DNA Templates for Fluorescent Silver Clusters and I-Motif Folding

Oct 19, 2009 - When compared with silver nanoparticles, silver clusters comprised of ∼101 atoms are distinguished by their strong fluorescence, and ...
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J. Phys. Chem. C 2009, 113, 19518–19524

DNA Templates for Fluorescent Silver Clusters and I-Motif Folding Bidisha Sengupta, Kerianne Springer, Jenna G. Buckman, Sandra P. Story, Oluwamuyiwa Henry Abe,† Zahiyah W. Hasan,‡ Zachary D. Prudowsky, Sheldon E. Rudisill, Natalya N. Degtyareva, and Jeffrey T. Petty* Department of Chemistry, Furman UniVersity, GreenVille, South Carolina 29613 ReceiVed: July 10, 2009; ReVised Manuscript ReceiVed: October 9, 2009

When compared with silver nanoparticles, silver clusters comprised of ∼101 atoms are distinguished by their strong fluorescence, and DNA directs and stabilizes particular types of clusters via base-specific interactions. Two main observations considered in this paper are the pH dependence of the fluorescence and the folded conformation of the oligonucleotide-cluster conjugates. Two i-motif forming oligonucleotides (dTA2C4)4 and (dC4A2)3C4 coordinate red and green emissive species, and these fluorescent species are favored in slightly acidic and basic solutions, respectively. The red emission is highest at pH 6, at which the i-motif forms of the oligonucleotides are also stable. When assessed by size exclusion chromatography, the oligonucleotide and cluster conjugate have similar global structures, which indicate that the DNA strands are similarly organized at this pH. The green emission is highest at pH 8-9. In these basic solutions, the oligonucleotide alone is unfolded, yet the green and red cluster-oligonucleotide conjugates have similar shapes. The pH-dependent fluorescence and the compact shapes of the cluster-oligonucleotide conjugates suggest that protons dominate DNA folding for the red emissive species, while the green emissive clusters themselves determine the shape of their DNA matrix. These studies provide the basis for understanding how specific base arrangements and environmental factors influence the formation of this new class of fluorescent nanomaterials. Introduction Relative to larger nanoparticles, clusters of noble metals have distinct physical and chemical properties.1,2 For instance, these metallic clusters have high fluorescence quantum yields because their sparser density of states favors radiative relaxation from electronically excited states.2,3 When compared with organic chromophores, silver clusters display distinctive electronic relaxation that has been utilized for high-sensitivity imaging when background interference is significant.4 Variations in the energy level structure depend on cluster stoichiometry, oxidation state, and geometry, as well as associated ligands, resulting in spectrally distinct clusters.2,5,11,12,14 Thus, having spectral properties that distinguish them from their larger nanoparticle counterparts, as well as organic chromophores, noble metal clusters are a new class of small, innocuous, and bright fluorophores whose spectral properties can be tuned.6,7 An important goal is to achieve stoichiometric and thus spectral control using ligands that bind with and inhibit aggregation.6,8-10 Nucleic acids are one example of a matrix with multiple ligands that can coordinate with small silver clusters.5,8,11-14 Previous studies have shown that Ag+ favors Lewis acid-base interactions with the electron-rich nitrogens and oxygens of the nucleobases.15 Silver clusters also exhibit a preference for the nucleobases. To illustrate, the insensitivity of fluorescence to salt suggests that interactions of clusters with phosphates is not significant.10 Direct evidence in support of complexation between the clusters and bases is inhibition of cluster formation by protonation of the N3 of cytosine and thymine.11,12 * To whom correspondence should be addressed. E-mail: jeff.petty@ furman.edu. † Current address: Allen University, Columbia, SC. ‡ Current addresss: Benedict College, Columbia, SC.

