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How Does Pyridine Actually Disassemble and Assemble Spherical Nucleic Acids? Yoon Hyuck Kim, Ju-Hwan Oh, Abigail Lytton-Jean, and Jae-Seung Lee Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b00005 • Publication Date (Web): 22 Mar 2017 Downloaded from http://pubs.acs.org on March 28, 2017
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Analytical Chemistry
How Does Pyridine Actually Disassemble and Assemble Spherical Nucleic Acids? Yoon Hyuck Kim,†,§ Ju-Hwan Oh,†,§ Abigail K. R. Lytton-Jean,*,‡ and Jae-Seung Lee*,† † Department of Materials Science and Engineering, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul, 02841, Republic of Korea ‡ David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
ABSTRACT: The spotlighted dual functions of pyridine as a denaturant and as a stabilizer for duplex DNA are thoroughly investigated using spherical nucleic acids (SNAs). At neutral pH, pyridine destabilizes the duplex interconnects of assembled SNAs, resulting in a gradual decrease in their melting temperature (Tm) as a function of the pyridine concentration. This result is in good agreement with the conventional role of pyridine as a powerful denaturant for free duplex DNA. On the contrary, the addition of pyridine dramatically increases the Tm of hybridized SNAs under acidic conditions, which could be a striking result of pyridine’s stabilizing effect for DNA duplex as previously suggested based on the pyridine-nucleobase interactions. After comprehensive and quantitative investigation based on the analysis of the sharp melting transitions of SNAs, however, we report that, in fact, the pH increase induced by pyridine is also an essential parameter accounting for pyridine’s DNA-stabilizing effects under acidic conditions. Importantly, we prove that pyridine, particularly at a low concentration, does not increase the Tm of hybridized SNAs even under acidic conditions, if the pH increase by pyridine is corrected to maintain the same initial pH.
Pyridine is a six-membered heterocyclic aromatic amine, and has been used as a key reagent in many aspects of nucleic acid chemistry.1,2 Pyridine is an essential solvent used for the solidphase oligonucleotide synthesis,3 and an efficient organocatalyst used for rapid chemical primer extension of nucleic acids.4 It is also one of the most frequently studied nucleobase analogues, providing a fundamental understanding of base-base interactions in duplexes.5-9 More importantly, pyridine is known as one of the most powerful organic denaturants for doublestranded nucleic acids.10,11 Although the exact mechanism has not been completely elucidated to date, pyridine is believed to induce the dehybridization of duplex DNA by (1) forming hydrogen bonds with nucleobases of DNA, or 'stacking' with the solvent-exposed nucleobases, and (2) further stabilizing the denatured form of DNA via hydrophobic interactions.10-12 Significantly, it is noteworthy that pyridine was recently reported to exhibit an unusual stabilizing property for the duplex form of DNA under acidic conditions, resulting in a substantial increase in melting temperature (Tm).13 To support and rationalize the experimental results, the presence of pyridine molecules in close proximity to the DNA duplex was proposed by 1- and 2dimensional NMR spectroscopic methods. Molecular orientations of pyridine as a binder in the groove of the duplex were also theoretically suggested by molecular dynamics (MD) simulations. This observation is surprising given pyridine’s 60-year history as a stabilizer. To further investigate the role of pyridine as a denaturant, we decided to validate this behavior in another class of nucleic acids. Spherical nucleic acids (SNAs) are a nanoparticulate form of nucleic acids, typically consisting of a gold nanoparticle core and densely immobilized DNA strands on the nanoparticle surface.14,15 SNAs exhibit distinct advantages over conventional
