Antibacterial Activity of DNA-Stabilized Silver Nanoclusters Tuned by

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Antibacterial Activity of DNA-Stabilized Silver Nanoclusters Tuned by Oligonucleotide Sequence Siamak Javani, Romina Lorca, Alfonso Latorre, Cristina Flors, Aitziber L. Cortajarena, and Álvaro Somoza ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 08 Apr 2016 Downloaded from http://pubs.acs.org on April 8, 2016

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ACS Applied Materials & Interfaces

Antibacterial Activity of DNA-Stabilized Silver Nanoclusters Tuned by Oligonucleotide Sequence Siamak Javani, Romina Lorca, Alfonso Latorre, Cristina Flors, Aitziber L. Cortajarena*, Álvaro Somoza*. Instituto Madrileño de Estudios Avanzados en Nanociencia (IMDEA Nanociencia), & Nanobiotecnología (IMDEA-Nanociencia), Unidad Asociada al Centro Nacional de Biotecnología (CSIC), 28049 Madrid, Spain KEYWORDS: Oligonucleotide, antibacterial, silver nanocluster, DNA, fluorescence

ABSTRACT Silver nanoclusters (AgNCs) stabilized by DNA are promising materials with tunable fluorescent properties, which have been employed in a plethora of sensing systems. In this report, we explore their antimicrobial properties in gram positive and gram negative bacteria. After testing 9 oligonucleotides with different sequence and length, we found that the antibacterial activity depends on the sequence of the oligonucleotide employed. The sequences tested yielded fluorescent AgNCs, which can be grouped in blue, yellow and red emitters. Interestingly, blue emitters yielded poor antibacterial activity whereas yellow and red emitters afforded an activity similar to silver nitrate. Furthermore, structural studies using circular dichroism indicate the 1 ACS Paragon Plus Environment


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formation of complexes with different stability and structure, which might be one of the factors that modulate their activity. Finally, we prepared a trimeric structure containing the sequence that afforded the best antimicrobial activity, which inhibited the growth of gram positive and negative bacteria in the sub-micromolar range.

INTRODUCTION DNA-stabilized silver nanoclusters (DNA-AgNCs) have recently emerged as new materials with interesting fluorescent properties since they are more stable to photobleaching than organic dyes and smaller than quantum dots or fluorescent proteins. These nanostructures are obtained upon reduction of a silver salt in the presence of oligonucleotides. They contain few atoms of silver (210) and have a small size (< 2 nm),1,2,3,4 close to the Fermi distance, which endows these structures with fluorescent properties. This phenomenon can be observed in a variety of metallic nanoclusters (e.g. Au, Cu, and Pt) protected with different ligands.5,6,7 In general, these structures present molecule-like properties, such as discrete energy levels, size-dependent fluorescent, good photostability, and biocompatibility. Their fluorescent properties depend on the size and shape of the cluster as well as their environment and are being evaluated as fluorescent probes.8,9 One of the most striking characteristics of DNA-AgNCs is the tunability of their fluorescent properties, which is an indication of the particular arrangement of the silver atoms.10,11 In this sense, it is possible to modulate their excitation and emission wavelength by the sequence of the oligonucleotide employed in their preparation. Although the fluorescent properties of DNAAgNCs cannot be predicted, emitters from blue to near-infrared have been reported, depending on the oligonucleotide sequence.3 2 ACS Paragon Plus Environment