To explore beyond primary interactions between the bases and clusters, this paper addresses the key issue of whether base arrangements in particular DNA structures guide the synthesis of specific types of fluorescent silver clusters. The studies were motivated by the concentration and pH dependence of the red emissive species that forms with dC4T4C4.12 Relative to a bluegreen emissive species that forms with this oligonucleotide, the red emissive species is favored at higher oligonucleotide concentrations, thus indicating that strand aggregation is coupled with cluster formation (Figure S1). In addition, maximum fluorescence occurs at pH 7, at which the N3 of cytosine and thymine are deprotonated and protonated, respectively. This degree of protonation suggests that protons are not influencing how individual bases interact with the clusters.11,12 A possible explanation for the pH and concentration dependence is that higher order DNA structures are involved in cluster formation, as polymorphic forms of DNA are dependent on the solution environment and base composition.16,17 The i-motif is one such form that is favored by cytosine-rich sequences, and it is distinguished by intercalation of two sets of base-paired strands (Scheme 1).18,19 Hemiprotonated cytosine-cytosine pairs are favored when the pH is slightly acidic to neutral and thus close to the equivalence point of the N3 of cytosine.20-22 Such a matrix could provide a favorable environment that stabilizes clusters with the bases while also protecting them from quenching by solvent.9 In this work, two cytosine-rich sequences (dTA2C4)4 and (dC4A2)3C4 with a common C4 i-motif core serve as templates for fluorescent silver clusters. Red and green emissive species are most prominent, and the secondary structure of their oligonucleotide matrix is interrogated using the pH dependence of fluorescence and using the structural information derived from size exclusion chromatography. The pH effect is investigated within the context of the oligonucleotide conformation, and a

10.1021/jp906522u CCC: $40.75  2009 American Chemical Society Published on Web 10/19/2009

DNA Templates for Fluorescent Silver Clusters SCHEME 1: Schematics of the Cytosine-Cytosine Base Pairing (Left) and the Strand Arrangement (Right) in the i-Motif Structuresa

a On the right is a representation of the intramolecularly folded i-motifs used in these studies. Each cytosine is represented by an arrow that shows the strand polarity. The curved lines depict the loops that connect the C4 i-motif core.

correlation is observed between the stability of the secondary structure and the intensity of red emission. The oligonucleotides were designed to allow intramolecular folding, and size exclusion chromatography studies show that the global structures of the cluster conjugate and the i-motif form of the oligonucleotide alone are similar. The green emission is strongest at higher pH. Although the oligonucleotide alone is unfolded in these basic solutions, size exclusion chromatography shows that the structures of red and green emissive species are similar. This similarity indicates that green emitting clusters promote intrastrand interactions that result in folding. Collectively, the observations suggest that multidentate coordination within particular DNA structures determines the favorability of forming specific types of fluorescent silver clusters. Experimental Section Silver clusters were prepared by combining AgNO3 (99.9995%, Alfa Aesar) and oligonucleotide (Integrated DNA Technologies) followed by addition of NaBH4 (99%, Sigma Aldrich) and by vigorous shaking for ≈1 min.10 The typical oligonucleotide concentration of 15 µM was used to conserve materials while still providing high fluorescence intensity. The typical Ag+/ oligonucleotide concentration was 16:1 with equal amounts of Ag+ and BH4-. Buffered solutions used 10 mM acid/conjugate base to maintain the pH, and the buffers were formate, cacodylate, phosphate, and borate. The reactions were conducted at 4 °C because this yielded the highest intensities for both red and green emissive species in the same solution. Spectra were typically recorded after allowing for overnight reaction. Stock solutions of the desalted oligonucleotides were prepared in water, and the concentration was determined using extinction coefficients at 260 nm of 248 500 M-1 cm-1 for (dTA2C4)4 and of 188 600 M-1 cm-1 for (dC4A2)3C4.23 Absorption spectra were acquired on a Cary 50 (Varian) using a scan rate of 600 nm/ min. Temperature-dependent absorbances were collected using a Cary 300 (Varian) using a temperature scan rate of 1 °C/min in 1 °C increments from 20 to 90 °C. At each increment, the temperature equilibrated for 3 min. The buffer contributions to the absorbance profiles were subtracted. Circular dichroism spectra were acquired with a J-710 spectropolarimeter (Jasco). The scan rate was 500 nm/min, and three consecutive spectra were averaged to produce the final spectrum. The temperature was manually varied using a RTE 7 Neslab water bath (Thermo