nucleic acids for reversible denaturation studies. SNAs typically exhibit very sharp melting profiles (full width at half maximum of the first derivative = ~ 1.5 oC) and Tms that are ~ 10 oC higher than those of regular DNA having the same sequence (Figure S1, see Supporting Information),16 based on the cooperative melting properties and enhanced binding properties. Moreover, the wavelength and intensity of their absorption bands are sensitively dependent on the interparticle distance.17 As a result, the dissociation properties of SNAs are quantitatively and further proportionally correlated with those of regular DNA, and can be used as a measure to sensitively and selectively quantify the dissociation properties of regular DNA. These unique chemical and physical properties are attributed to (1) the multiple duplex DNA interconnects between hybridized SNAs and (2) intense optical properties of gold nanoparticles based on their surface plasmon resonance. These features make SNAs stand out as a highly attractive platform for fundamental investigations of nucleic acid hybridization properties. Herein, we present a thorough investigation of the assembly properties of SNAs in the presence of pyridine, and explain the two contrasting effects of pyridine on the assembly of SNAs. Quantitative analysis was precisely made possible based on the sharp melting transitions of SNAs. EXPERIMENTAL SECTION Materials and Instrumentation. Pyridine (Cat. # 494410), 2-aminopyridine (Cat. # A76997), 3-aminopyridine (Cat. # A78209), 4-aminopyridine (Cat. # 275875), 4-methylpyridine (Cat. # 239615), pyridoxine hydrochloride (Cat. # 47862), pyridoxine (Cat. # P5669), NaCl (Cat. # S6546), citric acid (Cat. #
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251275), sodium citrate tribasic (Cat. # S4641), sodium phosphate monobasic (Cat. # 71505), sodium phosphate dibasic (Cat. # S7907), Tween 20 (Cat. # P9416), gold(III) chloride trihydrate (HAuCl4·3H2O, 99.9%, Cat. # 520918) and dithiothreitol (Cat. # 43815) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The other pyridine derivatives such as 2,3-dimethyl pyridine (Cat. # L0063), 2,4- dimethyl pyridine (Cat. # A0731), 2,4,6-trimethylpyridine (Cat. # T0716), 2-amino-4-methylpyridine (Cat. # A0402), 5-ethyl-2-aminopyridine (Cat. # E0142) and 6-amino-2,4-lutidine (Cat. # A0731) were purchased from Tokyo Chemical Industry (Tokyo, Japan). The illustra NAP-5 Columns was purchased from GE Healthcare (Little Chalfont, UK). The HPLC-purified thiol DNA sequences (s1: 5' HS-A10ATTATCACT 3'; s2: 5' HS-A10-AGTGATAAT 3') were purchased from Genotech Corp. (Daejeon, Republic of Korea). The pH of the solutions used in the experiments was measured by ORION 5 STAR (Thermo Fisher Scientific Inc., Waltham, MA, USA). An Agilent8453 UV-vis Spectrophotometer (Agilent Technologies; Santa Clara, CA, USA) and NanoDrop 2000 UVVis Spectrophotometer (Thermo Fisher Scientific Inc.; Waltham, MA, USA) were used for UV-vis spectroscopic measurements. Synthesis of gold nanoparticles (AuNPs). HAuCl4 solution (0.254 mM, 50 mL) was heated to the boiling point followed by rapid injection of sodium citrate (Na3C6H5O7, 38.8 mM). In a few minutes, the color of the solution changed from pale yellow to dark red, indicating the complete synthesis of 15 nm AuNPs. The solution was stirred for 10 min, and slowly cooled to 25 oC with stirring. Synthesis of spherical nucleic acids (SNAs). The thiolated DNA sequences (s1 and s2) were deprotected with dithiothreitol (0.1 M, 170 mM phosphate buffer, pH 8.0), and further purified using an illustra NAP-5 column. The deprotected DNA sequences were combined with 1 mL of as-synthesized 2 nM AuNPs (final [DNA] = ~ 4.8 μM). The mixture was buffered with phosphate (pH 7.4, 10 mM phosphate, 0.5 M NaCl, 0.01% SDS), and incubated for 12 hours at 25 oC. To remove the unconjugated DNA, the mixture was centrifuged at 8000 rpm for 20 min. The supernatant was then discarded, and the remaining SNAs were redispersed in aqueous 0.01% Tween 