A Silver-Specific DNAzyme with a New Silver Aptamer and Salt

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A Silver-Specific DNAzyme with a New Silver Aptamer and Salt-Promoted Activity Runjhun Saran, Kimberly Kleinke, Wenhu Zhou, Tianmeng Yu, and Juewen Liu Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b01131 • Publication Date (Web): 27 Mar 2017 Downloaded from http://pubs.acs.org on March 29, 2017

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A Silver-Specific DNAzyme with a New Silver Aptamer and Salt-Promoted Activity

Runjhun Saran, Kimberly Kleinke, Wenhu Zhou, Tianmeng Yu and Juewen Liu*

Department of Chemistry, Waterloo Institute for Nanotechnology, University of Waterloo, Waterloo, Ontario, N2L 3G1, Canada Email: [email protected]

Funding Funding for this work is from The Natural Sciences and Engineering Research Council of Canada (NSERC, 386326).

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Abstract Most RNA-cleaving DNAzymes require a metal ion to interact with the scissile phosphate for activity. Therefore, few unmodified DNAzymes work with thiophilic metals due to their low affinity to phosphate. Recently, an Ag+-specific Ag10c DNAzyme was reported via in vitro selection. Herein, the Ag10c is characterized to rationalize the role of the strongly thiophilic Ag+. Systematic mutation studies indicate that the Ag10c is a highly conserved DNAzyme and its Ag+ binding is unrelated to C-Ag+-C interaction. Its activity is enhanced with Na+ concentrations of buffer. At the same metal concentration, the activity ranks Li+>Na+>K+. The Ag10c binds one Na+ and two Ag+ ions for catalysis. The pH-rate profile has a slope of ~1 indicating a single deprotonation step. Phosphorothioate substitution at the scissile phosphate suggests that Na+ interacts with the pro-Rp oxygen of the phosphate, and dimethyl sulfate (DMS) foot printing indicates that the DNAzyme loop is a silver aptamer binding two Ag+ ions. Therefore, Ag+ exerts its function allosterically, while the scissile phosphate interacts with Na+, Li+, Na+ or Mg2+. This work suggests the possibility of isolating thiophilic metal aptamers based on DNAzyme selection, and it also demonstrates a new Ag+ aptamer.


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DNAzymes are DNA-based catalysts (also known as deoxyribozymes and catalytic DNA),1-6 and metal ions are an indispensable part of DNAzyme catalysis.7-10 The first DNAzyme was reported in 1994 for RNA cleavage in the presence of Pb2+.11 Since then, many DNAzymes were isolated for specific metals such as Zn2+,12 Mg2+,13 Ca2+,14 UO22+,15 Cu2+,16 Hg2+,17 Cd2+,18 and trivalent lanthanides.19-22 These studies have led to the widespread application of DNAzymes in analytical chemistry,23-26 and nanotechnology.27 For these DNAzymes, however, it is quite challenging to extract metal aptamers from them, despite their excellent metal specificity. A few Na+-specific RNA-cleaving DNAzymes were reported recently.28-31 For example, the NaA43 DNAzyme reported by Lu and co-workers is active with Na+ alone.28 A portion of its sequence is identical to a lanthanide-dependent DNAzyme we previously reported.19 A careful study identified a common Na+ aptamer in these two DNAzymes.29,32-34 This is the first welldefined metal aptamer embedded within in vitro selected DNAzymes. Recently, we reported a silver-specific DNAzyme named Ag10c.35 At pH 6, it has a saturated cleavage rate of 0.42 min-1, which is faster than most other DNAzymes under the same condition. Its high efficiency is surprising since no divalent metal ions were involved. Ag+ is a strongly thiophilic metal and this is the first report that such thiophilic metals can activate a nonmodified DNAzyme.12,16-18 To understand its intriguing activity, we herein performed a systematic biochemical characterization by mutations, varying ionic strength and pH, phosphorothioate modification and footprinting. We gained interesting insights into metal binding, and a new Ag+ aptamer was identified to be responsible for its Ag+ specificity, which is independent of the well-known C-Ag+-C base pair.36


