A DNA-Encapsulated and Fluorescent Ag106+ Cluster with a Distinct

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A DNA-Encapsulated and Fluorescent Ag Cluster with a Distinct Metal-like Core 106+

Jeffrey T. Petty, Mainak Ganguly, Ian J. Rankine, Daniel M Chevrier, and Peng Zhang J. Phys. Chem. C, Just Accepted Manuscript • Publication Date (Web): 23 Jun 2017 Downloaded from http://pubs.acs.org on June 23, 2017

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The Journal of Physical Chemistry

A DNA-Encapsulated and Fluorescent Ag106+ Cluster with a Distinct Metal-like Core Jeffrey T. Petty,*†o Mainak Ganguly, † Ian J. Rankine,†

Daniel M. Chevrier‡o and Peng Zhang*‡

†Department of Chemistry Furman University Greenville, SC 29613 ‡Department of Chemistry Dalhousie University Halifax, NS, Canada B3H 4R2

ABSTRACT: Silver cluster - DNA complexes are optical chromophores, and pairs of these conjugates can be toggled from fluorescently dim to bright states using DNA hybridization. This paper highlights spectral and structural differences for a specific cluster pair. We have previously characterized a cluster with low emission and violet absorption that forms a compact structure with single-stranded oligonucleotides. We now consider its counterpart with blue absorption and strong green emission. This cluster develops with a single-stranded/duplex DNA construct and is favored by low silver concentrations with ≲8 Ag+:DNA, an oxygen atmosphere, and neutral pH. The resulting cluster displays key signatures of a molecular metal with well-defined absorption/emission bands at 490/550 nm, and with a fluorescence quantum yield of 15% and lifetime of 2.4 ns. The molecular cluster conjugates with the larger DNA host because it chromatographically elutes with the DNA and it exhibits circular dichroism. The silver cluster is identified as Ag106+ using two modes of mass spectrometry and elemental analysis. Our key finding is that it adopts a low-dimensional shape, as determined from a Ag K-edge extended X-ray absorption fine structure analysis. The Ag0 in this oxidized cluster segregates from the Ag+ via a sparse number of metal-like bonds and a denser network of silver-DNA bonds. This structure contrasts with the compact, octahedral-like shape of the violet counterpart to the blue cluster, which is also a Ag106+ species. We consider that the blue- and violet-absorbing clusters may be isomers with shapes that are controlled by the secondary structures of their DNA templates.

Introduction As bulk noble metals shrink to nanoparticles and clusters, a diverse suite of chromophores develops.1 These spectra emerge because the continuum of electronic energy levels segregates into bands of states and ultimately into discrete levels.2-4 For noble metal clusters with ~10 atoms, their diverse spectra and photophysical characteristics offer the prospect of a new class of optical labels and reporters, and we are particularly interested in silver clusters that develop with DNA hosts.5-11 Their distinctive

optical properties are imprinted by the cluster stoichiometry, as single-atom changes in silver and gold clusters produce spectra that span the ultraviolet to near-infrared regions.12-14 Specific species are stabilized via electronrich ligands that trap nascent, atomically-distinct clusters when cationic metal precursors are chemically reduced. For example, alkyl thiols tune the sizes of 1-3 nm diameter gold clusters via the initial thiol:gold precursor ratio and the reaction specificity via the thiol substituents.15-16 These cluster-ligand complexes can be synthe-

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sized with atomic precision, which has enabled in-depth characterization of structure and electronic properties through crystallographic, spectroscopic, and computational methods.17-20 Furthermore, ligands encapsulate reactive metal clusters and create chemically stable complexes in solution. This protective shell is modular and functionalizes the metal chromophore for specific tasks such as phase transfer, analyte recognition, targeted interactions, and catalysis.8, 21-22 Our studies utilize DNA strands with ~10-30 nucleobases that form molecular silver clusters in aqueous solution.23 The electron-rich heteroatoms in DNA nucleobases coordinate silver clusters with ~10 atoms, and the resulting adducts are optical chromophores whose spectra are tuned by the DNA sequence and structure.24-33 These composite chromophores absorb across the visible to near-infrared spectral regions with extinction coefficients ~105 M-1cm-1, fluorescence quantum yields ~30%, and fluorescence lifetimes ~2 ns.30, 34-35 Thus, these fluorophores can be efficiently excited and strongly emit, even in biological environments with high autofluorescence backgrounds.36-38 The DNA-cluster conjugates have hydrodynamic radii ~1 nm, sizes that are smaller than semicrystalline quantum dots labels and comparable to organic dyes. 30, 39-40 41-42 Oligonucleotides also functionalize their cluster adducts, and our studies focus on DNA-cluster complexes that are fluorescent sensors.43-55 When a DNA host is lengthened, it can bind not only a silver cluster but also a target analyte, as exemplified by oligonucleotides with 5’ sequences that stabilize specific cluster chromophores and 3’ sequences that hybridize with target complementary strands.7-8 These complements bind specifically and strongly but are not simply passive binders. They can reshape cluster environments by switching the DNAbound cluster from a dim to highly fluorescent species with as much as ~500-fold stronger emission.46, 56-57 Thus, DNA-silver cluster conjugates signal specific DNA analytes via spectral and photophysical changes that rival those of other optical DNA sensors such as molecular beacons.58-59 Moreover, these cluster-based sensors are spectrally tunable, conveniently synthesized in-situ without purification, and cost effective. While molecular beacons fluorescence when target analytes separate the proximal and quenched organic dyes, silver cluster chromophores take a different route because they are single molecules within an encapsulating DNA matrix.36, 60-61 Our goal is to better understand and utilize this alternative mechanism of fluorescence signaling, so we have studied a specific pair of clusters: one with low emission and violet absorption and its partner with strong green emission and blue absorption.39, 61-62 Previously, the stoichiometry, charge, and structure of the violet species were measured using mass spectrometry along with X-ray absorption and optical spectroscopies.61 Now, this analysis is extended to its fluorescent variant. We studied the synthetic conditions that favor this species and characterized its fluorescence. Importantly, this fluorescent cluster has the identical stoichiometry and oxidation state as its violet counterpart but has a distinct shape. We consider how cluster