20. The washing process was repeated 3 times. Reversible assembly properties of SNAs in the presence of pyridine and pyridine derivatives. Two complementary SNAs (s1 and s2) were combined and allowed to hybridize for 12 hours at 25 oC (final [SNA] = 1 nM) under various buffer conditions (see below for detailed buffer conditions). The pyridine and its derivatives were injected into the hybridized SNAs and allowed to interact with the SNAs for 30 min. The dehybridization of the hybridized SNAs were monitored using UVVis spectroscopy by measuring the change in extinction at 525 nm as a function of temperature (1 oC / min) with homogeneous stirring Measurement of pH. The pH of the aqueous solutions of the reaction mixtures was determined using an Orion 5-Star Plus multi-parameter meter (Thermo Scientific, UK) by directly placing the electrode in the solution (8 mL) in triplicate. The pH values were obtained to two significant figures. Buffer conditions. Fig. 1: (1) pH 4.2, 200 mM citrate buffer ([Na+] = 640 mM), (2) pH 4.2, 200 mM citrate buffer ([Na+] = 300 mM), (3) pH 5.0, 200 mM citrate buffer ([Na+] = 640 mM);
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Fig. 2: (1) pH 7.4, 200 mM phosphate buffer ([Na+] = 300 mM), (2) pH 4.5, 200 mM citrate buffer ([Na+] = 300 mM); Fig. 4: pH 4.5, 200 mM citrate buffer ([Na+] = 300 mM), 50 mM pyridine derivatives; Fig. 5: (1) pH 4.2 - 5.8, (citric acid + sodium phosphate dibasic) buffer ([Na+] = 400 mM), (2) pH 5.8 - 7.8, phosphate buffer ([Na+] = 400 mM); We duplicated pH 5.8 using both types of buffers, whose experimental pH values were confirmed to be identical. RESULTS AND DISCUSSION We first examined the pH-dependent role of pyridine for three sets (Set A, B, and C) of two complementary SNAs (s1 and s2, see Materials and Instrumentation) at various pH values (< 7.0) and [Na+]s. Thermal denaturation experiments were performed in the absence (‘Blank’) and presence (‘Py’) of pyridine. The Tms were obtained from the melting transitions, providing a quantitative measure of the duplex DNA stability between the SNAs.18 In Set A (pH 4.2, [Na+] = 0.64 M), the Tm was 36.3 oC in the absence of pyridine, which dramatically increased to 43.6 o C (Δ Tm = 7.3 oC) after the addition of pyridine (Figure 1a). This observation demonstrates that pyridine stabilizes not only conventional duplex DNA but also hybridized SNAs under acidic conditions.13 Because of the basicity of pyridine (pKb = ~ 8.8), however, the pH of the mixture after the addition of pyridine was elevated from 4.2 to 4.4 despite the 200 mM buffer concentration compared to 30 mM pyridine. To accurately analyze the stabilizing function of pyridine under acidic conditions, we corrected the pH to 4.2 using diluted HCl solution ('Py+HCl'). Interestingly, the denaturation experiment of the SNAs after the addition of pyridine with pH correction showed a negligible increase in the Tm from that of Blank (Δ Tm = 0 oC), indicating that the duplex stability did not change even in the presence of pyridine. In addition, we obtained the Tm of Blank after its pH was increased to 4.4 using diluted NaOH solution (‘Blank+NaOH’). Noticeably, the Tm increased to 43.1 oC without pyridine, similar to the experiment in the presence of pyridine without the pH correction. This observation evidently demonstrates that the Tm increases mainly due to the pH increase, not the presence of pyridine. In Set B (Figure 1b), we prepared Blank at the same pH (4.2) but at a lower [Na+] (0.3 M), and added pyridine at 60 mM, which is two times higher than the pyridine in Set A. The Tm of Blank was 33.0 oC, and increased to 44.8 oC after the addition of pyridine, exhibiting a larger ΔTm (11.8 oC) and a larger ΔpH (0.3 units) than that of Set A because of the higher [pyridine]. After pH correction, however, the Tm returned to 33.5 oC, nearly identical to the 33.0 o C Tm seen in the absence of pyridine. As expected, the Tm of Blank+NaOH after the pH increase to 4.5 was obtained at 