Green Synthesis of Gold Nanoparticles Coupled with Nucleic Acid

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Green Synthesis of Gold Nanoparticles Coupled with Nucleic Acid Oxidation Tatsuki Kunoh,†,‡ Minoru Takeda,§ Syuji Matsumoto,†,‡ Ichiro Suzuki,§ Mikio Takano,†,‡ Hitoshi Kunoh,†,‡ and Jun Takada*,†,‡ †

Core Research for Evolutionary Science and Technology (CREST), Japan Science and Technology Agency (JST), and ‡Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan § Graduate School of Engineering, Yokohama National University, 79-5 Tokiwadai, Hodogaya-ku, Yokohama 240-8501, Japan S Supporting Information *

ABSTRACT: Green synthesis of metal nanoparticles, especially gold nanoparticles (AuNPs), has attracted the great interest of scientists and engineers in the medical and pharmaceutical fields; thus, a variety of ecofriendly, energy- and cost-saving techniques have been developed. In this study, we found that cells of Leptothrix (iron-oxidizing bacteria) released extracellular RNA some of which could exist as a constituent of the cell-enclosing sheaths. As a part of studies of metal encrustation in the sheaths, here we show that RNA prepared from the Leptothrix cells can reduce Au(III) and spherical AuNPs eventually form when an aqueous HAuCl4 solution is added under ambient conditions. RNA and DNA of other organismal origins have the same ability. Of the nucleosides and nucleobases, only guanosine and guanine can form AuNPs. The DNA moiety, 2′-deoxyguanosine (dG) (used as a reference material), forms AuNPs when mixed with HAuCl4 solution, but 8-hydroxy-2′-deoxyguanosine (8OHdG) does not, indicating that AuNP formation evidently depends on the reduction potential of the guanine moiety, not the sugar moiety. This finding is the first demonstration that spherical AuNPs of ca. 5 nm diameter can be obtained by simply adding guanine to HAuCl4 solution at ambient temperature; no other chemicals or physical treatments are needed. KEYWORDS: Green synthesis of AuNPs, Nucleic acids, Guanine, Nucleic acid oxidation, 8-OHdG, RNA in Leptothrix sheaths

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INTRODUCTION

through infiltration and/or capping and are often toxic to organisms including humans, the use of toxic chemicals to produce AuNPs greatly limits applications in biomedical fields, especially for clinical purposes.1,3,18 To avoid such risky and costly processes to synthesize AuNPs, scientists and engineers are focusing on green chemistry to harness the biological potential of physicochemical interactions of organisms with surrounding inorganics, such as redox interactions. For example, extracts from organisms (lemongrass, tea, and brown algae),24−26 human and microbial living cells,27−31 and biomolecules such as proteins, sugars, and adenine32−34 have been reported to be beneficial for producing AuNPs through the reduction of cationic Au(III). To take one instance, Zhang et al.4 biosynthesized nanoscale Au−Ag alloy

In recent years, metal nanoparticles such as gold, silver, platinum, and copper have attracted the attention and interest of engineers and scientists for specific physical and chemical properties such as their size-dependent surface-plasmon response.1,2 Gold nanoparticles [actually, nanoclusters of gold nanoparticles (hereafter referred to as AuNPs)] and nanorods have promising applications particularly in fields such as biology,1 biomedicine,3−7 and catalysis.8 Diverse physical techniques such as thermolysis and irradiation with ultrasound, microwaves, γ-rays, or electron beams9−14 are often used to synthesize AuNPs, but they can also be produced chemically through the reduction of cationic Au(III) by a reducing agent such as sodium citrate, ascorbic acid, or sodium boron hydride.15−17 During these processes, undesirable aggregation of AuNPs is prevented by additives such as thiolate surfactants for dispersion in liquid and metal oxides as solid supports.18−23 Since such chemicals may contaminate the finished AuNPs © XXXX American Chemical Society

Received: July 31, 2017 Revised: September 8, 2017 Published: October 5, 2017 A

DOI: 10.1021/acssuschemeng.7b02610 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