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shape and structure may be linked with metal-based fluorescence. Experimental methods We followed the experimental protocol from our earlier studies, and the key aspects are summarized.39, 63-64 Our studies focused on two DNA strands with the following sequences: CCCCACCCT-CCCGCCTTTTGGCGGG (S1-Hp) and CCCCAACTCC-CCCGCCTTTTGGCGGG (S2-Hp), where the strand polarity is 5’ → 3’. The 5’ regions (bold) were designed to favor the blue-absorbing/green-emitting clusters via their cytosine-rich sequences.30, 65 The 3’ regions (italics) were designed to fold into hairpins with 6 base pair stems and dT4 loops (Figure 1B). The clusters were typically prepared using 30 µM oligonucleotide with 8 equivalents of Ag+ (AgNO3, Acros) and 4 equivalents of BH4- (NaBH4, Aldrich) in a 20 mM ammonium acetate buffer at pH = 7 with 500 psi O2 for 1 h. Absorption spectra were acquired with a Cary 50 (Varian), circular dichroism spectra were measured with a DSM 17 CD spectrophotometer (Olis), and reversed-phase chromatograms were collected with a Prominence high performance liquid chromatography system (Shimadzu). Solutions for mass spectrometry were dialyzed with 102 – 104 volumes of buffer and then diluted to ~1 µM oligonucleotide for infusion. Mass spectra were acquired in negative ESI mode using a Q-TOF G2-S (Waters) following our earlier procedure.39, 61 Mass spectra were acquired in positive ESI mode using a Q ExactiveTM Plus Mass spectrometer (Thermo Scientific) via the HESI-II source (Thermo Scientific). The complexes were diluted in a 5 mM ammonium acetate buffer at pH = 7. The sample was directly infused at a rate of 20 μL/min. The spectra were acquired with a sheath gas flow of 12 units, auxiliary gas of 3 units and no sheath gas flow, a spray voltage of 2.9 kV and a capillary temperature of 120 oC. The automatic gain control (AGC) targets of 1×106 with a maximum injection time of 1000 ms, and 10 microscans were combined per spectrum. Spectra were averaged using Qual Browser Ver. 3.0.63 (ThermoScientific) and deconvoluted manually. Ag K-edge (25514 eV) extended X-ray absorption fine structure (EXAFS) spectra of Ag-DNA conjugates in buffered solution (~1 mM concentration of the Ag-DNA species) were collected at the CLS@APS beamline (Sector 20BM) at the Advanced Photon Source, Argonne National Laboratory (IL, USA). Experiments were conducted at room temperature in solution-phase and under ambient conditions. EXAFS data were collected in fluorescence mode using a 12-element fluorescence detector. Multiple scans were averaged to achieve minimal experimental noise in the late k-region of the EXAFS data. Raw EXAFS data were background subtracted, energy referenced, normalized, and further transformed to k- and R-space (FT-EXAFS) using the WinXAS3.1 package (see Figure S16 for k-space). Simulated scattering paths used to fit Ag Kedge EXAFS data were generated using FEFF8.2 computational software.66 Reported uncertainties for EXAFS fitting results were computed from off-diagonal elements of the correlation matrix, which were weighted by the square