45.2 o C, indicative of predominant effect of pH, not pyridine, on the Tm. The Tms of the four mixtures (Blank, Py, Py+HCl, and Blank+NaOH) of Set C (pH 5.0, [Na+] = 0.64 M; Figure 1c) were obtained at 51.5, 52.5, 51.4, and 53.1 oC, respectively. Significantly, the results obtained with the three independent experimental sets provide comprehensive evidence indicating that pyridine mainly changes the pH of the solution, and is less likely to stabilize the duplex DNA interconnects between the SNAs due to another stabilizing mechanism under the conditions studied (Figure 1d). In general, protonation and deprotonation of duplex DNA and hybridized SNAs have substantial influence on their stability. Duplex DNA and hybridized SNAs
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Figure 2. A plot showing the Tm of the hybridized SNAs as a function of the [pyridine] at pH 4.5 and 7.4.
Figure 1. Melting transitions of the hybridized SNAs before and after the addition of pyridine, after the pH correction using HCl, and pH-adjusted Blank using NaOH for the three experimental sets (Set A: (a), Set B: (b), and Set C: (c)). The Tms of each set are obtained from the melting transitions and compared in bar graphs on the right-hand side. The initial pH, [Na+], and [pyridine] of each set are (a) pH 4.2, 0.64 M, and 30 mM, (b) pH 4.2, 0.3 M, and 60 mM, and (c) pH 5.0, 0.64 M, and 30 mM, respectively. Note that the pH was measured when the solutions were at equilibrium after combining the components. (d) Schematic illustration of the duplex-stabilizing effect of pyridine under acidic conditions.
exhibit the highest stability and thus the highest Tm at neutral pH owing to the most possible Watson-Crick base pairing. As the pH increases or decreases from ~ 7, however, the nucleobases are either protonated or deprotonated, resulting in a decrease of base pairing, destabilization of duplex DNA and hybridized SNAs, and eventually a decrease in Tm. This pH-dependency of Tm is also effective with various forms and additives in association with DNA, such as DNA triplex, Hoogsteen base pairing, metal ions, intercalators, and mismatches.19-23 Therefore, the increased Tm seen with higher pH is because of
the deprotonation of protonated nucleobases, which leads to an increase of Watson-Crick base pairing.24,25 After observing the effect of the pH correction, we further systematically investigated the assembly properties of SNAs (s1 and s2) in the presence of pyridine by conducting their thermal denaturation without pH correction under acidic and neutral conditions (pH 4.5 and 7.4, respectively; Figure S2, see Supporting Information). Initially without pyridine, the Tms obtained were 38.5 oC and 41.7 oC at pH 4.5 and 7.4, respectively (Figure 2). The lower Tm at pH 4.5 is due to the protonation of nucleobases and loss of Watson-Crick base pairing.24,25 The Tms at various [pyridine]s were obtained at each starting pH (without pH correction after pyridine addition). As expected, at pH 4.5 the Tms gradually increase as the [pyridine] increases to ~ 50 mM. This is mainly due to the pH increase imparted by the presence of pyridine (Table 1). Pyridine’s stabilizing effect is also observed at higher [pyridine]s (> 100 mM).13 However, the Tm slightly decreases at 250 mM, presumably because the concentration of unprotonated pyridine is high enough to exhibit Table 1. Experimentally obtained pH values as a function of the [pyridine] under acidic (initial pH = 4.5) and neutral (initial pH = 7.4) conditions.a [pyridine] (mM)
Experimental pH (Initial pH = 4.5)
Experimental pH (Initial pH = 7.4)
0
4.5
7.4
50
4.8
7.4
100
5.0
7.3
150
5.1
7.3
200
5.2
7.4
250
5.3
7.5
a The
solutions were buffered using 200 mM citrate (initial pH = 4.5) or 200 mM phosphate (initial pH = 7.4). In both cases, the [Na+] was 300 mM. Note that the pH increased as the [pyridine] increased under acidic conditions, while the pH remained almost the same regardless of the [pyridine] under neutral conditions.