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Materials and Methods Chemicals. All of the DNA samples were purchased from Eurofins (Huntsville, AL) and Integrated DNA Technologies (Coralville, IA). Their sequences and modifications are listed in Supplementary Table S1. All the metal salts, buffers, dimethyl sulfate (DMS), βmercaptoethanol and piperidine were from Sigma-Aldrich. Activity assays. The DNAzyme complex was formed by incubating the substrate and enzyme strands at 90 °C for 2 min and then slowly cooling to 4 °C. For a typical gel-based activity assay, a final concentration of 0.4 µM FAM-labeled substrate and 0.8 µM enzyme were added to 50 mM MOPS buffer (pH 7 and 7.5) or 50 mM MES buffer (pH 6 and 6.5) containing up to 1 M NaNO3 as indicated. At room temperature, a final concentration of 0.5 - 10 µM Ag+ was added to initiate the cleavage reaction. The reaction was quenched by transferring 10 µL aliquots of the sample to 10 µL urea (8 M) at designated time points. The cleaved products were separated using 15% denaturing polyacrylamide (PAGE) gel and analyzed by a Bio-Rad ChemDoc MP imaging system. For determining the rate of cleavage, the data obtained were fitted according to the firstorder rate equation Yt = Yₒ + a(1-e-bx), where Yt and Yₒ are cleavage fractions at a given reaction time t and 0 min, respectively and b is the observed rate constant. DMS footprinting was performed following previously published protocols.29 Briefly, the 3′end FAM labeled enzyme (Ag10c-FP-FAM) and the non-cleavable substrate were used. The DNAzyme complex ([Ag10c-FP-FAM] = 4 µM, [Sub-dA] = 20 µM) was formed by annealing in 50 mM MOPS buffer (pH 7.5) containing 0 or 200 mM NaNO3. Before starting the footprinting, the DNAzyme complex (10 µL) was incubated with a final concentration of 0, 10, 40, 60, 80, 100 and 200 µM of a metal ion such as Ag+ , Hg2+ , Cd2+ , Ni2+, Tl+, Zn2+, Mg2+ or Ca2+ for 15


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min. To methylate the guanosine, 1 µL of freshly prepared 4% DMS was added to the 10 µL DNAzyme-metal complex, followed by 15 min incubation in dark. Then, the reaction was quenched by adding 200 µL solution containing β-mercaptoethanol (1 M) and NaOAc (600 mM, pH 5.2) followed by ethanol precipitation and cleavage by 10% piperidine. Finally, the products were separated by 15% dPAGE for analysis.

Results and Discussion The Ag10c DNAzyme has a well-defined structure. The secondary structure of the Ag10c DNAzyme is shown in Figure 1A.35 Its substrate strand has a single RNA linkage (rA, riboadenine) that serves as the cleavage site. The enzyme strand binds the substrate via two base paired regions. In the presence of Ag+, the substrate is cleaved into two pieces. The catalytic core folds into a hairpin flanked by two single-stranded loops.

Figure 1. The secondary structure of (A) the wild-type Ag10c DNAzyme and (B) the modifications made to the hairpin. Each base in the loops is numbered from 1 to 17. Cleavage


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activity of the mutants with modifications to (C) the hairpin region, (D) the cytosines in the loop, and (E) the other nucleotides. The rates were determined in 50 mM MOPS, pH 7.5, 200 mM NaNO3 with 10 µM Ag+. The fold of activity drop of the mutants is color coded to be 1000-fold. Therefore, all these cytosines are critical for activity. 6 ACS Paragon Plus Environment

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While this study confirms the crucial role of these cytosines, it argues against C-Ag+-C base pairing. If C-Ag+-C were required for activity, mutating one of the C to G should maintain the base pair and thus activity. However, all the C-to-G mutations inhibit the activity. Furthermore, C also binds Ag+ together with T.39 There are only two thymines in the enzyme loops. For the same reason mentioned above, it is unlikely that C-Ag+-T base pairs are formed, since otherwise the C-to-A mutation should maintain the activity. Altogether, the role of Ag+ should be more sophisticated than such simple mismatch stabilization.40 It was recently reported that Ag+ can also be inserted between two guanines, and such binding does not disrupt the G-C Watson-Crick base pairing.41 Since all the bases can coordinate with Ag+, the exact way of Ag+ binding in Ag10c remains unclear, and we only ruled out the most well-known C-Ag+-C(T) base pairs. As will be discussed later, the binding of these two silver ions is cooperative, but it is unlikely that they stabilize a typical duplex structure. For the rest of the nucleotides, we tested A-to-G, G-to-A, and T-to-C mutations (Figure 1E). The ability to tolerate purine-to-purine or pyrimidine-to-pyrimidine mutations indicates the possibility of a structural role of the bases. Mutations to the T1, G6, A7 and A14 do not affect the activity. Further deletion experiments indicate that T1 is critical, while deleting G6G7 together decreases the activity only by 10-fold and deleting A14 does not affect the activity. Apart from these four nucleotides, mutations to the rest were more detrimental. Overall, this is a well-defined enzyme and most of the nucleotides cannot be randomly changed.

