To further understand the effect of Ph Bn and Napht Bn modifications, we evaluated also the performance of split DNAzymes where these moieties were added after the T12, T16, T25, T27 and T29 positions instead of replacing those final residues. In each case, the activity of the corresponding nonmodified split DNAzymes was evaluated as control.

The Mg 2+ -dependent catalytic ability was first evaluated on the parent Dz1, which showed a high cleavage rate (k = 0.237 ± 0.045 min -1 ) and a percentage of RNA cleaved (A) of around 90 % in accordance with previously reported data, [16] while no reaction could be observed in the absence of Mg 2+ ions (Table 1, entry 1, Figure 3A). We started our study with the unmodified split Dz2, which showed no cleavage after 2 h of incubation. This result is coherent with previous studies, which demonstrated that nonbridging phosphate oxygens located between C11 and T12 are involved in direct coordination of Mg 2+ ions (Table 1, entry 2). [12c] Consequently, it was not surprising that the replacement of C11 by a ribocytosine and T12 by T Bn did not restore the activity (Table 1, entry 3). It is interesting to note that, if the DNAzyme was split between C15 and T16 (Dz3), the enzyme is still active, although a 6-fold reduction of the cleavage rate compared to Dz1 was observed (k = 0.039 ± 0.003 min -1 , Table 1, entry 4). However, replacing C15 by a ribocytosine and T16 by T Bn significantly restored activity (k = 0.127 ± 0.045 min -1 , A = 94%), which implies that the two fragments can reconstitute more than half of the active enzyme by formation of a boronate internucleoside linkage instead of the natural phosphodiester (Table 1, entry 5). Interestingly, joining of the fragments occurs in a region where flexibility is important. A control experiment with the fragment carrying the terminal C15 and the T Bn modified fragment confirmed the requirement of having a diol system for boronate formation and to activate the split DNAzyme (k = 0.048 ± 0.007 min -1 , Table 1, entry 6). Among the other evaluated Dz3based systems (Table 1, entries 7 and 8), only the one with a fragment bearing an additional Napht Bn unit after T16 displayed an activity higher than that of the non-modified split Dz3 (k = 0.068 ± 0.004 min -1 ) but still twofold lower than with the T Bn modified fragment. When the DNAzyme is split at the junction of the catalytic core and the flanking arm (Dz4), an almost complete loss of activity is observed (k = 0.005 ± 0.001 min -1 , Table 1, entry 9), most probably due to the incapacity of the DNAzyme to adopt its active structure. As it was observed with Dz3, the replacement of C24 by a rC residue and T25 by T Bn in Dz4 (Table 1, entry 10, A = 69 %) restores the activity remarkably (k = 0.094 ± 0.017 min -1 ). All other modifications increase the cleavage rate by varying degrees (Table 1, entries 11-14), the incorporation of TPh Bn within the flanking arm being the best candidate after T Bn (k = 0.045 ± 0.005 min -1 ).

When the cut is positioned within the flanking arm between C26 and T27, the non-modified split DNAzyme Dz5 (Table 1, entry 15) recovers some activity (k = 0.087 ± 0.012 min -1 ), although only three base pairs of the 5'-fragment are complementary to the RNA substrate. Here again, the formation of a boronate internucleoside linkage significantly restores activity when C26 and T27 are replaced by a ribocytosine and T Bn respectively (Table 1, entry 16, k = 0.141 ± 0.052 min -1 , A = 85 %). Surprisingly, in this specific configuration the highest cleavage rate is obtained, when Ph Bn is added after T27. Activity of this split variant is nearly as high as that of the parent Dz1 (Table 1, entry 19, k = 0.204 ± 0.058 min -1 ), although the percentage of total RNA cleavage is somewhat lower (A = 72%). The exact nature of the activity increase observed with Y = TPh Bn in Dz5 remains unclear. It is well known that the formation of boronate esters is favored with aromatic boronic acids compared to aliphatic ones. [6] Hence, the rather high flexibility of the 3'terminal nucleotides of the 5'-fragment in Dz5 might be counterbalanced by the rigidity of the aromatic boronate ester, formed when Y = TPh Bn . By contrast, when Ph Bn replaces T27 (Y = Ph Bn ), the activity is reduced by one order of magnitude (Table 1, entry 17, k = 0.019 ± 0.003 min -1 ), most likely because the structure of the resulting DNAzyme becomes strained due to the missing nucleotide T27. Lastly, split DNAzyme Dz6 is characterized by a cut between C28 and T29, such that five base pairs at the 3'-terminus of the 5'fragment remain for binding to the substrate. Surprisingly, activity of this split variant is somewhat lower than activity of non-modified split Dz5, despite the two additional base pairs in Dz6 (Table 1, entry 21, k = 0.059 ± 0.007 min -1 , A = 48 %). However, when C28 and T29 are replaced with rC and T Bn , respectively, a remarkable increase of the cleavage rate is observed (Table 1, entry 22, k = 0.211 ± 0.037 min -1 ), almost as high as for the parent Dz1. All other modifications did not increase the activity compared to the unmodified split Dz6. Just Ph Bn , when added after T29 slightly increased the cleavage rate (Table 1, entry 25, k = 0.085 ± 0.013 min -1 ).