J. Phys. Chem. C, Vol. 113, No. 45, 2009 19519 Scientific). The fluorescence spectra were acquired using a Fluoromax 3 (Horiba Jobin Yvon). The scan rate was 300 nm/ min with bandwidths of 2 nm for both the excitation and emission. The integration times were 1 s, and the fluorescence intensities are reported in counts/s (cps). All spectra were collected using quartz cuvettes with 1 cm pathlengths. Size exclusion chromatography used 300 × 7.8 mm i.d. Polysep GFC 3000 and GFC 2000 columns (Phenomenex) on a Prominence HPLC system (Shimadzu). These columns allow basic solutions, so that the effect of pH on the conformation of the oligonucleotides and cluster conjugates could be investigated. A SPD20A UV-visible detector (Shimadzu) and a LS-40 fluorescence detector (Perkin-Elmer) were used, and the signals are reported in mV (UV-visible) and counts per second (fluorescence). The time difference between the two detectors was determined from the absorbance (260 nm) and the emission (λex ) 307 nm, λem ) 370 nm) for the oligonucleotide 5′-CAGCA*GCAG-3′, where A* is 2-aminopurine. The injection volume was 20 µL. Three or more chromatographs were acquired to determine an average retention time with a standard deviation. For the thymine oligonucleotides dT5, dT10, dT15, dT20, dT30, the averages and standard deviations were used in the linear fit to relate the retention times to the molecular masses. The molecular masses and relative molecular masses were determined using standard error propagation methods.24 Dilutions do not influence the retention times. Results Oligonucleotide Secondary Structure. Both oligonucleotide concentration and solution pH influence silver cluster formation with the cytosine-rich oligonucleotide dC4T4C4.12 These earlier studies showed that a blue-green emitting species is observed at lower oligonucleotide concentrations while a red emitting species only becomes significant at higher concentrations. This observation suggests that strand aggregation is coupled with cluster formation (Figure S1). In addition, the maximum fluorescence intensity occurs at pH 7, at which the N3 of cytosine (pKa ) 4.5) and thymine (pKa ) 9.5) are primarily in deprotonated and protonated states, respectively. This distinction between the pKa’s and the optimal pH for fluorescence suggests that the degree of protonation of the individual bases is not influencing cluster formation.11,12 One possibility is coordination by multiple bases within a higher-order DNA structure. Because i-motif structures depend on protons, two cytosine-rich oligonucleotides (dTA2C4)4 and (dC4A2)3C4 are considered in these studies. These are related to dA2C4 and dC4A2 that form termolecular i-motif structures with C4 cores that have similar structures.25 For these studies, intramolecular i-motifs were formed by integrating four shorter sequences into a single oligonucleotide. The loop and flanking bases are varied to consider the role of the common i-motif core. To interpret spectroscopic and chromatographic measurements for the intramolecular i-motifs, the constituent sequence dTA2C4 of (dTA2C4)4 was investigated. Consistent with prior studies of dA2C4, dTA2C4 also adopts a termolecular i-motif structure.25 At pH 5, two peaks are present in the chromatogram, and their different retention times suggest differences in the degree of strand aggregation (Figure 1). The most retained species is favored by heating to 90 °C, which indicates that noncovalent interactions in the aggregate are disrupted to form the monomer. Further support for the monomeric form of dTA2C4 is obtained from molecular mass measurements (Figure 1). Thymine oligonucleotides were used to relate observed retention times to molecular mass, as these homooligonucleotides favor single-