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The main applications of DNA-AgNCs are focused on the generation of sensors for assorted structures such as small molecules, proteins, DNA, mRNA, and microRNAs.2,3,4,12,13 Interestingly, these nanostructures have shown very low toxicity14 and, therefore, are promising candidates for in vivo applications. Despite the large number of reports on DNA-AgNCs during the recent years, none deals with the antimicrobial properties of this novel material. Indeed, the search for new and unconventional antibiotics is a crucial challenge, as antibiotic resistance has become a major threat to public health globally, and can turn simple infections into deaths. The resistance to current antibiotics is due to the exceptional capability of microorganisms to adapt and survive, combined with the misuse of antibiotics during the last decades. In addition, most of the antibiotics currently available were developed in the 20th century, between the 40’s and 60’s and bacteria have evolved quickly resistance to them. Since then, the number of new antibiotics has been limited, and only three new classes of antibiotics have been approved since 2000.15 Due to the lack of new therapeutic weapons and the fast evolution of the current bugs we are facing a global risk, which has been highlighted by the World Health Organization in a recent report.16 One explanation for the lack of new antibiotics is that the traditional methods employed to develop them have not been very effective. In this sense, the scientific community is exploring a plethora of new approaches to tackle this problem,17,18 such as using uncultured microorganisms,19 predatory bacteria,20 phages,21 phagemids,22 gene-editing enzymes,23 antimicrobial peptides,24 and metals.25

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In the case of metals, they have been used as salts or also as their metallic derivatives such as silver,26 zinc27 or copper.28 Among these, silver is the most employed, which antimicrobial properties were exploited in the ancient times to keep water fresh and prevent infections.29 Nowadays the bactericidal properties of silver are used in a variety of applications, such as bandaids, water purification systems or food packaging among others. However, silver salts used directly have toxic effects, and, therefore, it is preferable to use systems that can gradually release silver cations, thus minimizing their toxic effect. For this reason, there is a great interest in the design of nanomaterials based on silver that can release enough Ag+ to control the growth of bacteria. In this sense, the use of silver nanoparticles to control different strains of bacteria has been extended in the last years.30,31 Studies on the antibacterial activity of silver nanoparticles suggest that the activity is due to the release of Ag+ from the nanostructure.32 Therefore, the nanoparticle works as a reservoir of silver cations that releases them over time, depending on the size of the particle and the capping agents employed, such as DNA from salmon milt.33 Another parameter that can also modulate the activity is the arrangement of Ag+ and Ag atoms in the nanostructure, which depends on the size and charge of the nanostructure.34 In a similar way, AgNCs are being used,35 were different stabilizing agents have been evaluated to control the release of Ag+ such as cellulose,36 silica nanospheres,37 silica composites38 and small organic molecules.39,40 However, the use of oligonucleotides to stabilize AgNCs can provide additional features, such as diversity, since different sequences can be prepared easily and employed in the preparation of AgNCs. What is more, the assembly properties of oligonucleotides can be exploited to prepare more complex structures. Regarding the mechanism of action of silver derivatives as antimicrobials, it is not completely clear and multiple mechanisms could be involved. For instance, silver salts can inhibit some key 4 ACS Paragon Plus Environment

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enzymes, reduce the level of antioxidants or even disrupt the cell membrane.25,26,30 In the case of AgNCs, they are able to intercalate in the DNA41 and can inhibit the activity of type II topoisomerases providing a complementary bactericidal effect to those obtained by silver salts and silver nanoparticles.42 However, the generation of Reactive Oxygen Species (ROS) is usually the most general process implicated in the bacterial response to silver derivatives.43 Herein we report the antibacterial activity of silver nanoclusters stabilized with DNA strands. After the evaluation of several strands, we found that the antibacterial activity of DNA-AgNCs depends on the DNA sequence employed in their preparation. The analysis of the structures by circular dichroism (CD) showed significant differences between the derivatives tested, suggesting that the final structure is key in the antimicrobial activity. EXPERIMENTAL SECTION Oligonucleotide Synthesis. The oligonucleotides were prepared using a MerMade4 DNA Synthesizer using commercial phosphoramidites and solid supports (Link Technologies). The alkyne derivative was obtained using a modified solid support prepared in our laboratory (SI). After solid-phase synthesis, the solid support was transferred to a screw-cap glass vial and incubated at room temperature for 16 h with 2 mL of ammonia solution (33%). Then, the supernatant was transferred by pipet to microcentrifuge tubes and the solid support and the vial were rinsed with water. The combined solutions were evaporated to dryness using an evaporating centrifuge. The samples were purified by polyacrylamide gel electrophoresis 20% and the oligonucleotides were eluted from gel fractions using an elutrap system. The solutions were desalted using an NAP-10 column and concentrated in an evaporating centrifuge.