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root of the reduced chi-squared value obtained from each simulated fit.67 The amount of experimental noise was also taken into consideration for each Fourier transformed R-space spectrum from 15−25 Å.68 Results DNA-Directed Synthesis DNA secondary structure dictates the spectra of pairs of silver cluster chromophores.8, 39, 53, 56-57, 62, 69-74 We first consider two forms of the oligonucleotide CCCCACCCCT – CCCGCCTTTTCC (Strand SI, Figure 1A).65 The 5’ cluster domain (bold) is cytosine-rich because this nucleobase stabilizes silver clusters, and the state of the neighboring 3’ recognition site (italics) controls the type of cluster adduct.27, 29,23 When this 3’ sequence binds with a complementary strand (Strand SII), the hybridized DNA favors a cluster with λmax ~ 490 nm. When this complement is extracted via its 5’ overhang and its perfectly matched partner (Strand SIII), the resulting singlestranded DNA yields a violet species with λmax = 400 nm. Thus, base pairing distinguishes distinct cluster coordination environments.71 In this paper, we focused on the blue-absorbing cluster and simplified its synthesis by replacing the intermolecular duplex with a stem-loop hairpin (Figure 1B). The stem length controls both the DNA stability and the cluster environment. Stems with 3 to 6 base pairs become progressively more stable, as reflected in the melting temperatures and enthalpy/entropy changes for unfolding (Figure S1A&B).75 In turn, DNA stability correlates with changes in the cluster environment (Figures 1B and S2). Longer duplexes produce stronger absorbance at 490 nm and suppress other absorptions. Thus, they favor the target cluster. Our studies focused on the constructs with 6 base pair stems, whose stability controls the cluster adduct. As found in previous studies, the cluster changes the stability of its DNA , the cluster stabilizes its DNA host (Figure S1C). Our studies focused on the 6 base pair stem as a common stem that was joined with two 5’ cluster domains S1 and S2. Both produce the target blue-absorbing cluster (Figure S2). The S1 sequence CCCCACCCCT was used because its contiguous cytosines allows us to better understand the DNA-cluster interaction.65 The S2 sequence CCCCAACTCC was used because it also stabilizes the violet cluster in an analogous single-stranded DNA.39, 61 The blue cluster that develops with the S1-Hp and S2Hp DNA constructs is favored by three reaction conditions. First, lower Ag+:DNA concentrations suppress alternate species (Figures 1C and S3). With 2-8 Ag+:DNA, a single absorption band at ~490 nm increases with the Ag+ concentration, and this trend suggests that only one type of cluster is thermodynamically favored at these relatively low Ag+ concentrations. With 16 Ag+:DNA, this preference shifts because the λmax shifts to ~ 505 nm and a shoulder develops at ~ 570 nm. These spectral changes suggest that concentrations ≳8 Ag+:DNA

saturate the initial binding site and populate an alternate site. With 32 Ag+:DNA, a new band with λmax ~ 450 nm develops. Its breadth contrasts with the sharper spectra at lower Ag+:DNA stoichiometries and thus suggests that the molecular clusters agglomerate into a larger nanoparticle. Our studies used 8 Ag+:DNA to focus on the blue cluster, and we propose that this relative concentration approximates the cluster stoichiometry. Ag+ binds DNA with an affinity constant of ~6 x 106 M-1, so ~99% of the Ag+ is bound to the DNA with our initial concentrations of 240 µM Ag+ and 30 µM DNA.39 These Ag+ have a local concentration of ~3 M within their DNA host and are expected to coalesce into a cluster following chemical reduction.39 Second, oxygen eliminates competing species

Figure 1: (A) Absorption spectra of the clusters that develop with two forms of the SI oligonucleotide. The blue cluster (red spectrum) with λmax~490 nm develops with the hybridized form SI-SII. The violet cluster (blue spectrum) with λmax~400 develops when this complement is extracted by the perfectly matched SIII via its 5’ toehold. (B) Absorption spectra of the S1-Hp/Cluster complex with 3 (black) and 6 (red) base pair stems. The black lines represent the stem-loop hairpin and red lines represent the CCCCACCCCT cluster domain. (C) Absorption spectra of the S1-Hp with 2 (black), 4 (blue), 8 (red), and 16 (green) Ag+:DNA. The dotted line shows that the λmax = 493 nm is constant up to 8 Ag+ but shifts to λmax~503 nm at 16 Ag+ :DNA, and a new band also develops at ~560 nm.

(Figure S4). Samples were prepared in atmospheres of air and oxygen with spectra collected after ~1 h, and oxygen produces a stronger absorption at 490 nm with relatively weaker absorptions at 450 and 540 nm. Both samples



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yield a common spectrum after ~10 h, and this time evolution suggests that this reaction is slower in air due to its lower oxygen concentration. Hydrogen peroxide yields analogous results, so we suggest that these oxidizing agents decompose alternate species and thereby favor a partially oxidized cluster.7,76 Third, neutral pH stabilizes the cluster (Figure S5). Solutions with pH = 6-8.5 yield similar clusters with λmax ~490 nm, but their absorbances diminish at higher pH. These differences may arise because the nucleobases have distinct pKa values.77 The N3 in cytosine deprotonates at neutral pH and thereby binds silver clusters.27 The N3 of thymine also deprotonates, but at higher pH and correspondingly yields a different cluster. However, acidifying back to neutral pH recovers the original species.28, 65 This reversibility suggests that non-protonated cytosines coordinate the blue cluster at neutral pH and that the deprotonated thymine disrupts this coordination environment at higher pH. In summary, these chemical studies highlight features of the blue-absorbing cluster that frame our subsequent discussion. Low Ag+:DNA concentrations support a molecular cluster, oxygen may partially oxidize the cluster, and neutral pH deprotonates cytosines that stabilize molecular silver clusters.