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Analysis of Sugar Composition in the GTPC Fraction. To examine the purity of RNA in the GTPC fraction, we analyzed the sugar composition (target: ribose) using gas chromatography (GC) as follows. The freeze-dried GTPC fraction (1 mg) was hydrolyzed in 1 mL of 2 M trifluoroacetic acid at 100 °C for 4 h, followed by evaporation. The sugars in the hydrolysate were derivatized to their corresponding alditol acetates as previously described.47,48 Alditol acetates were identified using a GC-2010 Plus (Shimadzu, Kyoto, Japan) equipped with flame ionization detector under the following conditions: column, DB-1 (ID 0.25 mm, L 30 m, SUPELCO, SigmaAldrich); carrier gas, He; temperature program, from 180 to 250 °C at a rate of 3 °C/min, and then kept at 250 °C for 10 min. Hydrolysis of RNA Prepared from SP-6. As mentioned already, the GTPC fraction was composed of ribose, a subunit of RNA, and thus, we refer to this fraction as bacterial RNA (bRNA). When necessary, bRNA was hydrolyzed by RNase A. For this purpose, 100 μg of RNase A (Nacalai Tesque, Kyoto, Japan) was added to 3 mL of the bRNA solution in UPW and incubated for 12 h at 37 °C. For assessing the efficacy of the RNase treatment, 20 μL of the solution was separated in 1% agarose, and the gel was then stained using GelRed Nucleic Acid Gel Stain (Biotium Inc., Hayward, CA, USA). RNA bands were visualized using the UV illuminator Dolphin-View2 (Kurabo, Osaka, Japan). Test Compounds. Baker’s yeast RNA, calf thymus DNA, nucleosides, and DNA-related analogues 2′-deoxyguanosine (dG) and 8-hydroxy-2′-deoxyguanosine (8-OHdG), and a nucleotide, guanine, were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). The poly G-DNA primer (45-mer) was synthesized by GeneDesign (Osaka, Japan). Formation of AuNPs Using Nucleic Acids and Analogues Mixed with HAuCl4 Solution. To test the ability of the respective test nucleic acids and related analogues to form AuNPs, 0.5 mL of 100 mM Au(III) chloride solution (pH 2.0) (hereafter HAuCl4 solution), which was prepared by suspending HAuCl4 (Sigma-Aldrich) in UPW, was added to 3.5 mL of a solution of the respective nucleic acids (final concentrations of 2.5−7.5 mg/mL), nucleosides, or guanine (final concentrations of 5−15 mg/mL) in 15 mL conical tubes wrapped with an aluminum foil to avoid the photochemical reactions and incubated at room temperature on a shaking mixer (SHM-2002, LMS, Tokyo, Japan) for 16 h. Then, specimens were centrifuged at 14 000g for 5− 30 min (Model 3750, Kubota, Tokyo, Japan) to collect the precipitates. When necessary, the supernatants were used to test AuNP formation. Electron Microscopy and ED Analysis. For STEM and TEM observations and XRD analysis of RNA and DNA solutions, the resultant precipitates were washed with UPW at least four times, then in 99.5% ethanol at least 4 times, then freeze-dried (EYELA FDU1200, Tokyo Rikakikai, Tokyo, Japan). For TEM observation of suspensions of other nucleic acid-related chemicals, the supernatants after a 30 min-centrifugation at 14 000g were transferred to new tubes and diluted 200−1000-fold with 99.5% ethanol. The ethanol suspensions were dropped onto Cu grids and air-dried before placement in the TEM column. For XRD analysis, the supernatants were freeze-dried. For reference, the nucleic acids, nucleosides and related analogues were suspended in UPW instead of HAuCl4 solution, then freeze-dried and used as negative controls for XRD analysis. The air-dried precipitates or freeze-dried supernatants were suspended in 99.5% ethanol using an ultrasonicator (Ultrasonic cleaner USK-3R, AS ONE, Osaka, Japan) and dropped onto a carboncoated Cu grid, then air-dried. Images were obtained with a TEM (JEOL 2100F, Tokyo, Japan). ED patterns were obtained using the electron diffraction mode of the same TEM. XRD Analysis. XRD patterns were obtained using an X-ray diffractometer (RINT-2500HF, Rigaku, Tokyo, Japan) with Cu Kα radiation (voltage: 40 kV; current: 200 mA) to examine the crystallinity of the air-dried precipitates or freeze-dried supernatants as described previously.39 Test nucleic acids, nucleosides and nucleobases were dissolved in UPW instead of HAuCl4 solution and treated similarly to the test materials and used as mock samples for the

using chloroplasts as reducers and found that chloroplast proteins attached to the alloy surfaces through free amino groups (NH2). However, the detailed mechanisms for Au(III) reduction by these biological materials in the context of redox reactions remain largely elusive: which molecules are responsible for interaction with Au(III) and its reduction to Au(0)? Among the organisms with promise for “green chemistry”, Leptothrix species, which are aquatic iron-oxidizing bacteria, produce microtubular organic−inorganic sheaths that encase their cells. The skeleton of an immature sheath, consisting of organic exopolymer fibrils of bacterial origin, is formed first, then the sheath becomes encrusted with aquatic-phase inorganic materials.35 Recently, we found that the expolymer fibrils excreted from L. cholodnii SP-6 (hereafter SP-6) can sorb Au(III), although the detailed mechanism remains elusive.36 Among the glycoconjugates (with uronic acids, amino acids, amino sugars) and lipids that comprise these fibrils,37,38 the amino sugars participate in sorption reactions with metallic cations through chelation with the functional groups NH2 and OH.11,39 We have also had an interest in metal sorption by nucleic acids, because the carbonyl group of the nucleobases guanine and thymine are binding sites for the formation of metal−DNA complexes.40−44 In most earlier methodologies concerning AuNP formation using biomolecules, irradiation with an electron beam or use of a reducing agents was required to reduce Au cations.11,41 Recently, we found that the sheath remnants of SP-645 contain RNA and that SP-6 cells release a remarkable amount of extracellular RNA into the culture medium (see Supporting Information Figure S1). This finding led us to postulate that, if RNA is one of the constituents of immature sheaths, extracellular RNA could be involved in metal encrustation of sheaths. Here, we first examined the ability of RNA prepared from Leptothrix cells to chelate Au(III), then searched for critical factor(s) in the RNA moiety that may be involved in the reduction of Au(III) and formation of AuNPs. Our findings have helped us to understand more deeply the process of AuNP formation by the moiety of nucleic acids and their related molecules.