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Figure 4. The relative Tms of the hybridized SNAs at 300 mM NaCl (Blank), and the mixtures of one of the pyridine derivatives and Blank. Note that the concentration of the pyridine derivatives was 50 mM, while the buffer concentration was 200 mM. Figure 3. Molecular structures of the thirteen pyridine derivatives. Note that A to J are designated following the IUPAC nomenclature, while K, L, and M are rather conventionally designated for simplicity's sake.
the conventional destabilizing properties for the hybridized SNAs. In contrast, at starting pH 7.4, the Tm decreased as a function of the [pyridine]. This is consistent with the established understanding that pyridine acts as a denaturant with genomic DNA and short oligonucleotides.10,13 We subsequently investigated the correlation between the duplex stabilization of pyridine under acidic conditions and the chemical properties of pyridine in relation to its molecular structure. Pyridine is nucleophilic and basic because the lone pair of pyridine's nitrogen atom does not participate in the aromatic π-electron system. The nucleophilicity and basicity of pyridine can be altered by the ring-substitution, specifically by the type, position, and number of substituents. For example, pyridines with alkyl and amine substituents are supposed to exhibit increased basicity in general, although the specific chemical mechanisms are different.26 In addition, 2,4-dimethyl pyridine was reported to much less stabilize free duplex DNA than pyridine did under acidic conditions, which was proposed to be the result of the bulkiness of 2,4-dimethyl pyridine and the corresponding steric hindrance against binding at duplex grooves for stabilization.13 Therefore, the effect of the fundamental chemical properties and the molecular structure of pyridine and its derivatives on the stabilization of hybridized SNAs under acidic conditions needs to be thoroughly investigated, which will help us understand how pyridine actually functions. For this purpose, we constructed a library composed of pyridine and its 12 derivatives with substituents including one (I), two (B, C, J), or three (D) methyl/ethyl groups, one amine group (F, G, H), both methyl and amine groups (E, K), and hydroxyl groups (L, M), all at various positions of the pyridine ring (Figure 3). Thirteen batches of hybridized SNAs were prepared under buffer conditions ([Na+] = (300 mM, pH = 4.5), combined with one of the pyridine derivatives in Figure 3 (50 mM), and examined for the
quantitative analysis of the SNA stabilization by obtaining the Tms (Figure 4; Figure S3, see Supporting Information). Interestingly, all the pyridine derivatives, except M, exhibited substantial increases in Tm under acidic conditions. The average ΔTm was approximately 5 oC, similar to the ΔTm obtained with pyridine (A: ~6 oC). Only M showed a decrease in Tm of 1.8 oC. We then looked at the resulting pH in each sample, after the addition of the pyridine derivatives, (Table 2). As expected, the pyridine derivatives A to L, regardless of the substituents, exhibited higher pH than Blank. The only exception was M, the hydrochloride salt form of pyridoxine (L). Unlike other pyridine derivatives including L, M decreases the solution pH from 4.5 to 4.2 (Table 2) because HCl, a very strong acid, is added simultaneously at the same concentration (50 mM). As a result, this pH decrease leads to a Tm decrease. Considering that pyridoxine itself (L) still exhibited a considerable increase in Tm (ΔTm = 4.2 oC), the discrepancy of the Tms obtained with L and M strongly supports the pH-dependent mechanism of the duplex stabilization, and strongly excludes the possibility of the effective molecular interactions of pyridine and pyridine derivatives at 50 mM with SNAs. In brief, the presence of HCl of M leads to the difference in pH and resulting Tms of L and M. To further investigate