A Molecular and DNA-Bound Cluster A molecular silver cluster is most distinctly signified by its fluorescence (Figure 2A). Silver differs from gold because it has a larger s-d energy gap, leading to electronic spectra with sharp transitions for molecular forms of sil-

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ver.78-79 Furthermore, silver clusters with DNA radiatively relax with strong emission in comparison to their bulk and nanoparticle forms.13, 30, 78, 80 The cluster studied here fluoresces at 550 nm when excited at 490 nm, has a fluorescence lifetime of 2.4 ns, and a quantum yield of 15% (Figures 2A, 2C, S6, and S7). The emission band does not shift with the excitation wavelength, and the excitation and absorption maxima coincide. These observations support a single type of DNA-bound cluster.23 This molecular cluster develops with a larger DNA host, as shown by chromatography and spectroscopy studies. Reversed phase chromatography resolves two types of S1-Hp strands (Figure S8). Both species absorb at λmax ~ 260 nm due to the nucleobases, but the leading species also absorbs at 490 nm due to the cluster. Thus, this latter species is ascribed to the cluster-DNA complex, which may elute earlier because silver clusters can alter DNA shape and charge.39, 70, 81 In addition, circular dichroism suggests that the DNA host alters its cluster adduct (Figures 2B and S9). A transition at 490 nm coincides with the cluster absorption and suggests that the cluster either becomes chiral or develops an induced response due the chiral DNA template.18 The anisotropy κCD gauges the intrinsic circular dichroism by normalizing the ellipticity using the absorbance, and this ratio is thus independent of the cluster concentration.82 The resulting κCD = 0.0004 is relatively low when compared against other DNA-bound silver clusters such as the violet cluster with κCD = 0.0012.39, 61, 83 This difference suggests that the blue cluster has a distinct structure and/or binding site.82 An Oxidized Ag106+ Cluster Strong fluorescence and a single, sharp absorption band for ≲8 Ag+:DNA signify a molecular silver cluster, and its stoichiometry was measured by mass spectrometry and elemental analysis. Our analysis focuses on the isotopic fine structure in the m/z spectra because this reveals both the cluster stoichiometry and charge (Figure 3).39, 84-85 The peak spacings and intensities are largely dictated by the two isotopes of silver - 107Ag (51.84%) and 109 Ag (48.16%). However, the peak envelops shift with the number of hydrogens. H+ cations condense onto the ionized phosphate backbone during the negative electrospray process and partially neutralize the overall negative charge of the complex. We particularly focus on how the numbers of H+ adjust with the numbers and charges of the silver adducts, and we first consider the simple case of Ag+ complexes with S1-Hp and their -6 charged ions (Figure S10). The oligonucleotide alone has 6 ionized phosphates and has the formula C246H312O158N87P25, which has 6 fewer H+ relative to its fully protonated neutral strand. This ion complexes with 1 - 6 Ag+ but with incrementally fewer H+. This balance suggests that the DNAAg+ ions maintain their -6 charges because the Ag+ adducts replace H+. We used the numbers of H+ in the DNA-cluster complexes to indirectly measure the cluster charge.35

Figure 2: Spectral and photophysical characterization of the silver cluster/S1ACS Paragon Hp complex. (A) Absorption (blue) and emission (red) spectra exhibit maxima at 490 and 550 nm, respectively. The inset describes the hairpin structure of S1-Hp host. (B) The circular dichroism spectrum determines a relatively low anisotropy κCD = 0.004. (C) The 550 nm fluorescence lifetime τFluor is 2.4 ns.