The Ag10c binds two Ag+ ions cooperatively. After confirming its secondary structure, we studied the effect of Ag+ by measuring the cleavage rate at various Ag+ concentrations. All the samples followed a first order kinetics (Figure 2A) and we calculated the cleavage rates (Figure 7 ACS Paragon Plus Environment

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2B). Below 1 µM Ag+, the activity is very low. With increase in [Ag+], the DNAzyme quickly activates, reaching saturation at >5 µM Ag+. This sigmoidal curve suggests multiple Ag+ ions binding cooperatively to the DNAzyme. To calculate the number of Ag+, a double log plot was made with up to 5 µM Ag+ (Figure 2C). It has a slope of 1.9, suggesting that the DNAzyme binds two Ag+ ions. We stopped at 5 µM since the cleavage activity approaches saturation beyond this concentration.

Figure 2. (A) Kinetics of the Ag10c DNAzyme in presence of various concentrations of Ag+. The data are fitted using the first-order kinetics model. (B) Cleavage rate of the Ag10c as a function of Ag+ concentration, exhibiting a sigmoidal curve fitted using the Hill equation, / , where Kd is the dissociation constant and h is the Hill co-efficient indicating positive co-operation (h > 1). (C) The double log plot for the same data in (B) at low Ag+ concentrations with a slope of 1.9 suggesting binding of two Ag+ ions. The reaction buffer contained 50 mM MOPS, pH 7.5, and 200 mM NaNO3.

Other than a few lanthanide ion dependent DNAzymes,20,22 most RNA-cleaving DNAzymes only use one metal ion for catalysis. In most DNAzymes, the metal interacts with the scissile phosphate as probed by phosphorothioate modification.16,18,42,43 However, Ag+ is a highly


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thiophilic metal and its affinity with the normal DNA backbone phosphate is very low. Therefore, Ag+ might exert a different function in the Ag10c DNAzyme.

Salt promotes cleavage activity. To further understand the Ag10c DNAzyme, we next changed the buffer conditions. Since DNA is a polyanion, salt concentration might influence its interaction with Ag+. We fixed the Ag+ concentration to be 10 µM and measured the rate as a function of NaNO3 concentration (Figure 3A). Interestingly, NaNO3 significantly promotes the activity by ~10-fold, and an apparent Kd of 87.6 mM Na+ was obtained. We also made a double log plot (Figure 3B), yielding a slope of 1.26. Therefore, one Na+ interacts with the enzyme to promote its cleavage activity (Figure 3B inset). To test whether this salt-accelerated activity is unique to the Ag10c, we also measured the cleavage of a few other RNA-cleaving DNAzymes including the 17E with Pb2+,13 Lu12 with Ce3+,44 and Tm7 with Tm3+.20 However, they were all inhibited by salt (Figure 3C). Salt has a number of effects on DNAzyme catalysis. First, at very low salt concentration, salt is often required for stabilizing the DNAzyme complex to ensure that its melting temperature is above room temperature. Around 20 mM Na+ is often sufficient for this purpose. Second, salt can screen the electrostatic interaction between metal ions and DNA, which can explain its inhibition effect in Figure 3C. When the stability of the DNAzyme complex is no longer a limiting factor for activity, the charge screening effect may take over. Finally, at even higher salt concentrations (e.g. ~1 M), salt ions may influence the structure of water and solubility of biopolymers.45 The slightly reduced activity of the Ag10c in Figure 3A at >500 mM Na+ might be related to this. This salt-dependent study also suggests that the mechanism of Ag10c is indeed different from most previously reported DNAzymes. 9 ACS Paragon Plus Environment

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This finding has led us to question whether Na+ is required in the Ag10c or not. For example, a lanthanide-dependent DNAzyme named Ce13d requires both a lanthanide and Na+.19,29 A few recently reported DNAzymes work with Na+ alone but not with other group 1A metals.28,30 To answer this question, we measured the Ag10c activity with Li+ or K+ in the presence of 10 µM Ag+ (Figure 3D). The Ag10c is active in all these salts. Therefore, these group 1A metals do not appear to play a specific chemical role (unlike in the case of Ce13d), and they only act as a general salt to raise the ionic strength. At the same metal concentration, Li+ gave the highest cleavage rate, which is attributable to its highest charge density to allow the strongest electrostatic interaction with DNA.