the correlation between the solution pH and the duplex stabilization, the Tms of the hybridized SNAs at various pHs in the absence and presence of pyridine were systematically examined (Figure 5a; Figures S4, S5, and S6, see Supporting Information). Without pyridine, the Tm increased as the pH increased until approximately 7.4. The maximum Tm (54.3 oC) was obtained under neutral conditions. Under acidic conditions the nucleobases are protonated and disrupt the hydrogen bonding.24,25 The same experiment was repeated in the presence of pyridine at 30 and 75 mM. Similarly, the highest Tms were obtained around pH 7.4 (54.6 and 54.4 oC, respectively) and were practically identical to the experiment without pyridine. The pH-Tm curves obtained at both [pyridine]s show that below pH 5.0, the Tm substantially increases as the [pyridine] increases (Figure 5a, green box); however, the Tm increment gradually diminishes as the pH increases, leading to an
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Table 2. Pyridine derivatives and their aqueous solution pHs. Note that only the hydrochloride salt form of pyridoxine decreased the pH than that of Blank. a Pyridine Derivatives (50 mM)
Experimental pH (Initial pH = 4.5)
Blank
4.5
A. pyridine
4.8
B. 2,3-dimethyl pyridine
4.7
C. 2,4- dimethyl pyridine
4.7
D. 2,4,6-trimethylpyridine
4.7
E. 2-amino-4-methylpyridine
4.8
F. 2-aminopyridine
4.7
G. 3-aminopyridine
4.7
H. 4-aminopyridine
4.8
I. 4-methyl pyridine
4.7
J. 5-ethyl-2-methylpyridine
4.7
K. 6-amino-2,4-lutidine
4.8
L. pyridoxine
4.7
M. pyridoxine · HCl
4.2
a
The solutions were buffered using 200 mM citrate (initial pH = 4.5) at 300 mM Na+.
almost identical Tm, over pH 5.0. Above pH 6 the Tms converge at ~ 54 oC (Figure 5a, orange box). Moreover, we note that a dramatic increase in Tm in Figure 5a was obtained when the pH increased under pyridine-free conditions. For example, the ΔTm was ~13 oC when the pH increased from 4.2 to 5.0 (Δ[H+] = 5.3 × 10-5 M) in the absence of pyridine, which is comparable to the ΔTm of ~13 oC when the [pyridine] increased from 0 to 75 mM (Δ[pyridine] = 7.5 × 10-2 M) at pH 4.2. In other words, from the viewpoint of molecular and ionic interactions, the change in [pyridine] required for the stabilization of duplex DNA corresponding to the ΔTm of ~13 oC is 1400 times as high as that of [H+], indicating that the duplex stability is dependent on pH rather than pyridine. This observation led us to first-hand measure the experimental pH of the SNA-pyridine mixtures. Even though the [buffer] was 200 mM, much higher than the [pyridine]s examined in this work, we still presumed the possibility of changes in pH owing to the basicity of pyridine, which was examined by measuring the pH of ten buffer solutions whose initial pH ranged from 4.2 to 7.8 (pH increment = 0.4) before and after the addition of pyridine (Figure 5b). Importantly, the pH of the mixtures undoubtedly increased in proportion to the [pyridine] despite the sufficient buffering capacity (ΔpH ≤ 1.0), particularly under acidic conditions (pH < 6.0) (Figure 5b, green box). Considering that a simple pH increase of only 0.8 from pH 4.2 by controlling buffer components induced a ΔTm of ~13 oC under pyridine-free conditions, the ΔTm of ~13 oC after the addition of pyridine (75 mM) at initial pH 4.2 should be mainly attributed to the pH increase of 0.8 induced by pyridine, rather than direct molecular interactions of pyridine with SNAs as proposed by previous literature.13 Above pH 6.0, both pHs before and after
Figure 5. (a) A plot presenting the Tm of the hybridized SNAs at an initial pH ranging from 4.2 to 7.8 (increment = 0.4) in the absence or presence of pyridine (30 and 75 mM). (b) The experimentally measured pH of the mixtures in Figure 5(a) as a function of the initial pH and the [pyridine]. The green box designates the data below pH 5.5, and the orange box above pH 6.0 (see the main text).