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EXPERIMENTAL SECTION

Preparation of RNA from Leptothrix Cells. Cells of Leptothrix cholodnii SP-6 (ATCC 51168) (hereafter SP-6) from frozen stock cultures were streaked onto MSVP agar plates46 and incubated at 20 °C for 7 days. Single colonies were transferred separately to 25 mL of MSVP and precultured on a rotary shaker at 20 °C and 70 rpm. After 2 days, 1−5 mL of the cell suspension (adjusted to 10 cfu/mL by densitometry) was added to 500 mL of MSVP in each of four 1 L flasks, followed by shake culture for 2−3 days. Cells were then harvested by centrifugation at 3400g for 5 min and washed with ultrapure water (hereafter UPW) at least 4 times. The resultant cell pellets were added to a mixture of 25 mL UPW and 25 mL of IsogenLS (composed of acid guanidinium thiocyanate and phenol; Nippon Gene, Tokyo, Japan), vigorously mixed by a vortex mixer (Scientific Industries, Bohemia, NY, U.S.A.), then rested for 5 min at room temperature. After 1 mL of chloroform (Nacalai Tesque, Kyoto, Japan) was added, the cell suspension was again mixed and rested, then centrifuged at 14 000g for 30 min. The resultant supernatant was carefully collected, dialyzed against 2 L of UPW at least four times using a Slide-A-lyzer dialysis cassette (Thermo Scientific, Waltham, MA, U.S.A.), and then freeze-dried. Because the combination of Isogen-LS and chloroform is the commercial reagent for RNA extraction, the final freeze-dried sample was expected to consist of RNA (tentatively referred to as the GTPC fraction). B

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Figure 1. Formation of Au-nanoparticles (AuNPs) by the acid guanidinium thiocyanate−phenol−chloroform (GTPC) fraction (= bRNA) in Au(III) chloride (HAuCl4) solution. (a) The clear yellow HAuCl4 solution (left) changed to turbid brown (middle) within 4 h after mixing with the GTPC fraction; a brown precipitate formed (right) after 16 h. (b) GC spectrum of sugar composition of GTPC fraction shows a prominent ribose peak. Based on this result, the fraction was regarded as bacterial RNA (bRNA) (left). Electrophoretic patterns of RNase A-treated (right lane) and -untreated bRNA (left lane, Mock) in agarose gel, indicating that bRNA was degraded but not completely (right). (c) STEM images (left) and ED patterns (right) of freeze-dried precipitates obtained from mixture of bRNA and HAuCl4 solution. Electron-dense particles scattered on the aggregated bRNA (left upper). The particles are nearly spherical in the enlarged image (left, lower). Scale bar = 10nm, upper; 5 nm, lower. ED patterns obtained from the particles (P1 and P2 in left upper image). From calculations using diffraction indices, Au(200) and Au(020) crystal faces were present in P1 and Au(200) in P2 (right upper and lower). (d) Histogram of AuNP diameters measured in STEM images (average 6.1 ± 1.4 nm). (e) XRD patterns of precipitates from bRNA-mixed HAuCl4 solution and UPW (Mock) after 16 h incubation, showing the presence of Au(111), Au(200), Au(220), Au(311), and Au(222) crystal faces in the former precipitate but not in the latter. 0.45 μm-membrane filter (DISMIC-03CP, Advantec, Tokyo, Japan) and analyzed with HPLC. Colorimetric Assay for 8-OHdG, a Marker of Oxidative DNA Damage. For this purpose, an EpiQuik 8-OHdG DNA damage quantification direct kit (colorimetric) (Epigentek, Farmingdale, NY, U.S.A.) was used. Briefly, 1−400 μg of calf thymus DNA in 400 μL of Tris-EDTA (TE) buffer (pH 8.0) was added to 50 μL of 100 mM HAuCl4 solution and incubated at room temperature for the indicated times, then ethanol-precipitated and air-dried (Figure 3b, c). After the air-dried DNA precipitates were dissolved completely in 50 μL of TE buffer by sonication for 5 min, 8 μL of the resultant DNA solution was used to detect 8-OHdG according to the manufacturer’s protocol. For colorimetric detection of the specimens, absorbance at 450 nm was measured by a Nanodrop 2000C spectrophotometer (Thermo Scientific).