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Figure 1. (Top) Size-exclusion chromatograms of a 50 µM solution of dTA2C4 and a 12.5 µM solution of (dTA2C4)4 in 20 mM cacodylate buffer at pH 5 and with 300 mM NaCl. For dTA2C4, two peaks are present in the chromatogram (dotted line) corresponding to an aggregated species with a shorter retention time (Peak A) and monomeric species with a longer retention time (Peak B). Heating the sample to 90 °C followed by cooling (dashed line) results in the conversion of the aggregated species to the monomer. The retention times from three separate measurements are 12.11 ( 0.08 min for the aggregate of dTA2C4, 13.93 ( 0.07 min for the monomer of dTA2C4, and 12.16 ( 0.02 min for (dTA2C4)4. (Bottom) Calibration curve used to relate the column retention time of the monomeric dTA2C4 species to the molecular mass. The single-stranded thymine oligonucleotides dT5, dT10, dT15, and dT30 were used as the size standards. Each data point is the average of three independent measurements with the standard deviation represented by the error bar. The position of the peaks from the chromatogram in the top panel is indicated by the letters A and B above the data points (crosses).

stranded and unfolded conformations.26 The similarity of the measured mass of 2030 ( 240 g/mol to the intrinsic molecular mass of 2025 g/mol suggests that the most retained species in the chromatogram is the monomeric form of dTA2C4. For the least-retained species, reference to the single-stranded thymine oligonucleotides provides a mass that is 2.5 ( 0.1 times the mass of the monomeric dTA2C4. Relative concentrations of the aggregate and monomer do not vary with flow time, indicating that aggregate dissociation is not significant during analysis. While relative mass measurements have been used previously to identify tetramers, other studies have observed mass discrep-

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Figure 2. (Top) Circular dichroism spectra of 5 µM solutions of (dTA2C4)4 in 10 mM formate (pH 3, 4, and 5), cacodylate (pH 6, 7) and borate (pH 8, 9) buffers at 25 °C. The dashed dotted line shows the one at 90 °C for the pH 6 solution. (Bottom) Circular dichroism spectra of 5 µM solutions of (dC4A2)3C4 using the same conditions.

ancies when using thymine oligonucleotides as standards.19,27 Both this latter and the current studies suggest that differences in the shapes of i-motif structures and thymine oligonucleotides influence their retention. Circular dichroism spectra depend on the base arrangements within different types of DNA secondary structures, and these measurements provide further support for i-motif formation by dTA2C4.23,28 The spectra exhibit bands with positive helicity at 288 nm and negative helicity at 256 nm at pH 4-5 range (Figure S2). These spectral positions and their relative ellipticities are consistent with base arrangements in other oligonucleotides that form i-motif structures.21,22,29 The spectra reflect an expected sensitivity to the proton concentration, as ellipticities and wavelengths shift outside the pH 4-5 range. Furthermore, the ellipticities diminish and the spectra shift at high temperature, as expected for noncovalent interactions. The present studies focus on cytosine repeats that are covalently linked, and intramolecular i-motif structures are supported by spectroscopic and chromatographic studies. Both (dTA2C4)4 and (dC4A2)3C4 exhibit transitions in the circular dichroism spectra that are similar to other i-motif forming oligonucleotides with respect to spectral positions, relative ellipticities, and pH sensitivity (Figure 2). The intramolecularly folded i-motifs are stable at higher pH values than the corresponding structure for the intermolecular i-motif of (dTA2C4)4, which suggests that an entropic penalty for i-motif formation