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Oligonucleotides were analyzed by MALDI-TOF, at the 'Centro Nacional de Biotecnología', a member of ProteoRed network. Synthesis of Silver Nanoclusters. To an aqueous solution of the corresponding oligonucleotide (25 µmol), six equivalents of AgNO3 were added. The mixtures were incubated at room temperature for 10 min. Then, six equivalents of a fresh solution of NaBH4 were added to each sample and vortexed for 1 min. The resulting solutions were incubated in the dark at room temperature for 6 h and the excess of reagents removed using Amicon centrifuge filters (3K). Antibacterial activity test. Escherichia coli DH5 alpha (ATCC:668369, Gram-negative) and Staphylococcus epidermidis (ATCC:6538p, Gram-positive) bacteria were chosen as bacterial models to study the antibacterial properties of DNA-AgNCs. The susceptibility of E. coli and S. epidermidis to DNA-AgNCs was determined by the growth curve method.44 Briefly, bacteria were cultured in Luria-Bertani (LB) medium (OD 650 nm= 0.1, which corresponds to about 8 x 107 bacteria/ml) and incubated with the different antimicrobial agents at 37 °C for 14 h. DNAAgNCs concentration was adjusted to 1.5 µM, 2.25 µM, 3 µM, 4 µM and 5 µM based on the DNA concentration in a final volume of 200 µl. Silver nitrate (AgNO3) and silver nanoparticles (AgNPs) were used at the same concentration of silver present in the DNA-AgNCs. Growth curves were determined by measuring optical density (OD) at 650 nm every hour during 14 h. Agar Disk Diffusion Test. Kirby-Bauer disk diffusion susceptibility test was performed to determine the antibacterial activity of complexes formed with Seq-1, Seq-2 and Seq-3. For this purpose, E.coli bacteria were cultured in LB agar medium. DNA-AgNCs (5 µmol) were placed on sterile paper discs, which were transferred to the agar plate and incubated at 37 °C for 24 h.


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The presence or absence of inhibition zone is used as an indicator of the sensitivity or resistance of the bacteria. Total Reflection X-Ray Fluorescence (TXRF). TXRF was used as the method to measure silver composition and concentration in the aqueous solution of DNA-AgNCs (Seq-1, Seq-2 and Seq-3). TXRF analysis of the DNA-AgNCs samples was performed at the X-Ray facility of the Servicio Interdepartamental de Investigación (SIDI) at the Universidad Autónoma de Madrid in a benchtop S2 PicoFox TXRF spectrometer from Bruker nano (Germany) equipped with a molybdenum X-ray source. Reactive Oxygen Species (ROS) Detection. E. coli and S. epidermidis bacteria (OD 650= 0.1) were treated with the corresponding silver derivative and incubated for 14 h in a microplate reader, where the OD was recorded every hour. At the end of the incubation time, cells were centrifuged down at 4 °C for 5 min, the supernatants removed and the pellets resuspended in water. The washing process was repeated three times. Then, 10 µl 2’,7’-dichlorofluorescein diacetate (DCFDA) (5 µM) was added to the pellets and the final volumes adjusted to 200 µL. The samples were incubated for 2 h at room temperature, with mild shaking (80 rpm) in the dark. The fluorescence of the samples was registered (λex= 488 nm, λem= 525 nm), where the increase in fluorescence is due to the increase of ROS.