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The mass spectra of the DNA/cluster samples at neutral pH reveal a pattern of four peaks, and their masses correspond to the S1-Hp oligonucleotide with 3, 4, 10, and 11 silvers (Figures 3A/B and S11). Four experiments distinguish these species and ultimately determine the stoichiometry and charge of the blue cluster. First, the adducts have different oxidation states, as revealed by the isotope distributions. The isotopologue masses and intensities for the -6 charged strands with 3 and 4 silvers are respectively modeled by the formulas C246H309O158N87P25Ag3-6 and C246H308O158N87P25Ag4-6 (Figures S12A&B). These ligated strands have 3 and 4 fewer H+ relative to the -6 charged oligonucleotide with the formula C246H312O158N87P25, respectively. So, the numbers of H+ and Ag+ compensate, which suggests that these DNA-bound silvers are fully oxidized.86 Because of the small numbers of silvers, these complexes are unlikely to form clusters and are thus individual Ag+ adducts with the DNA. The peak envelops for the strands with 10 and 11 silvers were similarly analyzed using the formulas C246H306O158N87P25Ag10 and C246H305O158N87P25Ag11, respectively (Figures 3C and S12C&D). These species have 6 and 7 fewer H+ relative to the -6 charged oligonucleotide, which suggests that these Ag10 and Ag11 clusters have net charges of +6 and +7, respectively. Thus, both species have 4 reduced silvers, and noble metal clusters with such small numbers of reduced silvers are optical chromophores.4, 87 Second, the charges of the clusters were independently measured using positive ion mode electrospray ionization mass spectrometry. This technique produces positively charged DNA ions via NH4+, which has a lower gas-phase proton affinity than the phosphates or basic functional groups in nucleobases.88 The positively charged DNA ions alter the electrostatic and chemical environment of the underlying, encapsulated cluster, and our studies probed the cluster stoichiometry and charge in this new environment. The prominent peaks are the +4 charged complexes of S1-Hp with 4, 10 and 11 silvers (Figure S13). The isotope distributions predict numbers of H+ and silver oxidation states that agree with the results from the negative electrospray experiments; i.e., the 4 silver complex has completely oxidized silvers whereas the 10 and 11 silver complexes have charges of +6 and +7, respectively. Thus, the two modes of mass spectrometry suggest that the Ag10 and Ag11 clusters are partially reduced with 6 and 7 Ag+, respectively, but both with 4 Ag0. Third, the distribution of the four silver species changes with pH. The samples were prepared at pH = 7 to favor the blue cluster and subsequently diluted in pH = 7, 7.5, 8.5, and 9 solutions (Figures 3A&B, S5, and S14). Higher pH disfavors the complexes with 3 and 4 silvers and concurrently favors the unligated, native oligonucleotide. This correlation suggests that the complexes with 3 and 4 Ag+ adducts dissociate at higher pH, as also shown by S1-Hp complexes with Ag+ alone (Figure S15). In contrast, higher pH retains DNA complex with 10 silvers, and this species becomes the dominant adduct at pH = 9 (Figures 3B). Thus, we suggest that the blueabsorbing and green-emitting cluster is the adduct with 6 Ag+ and 4 Ag0. Higher pH also disfavors the 11 silver adduct. Like the Ag10 cluster, this species also has 4 reduced

Figure 3: (A&B) Mass:charge spectra of the -6 charge states of the S1Hp with silver at pH = 7 and 9, respectively. The pH = 7 buffer forms four species with 3, 4, 10, and 11 silvers. The pH = 9 buffer favors the S1-Hp oligonuncleotide and its complex with 10 silvers. (C) Expanded view of the -6 charged ion of the Ag10/S1-Hp complex (red spectrum). The solid lines represent the predicted distributions based on the formulas with H305 (blue), H306 (black), and H307 (green). The spectrum with H306 gives the best prediction based the uncertainties in the masses (σMass) and the intensities (σInt).

silvers but with 7 Ag+, so higher pH may eliminate the additional Ag+ and produce the favored 6 Ag+/4 Ag0 species. Fourth, the blue cluster was chemically isolated and studied by elemental analysis.40 The cluster-DNA complex was first separated and isolated from its unlabeled DNA counterpart via reversed phase chromatography, and this purified product was analyzed via inductively coupled plasma atomic emission spectroscopy (Figure S8). The emission intensities at 213.67 and 328.07 nm yielded the concentrations for phosphorus and silver, respectively, with phosphorus acting as the elemental surrogate for DNA. These relative amounts determined an empirical stoichiometry of 9.3 ± 0.3 Ag/DNA, which supports the mass spectral results. In summary, these mass spectral and elemental analysis studies suggest that the blue-absorbing/green-fluorescent silver cluster has 6 Ag+ and 4Ag0. These DNA-bound silvers are expected to agglomerate into a single cluster due to their proximity



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with the DNA scaffold. This assertion is supported because oligonucleotides with different lengths yield common clusters with the same spectra and same silver stoichiometries.69 Altered Core The fluorescent cluster is Ag106+, and it violet partner has the same stoichiometry and charge.61 Yet, the two cluster are distinct chromophores, so their structures were studied using the Ag K-edge extended X-ray absorption fine structure (EXAFS) technique (Figures 4 and S16).89-90 We first consider the S2-Hp/blue cluster complex because a similar host stabilizes the violet cluster – both DNA strands have the same cluster domain CCCCAACTCC but have duplex vs open recognition sites, respectively (Figure S16). Both species exhibit 3 distinct scattering paths for the photodetached electron from the silver, and the spectra were fit to determine bond distances, coordination numbers, and Debye-Waller factors (Figure 4 and Table 1). The first peak is ascribed to silvernucleobase bonding because the associated bond distances of 2.1 – 2.2 Å is typical for Ag+- and Ag0-nucleobase complexes.91-95 EXAFS fitting cannot discriminate Ag-N vs. Ag-O scattering paths, and prior experimental and theoretical studies support cluster complexes with both heteroatoms.27-29, 96 The second scattering peak is as-

Figure 4. (A) Ag K-edge EXAFS of the Ag/S1-Hp conjugate fitted with three individual scattering paths (shown separately). (B) Ag Kedge EXAFS of the Ag/S2-Hp conjugate (green) and the Ag/S2 DNA (purple).