Figure 3. (A) The activity of the Ag10c DNAzyme with increasing concentration of NaNO3 in the presence of 50 mM MOPS, pH 7.0 with 10 µM Ag+. (B) The double log plot based on the data in (A). Inset: the low Na+ concentration data fit to a straight line with a slope of 1.26. (C) Percentage of cleavage of the Lu12, 17E and Tm7 DNAzymes with 1 µM Ce3+, 1 µM Pb2+ and 10 µM Tm3+, respectively, in 30 min in the presence of various concentrations of NaNO3 in 50 10 ACS Paragon Plus Environment

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mM MOPS, pH 6.0. The activity of the Ag10c with increasing concentrations of (D) KNO3, NaNO3, LiNO3 and (E) MgSO4 in 50 mM MOPS, pH 7.5 and 10 µM Ag+.

In RNA cleavage, metal ions might play the following roles. First, the metal bound water after deprotonation might act as a general base to help deprotonate the 2′-OH nucleophile. Second, metals may interact with the scissile phosphate and stabilize the transition state phosphorane, which is highly negatively charged. We suspect that the role of Na+ (or Li+/K+/Mg2+) here is the latter since it is unlikely for the thiophilic Ag+ to play such a role. Ag+ is a very soft metal of low affinity with the phosphate. The fact that Li+ works the best also supports this hypothesis. Monovalent Na+ does not bind to phosphate tightly, and it needs a few hundred mM to achieve the optimal activity. For comparison, divalent metal ions such as Mg2+ work optimally at ~10 mM concentrations,46-49 while trivalent lanthanides work optimally even at nM concentrations due to very strong phosphate binding.20,22 To further support our hypothesis of phosphate binding, we also used Mg2+ in addition to 10 µM Ag+ (Figure 3E). Indeed, ~5 mM Mg2+ could accelerate the Ag10c activity to the same as that by 200 mM of Li+. Therefore, group 1A metals are not absolutely required and its charge screening role can be replaced by a group 2A metal as well. Taken together, Ag+ does not interact with the scissile phosphate of the Ag10c (as will be discussed later, it interacts with some nucleobases of more covalent nature), and thus salt does not compete with its binding to the DNAzyme. Most other DNAzymes rely on the metal cofactor binding to the scissile phosphate via electrostatic interaction, and salt can inhibit the reaction. In the Ag10c, the phosphate is activated by the buffer salt.


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Effect of pH. After studying the effect of salt, we further probed the mechanism by varying pH. Below pH 5.5, the rate is slower than 0.01 min-1 and thus we did not measure lower than pH 5.5. The cleavage rate increases with pH and starts saturating as the pH approaches 8 (Figure 4A). The rise in log (rate) increases linearly with pH between 6.0 and 7.0 with a slope ~1 (Figure 4B). This suggests a single deprotonation step in the cleavage. This trend has been seen typically in most RNA-cleaving DNAzymes. The first pKa of Ag+ is around 10.0, and thus in the pH range we tested, Ag+ is a fully hydrated ion and the rate change we probed is unlikely to be related to the hydrolysis of Ag+. A critical step of the RNA cleavage reaction is the nucleophilic attack of the scissile phosphate by the 2′-OH nucleophile. The pH profile probed here is likely due to a nucleobase with shifted pKa (e.g. by interacting with Ag+) acting as a general base and helping in the deprotonation of the 2′-OH to make it a stronger nucleophile, which has been commonly seen in ribozymes.8

Figure 4. The log of the cleavage rate of Ag10c plotted against pH (A) over a wide pH rage and (B) a narrow linearly rising pH range. In (B), the pH-log (rate) slope is 1.15. The rates are calculated in presence of 50 mM MES or MOPS to cover the pH range with 200 mM NaNO3 and 10 µM Ag+.