RESULTS AND DISCUSSION To study the antimicrobial properties of fluorescent DNA-AgNCs we selected three representative systems previously reported by Dickson and coworkers, which fluorescent emission are in the blue, yellow and red region.3,45 These derivatives are easily obtained by 7 ACS Paragon Plus Environment


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incubation of the required oligonucleotide with silver nitrate followed by reduction with sodium borohydride (Scheme 1). The samples were kept in the dark for 6 h and then washed using Amicon centrifuge filters to remove the excess of silver nitrate and sodium borohydride. DNA-AgNCs emitters (1, 2 and 3) were excited at 370 nm, 480 nm and 560 nm, showing their emission at 475 nm, 572 nm, and 630 nm, respectively, confirming the formation of AgNCs. Control experiments carried out without DNA strands did not produce any fluorescent material, highlighting the role of DNA in the generation of AgNCs as previously reported.3,45

Scheme 1. Schematic representation of DNA-AgNCs preparation. The DNA sequences employed are shown inside the oligonucleotides. (1)Blue, (2) Yellow, (3) Red emitters. These DNA-AgNCs were incubated with E. coli and their antibacterial activity was evaluated using the growth curve method, monitoring the optical density at 650 nm. In the control experiment, where no antibacterial agent was added, bacteria growth was observed after 2 h of incubation. However, the growth was clearly delayed when DNA-AgNCs were present in the solution (Figure 1). Remarkably, we found that the sequence employed in the preparation of AgNCs has a relevant role in the extent of antibacterial activity. Particularly, the best inhibition was obtained with the red emitter (Seq-3), followed by the yellow emitter (Seq-2), and the blue emitter, which has the smallest inhibitory activity (Seq-1).


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This behavior was confirmed using a complementary method to evaluate the antimicrobial activity, the Kirby-Bauer disk diffusion susceptibility test. In this test, a paper disk containing the reagent is placed on an agar plate where bacteria can grow. The reagent from the disk diffuses and prevents the growth of the bacteria around it, leaving a clear inhibition area. In the inset of Figure 1 the effect of the different samples can be compared. As in the previous method, the Seq3 showed the best antimicrobial activity. Similar experiments were performed on Staphylococcus epidermidis, a gram-positive bacterium present in human skin and mucous membranes. The results obtained are comparable to the experiments done with E. coli and can be found in the supplementary material (Figures S3-S8).

Figure. 1 E. coli growth curves in the presence of three DNA-AgNCs at 3 µM. Black line: without DNA-AgNCs. Blue line: Seq-1. Yellow line: Seq-2. Red line: Seq-3. The experiments were done in triplicates and the error bars represent the standard deviation. Inset: Kirby-Bauer disk diffusion susceptibility test. A) Water. B) Seq-1. C) Seq-2. D) Seq-3. The antibacterial effect is confirmed by the presence of a zone without bacteria surrounding the disks with DNAAgNCs. One explanation for the different activity observed with the three sequences tested would be the presence of different amounts of silver. In this regard, it is known that cytosine binds silver better 9 ACS Paragon Plus Environment


than the other nucleobases.46 Thus, since Seq-3 has more cytosines than the other sequences, the amount of silver in this sample could be potentially higher, leading to better antimicrobial activity. However, quantification of silver in the different samples using Total Reflection X-Ray Fluorescence (TRXF) revealed that Seq-1 contained more silver than Seq-2 and Seq-3 derivatives, which contained almost the same amount (Figure 2).

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Figure 2. Quantification of silver of the DNA-AgNCs by TXRF. The data shown is the average of three independent measurements, and the error bars represent the standard deviation. ANOVA test, P < 0.05. The differences observed are statistically significant.

These results indicate that the activity of DNA-AgNCs is not only due to the amount of silver, and other factors might modulate the antibacterial effect (e.g. sequence, structure, Ag arrangement). Since the sequence of DNA-AgNCs plays a role in their antimicrobial activity, we decided to evaluate additional sequences aiming to understand better their effect on the bactericidal properties. We selected sequences of different lengths that have been reported to yield fluorescent DNA-AgNCs with emission in the blue (Seq-1, Seq-4 and Seq-5), yellow (Seq-2, 10 ACS Paragon Plus Environment

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Seq-6 and Seq-7) and red (Seq-3, Seq-8 and Seq-9) region.3 Particularly, the sequences of the oligonucleotides tested in this work are summarized in Table 1.