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X-ray absorption near-edge structure (XANES) spectra have energy minima after the absorption edge at 25.545 and 25.550 keV for violet and blue clusters, respectively (Figure S16). This difference signifies structural differences, as observed for other molecular clusters.102-103 Second, in relation to the violet species, the blue cluster exhibits relatively weaker Ag-Ag1 scattering and thus a distinct metal-like core (Table 1). The two cores have identical Ag-Ag1 bond distances within experimental error (2.73-2.74 Å), but their coordination numbers differ by ~three-fold. The higher coordination number of 2.2 for the violet cluster is consistent with an octahedral arrangement of 6 silvers (Figure S16B).61 The lower coordination number of 0.7 for the blue cluster can be explained with a 4 atom core, and linear, rhombus, and tetrahedral arrangements are possible within the uncertainty of our fitting (Supplemental Information, Section II). Third, a reorganized core may also be reflected in the silver-DNA interaction (Table 1). The violet cluster has relatively high Ag-Ag1 coordination but correspondingly low Agnucleobase coordination, and the Ag-nucleobase coordination number of 1.0 signifies intermittent contacts between the Ag+ and it 20-nucleobase polymeric ligand. Conversely, the blue cluster has lower Ag-Ag1 coordination vs higher Ag-nucleobase coordination, and the Agnucleobase coordination number of 1.8 suggests that diminished contacts within the core are balanced by greater contacts with the DNA. To further understand this interaction, we consider the S1-Hp complex because of its sequence and structure. The S1 cluster domain (C4AC4T) has a repeated pattern of contiguous cytosines, whose non-protonated N3 sites coordinate silver at neutral pH.27 This sequence is also folded, as shown by chromatographic and structural studies.65 We propose that the silvernucleobase coordination number of ~2 develops because 2 cytosines coordinate 1 silver, the prominent structure of a range of Ag+-DNA complexes.104 Cytosines have a several electron-rich binding sites, so a range of coordination geometries could allow a 2 cytosine:1 silver arrangement.96 In summary, this balance between silver-silver and silverDNA contacts supports a distinct core for the blue cluster vs its violet counterpart. Discussion

signed to metallic silver-silver bonding (Ag-Ag1 in Table 1). The fitted bond distance of 2.74 Å is similar to that in bulk silver and in the reduced cores of thiolate-silver complexes.97-100 Because the Ag106+ is largely oxidized, this metallic bonding suggests that the reduced silver atoms coalesce and sequester away from the oxidized silvers.17 The third scattering peak may develop because of longrange silver-silver bonding (Ag-Ag2 in Table 1).97, 101 A fitted bond distance of ~3.3 Å matches the sum of two van der Waals radii for silver and suggests a weak interaction between Ag+-Ag+/Ag0.97 This analysis indicates that the two clusters share the same gross structures: a reduced, metal-like core that is encapsulated by a Ag+-DNA shell. However, their spectra signify three structural differences. First, the Ag K-edge

Silver clusters can be switched from dim to bright fluorophores via DNA hybridization and are thus optical DNA sensors with high signal contrast and tunable spectra.7-8, 46, 71 To better understand this enhancement mechanism, we have focused on a specific spectral pair. The precursor cluster forms in a single-stranded DNA and has a sharp violet absorption band with λ ~ 400 nm and weak emission. Its counterpart develops when the oligonucleotide host hybridizes with a short complementary strand to yield a duplex with a dangling 5’ single-stranded cluster domain. This new chromophore is now a fluorophore with a blue absorption/excitation band at λ ~ 490 nm and 60-fold stronger green emission at λ ~ 550 nm (Figures 2, S6, S7).39, 61-62 Because these two silver clusters have a small number of atoms, their valence electronic structure