XRD analysis. The specimens were fixed on a zero background sample holder and scanned continuously from 10° to 90° (2θ value) at 3°/ min. The XRD pattern of the zero background sample holder was also measured. Detection of Chemical Modification of dG as a Reference to Monitor Modification of Chemical Properties of DNA. To examine whether dG was chemically modified in HAuCl4 solution, we detected the dG peak using HPLC and the following conditions: column, 5C18-PAQ (4.6 × 150 mm, Nacalai Tesque); mobile phase, 22 mM phosphate buffer (pH 7.0); flow rate, 1 mL/min; column temperature, 30 °C; detection, absorbance at 260 nm. For the redox reaction of Au(III) with dG, 0.1 mL of 10 mM HAuCl4 solution was added to 0.7 mL of 0.6 mM dG solution followed by incubation at 25 °C. The reaction was monitored by sampling 0.1 mL of the suspended solution at intervals of 30−120 min; the sample was passed through a C

DOI: 10.1021/acssuschemeng.7b02610 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

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Figure 2. Formation of Au-nanoparticles (AuNPs) by incubation of RNA nucleosides with Au(III) chloride (HAuCl4) solution. (a) Colors of HAuCl4 solution after mixing with either of four RNA nucleosides (adenosine, uridine, guanosine, or cytidine). The mixture started to change color within 1 min after mixing with guanosine and after 1 h with adenosine, but remained unchanged until 1 h after mixing with uridine and cytidine. The precipitate (bottom, arrowheads) and supernatant were obtained by centrifugation at 16 h. (b) TEM image of electron-dense spherical particles on surface of aggregated guanosine obtained from supernatant after 16 h incubation (upper). Scale bar = 5 nm. The ED pattern obtained from an electron-dense particle shows several spots (arrows) reflecting the presence of metallic Au (lower). (c) Histogram of AuNP diameters measured from the TEM images (average 3.5 ± 0.8 nm). (d) TEM image showing absence of electron-dense particles on surface of aggregated adenosine obtained from supernatant (left). Scale bar = 5 nm. The ED pattern obtained from the aggregated adenosine lacked signals for metallic Au (right). (e) XRD patterns from supernatant and precipitate from guanosine-mixed HAuCl4 solution after 16 h incubation: only the supernatant showed the presence of Au(111), Au(200), Au(220), Au(311), and Au(222) crystal faces. □: unidentified peaks. (f) XRD patterns from supernatant and precipitate from adenosine-mixed HAuCl4 solution after 16 h incubation. Note lack of signal for Au in both specimens. Mock = guanosine or adenosine mixed with UPW. XRF Analysis. For determining the atomic percentage (hereafter atomic %) of Au in test specimens, freeze-dried precipitates were packed into small aluminum pans for elemental analysis using an Orbis micro X-ray fluorescence (XRF) analyzer (Ametek, Berwyn, PA, USA). Quantitative data for the target element in specimens were expressed as atomic %, defined as the percentage of the theoretical intensity of characteristic X-ray fluorescence from Au per a total characteristic X-ray fluorescence from all constitutive elements, which

is calculated using physical constants and instrumental parameters by the fundamental parameter method.49 Atomic % of Au was expressed as the mean (±SE) of 10 spots.

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RESULTS AND DISCUSION Formation of AuNPs by Incubation of Leptothrix RNA in Au(III) Chloride Solution. To examine whether the

D

DOI: 10.1021/acssuschemeng.7b02610 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