Table 1. Sequences evaluated for the preparation of AgNCs. name

sequence

Seq-1 5´-TTTTCCCCTTTT-3 Seq-2 5´-CCCTTAATCCCC-3´ Seq-3 5´-CCCCCCCCCCCC-3´ Seq-4 5´-CCCTTTAACCCC-3´ Seq-5 5´-CCCCTTTTCCCC-3´ Seq-6 5´-GGGTTAGGGTCCCCCCACCCTTACCC-3´ Seq-7 5´-GGGTGGGTCCCCCCACCCACCC -3´ Seq-8 5´-CCTCCTTCCTCC-3´ Seq-9 5´-AGGTCGCCGCCC-3´

All the AgNCs prepared with these sequences reported some antimicrobial activity, which varied depending on the sequence employed (Figure 3). Among them, Seq-3, which is composed only by cytosines, yielded the best activity. We, therefore, plotted the antibacterial activity together with the cytosine content (Fig. 3D). However, we could not find a clear correlation between the activity and the number of cytosines. For instance, Seq-3, Seq-6 and Seq-7 have the same amount of cytosines but different antimicrobial activity. On the other hand, Seq-2 has fewer cytosines than Seq-5, Seq-6, Seq-7, and Seq-8 but has better antimicrobial activity.



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Figure 3. E. coli growth curves in the presence of DNA-AgNCs at 3 µM. A) Blue emitters, B) Yellow emitters, C) Red emitters, D) Representation of antimicrobial activity and number of cytosines for each sample. Controls show the bacteria growth without DNA-AgNCs. A more revealing comparison can be made regarding the emission color of the clusters. It seems clear that blue emitters have the lowest activity (Figure 3A), whereas yellow and red emitters showed an increased activity (Figures 3B and 3C), where the red emitter obtained with Seq-3 presents the highest activity. This observation suggests that the factors that affect the emission color of the clusters (which are not clearly understood at this point but are mainly related to the size, structure and environment of the clusters), are also important in the antibacterial properties of DNA-AgNCs. It is known that the fluorescence of AgNCs highly depends on the stabilizing molecules,47 however, despite


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the advances made in understanding the relation between oligonucleotide sequence and the structure and composition of DNA-stabilized AgNCs, it is still an unresolved issue.48 To further test the effect of the secondary DNA structure, as well as stability, we carried out CD studies to compare these parameters before and after the formation of the DNA-AgNCs (Figure 4).

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Figure 4. CD of the samples Seq-1, Seq-2 and Seq-3 before (A, C and E respectively) and after (B, D and F respectively) the generation of AgNCs. In the case of Seq-1 the CD spectra showed the characteristic peaks of a single stranded DNA with a positive band at 280 nm and a negative one around 255 nm,49 and the temperature did not affect the spectra significantly, suggesting that the strand was already unfolded (Figure 4A). On the other hand, Seq-2 and Seq-3 showed the characteristic peaks of an i-motif with a dominant positive band around 290 nm and negative one close to 260 nm (Figures 4C and 4E).50,51 These i-motif structures were destabilized as the temperature increased to yield the standard single stranded DNA spectrum shown in Figure 4A. In the case of Seq-2, the i-motif was denatured at lower temperatures (55 °C) compared with Seq-3 (75 °C). In both instances the 13 ACS Paragon Plus Environment


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generation of AgNCs induced significant changes in the CD spectra, with negative bands around 215 nm and 270 nm and positive one near 240 nm (Figures 4D and 4F). In the case of Seq-3 the bands were more pronounced and the band at 290 nm, characteristic of an i-motif, disappeared completely. It is worth highlighting the high stability of the structures obtained with both sequences, whose CD spectra did not change along the different temperatures tested. The derivative obtained with Seq-3 presents larger CD signals at 220 nm and 270 nm, suggesting a more structured conformation, whereas the intensity obtained with Seq-2 is smaller and in the case of Seq-1 there is no significant CD signal. These differences in the secondary structure and stability might be related to the different antibacterial activity observed because the CD spectra of the DNA-AgNCs samples show a trend that matches the activity observed (Figure 4B, 4D and 4F). We also tested the stability of the DNA-AgNCs against DNase I and we observed that the sample prepared with Seq-1 was the less stable. Whereas, Seq-2 was slightly more stable than the sample prepared with Seq-3 (Figure S13). These results suggest that the stability of the complex plays a role in the final activity, where the most stable DNA-AgNCs present the highest activity. This observation is in line with previous reports where partially oxidized AgNCs are less active than freshly prepared AgNCs.52 In our case, the oligonucleotides might prevent the oxidation of the AgNCs till they reach the bacteria. Despite this variety of studies, we could not find a unique explanation of the sequencedependence activity of DNA-AgNCs. In our opinion, multiple parameters might be playing a role, such as silver content, stability, secondary (and tertiary) structure, and even the uptake by