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and optical spectra depend on the cluster composition, charge, and structure.13, 105 We have studied these factors using optical and X-ray absorption spectroscopies, mass spectrometry, and thermodynamic studies.62, 65 Reversibility is a core feature of the violet ↔ blue cluster transformation. The blue converts to the violet cluster when the complement is isothermally extracted (Figure 1A). Additionally, these chromophores interconvert by changing temperature, and an isosbestic point between their absorption bands supports a two-state equilibrium.62, 65 Enthalpy and entropy changes determined from van’t Hoff analysis suggest that the blue cluster changes to the violet species when the complement denatures from the recognition site. The blue cluster recovers when the complement reanneals. Furthermore, the clusters reversibly transform when the reagents are diluted 10-fold.62 Because dilute solutions would impede intermolecular reactions such as agglomeration and oxidation/reduction, this concentration independence suggests that the cluster stoichiometry and charge are preserved during the accompanying changes in DNA structure. DNA structural and cluster spectral changes may also be coupled by changing the solvent.83 Our mass spectrometry, optical spectroscopy, and Xray absorption spectroscopy studies establish an even stronger connection between the two chromophores. Both clusters have the same molecular formula Ag106+.61 The stoichiometry and charge of the blue cluster were determined via negative and positive mode electrospray ionization mass spectrometries, and the stoichiometries were substantiated by elemental analysis of a chromatographically purified sample and by the Ag+-dependent absorption spectra (Figures 1, 3, S8, and S10-S14). The blue and violet clusters also retain similar structures with a metal-like core within a Ag+ - DNA shell, as determined from the bond distances, coordination numbers, and Debye-Waller factors derived from the Ag K-edge EXAFS spectra (Figures 4 and S16). However, the two clusters are distinct from two experimental perspectives. First, the circular dichroism anisotropy is ~3-fold lower for the blue vs. the violet cluster, and their distinct chiralities suggest that the two clusters have different binding sites and/or structures within their respective DNA hosts (Figures 2B and S9).83, 106 Second, X-ray absorption spectra discern distinctions in the spectral and structural fingerprints (Figure 4). Relatively low Ag-Ag1 and relatively high Agnucleobase coordination numbers indicate less metallic and more silver-DNA bonding in the blue vs. the violet cluster, and we suggest a metal-like core with 4 reduced silvers. Quantum mechanical calculations have identified a longitudinal mode in linear clusters whose energy gap changes with the number of silvers.107 This scaling law is followed by a range of purified silver-DNA complexes, and the size and spectra of our Ag40 blue cluster fits this trend.35 Such a structure may be imprinted by its DNA host, and the 4 Ag0 may be arranged by the S1 cluster domain. We chose the S1-Hp template because its cluster domain has two C4 segments that are folded.65 This set of 4 juxtaposed cytosines could coordinate the Ag0 via their

N3 heteroatoms, as suggested by prior studies of Ag+ cytosine complexes.27, 29 The host DNA structure resolves the spectra of the violet and blue clusters, and we suggest that hybridization defines distinct cluster binding sites. Open nucleobases coordinate Ag+ and silver clusters but also H+, and these adducts compete for heteroatom binding sites, as indicated by two experiments.24, 29-30, 32, 108 First, hybridization modulates cluster emission.71 The DNA templates have a 5’ cluster domain and a 3’ recognition site, and emissive clusters form when the complementary strand hybridizes with the latter recognition site. However, cluster emission diminishes when longer complements invade the 5’ cluster domain, which suggests that base pairing obstructs cluster binding sites. Second, these sites can be targeted via changes in pH. 27-28 To illustrate, the homooligonucleotides dT12 and dC12 yield fluorescent clusters for solutions with pH ≳ 10 and 4.5, respectively. These pH values match the pKa for the N3 in these two nucleobases, which suggests that protonated nucleobases inhibit cluster formation.77, 109 Our studies used a hybridized DNA construct to synthesize a specific blueabsorbing/green-emitting silver cluster, but this preference depends on the DNA secondary structure. The length and stability of the duplex component determine the yield of this product (Figures 1B and S1). More importantly, a single-stranded variant of this DNA forms the violet counterpart to the blue cluster (Figure 1A). Such oligonucleotides encapsulate their violet adducts via multiple silver-nucleobase contacts.39, 61-62 We suggest that these contacts are broken via base pairing with the short complementary strand, so hybridization forces the violet cluster to the relatively short S1/S2 cluster domains within the larger S1-Hp/S2-Hp constructs (Figure 4B). This new coordination environment may reorganize the cluster structure without changing its stoichiometry and charge. This hypothesis is supported by thermodynamic studies that shifted the registration of the complementary strands within the recognition site.62 From the pioneering work of Faraday to today, our understanding of the relationship between the electronic spectra of nanoparticles and their sizes has greatly improved.110-111 The valence electronic states of smaller molecular clusters also depend on size/stoichiometry but also structure.4, 87 Different isomers can be separated by shallow wells, so minor changes in the temperature and/or coordination environment impact the electronic state organization.13, 112-115 For example, two spectrally distinct forms of a Au28 species are controlled by the alkyl substituents of the thiol ligands, and this structural transformation reorganizes the interfacial Au-S staple motifs.114 We have focused on a pair of silver clusters with dramatic electronic changes: an ~ 90 nm shift in the absorption and ~60-fold stronger 550 nm emission. We suggest that these stark spectral differences arise because the DNA ligands prescribe distinct coordination environments. Thus, we are motivated to better understand how DNA structure and cluster coordination are linked and hope to