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obtained again by TEM and XRD when HAuCl4 solution was adjusted from its original pH 2.0 to 7.0 (see Supporting Information Figure S4), indicating that as for RNA, DNA could reduce Au(III) to Au(0) without needing a reducing agent, even at neutral pH. Potential of Guanosine and Guanine to Form AuNPs. Subsequently, we examined which nucleoside(s) of RNA were responsible for reducing Au(III) to Au(0). After mixing each of the nucleosides (adenosine, guanosine, uridine, and cytidine) with HAuCl4 solution, the yellow mixture of guanosine and adenosine turned brown within 1 min and 1 h, respectively, while that of uridine and cytidine did not change for 16 h (Figure 2a). A small amount of precipitate was obtained from the guanosine mixture by 16 h (Figure 2a, arrow). Unexpectedly, electron-dense particles were not found in this precipitate by TEM (see Supporting Information Figure S5a left), and the subsequent XRD did not detect any peaks corresponding to metallic Au (Figure 2e). Nevertheless, X-ray fluorescence (XRF) analysis detected the peaks of Au and approximately 90 atomic % of Au in this precipitate (see Supporting Information Figure S5b left). Based on these data, we suspected that AuNP-holding guanosine might be in the colloidal state whereby it could not precipitate under the present centrifugation conditions. To be certain, we diluted the supernatant with ethanol, then air-dried it for TEM and ED or freeze-dried it without the ethanol dilution for XRD. As expected, the electron-dense particles 2−6 nm in diameter (average 3.5 ± 0.8 nm) were observed on the surface of the aggregated guanosine by TEM (Figure 2b upper, c), similar to the cases for the RNAs and DNA (Figure 1c and see Supporting Information Figures S2c and S3b). The ED patterns showed several spots indicating metallic Au: Au(111), Au(220), and Au(311) crystal faces (Figure 2b lower). Consistently, XRD analysis detected peaks derived from metallic Au: Au(111), Au(200), Au(220), Au(222), and Au(311) (Figure 2e). These results showed that guanosine can reduce Au(III) and eventually form AuNPs, but the AuNPholding guanosine was suspended in the mixture. Why are the AuNPs holders found in the supernatant but not in the precipitate? We assumed that the polymeric structure of the holders may be necessary for precipitation of AuNPs holders under the present centrifugation conditions. This assumption was supported when poly G DNA-primer (= a polymer of 2′-deoxyguanosine [dG]; hereafter poly dG) was incubated with HAuCl4 solution, then centrifuged (see Supporting Information Figure S6). In a TEM image, electron-dense AuNPs of 3−9 nm diameter (average 4.9 ± 1.0 nm) were scattered on the aggregated poly dG in the precipitate (see Supporting Information Figures S6b−d). These results strongly suggest that polymerization of dG through a phosphate group might be important for precipitation with AuNPs. In a study on the formation of ATP-mediated capping of the surface of AuNPs, negatively charged phosphate groups were proposed as the mode of interaction between ATP and AuNPs.18 Interestingly, no electron-dense particles were observed even on the amorphous-looking aggregated adenosine prepared from either supernatant (Figure 2d left) or precipitate (see Supporting Information Figure S5a middle). Accordingly, neither ED (Figure 2d right) nor XRD (Figure 2f) analyses showed any spots, rings, or peaks that would indicate the existence of metallic Au on both specimens. Nevertheless, XRF analysis detected approximately 92 atomic % of Au in the

fraction prepared from SP-6 cells by the acid guanidinium thiocyanate-phenol-chloroform method (hereafter GTPC fraction) can interact with Au(III) cations, the fraction was mixed with an Au(III) chloride solution (hereafter HAuCl4 solution). Within 4 h, the mixture turned brown and turbid (Figure 1a middle). After four washes with ultrapure water (hereafter UPW), the brown precipitate was obtained from the mixture (Figure 1a right), suggesting that the GTPC fraction and Au cations most likely converted to an insoluble GTPC− Au(III) complex. Although the GTPC method is frequently used to extract RNA from a variety of cells, the GTPC fraction is often contaminated with diverse saccharides of cell origin.50 We therefore examined the purity of the RNA in the fraction by targeting ribose in the GTPC fraction by gas chromatography (GC). A single prominent peak corresponding to ribose was detected at a retention time of 13−14 min (Figure 1b left). When the GTPC fraction was then treated with RNase A, an endonuclease of RNA, the flow pattern on the agarose gel, the bands in the mock disappeared almost completely after the RNase treatment (Figure 1b right). Based on these results, we concluded that the GTPC fraction was primarily RNA and thus refer to it as bacterial RNA (bRNA) hereafter. Scanning transmission electron microscopy (STEM) of the precipitate formed in the mixture of bRNA and HAuCl4 solution revealed abundant electron-dense, spherical, monodisperse particles on the surface of the aggregated bRNA (Figure 1c left). The diameter of over 300 particles, estimated from STEM images, ranged between 2 and 10 nm (average 6.1 ± 1.4 nm), and 86.7% of the particles were 4−8 nm in diameter (Figure 1d). The electron diffraction (ED) patterns obtained individually from arbitrarily selected particles P1 and P2 (Figure 1c left) showed well-defined spots indicating good crystallinity, which were assigned to Au(200) and Au(020) with d200 = 0.203 nm and d020 = 0.181 nm (Figure 1c right). Consistent with this ED outcome, X-ray diffraction (XRD) analyses showed Au(111), Au(200), Au(220), Au(311), and Au(222) peaks at 2θ values of 38.1°, 44.3°, 64.5°, 77.5°, and 81.6°, respectively (Figure 1e). Therefore, we considered that the bRNA had the potential to reduce Au(III) to Au(0) and eventually to form AuNPs. To confirm this potential, we tested the ability of baker’s yeast (hereafter yRNA) to reduce Au cations to metal forms for reference (see Supporting Information Figure S2). Results similar to those in Figure 1 were obtained: brown precipitates formed within 5 min when yRNA was mixed with HAuCl4 solution; electron-dense particles were observed on the aggregated yRNA with transmission electron microscopy (TEM); particle diameters were 3−8 nm (average 5.6 ± 0.9 nm); XRD peaks were ascribed to Au(111), Au(200), Au(220), Au(311), and Au(222). Results apparently showed that yRNA was able to reduce Au(III) to Au(0) as found for bRNA. Next, we examined the Au(III)-reducing ability of DNA by mixing calf thymus DNA with HAuCl4 solution. As illustrated in Supporting Information Figure S3, results very similar to those for the RNAs (Figures 1 and Supporting Information Figure S2) were obtained by TEM, ED, and XRD. Here we also examined the possible interference of pH with reduction of Au(III) to Au(0) by DNA, because an earlier paper41 reported that, in a mixture of salmon sperm DNA in HAuCl4 solution at pH 5.6, an Au(III)−DNA complex was generated by chelating Au(III) at the sites of O6 and N7 in the guanine moiety and that they needed a reducing agent, hydrazine, to obtain AuNPs. Results similar to Supporting Information Figure S3 were E