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the cells. In this sense, it has been recently reported that the use of a DNA nanostructure enhances the delivery of an antibiotic (Actinomycin D) into bacteria, improving its activity.53 For further studies, we focused on AgNCs prepared with Seq-3, as well as a trimer thereof (see SI for details on the trimer). First, we carried out a dose-response study using five different concentrations as illustrated in Figure 5. Inhibition of bacterial growth showed a clear dose-dependent response, where the higher the concentration of the DNA-AgNCs employed, the larger the antimicrobial inhibition activity. With the highest concentration (5 µM) the DNA-AgNCs were able to suppress the growth of the bacteria for at least 14 h.

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Time (h) Figure 5. E. coli growth curves in the presence of AgNCs stabilized with Seq-3 at different concentrations. Control: Bacteria growth without DNA-AgNCs. Then, we compared the activity of this derivative with related silver species, such as silver cations and silver nanoparticles, which are known to have antibacterial activity. We employed the silver nitrate used in the preparation of AgNCs as a source of silver cations. Silver nanoparticles were prepared by reduction of silver nitrate with sodium borohydride, following



the same procedure as for the preparation of AgNCs, but without the addition of DNA. In this case, the nanoparticles obtained showed their characteristic absorption band at 390 nm (Figure S2). The same amount of silver nitrate was used in the preparation of the three samples. As shown in Figure 6, AgNCs stabilized with Seq-3 and silver nitrate were found to have better activity than AgNPs. The excellent activity obtained with DNA-AgNCs might be due the combination of different mechanisms of action, such as the intercalation of AgNCs in the DNA42 and the release of silver cations.32

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Time (h) Figure 6. E. coli growth curves in the presence of AgNCs stabilized with sequence 3 (Seq-3) at 3 µM, silver nanoparticles (AgNPs) and silver nitrate (AgNO3). The concentration of silver in all samples was 9.8 µM. The experiments were done in triplicates and the error bars represents standard deviation. Then, we investigated the production of ROS using the fluorescent probe 2',7' – dichlorofluorescein diacetate (DCFDA), which is sensitive to several ROS, including hydrogen


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peroxide, hydroxyl radicals, and peroxynitrite. The fluorescein derivative is oxidized by the ROS species to 2’,7’-dichlorofluorescein (DCF), which is a fluorescent compound. ROS production was evaluated in E. coli and S. epidermidis bacteria after the incubation of DNA-AgNCs stabilized with Seq-3 for 14 h in the dark. The experiment showed an increase in bacterial ROS production as a response to the presence of DNA-AgNCs (Figures S9 and S10). Thus, we speculate that the antimicrobial activity of DNAAgNCs could be partially due to oxidative damage. Motivated by the excellent properties of the AgNCs generated with the Seq-3 as antibacterial agent, we decided to prepare a modified derivative aiming to increase their inhibitory activity. In this regard, we have recently reported the use of oligonucleotide trimers in the preparation of silver nanoclusters, which increased fluorescence and stability.54 Therefore, we wanted to explore whether a trimer of the Seq-3 would show enhanced antibacterial activity. To obtain the desired trimer a click reaction between a triazide derivative and an alkynemodified oligonucleotide was carried out. The resulting trimer was purified by gel electrophoresis and used in the preparation of AgNCs. Incubation of this compound with silver nitrate followed by reduction with sodium borohydride yielded the fluorescent silver nanoclusters (Figure 7).