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Phosphorothioate modification to probe metal binding. To further probe metal binding, we also introduced a phosphorothioate (PS) modification at the scissile phosphate. PS refers to replacing one of the non-bridging oxygen atoms in the backbone phosphate by sulfur. Once a PS is introduced, the phosphorus becomes a chiral center with two stereoisomers (called Rp and Sp). A scheme of the cleavage site dinucleotide junction in normal phosphodiester (PO), Rp and Sp substrates is shown in Figure 5A. With 10 µM Ag+ and 200 mM NaNO3, the PS substrate has only about half of the cleavage yield of the normal PO substrate (Figure 5B), while its cleavage rate (0.37 ± 0.02 min-1) is quite similar to the PO substrate (0.42 ± 0.01 min-1). This lower cleavage yield of the PS substrate suggests that only one of the isomers is cleavable, which is quite typical for RNA cleavage by ribozymes and DNAzymes.42,50-52 We then further separated these two isomers following previously established protocols,18 and measured their cleavage separately (Figure 5C). The cleavage yield was significantly higher with the Sp substrate than with the Rp one, which again is consistent with all previous reports.42,50-52 Their average yield also agrees with that of their mixture (Figure 5B, green trace). A careful examination of the kinetic profile indicates that the Rp has an initial fast cleavage (but only up to ~15% yield) followed by a much slower rise, while the Sp substrate has a more normal kinetic profile. Therefore, the cleavage of the Rp substrate was fitted with a tworate model (R2 = 0.99) yielding a faster rate of 1.17 ± 0.22 min-1 a slower rate of 0.037 ± 0.03 min-1. Note this faster rate may be an over-estimation due to its very low cleavage yield. The Sp substrate was fitted with a normal single-rate of 0.37 ± 0.01 min-1 (R2 = 0.94). Typically, for such PS-modified experiments, a thiophilic metal such as Cd2+ or Mn2+ is used to rescue the activity of the inactive isomer (e.g. the Rp). However, our system requires Ag+, which has a much stronger thiophilicity than Cd2+ or Mn2+. In this sense, our system is already 13 ACS Paragon Plus Environment

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under the rescued condition, and the thiophilicity of Ag+ can cause complication in data analysis. For example, the initial fast phase of the Rp substrate can be attributed to the following factors due to the extremely strong thiophilicity of Ag+: 1) direct cleavage of the PS substrate, 2) desulfurization of the substrate from PS to PO, which is then cleaved by the DNAzyme, and 3) direct cleavage by the DNAzyme.53-56 Note that 1) and 2) are impossible for Cd2+ or Mn2+ under ambient conditions but take place with Hg2+, Tl3+ and Ag+. To measure the amount of Ag+ induced direct cleavage, we incubated 0.4 µM PS substrate with 10 µM Ag+ for 1 h, where only 2% direct cleavage was observed (Figure 5D). Next, to measure the amount of Ag+-induced desulfurization, we again incubated the PS substrate with Ag+ but for various time periods up to 60 min. At each time point, an aliquot was taken out and the reaction was stopped by chelating Ag+ with 1 mM β-mercaptoethanol. Then, the reaction mixture was annealed with the Tm7 DNAzyme followed by the addition of 10 µM Er3+. The purpose was to examine the kinetics of desulfurization by Ag+. The amount of cleavage by Tm7 after 1 h was measured (Figure 5E). As the Tm7 cleaves only 3′-5′ PO substrate but not PS substrate,20 the measured cleavage suggests that Ag+ desulfurizes the PS substrate into the cleavable PO substrate mainly within the first two minutes but there is a slow yet noticeable rise in desulfurization, taking place at least till 1 h. Therefore, the slower phase in Figure 5C (red dots between 10 min and 4 h) is attributable to the slow phase of desulfurization followed by the Ag10c DNAzyme cleavage. Our data indicates that at least 35% of the PS was desulfurized to the 3′-5′ PO substrate in 1 h and a similar amount should isomerized to the non-cleavable 2′-5′ PO.54 Considering the cleavage yield by the Tm7 is not 100% in 1 h, we reason that most of the PS substrate was desulfurized by Ag+. Note that during desulfurization, the concentration of Ag+ is also decreased, and this also reduces the cleavage rate. At the same time, Ag+ can act as a rescue metal to bind to the sulfur, and this can