Important results have emerged from this study. The different non-modified split systems display various degrees of cleavage activity (Figure 3A). This was surprising, since one may expect that, if split into fragments, the catalytically competent conformation is difficult if not impossible to adopt. This would apply in particular to variants Dz2, Dz3 and Dz4, where the split is located within the catalytic loop (Fig. 2C). Yet, activity was observed for the non-modified split variants of Dz3 and Dz4 (Table 1, Figure 3). Just Dz2 showed high sensitivity when split in fragments, no activity was observed, neither for the nonmodified split variant, nor for the modified variants with potential for boronate formation (Table 1, Fig. 3A). Thus, while many studies have been devoted to the analysis of functional sequence requirements within the catalytic core of the 10-23 DNAzyme, [12] we here confirm and extend previous results [16c, 17] that even if perturbed, non-modified split systems are able to maintain some level of functionality. Furthermore, we have demonstrated that activity of split DNAzymes can be significantly restored by boronate ester formation, not requiring any kind of additional chemical or enzymatic activation. This is a clear advantage over other split systems that, in order to become functional, rely on chemical or enzymatic ligation of the assembled fragments. [14] While the potential of Ph Bn and Napht Bn remains limited, T Bn stands out as an efficient bioisosteric analogue of natural thymidine, even when located in non-Watson-Crick base paired regions as demonstrated with Dz3 (Figure 3B-E).

Although the formation of boronate esters can be observed in neutral aqueous media, it is well known that the association between boronic acids and cis-diols is favored at higher pH. This effect is explained by the formation of an hydoxyboronate complex resulting from the rehybridization of the boron atom from sp 2 to sp 3 and a substantial release of angle strain around the boron center. [START_REF] Hall | Boronic acids[END_REF] Thus, we decided to evaluate the cleavage activity of Dz4 from pH 5.5 to pH 9.6. At pH 5.5 neither the parent Dz1 nor its split Dz4 analogues (X = T or T Bn , Y = C or dC) were found to be active. However, at pH 9.6 we were pleased to observe a slight increase of cleavage rate of the T Bn modified split Dz4 (Figure 3F, k = 0.096 ± 0.017 min -1 at pH 9.6 vs k = 0.094 ± 0.017 min -1 at pH 8.6). Moreover, this effect was accompanied by increase of the percentage of cleaved substrate from 65% to 95% after 2h of incubation, further demonstrating the ability of the noninvasive nature of borono-modified systems to control DNAzyme activity.

In conclusion, we report here a new concept for the control of activity of split DNAzymes, through the use of two fragments, one modified with a boronic acid at the 5'-end and the other with a cis-diol at the 3'-end. In the presence of a RNA substrate, the assembly of the two non-functional fragments brings the two moieties in close proximity and a boronate internucleoside linkage is formed spontaneously. This leads to stabilization of the DNAzyme structure and restored activity for cleavage of the RNA substrate. By varying the position of the boronate linkage we were able to characterize the key elements and the optimum split site for maintaining high levels of activity. In the long term, the reversible formation of boronate esters and the tunable boronic ester-boronate equilibrium may be a promising concept to control functional nucleic acids for the development of valuable biosensing platforms.

Figure 1 .Figure 2 .

 12 Figure 1. Schematic representation of boronic-acid mediated assembly of nucleic acids

Figure 3 .

 3 Figure 3. Time courses of cleavage reactions under single-turnover conditions analyzed by a LICOR DNA sequencer. A) Parent DNAzyme Dz1 and split DNAzymes Dz2-Dz6 modified at the 3'-and 5'-extremity with a ribocytosine and T Bn respectively. DNAzyme activities of non-modified and modified split DNAzymes B) Dz3, C) Dz4, D) Dz5, E) Dz6. F) pH-dependency of Dz4.

Table 1 .

 1 Observed rate constants and percentage of total RNA cleaved of 10-23 DNAzymes Dz1 and split DNAzymes Dz2-Dz6.

	Entry	DNAzyme	X	Y	k [min-1]
	1	Dz1	-	-	0.237 (±0.045)	92
	2 3	Dz2	C rC	T T Bn	inactive inactive	--
	4		C	T	0.039 (±0.003)	82
	5		rC	T Bn	0.127 (±0.011)	94
	6	Dz3	C	T Bn	0.048 (±0.007)	83
	7		rC	Nahpt Bn	0.050 (±0.005)	88
	8		rC	TNapht Bn	0.068 (±0.004)	83
	9		C	T	0.005 (±0.001)	34
	10		rC	T Bn	0.094 (±0.017)	69
	11 12	Dz4	rC rC	Ph Bn Nahpt Bn	0.021 (±0.001) 0.026 (±0.001)	41 45
	13		rC	TPh Bn	0.045 (±0.005)	55
	14		rC	TNapht Bn	0.021 (±0.004)	39
	15		C	T	0.087 (±0.012)	63
	16		rC	T Bn	0.141 (±0.052)	85
	17 18	Dz5	rC rC	Ph Bn Nahpt Bn	0.019 (±0.003) 0.016 (±0.006)	44 52
	19		rC	TPh Bn	0.204 (±0.058)	72
	20		rC	TNapht Bn	0.033 (±0.004)	53
	21		C	T	0.059 (±0.007)	48
	22		rC	T Bn	0.211 (±0.037)	71
	23 24	Dz6	rC rC	Ph Bn Nahpt Bn	0.045 (±0.005) 0.052 (±0.008)	62 62
	25		rC	TPh Bn	0.085 (±0.013)	74
	26		rC	TNapht Bn	0.057 (±0.015)	79

a A (%)

a Observed rate constants of DNAzymes under single-turnover conditions (2 h reaction at 25 °C, 50 mM Tris buffer (pH 8.6), 20 mM MgCl2, 20 nM of substrate, and 2 μM of each Dz fragments). First-order rate constants (k) were fit to [S]=A

(1-e -kt )

, where [S] is the fraction of substrate uncleaved at time t, and A is the percentage of total RNA cleaved after 2h of incubation.

[16]

Acknowledgements

The Agence Nationale de la Recherche and the Deutsche Forschungsgemeinschaft are gratefully acknowledged for financial support (ANR PCRI "TEMPLAR"-16-CE92-0010-01; DFG MU1396/11).