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Figure 7. A) Representation of the synthetic process employed in the preparation of trimers. A click reaction between the alkyne and the azides yield the required trimer that is used in the formation of AgNCs. B) Bacteria growth inhibited by the presence of the DNA-AgNCs stabilized by trimers prepared with Seq-3 at different concentrations in E. coli. The trimer system showed excellent inhibition of bacteria growth at low concentration (0.75 µM or 1.5 µM) and even killing all bacteria at 2.25 µM (Figure 7 and S11). Remarkably, when the trimer at 0.75 µM was used against S. epidermidis the antimicrobial activity was similar to that obtained with the single strand system at 4 µM (Figure S12), highlighting the potential of this kind of derivatives in the development of novel antimicrobials. It is worth mentioning that the single stranded oligonucleotides bearing the alkyne modification showed the same activity as unmodified ones (results not shown). We believe that the globular arrangement of the trimers, as suggested previously,54 might stabilize the overall structure and improve the interaction with bacteria. CONCLUSIONS In summary, we have reported the antibacterial activity of AgNCs stabilized with modified oligonucleotides in gram-positive and negative bacteria (S. epidermidis and E. coli). Remarkably, the activity depends on the sequence selected, showing better growth inhibition when Seq-3 was employed, although it has lower silver content than other sequences. Based on these results sequences that can provide red emitters are more likely to have good antimicrobial activity. Our CD experiments indicate that the structure of the DNA involved in the stabilization of the AgNCs plays a role in the activity, where the most structured derivative showed higher antimicrobial activity. 18 ACS Paragon Plus Environment

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Finally, the preparation of a trimer containing Seq-3 yields a strong antimicrobial agent, which can delay the growth of the bacteria in the sub-micromolar range. Remarkably, this structure requires five times less concentration to achieve the same activity with AgNCs stabilized with oligonucleotides. The efficient growth inhibition in gram positive and negative bacteria, the easy preparation and the tunability of AgNCs stabilized with oligonucleotides, make these nanostructures a convenient tool for the development of new antibacterial agents. Also, the fluorescent properties of DNAAgNCs could be exploited to track the delivery in vivo of these antimicrobial agents using suitable carriers.

ASSOCIATED CONTENT Supporting Information. Data obtained with S. epidermidis. Synthetic procedures for the preparation of modified solid support, and trimers. ROS data. Experimental details of DNase I assay. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding authors E-mail: [email protected]. Phone: +34 912998856. Fax: +34 912998730 E-mail: [email protected]

ACKNOWLEDGEMENTS



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This work was supported by the following grants from the Spanish Ministry of Economy and Competitiveness (SAF-2010-15440, SAF2014-56763-R, BIO2012-34835, MAT2012–34487, RyC2011–07637) FP7-PEOPLE-2011-CIG grant number: 303620, BIONANOTOOLS (IRG246688) and IMDEA Nanociencia. RL thanks Chilean Ministry of Education CONICYT (PhD Scholarship: BECA CHILE), for financial support.

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Silver nanoclusters (AgNCs) stabilized by DNA are promising materials with tunable fluorescent properties, which have been employed in a plethora of sensing systems. In this report, we explore their antimicrobial properties in gram positive and gram negative bacteria. After testing 9 oligonucleotides with different sequence and length, we found that the antibacterial activity depends on the sequence of the oligonucleotide employed. The sequences tested yielded fluorescent AgNCs, which can be grouped in blue, yellow and red emitters. Interestingly, blue emitters yielded poor antibacterial activity whereas yellow and red emitters afforded an activity similar to silver nitrate. Furthermore, structural studies using circular dichroism indicate the formation of complexes with different stability and structure, which might be one of the factors that modulate their activity. Finally, we prepared a trimeric structure containing the sequence that afforded the best antimicrobial activity, which inhibited the growth of gram positive and negative bacteria in the sub-micromolar range.


Antibacterial Activity of DNA-Stabilized Silver Nanoclusters Tuned by

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