the addition of pH were almost identical (Figure 5b, orange box), which can account for the convergence and consistency of the Tms regardless of the initial pH above 6.0 (Figure 5a, orange box). As a denaturant, pyridine primarily breaks the hydrogen bonds of duplex DNA to separate into single strands. It has a dielectric constant higher than that of urea, and is more hydrophobic than formamide or urea owing to its aromaticity, all of which are suitable for explaining the denaturing function.27,28 Unlike other representative denaturants, however, pyridine not only interacts with DNA by itself, but also increases the solution pH as a base (pH < ~7), which independently affects the stability of duplex DNA. In fact, the effects of pyridine and a few other heterocyclic amines on the pH and the hybridization efficiency were also previously observed in microelectronic
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chip systems.29 Under acidic conditions created by the electrolysis of water at the anode, pyridine, 2,4,6-trimethylpyridine (A and D in Figure 3, respectively), imidazole, and histidine increased the pH close to 7, and consequently resulted in the increased hybridization of surface-immobilized duplex DNA. In contrast, other acidic amino acids rarely affected the hybridization. This previous observation strongly supports our assertion by emphasizing the importance of two analytical issues: (1) the first-hand measurement of the experimental pH, and (2) the universal effect of pyridine on the acidic pH for both densely packed particulate DNA and sparsely distributed independent one. Our study clearly reveals that one of the most powerful denaturants of DNA, acidic pH, is not immune to the basicity of pyridine in the context of SNAs, which, unfortunately, was disregarded in recent contradictory research about duplex DNA.13 The Tm of hybridized SNAs is highly sensitive to minor changes in pH under acidic conditions. Importantly, the stabilization of SNAs in the presence of pyridine at a low concentration, under acidic conditions, is rather due to the pH changes induced by pyridine, than direct molecular interactions of pyridine with SNAs. Although other non-charged DNA denaturants, such as formamide and urea, are not applicable to this pH-dependent duplex stability issue,28,30,31 it would be essential in the future to carefully examine a potential duplex DNA denaturants in relation to their capability to change the pH.
ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: xx.xxxx/acs.anal-chem.xxxxxx. Melting profiles of SNAs under various conditions (PDF)
AUTHOR INFORMATION Corresponding Author *E-mail:
[email protected] Phone: +82-2-3290-3267, Fax: +82-2-928-3584 *E-mail:
[email protected] Phone: +1-617-324-4281
Author Contributions §These authors contributed equally.
Notes The authors declare no competing financial interest.
ACKNOWLEDGMENTS This work was supported by the NRF funded by the Korean government, MSIP (NRF-2015R1C1A1A01053865, NRF2015M3A9D7031015, NRF-2016R1A5A1010148, and NRF2016R1E1A202073).