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Figure 3. Retention times determined from size exclusion chromatography for the oligonucleotides and their cluster conjugates at pH 8 (top) and 6 (bottom). The retention times are expressed relative to dT15 to facilitate comparisons between the two solutions. The vertical line demonstrates the distinction between the unfolded oligonucleotide at pH 8 relative to the clusters and the folded i-motif. Three separate measurements were performed to statistically confirm these similarities and distinctions. (Left - (dTA2C4)4) At pH 8, the relative retention time of the oligonucleotide is 0.9551 ( 0.0053 while the green emissive species is 0.9883 ( 0.0056. For pH 6, the relative retention time of the oligonucleotide is 0.9932 ( 0.0025 and the red emissive species is 0.9896 ( 0.0029. (Right - (dC4A2)3C4) At pH 8, the relative retention times are 0.9676 ( 0.0062 for the oligonucleotide and 0.9993 ( 0.0018 for the green emitting cluster. For pH 6, the oligonucleotide is 0.9915 ( 0.0026 and the red emitting cluster is 0.9970 ( 0.0031.

is reduced in an intramolecularly folded species. Elevated temperatures also destabilize the secondary structure, and recovery of the spectral response with cooling indicates that refolding to form the favored secondary structure is rapid.30 For (dTA2C4)4, the influence of temperature was also investigated using absorption spectroscopy (Figure S3). Absorbances at 265 and 295 nm exhibit hyperchromic and hypochromic changes, respectively, as the temperature increases, which is again consistent with other i-motif forming oligonucleotides.31 Using spectra of the denatured oligonucleotide at 90 °C and folded oligonucleotide at 20 °C, the resulting thermal difference spectrum supports i-motif formation at low temperatures.28 In chromatograms for both oligonucleotides, only one species is observed (Figures 1 and 3). For (dTA2C4)4, this species has a similar mobility when compared with the aggregated form of dTA2C4, suggesting that both species have similar strand arrangements and structures (Figure 1). To further understand the role of shape on mobility, measurements on (dTA2C4)4 were conducted at pH 8, where circular dichroism spectra indicate that the i-motif structure is destabilized by the deficiency of protons (Figures 2 and S4). Relative to single-stranded thymine oligonucleotides, (dTA2C4)4 has a molecular mass of 8530 ( 970 g/mol. The similarity of this measurement relative to the intrinsic mass of 8270 g/mol indicates that (dTA2C4)4 is unfolded at this pH. This behavior sharply contrasts with the mobility at lower pH where the oligonucleotide adopts an i-motif structure (Figure 3). The above observations indicate that (dTA2C4)4 and (dC4A2)3C4 adopt i-motif structures that are dependent on the

pH, and the goal of these studies is to consider the role of this particular secondary structure on silver cluster formation. Cluster-Oligonucleotide Characterization. The dominant emission from reduction of Ag+ bound to (dTA2C4)4 and (dC4A2)3C4 occurs in the green and red spectral regions (Figure 4). For both templates, red emission is most prominent with λex ) 560 nm and λem ) 625 nm. For (dTA2C4)4, the green emission is characterized by maxima at λex ) 460 nm and λem ) 560 nm, while the maxima are at λex ) 500 nm and λem ) 570 nm for (dC4A2)3C4. These spectral differences could be related to the flanking and loop base sequence adjacent to the i-motif core region. Weak emission was also observed in the far-red region (λex ) 640 nm/λem ) 690 nm), but this species could not be characterized by chromatography and is not discussed further. The fluorescence intensities increase as the oligonucleotide concentration increases up to 25 µM and as the relative concentration of Ag+/oligonucleotide increases up to 16:1 (Figure S5). The decreasing fluorescence intensity at higher oligonucleotide and silver cation concentrations suggests that larger clusters are preferentially formed relative to the smaller, more fluorescent clusters. Our earlier study showed that red emission increased with DNA concentration, and this difference is attributed to intermolecular vs the presently used intramolecular cluster templates.12 The relationship between the initial silver cation stoichiometry and the resulting silver cluster stoichiometry is obscure because of the formation of other types of less emissive clusters. One indication of alternate clusters is transitions in the absorption spectra that have no associated