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also contribute to the cleavage. These overlapping factors make quantitative dissection of cleavage contributions nearly impossible. The Sp substrate can also be acted upon by these mechanisms. Its much higher cleavage yield than the Rp indicates that the Sp substrate can be cleaved with a similar mechanism as the normal PO substrate. It has only reached 60% cleavage yield likely also due to the desulfurization and isomerization to the inactive 2′-5′ substrate. While quantitative discussion is obscured by the strong thiophilicity of Ag+, the higher yield of the Sp can still allow us to assign the metal binding site since we know that only one metal is involved in phosphate binding here. Since we already ruled out the binding of Ag+ to the scissile phosphate, we reason that Na+ is used by the DNAzyme for this purpose. This is also consistent with the above study using Li+ and K+ and Mg2+ based on their charge density. These metals interact with the pro-Rp oxygen in the phosphate. The effect of monovalent metals in the HDV ribozyme was recently studied, where the cation charge density was also articulated.50


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Figure 5. (A) The structure of the cleavage site dinucleotide junction in a PO, Rp and Sp substrates. The kinetics of Ag10c cleavage of (B) the PO and PS (mixture of Rp and Sp) substrates, and (C) separated Rp and Sp substrates. (D) A gel image showing that, upon incubation of PS substrate with 10 µM Ag+ for 1 h, only 2% of the PS substrate was cleaved. (E) After various time (0, 2, 10, 30, 60 min) of incubating the PS substrate with Ag+, the sample was treated with β-mercaptoethanol. Then the product was hybridized with the Tm7 DNAzyme. The cleavage yield after 1 h with 10 µM Er3+ is plotted here (buffer: 50 mM MOPS, pH 7.5, 50 mM NaNO3).

A silver aptamer. All the above experiments indicate that Na+ interacts with the scissile phosphate. As such, the role of Ag+ still remains elusive. We hypothesize that the DNAzyme loop might be a silver aptamer. To confirm this, we resorted to DMS foot printing. For this purpose, the DNAzyme complex in Figure 6A was used by labeling the enzyme strand instead of the substrate. DMS methylates the N7 position of guanine conferring instability. This results in opening of the purine ring vulnerable to displacement and β-elimination by a base such as piperidine.57 If a guanine is protected by folding into a particular structure (i.e. N7 inaccessible to DMS), it might be protected from methylation and thus cleavage. This does not mean that the guanine has to directly participate in metal coordination. As long as the structure is folded and the N7 position is inaccessible, a protection should be observed. If a treated DNA is analyzed using PAGE, each exposed guanine in the DNA would produce a band in the gel. To simplify our gel interpretation, all the guanines in the substrate binding arms of the enzyme were replaced by cytosines, and the corresponding substrate strand was also mutated to still maintain the base pairing. The nucleotides in the hairpin loop were also 16 ACS Paragon Plus Environment

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replaced by thymines, while the guanines in the hairpin stem were kept as controls for the experiment. The substrate was the non-cleavable all-DNA substrate.

Figure 6. (A) The Ag10c DNAzyme complex used for DMS foot printing with a FAM labeled in the enzyme strand. Gel images of the Ag10c DNAzyme in presence of (B) increasing concentration of Ag+ and (C) 165 µM of different metals in the DMS foot-printing experiment. The above experiments were done in the presence of 200 mM NaNO3.

When titrated with increasing concentration of Ag+, we found the G3, G4, G6, G10, G11, G12 and G15 to be more and more protected, while the two guanines in the hairpin stem (GS1, GS2) and G17 remain unaffected (Figure 6B). Strong participation of G3, G4, G11 and G12 in the binding pocket of Ag+ also corresponds well with the >1000-fold drop in the activity upon their mutation and also indicates that they may be directly involved in Ag+ binding. It is interesting to note that the G17 was not protected from DMS but it is critical for the catalytic activity of Ag10c (>1000 fold drop in rate upon mutation), suggesting it might have a direct role in catalysis but might not be part of the aptamer. When other metal ions were used (Figure 6C), no such protection was observed, further supporting the specific binding to Ag+. This experiment 17 ACS Paragon Plus Environment