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Figure 3. Detection of chemical modification of 2′-deoxy guanosine (dG) and generation of 8-hydroxy-2′-deoxyguanosine (8-OHdG) from calf thymus DNA during incubation in Au(III) chloride (HAuCl4) solution. (a) HPLC analysis of dG after 0 to 240 min in HAuCl4 solution. One dG peak was detected at a retention time of approximately 18 min at each time. Note that its absorbance level was quite high at time 0 but was much lower from 30 to 240 min and that an additional unidentified peak appeared at a retention time of approximately 19.5 min (arrows). (b) Specific antibody-based colorimetric detection of 8-OHdG production from various concentrations of DNA incubated in HAuCl4 solution for 12 h. DNA dissolved in UPW was used as the mock sample. (c) Time-course analysis of 8-OHdG production after incubation of 40 ng of DNA in HAuCl4 solution.

Failure of AuNP Formation by Hydroxylated Guanine Molecule. We suspected that structurally modified guanine analogues might lack the potential to reduce Au(III) and eventually form AuNPs. To check this possibility, we focused on commercially available 8-hydroxy-2′-deoxyguanosine (8OHdG), because the generation of 8-OHdG from dG is often used as a marker of oxidative damage of DNA and is readily detected by a specific antibody-based enzyme-linked immunosorbent assay.51 At first, we mixed dG with HAuCl4 solution and then monitored generation of the byproducts over time using high performance liquid chromatography (HPLC) (Figure 3a). Immediately after mixing (0 min), a prominent peak corresponding to dG was detected at a retention time of approximately 18 min. Intriguingly, at 30 min and later, the peak of dG reduced drastically and an additional peak appeared at a retention time of approximately 19.5 min (Figure 3a, indicated as unidentified), suggesting that HAuCl4 solution could modify the chemical property of dG and eventually generate an unidentified byproduct. What is the origin of this peak? At present, we infer that the peak could correspond to 8OHdG because 8-OHdG was detected when calf thymus DNA having dG as one of the nucleoside moieties was mixed with HAuCl4 solution (Figure 3b, c) as explained below, and, in addition, the retention time of 8-OHdG in HPLC analysis was reported to be longer than that of dG,52 although further studies are certainly needed before making a conclusion. If dG is chemically modified after mixing with HAuCl4 solution, theoretically, 8-OHdG is supposed to be generated. To verify such a possibility, various quantities of calf thymus DNA (2, 40, and 80 ng/mL) were incubated in HAuCl4 solution for 12 h, and the production of 8-OHdG was monitored using a colorimetric assay with an 8-OHdGrecognizing antibody. Absorbance at 450 nm remained low (ca. 0.5) when calf thymus DNA was dissolved in UPW

precipitate (see Supporting Information Figure S5b middle), suggesting that unlike guanosine, adenosine could not form AuNPs, although it interacted with Au cations. Wei et al.34 reported that the adenine−HAuCl4 solution hybrid colloidal particles were in a mixed-valence Au(I)/Au(III) state. At present, we infer that their evidence probably accounts for the detection of Au by XRF but lack of detection of metallic Au in the present adenosine-mediated precipitate by XRD and ED. Considering the difference in molecular structure between guanosine and adenosine, we speculated that the guanine moiety could be significant for formation of AuNPs. To verify this speculation, we mixed guanine with HAuCl4 solution. The mixture became turbid within 1 min and turned brown within 6 h (see Supporting Information Figure S7a). The precipitate and supernatant obtained from the 16 h specimens were then analyzed further. TEM showed that the electron-dense particles were not found on the surface of the precipitate (see Supporting Information Figure S5a right) but were evidently present on the surface of aggregated guanine prepared from the supernatant (see Supporting Information Figure S7b upper two). These particles were 3−10 nm in diameter (average 5.7 ± 1.4 nm; see Supporting Information Figure S7c). The ED pattern showed rings and spots corresponding to Au(111), Au(200), Au(220), and Au(311) (see Supporting Information Figure S7b lower). Consistent with the case of guanosine (Figure 2e), XRD analysis also detected peaks of Au(111), Au(220), Au(222), and Au(311) in the supernatant but not in the precipitate specimen (see Supporting Information Figure S7d). However, as for guanosine, XRF analysis detected approximately 94 atomic % of Au in the precipitate (see Supporting Information Figure S5b right). Based on these data, among the nucleobases, guanine was considered to be responsible for reducing Au(III) to Au(0) and eventually forming AuNPs in the suspension. F

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Scheme 1. Construable Mechanism of 8-OHdG Generation Coupled with Au(III) Reduction Based on Earlier Proposals for Formation of an Au(III)−DNA Complex and for Oxidative DNA Damage to the Guanine Moietya

a

Initially, a chelating adduct by binding Au(III) to guanine moiety through N7 and O6 (a), or a radical adduct by interacting of through guanine C8 (b) is generated. Secondly, the encounter of (a) with HO• or (b) with Au(III) leads to form a chelating and radical adduct (c). Then, the electron abstraction occurs from guanine N7 to Au(III) and eventually reduces Au(III) to lead 8-OHdG generation (d).