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Figure 4. Fluorescence intensities as a function of pH for the red (open square) and green (crosses) emitting clusters that form with (dTA2C4)4 (top) and (dC4A2)3C4 (bottom). The emission spectra associated with the intensity maxima are shown to the right.

fluorescence (Figure S6). Preliminary size exclusion chromatography studies indicate that the cluster-oligonucleotide conjugates and the folded oligonucleotide alone have similar retention times, which suggests that small numbers of silver atoms are bound with the DNA. The overall effect of clusters on DNA conformation was evaluated using circular dichroism, which is sensitive to the base arrangements within different types of DNA secondary structures (Figure S7).23 For (dTA2C4)4 at pH 6, bound clusters decrease the positive ellipticity at 288 nm. Otherwise, the magnitude of the band with negative ellipticity and the spectral position of both transitions are similar to the oligonucleotide alone, thus indicating that the template adopts a similar conformation with and without clusters. At higher pH, clusters have a distinctive effect on the (dTA2C4)4. Relative to unfolded DNA strands at pH 8, cluster-DNA conjugates shift the circular dichroism response, which is indicative of a clusterinduced change in conformation. To provide a further reference point for interpreting these spectral measurements, the dT12 template for blue-green emitting clusters was evaluated. Earlier studies established that this oligonucleotide and its cluster complexes adopt conformations expected for a semiflexible polymer.12 The circular dichroism spectra are distinguished from those with (dTA2C4)4 by the overall change in the relative

magnitude of the bands with positive and negative ellipticity, thus suggesting that the conformations of these two oligonucleotides with silver clusters can be distinguished. Because these measurements represent the net effect of the different types of clusters on the oligonucleotides, the subsequent discussion in this paper focuses on more specific characterization of the fluorescent clusters using the pH dependence of cluster fluorescence and size exclusion chromatography coupled with fluorescence detection. One factor that distinguishes the red and green emission bands is dissolved oxygen. Over a five day period, the intensity of the red emission decreases while the intensity of the green emission increases (Figure S8). The time dependence suggests that decomposition of BH4- changes the reducing capacity of the solution. To investigate the role of oxygen in this transformation, solutions were saturated with oxygen, air, and nitrogen (Figure S8). The reduction in intensity of the red emission as the concentration of O2 increases indicates that the species is reduced, a conclusion that is consistent with earlier studies of a red emitter.11 Green emission is stronger in air relative to N2, but no further enhancement is observed when the solution is saturated with O2. This trend suggests that the effect of O2 saturates at lower concentrations.