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develop a useful and potentially powerful optical DNA sensor. Conclusion Our studies address silver clusters that transform from dim to bright fluorophores. We have focused on a blueabsorbing/green-emitting cluster that is bound to a hybridized DNA construct. This cluster is related to a violet-absorbing but weakly emitting cluster that binds with a purely single-stranded DNA. These two species have the same silver stoichiometry and charge and reversibly interconvert but have distinct structures. In relation to the violet cluster with an octahedral-like structure, the blue species is more extended with sparse silver-silver bonding and extensive silver-nucleobase bonding. These structural differences may change the organization of the valence electrons in the silver clusters and thus alter their photophysical signatures. These studies also highlight how DNA hybridization can reshape the cluster binding sites and suggest that the well-established rules for DNA base pairing could be leveraged to fine-tune the optical properties of DNA-bound silver clusters.

ASSOCIATED CONTENT The supporting information contains 17 figures describing absorption spectra of the cluster with four stem-loop hairpins, absorption spectra of S1-Hp and S2- complexes with the blue cluster, absorption spectra of the S1-Hp with varying + amounts of Ag , absorption spectra of S1-Hp/cluster complexes with and without oxygen, absorption spectra of the S1Hp/cluster complex at different pH values, emission spectra of the S1-Hp/cluster complex, fluorescence lifetime measurements, reversed phase chromatograms of the S1Hp/cluster samples, circular dichroism spectra of the S1-Hp and S2-Hp complexes, mass spectra of S1-Hp with and with-

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+

out Ag , mass spectra of the -5, -6, -7, and -8 charge states of the S1-Hp/cluster complex, mass spectra that show how the intensities distributions shift with the numbers of hydrogens, positive ion mode mass spectra of the S1-Hp/cluster complex, mass spectra at different pH values, mass spectra of S1+ Hp/Ag complexes at pH = 7 and 9, Ag K-edge k-space of S1Hp and S2-Hp Ag10 blue clusters and FT-EXAFS of Ag10 S2-Hp cluster with simulated three-shell fit, and Ag K-edge FTEXAFS and XANES spectra for Ag10 blue clusters and Ag10 violet clusters. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author Email: [email protected] Phone: 864-294-2689

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. oThese authors contributed equally. ACKNOWLEDGMENT Technical support at CLS@APS (Sector 20-BM, APS) facilities from Dr. Robert Gordon and Dr. Zou Finfrock is acknowledged. We greatly appreciate the assistance of David Smalley with the mass spectrometry and conversations with R. Dickson. We also thank the reviewers for their insights and suggestions. We thank the National Science Foundation (CHE1611451), National Institutes of Health (1R15GM102818), and the Furman Advantage program. D.M.C. is supported by the NSERC CGS-Alexander Graham Bell scholarship. P.Z. acknowledges the NSERC Discovery Grant for funding. CLS@APS facilities (Sector 20) at the Advanced Photon Source (APS) are supported by the U.S. Department of Energy (DOE), NSERC Canada, the University of Washington, the Canadian Light Source (CLS), and the APS. Use of the APS is supported by the DOE under Contract DE-AC02-06CH11357.


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Table 1. Ag K-edge EXAFS fitting results for Cluster-DNA conjugates.a

Scattering 6+

Ag10 -Single DNA Violet Cluster

Stranded

6+

Ag10 -S1-Hp DNA Blue/Green Cluster

DNA Blue/Green Cluster

σ2 (Å )

ΔE0 (eV)

1.0(1)

2.24(1)

0.004(2)

-6(1)

Ag-Ag

1

2.2(3)

2.743(8)

0.011(1)

-6(1)

Ag-Ag

2

3.6(1.7)

3.37(2)

0.026(7)

-6(1)

Ag-Nucleobase

1.8(7)

2.14(3)

0.011(6)

-7(3)

Ag-Ag

1

0.7(4)

2.75(2)

0.004(5)

-7(3)

Ag-Ag

2

3(3)

3.34(6)

0.02(1)

-7(3)

Ag-Nucleobase 6+ Ag10 -S2-Hp

R (Å)

Shell Ag-Nucleobase

61

2

CN

2.2(7)

2.15(2)

0.012(5)

-8(2)

Ag-Ag

1

0.8(3)

2.73(1)

0.004(4)

-8(2)

Ag-Ag

2

4(4)

3.30(5)

0.03(1)

-8(2)

a CN are the coordination numbers, R are the bond distances, σ2 are the Debye-Waller factors, and ΔE0 is the shift in absorption edge energy from simulation to experimental results.


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A DNA-Encapsulated and Fluorescent Ag106+ Cluster with a Distinct

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