Figure 4. Potential of Au-nanoparticle (AuNP) formation by 8-hydroxy-2′-deoxyguanosine (8-OHdG) and 2′-deoxy guanosine (dG) in HAuCl4 solution. (a) The visual appearance of samples after dG (middle) or 8-OHdG (right) was mixed with HAuCl4 solution. Only the mixture with dG turned brown within 5 min. The supernatant was obtained by centrifugation of the 16-h sample. (b) TEM images (upper) and ED pattern (lower) of the supernatant collected from dG− or 8-OHdG−HAuCl4 solution at 16 h. Electron-dense particles were observed on the aggregated dG (upper left) but not on the aggregated 8-OHdG (upper right). Scale bar = 5 nm, left; 2 nm, right. Consistently, spots indicating the presence of metallic Au were detected on the aggregated dG (lower left, arrows) but not on the aggregated 8-OHdG (lower right). (c) Histogram of AuNP diameters measured from the TEM images (mean 4.4 ± 0.9 nm). (d) XRD analyses of supernatant and precipitate obtained from dG-suspended HAuCl4 solution. A suspension of dG in UPW was used as a negative control (Mock). The presence of metallic Au was indicated only in the HAuCl4 supernatant. □: unidentified peaks.

G

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eventual applications. In contrast, the nucleic acid-, nucleoside-, or nucleobase-mediated spherical AuNPs in this study were very close in size (mean diameter: 3.5−6.2 nm). Although small nanocrystals tend to aggregate because of van der Waals attraction,18,34 the AuNPs formed by the present methods did not, judging from a series of TEM images. These properties are critical for medical and/or pharmaceutical purposes. As far as we know, this is the first report concerning AuNP formation using a guanine moiety without any additional reducing reagents or physical stresses. To date, broad technological strategies have been studied toward developing AuNPs in the fields of optical sensing and imaging, drug delivery, cancer therapy, and so on.3,56 To exploit the homologous shape and size, dispersibility, and easy production of the AuNPs generated for the medical and pharmaceutical fields, we also need to develop methods to recover AuNPs from the carrier and modify their surfaces to provide affinitive and reactive properties to AuNPs and study their toxicity to target animal cells57 and other essential basic issues such as the stability of AuNPs in various salt solutions that are used routinely for biological applications. The present findings on the potential of bRNA to attract and reduce Au cations may open a new approach to harness the mechanisms of metal encrustation of Leptothrix sheaths in aquatic environments, because RNA is detected in sheaths (see Supporting Information Figure S1).

(Mock) but the absorbance increased when increasing quantities of DNA were dissolved in HAuCl4 solution (Figure 3b), indicating the generation of 8-OHdG within DNA in HAuCl4 solution (pH 2.0). In addition, 8-OHdG was detectable within 0.5 min and continued to increase for 1 h after a fixed amount of DNA (40 ng/mL) was added to HAuCl4 solution (Figure 3c). These results strongly indicated that DNA was oxidatively damaged in HAuCl4 solution, resulting in the production of 8-OHdG (see Scheme 1). Keeping these basic outcomes in mind, we separately mixed dG and 8-OHdG in HAuCl4 solution. Expectedly, the dG mixture turned brown within 5 min, while the 8-OHdG mixture did not change for 16 h (Figure 4a). After centrifugation of each mixture after 16 h, the supernatants were viewed with TEM. As illustrated in Figure 4b (upper left), electron-dense particles 2−7 nm in diameter (mean 4.4 ± 0.9 nm) (Figure 4c) were scattered on the aggregated dG, but no particles were seen on the aggregated 8-OHdG (Figure 4b upper right). The ED analysis detected rings and spots corresponding to Au(111), Au(200), Au(220), and Au(311) in the dG sample but not in the 8-OHdG (Figure 4b). The XRD analysis also detected Au(111), Au(200), Au(220), Au(222), and Au(311) peaks in the supernatant but not in the precipitate of dG (Figure 4d), similar to the case of guanosine (Figure 2e) and guanine (see Supporting Information Figure S7d). All these data evidently show that the guanine moiety hydroxylated at C8 cannot reduce Au(III) and thus cannot form AuNPs. According to earlier reports for the formation of an Au(III)− DNA complex40−42 and oxidative DNA damage of guanine moiety,53,54 either a chelating adduct is initially generated by the binding of Au(III) to the guanine moiety through N7 and O6, or a radical adduct is generated by interaction through guanine C8 [see Scheme 1a and b]. As the second step, the encounter of the former adduct with HO• or the latter adduct with Au(III) leads to the formation of a chelating and radical adduct [see see Scheme 1c]. Then, the electron abstraction from guanine N7 to Au(III) [see see Scheme 1d] eventually reduces Au(III) to Au(II) and leads to 8-OHdG generation. The generated Au(II) then likely accepts an electron in a similar manner and is converted to Au(I) and thereby to Au(0). Therefore, it seems most likely that electron transfer from guanine C8 to binding Au cations adjacent to guanine C6 and N7 holds a key for reduction of Au cations to metallic Au. The most important oxygen-free radical causing damage to basic biomolecules is the hydroxyl radical (HO•), which can be generated by diverse mechanisms, especially by the Fenton reaction involving hydrogen peroxide and by metals, and other endogenous and exogenous reactive oxygen species.53 However, in this proposed model, how and when initial HO• generation occurs in HAuCl4 solution are still unknown. Accordingly, further studies are certainly required to validate this model. As mentioned already, a variety of chemical, physical, and biological techniques have been used to produce AuNPs. However, the size and shape of the particles were quite variable, and sometimes the particles readily coagulated, especially those synthesized biologically or chemically. For example, irregular shapes and a range of sizes (20−85 nm in diameter) were synthesized using lemongrass extract,55 relatively large particles (ca. 310 nm in diameter) were generated in an adenine− HAuCl4 solution mixture,34 and near-spherical AuNPs with 45−80 nm diameter were produced using DNA/hydrazine.41 Such variations often slow the development of techniques and