DNA Templates for Fluorescent Silver Clusters The solution pH also differentiates the red and green emissive species that form with (dTA2C4)4 and (dC4A2)3C4, and the spectral and structural properties of the red emissive species are considered first (Figure 4). Using buffered solutions, the most intense fluorescence occurs at pH 6, and suppressed fluorescence away from this optimal pH suggests that the cluster environment changes (Figure 4). Only small changes are observed when different buffers are used at a constant pH, which suggests that the proton concentration is responsible for the changes in fluorescence (Figure S9). The spectroscopic and chromatographic measurements for (dTA2C4)4 alone demonstrate that this optimal pH also favors i-motif folding in the oligonucleotides, which suggests that the DNA-bound clusters are encapsulated within i-motif structures (Figures 2 and 3). To further substantiate how the proton concentration influences the clusters, the solution pH was changed after the clusters were prepared using the (dTA2C4)4 template (Figure S10). On the basis of the circular dichroism spectra, the solution pH was changed to sufficiently high and low values to disrupt the secondary structure of the oligonucleotide (Figure 2). A decrease in the fluorescence intensity accompanies both the increase in pH to 10 and the decrease in pH to 3. Returning these solutions to pH 6 results in an increase in the intensity. The recovery is not complete, suggesting the exposed silver clusters react in the aqueous environment when the oligonucleotide unfolds. Importantly, the equivalent response when the pH is changed in both directions suggests that the secondary structure of the oligonucleotide-cluster conjugate is altered, thereby influencing the cluster environment. Using fluorescence detection, the oligonucleotide-cluster conjugates were structurally characterized using size exclusion chromatography, and the similarity of their retention times relative to the oligonucleotides alone indicate that both species have a similar strand organization (Figure 3). The environment of the red emitting cluster was further evaluated using the constituent dTA2C4 that forms an intermolecular i-motif (Figure 1). The spectra are similar with both dTA2C4 and (dTA2C4)4, thus indicating that both red emitting clusters are stabilized by similarly arranged bases (Figure S11). Green emission was not observed for the shorter oligonucleotide. Size exclusion chromatography studies show similar retention times for the red emissive species that forms with dTA2C4 and the corresponding intermolecular i-motif structure (Figure S11). Given the greater stringency on assembling separate strands, the similar retention times provide strong evidence in support of an i-motif matrix. Collectively, a folded conformation of oligonucleotide-cluster conjugates, a correlation between i-motif stability and cluster fluorescence, and similar environments for intermolecular and intramolecular complexes indicates that this red emitting silver cluster is protected within i-motif structures. The green emissive species is most favored at pH 9 for (dTA2C4)4 and at pH 8 for (dC4A2)3C4. At these high pH values, spectroscopic and chromatography studies indicate that the oligonucleotides are unfolded with a conformation that is similar to single-stranded DNA (Figures 2 and 3). As with the red emission, the green emission is also sensitive to the proton concentration, and only small changes are observed when buffers are varied at constant pH (Figure S9). Changing the solution pH after forming the clusters again suggests that changes in the secondary structure of the DNA matrix influence the cluster environment (Figure S10). The (dTA2C4)4 template was used for these studies, and a decrease in fluorescence accompanies a decrease in the pH to 3, and the intensity recovers upon cycling back to pH 6. When the pH increases from 6, the intensity

J. Phys. Chem. C, Vol. 113, No. 45, 2009 19523 increases to a maximum at pH 9 and then reverts upon returning the pH back to 6. This trend tracks the behavior observed with buffers, again supporting the affect of protons on these changes. A net increase in fluorescence is attributed to two effects. First, a concomitant decrease in red emission suggests that pH cycling promotes conversion of red to green emissive species. Second, rapid DNA refolding by changing pH could access alternative conformations that influence the base arrangement for the clusters. This latter possibility is supported by a blue-shift in emission after complete pH cycle. After two days, the emission wavelength red-shifts, suggesting that the DNA template for the green-emitting cluster slowly adjusts to its original conformation. To study the nature of the secondary structure, fluorescence detection of the green emitting species was used for size exclusion chromatography studies (Figure 4). Comparisons with the studies at pH 6 are facilitated by using the relative retention times derived from a dT15 reference. The oligonucleotides alone adopt an unfolded conformation at pH 8, whereas the complex with the green emitting species has a longer retention time and thus more compact structure. Furthermore, the similar retention times of the red and green cluster conjugates, as well as the i-motif form of the oligonucleotide, suggests that all three species have similar global structures. Discussion The goal of these studies was to determine if fluorescent silver clusters are influenced by the base arrangement in particular secondary structures of DNA. Two cytosine-based oligonucleotides (dTA2C4)4 and (dC4A2)3C4 were investigated as DNA templates because they form intramolecular i-motif structures. The base arrangement and strand folding were investigated by optical spectroscopy and size exclusion chromatography. Attention was focused on the i-motif structure because of the importance of cytosine in silver cluster formation. To illustrate, over 2000 oligonucleotides comprised of adenine, thymine, and cytosine were evaluated using arrays, and this broad survey showed that cytosine is prevalent in sequences for a range of silver clusters.5 In addition, oligonucleotides with different numbers of cytosines show variations in the fluorescence spectra, suggesting that cluster formation is influenced by coordination with multiple bases.14 The oligonucleotides (dTA2C4)4 and (dC4A2)3C4 are templates for red and green emitting silver clusters. Two observations provide insight into the secondary structure of the oligonucleotide-cluster conjugates: the fluorescence spectra depend on pH and the DNA folds around and encapsulates the clusters. While protons could bind with the clusters, their effect is postulated to be an indirect effect on the DNA matrix, as cytosine-rich sequences favor self-pairs, DNA folding is observed using size exclusion chromatography, and cluster fluorescence tracks the pH dependence of i-motif stability. The maximum red intensity occurs at pH 6, where the i-motif form of the oligonucleotide is most stable. Away from this favored pH, the decrease in the intensity suggests that coupled unfolding of the i-motif matrix is detrimental to the emission and stability of the clusters. Fluorescence is favored at the higher pH range for i-motif formation, which suggests a balance is maintained between protons that stabilize i-motif structures and that also compete with clusters for binding sites. Size exclusion chromatography shows that the cluster-DNA complex has the same retention time and hence the same global structure as the i-motif form of the oligonucleotide alone. Thus, the evidence supports an i-motif matrix for the red emitting cluster. Relative to the red emitter, the green emitting cluster is favored in more oxidizing environ-