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not only indicates the existence of a highly specific Ag+ aptamer, but also supports that it is the role of Na+ rather than Ag+ to interact with the scissile phosphate. We also quantitatively measured the protection of the guanines as a function of silver concentration (Figure S1 in Supporting Information). The measured Kd values range from 45–65 µM Ag+ for the G residues involved in the binding pocket. This Kd is higher than that measured from its cleavage activity of ~4 µM Ag+ in Figure 2B. We attribute this to the different DNAzyme concentrations used for these assays. Only 400 nM of Ag10c complex was used in the gel based activity assay, while 4 µM of Ag10c was used here in the footprinting experiment. The 4 µM DNAzyme would require at least 8 µM Ag+ to bind, thus leading to a much higher apparent Kd. We also measured an apparent Kd of ~200 nM Ag+ from a Ag10c-based fluorescent biosensor.35 In that case, the signal was generated from substrate cleavage followed by release of the cleaved fragment. In that case, the Ag10c concentration was only 50 nM. These differences have led to a much lower Kd for the sensor. Further discussion. In this study, we started with biochemical characterization of the Ag10c DNAzyme, which was recently selected in our lab and was engineered into a highly sensitive and selective biosensor for Ag+.35 Many interesting observations were made here. First, it confirms the possibility of selecting DNAzymes for soft thiophilic metals using just the natural DNA nucleotides. So far, only a few modified DNAzymes were reported for thiophilic metals, such as the Hg2+-specific DNAzyme from the Perrin lab involving a few modified nucleotides.17 We introduced a PS modification at the scissile phosphate to obtain DNAzymes for Cd2+ and Cu2+.16,18,58 The Ag10c is the first in vitro selected unmodified RNA-cleaving DNAzyme that cleaves efficiently in the presence of a thiophilic metal. In this work, we rationalized the role of Ag+ and this method is likely applicable also to other soft metals. 18 ACS Paragon Plus Environment

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Biochemistry

Second, we established that this Ag10c DNAzyme uses two metals: Na+ (or other group 1A metals or Mg2+) binding to the phosphate, and Ag+ binding to the aptamer loop. This is the second such example. The first one is the Ce13d DNAzyme, which uses Ce3+ (or other lanthanides) to bind to the phosphate and it also has an aptamer loop for Na+.29 These are the only examples of aptamers embedded in DNAzymes obtained via in vitro selection, and they are different from those intentional aptazymes selections starting with an existing enzyme scaffold.59,60 With such fundamental understanding in this work, we should be able to intentionally adjust DNAzyme selection conditions to obtain more such metal aptamers. Third, in our original selection, only 25 mM Na+ was included in the buffer.35 In retrospect, this was a poor choice. Indeed, we only obtained ~0.1% of active Ag10c sequences in our final library via extensive deep sequencing analysis. Had the selection been carried out in a much higher Na+ concentration buffer, we might have obtained more such active sequences from the selection. After knowing this, one can intentionally use a high salt and promote the formation of such aptamers in DNAzymes selection. Fourth, this work has provided a new aptamer for Ag+ and this aptamer is completely different from the well-known C-Ag+-C structure.36 Therefore, for a given metal, there are many solutions for producing nucleic acid sequences. Such a complex binding structure is reminiscent of metal binding aptamers in riboswitches,61 where the aptamers are coupled with functions related to regulation of gene expression. Now, we can evolve such more complex sequences in vitro, which will give us more insights into the metal binding ability of nucleic acids. Finally, silver is an important analyte, and this aptamer will likely to be useful for developing silver sensors. The binding of two metals produces a sigmoidal response curve, and such a response might also be analytically useful. 19 ACS Paragon Plus Environment

Biochemistry

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Conclusions In summary, we systematically characterized a new Ag+-specific RNA-cleaving DNAzyme. The mutation studies argued against the formation of C-Ag+-C in our DNAzyme, and thus this is not a simple aptazyme that can be obtained via rational design. Through an Ag+-dependent activity assay, we confirmed that two Ag+ ions bind cooperatively. In addition, a group 1A or 2A cation is also needed for activity, where metals with a higher charge density are more effective for their role to interact with the pro-Rp oxygen of the scissile phosphate. Therefore, through this study a new theme of DNAzyme catalysis has emerged which may be used by soft metals to escape interaction with the scissile phosphate and yet confer effective catalysis. It has opened up the platform to explore and select novel soft-metal dependent DNAzymes. Finally, using DMS foot printing, we confirmed a well-defined silver aptamer that can fold into a compact structure by binding Ag+. Supporting Information. DNA sequences and quantification of DMS footprinting data. This material is available free of charge via the Internet at http://pubs.acs.org.

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For Table of Content Graphics Only

A Silver-Specific DNAzyme with a New Silver Aptamer and Salt-Promoted Activity

Runjhun Saran, Kimberly Kleinke, Wenhu Zhou, Tianmeng Yu and Juewen Liu


A Silver-Specific DNAzyme with a New Silver Aptamer and Salt

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