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CONCLUSIONS Based on the experimental outcomes, we propose that the structural modification from C−H to C−OH at guanine C8 plays a critical role for Au(III) reduction and formation of AuNPs: (i) Mixing bRNA, yRNA, or DNA solutions with HAuCl4 solution results in AuNP formation in the precipitates of the respective aggregated nucleic acids (Figure 1 and see Supporting Information Figures S2, S3). (ii) Guanosine and guanine cause AuNP formation, but sugar components of nucleic acids do not (Figure 2 and see Supporting Information Figure S7). (iii) Polymeric RNA, DNA, and DNA primer carrying AuNPs can be precipitated in HAuCl4 solution using the present centrifugation conditions, but monomers such as guanosine and guanine are not sufficient (Figures 1 and 2 and see Supporting Information Figures S2, S3, S5−7). (iv) A chemical property of dG is modified when dG is mixed with HAuCl4 solution, which could be related to production of 8OHdG (Figure 3). (v) The C8-hydroxylated dG (= 8-OHdG) fails to form AuNPs when mixed with HAuCl4 solution (Figure 4). The results indicate that fine spherical AuNPs are produced through the oxidation of the guanine moiety at C8 without the need for any reducing agents or physical stresses.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.7b02610. Figure S1. Presence of RNA in the SP-6 sheath remnants and extracellular release of remarkable amount of RNA from SP-6 cells in MSVP culture medium. Figure S2. Formation of AuNPs after incubation of yRNA with HAuCl4 solution. Figure S3. Formation of AuNPs by incubation of calf thymus DNA with HAuCl4 solution. Figure S4. AuNP formation by DNA in buffered HAuCl4 H

DOI: 10.1021/acssuschemeng.7b02610 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

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solution. Figure S5. AuNPs were not detected on the surfaces of precipitates collected from guanosine−, adenosine−, or guanine−HAuCl4 solution. Figure S6. AuNPs precipitated with poly G DNA primer in HAuCl4 solution. Figure S7. Formation of AuNPs by incubation of guanine with HAuCl4 solution (PDF)

AUTHOR INFORMATION

Corresponding Author

*Phone/Fax: +81-86-251-8106. E-mail: [email protected]. ac.jp. ORCID

Tatsuki Kunoh: 0000-0002-8423-2903 Author Contributions

T.K. and M.Takeda conceived the overall experimental strategy, performed all physiological and microscopic experiments, and wrote the manuscript. S.M. did the TEM imaging and ED analyses. STEM imaging and some ED analyses were performed by the Kobelco Research Institute (Kobe, Japan). I.S., M.Takano., H.K., and J.T. developed the original concept of the project and/or provided technical advice. Funding

This study was financially supported by JST-CREST (2012− 2017) (J.T.) and a Grant-in-Aid for Scientific Research (C) 16K07714 of JSPS (M.Takeda). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We appreciate professors Yoshihiro Kusano, Tomoki Shiraishi, and Kazuhiro Toyoda for valuable comments. We also thank Drs. Makoto Nakanishi, Tomonari Kasai, and Katsunori Tamura and Ms. Mika Yoneda and Keiko Toyoda for technical supports. We acknowledge Dr. Beth E. Hazen for reviewing and editing the manuscript.

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