REFERENCES
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(1) Kochetkov, N. K.; Budovskiı̆ , E. I. Organic chemistry of nucleic acids; Plenum Press: New York,, 1972. (2) Bloomfield, V. A.; Crothers, D. M.; Tinoco, I. Physical chemistry of nucleic acids; Harper & Row: New York,, 1974, p x, 517 p. (3) Van Vranken, D. L.; Weiss, G. A. Introduction to bioorganic chemistry and chemical biology; Garland Science: New York, 2013, p xvii, 486 p. (4) Rothlingshofer, M.; Kervio, E.; Lommel, T.; Plutowski, U.; Hochgesand, A.; Richert, C. Angew. Chem., Int. Ed. 2008, 47, 6065-6068. (5) Benner, S. A. Accounts Chem. Res. 2004, 37, 784-797. (6) Mishra, B. K.; Arey, J. S.; Sathyamurthy, N. J Phys Chem A 2010, 114, 9606-9616. (7) Hwang, G. T.; Hari, Y.; Romesberg, F. E. Nucleic Acids Res. 2009, 37, 4757-4763. (8) Stangret, J.; Savoie, R. J Mol Struct 1993, 297, 91-102. (9) Bathini, Y.; Rao, K. E.; Shea, R. G.; Lown, J. W. Chem. Res. Toxicol. 1990, 3, 268-280. (10) Levine, L.; Jencks, W. P.; Gordon, J. A. Biochemistry 1963, 2, 168-175. (11) Perez, A.; Orozco, M. Angew. Chem., Int. Ed. 2010, 49, 48054808. (12) Bueren-Calabuig, J. A.; Giraudon, C.; Galmarini, C. M.; Egly, J. M.; Gago, F. Nucleic Acids Res. 2011, 39, 8248-8257. (13) Portella, G.; Terrazas, M.; Villegas, N.; Gonzalez, C.; Orozco, M. Angew. Chem., Int. Ed. 2015, 54, 10488-10491. (14) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. Nature 1996, 382, 607-609. (15) Service, R. F. Science 2015, 349, 1150-1151. (16) Jin, R.; Wu, G.; Li, Z.; Mirkin, C. A.; Schatz, G. C. J. Am. Chem. Soc. 2003, 125, 1643-1654. (17) Sonnichsen, C.; Reinhard, B. M.; Liphardt, J.; Alivisatos, A. P. Nat. Biotechnol. 2005, 23, 741-745. (18) A melting temperature is typically obtained from the temperature where the maximum of the first derivative of the melting transition occurs. (19) Brown, T.; Leonard, G. A.; Booth, E. D.; Kneale, G. J. Mol. Biol. 1990, 212, 437-440. (20) Escude, C.; Mohammadi, S.; Sun, J. S.; Nguyen, C. H.; Bisagni, E.; Liquier, J.; Taillandier, E.; Garestier, T.; Helene, C. Chem. Biol. 1996, 3, 57-65. (21) Williams, M. C.; Wenner, J. R.; Rouzina, L.; Bloomfield, V. A. Biophys. J. 2001, 80, 874-881. (22) Miyake, Y.; Togashi, H.; Tashiro, M.; Yamaguchi, H.; Oda, S.; Kudo, M.; Tanaka, Y.; Kondo, Y.; Sawa, R.; Fujimoto, T.; Machinami, T.; Ono, A. J. Am. Chem. Soc. 2006, 128, 2172-2173. (23) Arya, D. P.; Micovic, L.; Charles, I.; Coffee, R. L.; Willis, B.; Xue, L. J. Am. Chem. Soc. 2003, 125, 3733-3744. (24) Wood, J. L. Biochem. J. 1974, 143, 775-777. (25) Hamaguchi, K.; Geiduschek, E. P. J. Am. Chem. Soc. 1962, 84, 1329-1338. (26) Morrison, R. T.; Boyd, R. N. Organic chemistry, 6th ed.; Prentice Hall: Englewood, Cliffs, N.J., 1992, p xxvi, 1279 p. (27) Herskovits, T. T. Arch. Biochem. Biophys. 1962, 97, 474-484. (28) Marmur, J.; Tso, P. O. P. Biochim. Biophys. Acta 1961, 51, 3236. (29) Edman, C. F.; Raymond, D. E.; Wu, D. J.; Tu, E. G.; Sosnowski, R. G.; Butler, W. F.; Nerenberg, M.; Heller, M. J. Nucleic Acids Res. 1997, 25, 4907-4914. (30) Blake, R. D.; Delcourt, S. G. Nucleic Acids Res. 1996, 24, 2095-2103. (31) Hutton, J. R. Nucleic Acids Res. 1977, 4, 3537-3555.
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