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ments and in more basic solutions with pH 8-9. This difference in the pH dependence of fluorescence of the two types of clusters suggests that the green emitting species is disfavored relative to the red emitting species by higher proton concentrations. In this high pH range, the oligonucleotide alone is unfolded but the silver cluster complexes have a more compact shape which is similar to both the red emitting cluster and the i-motif form of the oligonucleotide. These similarities of the global structures suggest that the green emitting cluster is entrapped within a structure that is similar to an i-motif. As opposed to the DNA matrix for the red emitting species, the local base arrangement for the green emitting cluster may differ because the intrastrand interactions are dominated by clusters as opposed to protons. The similarities using the (dTA2C4)4 and (dC4A2)3C4 templates indicate that common types of clusters form within a common i-motif core. The small differences in the spectra suggest that loop and flanking bases could be used to adjust the environment of the clusters.32 We are currently evaluating secondary structures of other cytosine-based sequences that also form red and green emitting clusters.5,14 Conclusions One mean of synthesizing fluorescent silver clusters is arresting agglomeration by coordination with the bases of DNA. The present studies consider whether the base arrangement within the DNA matrix influences the types of clusters that form. Attention was directed toward the i-motif form of two cytosinerich oligonucleotides (dTA2C4)4 and (dC4A2)3C4. In addition to the sequence requirement of repeated cytosines, the intercalated strand arrangement is also favored in the slightly acidic to neutral pH range. Two prominent clusters form with both oligonucleotides, and the red emissive species has a global structure and a pH sensitivity that is consistent with an i-motif matrix for the cluster. The green emissive species is favored at higher pH where the DNA template alone is unfolded, yet the cluster-DNA conjugate is structurally similar to the folded i-motif. This distinction suggests that protons stabilize the DNA matrix for the red species while the clusters promote folding around the green species. These results suggest that polymorphic forms of DNA can serve as reaction templates for the synthesis of novel fluorescent nanomaterials. Acknowledgment. We thank the National Institutes of Health (R15GM071370 and P20 RR-016461 (from the National Center for Research Resource)), the National Science Foundation (CHE-0718588 and CBET-0853692), and the Henry Dreyfus Teacher-Scholar Awards Program for support. We thank J. and S. Wheeler for assistance with the chromatography and A. Spaugh for the generous donation of a fluorescence detector. Supporting Information Available: Fluorescence emission spectra of dC4T4C4; circular dichroism spectra of dTA2C4; thermally dependent spectra of (dTA2C4)4; size exclusion chromatograph of (dTA2C4)4 at pH 8; concentration dependence of fluorescence spectra of (dTA2C4)4; absorption and circular dichroism spectra of cluster conjugates with (dTA2C4)4; time,

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