Unique Thermal Stability of Unnatural Hydrophobic Ds Bases in

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Unique Thermal Stability of Unnatural Hydrophobic Ds Bases in Double-Stranded DNAs Michiko Kimoto, and Ichiro Hirao ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.7b00165 • Publication Date (Web): 13 Jul 2017 Downloaded from http://pubs.acs.org on July 13, 2017

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ACS Synthetic Biology

Unique Thermal Stability of Unnatural Hydrophobic Ds Bases in Double-Stranded DNAs Michiko Kimoto1,2 and Ichiro Hirao1,2,* 1

Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, #09-01, Singapore 138669, 2RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan 1

Current address

ABSTRACT: Genetic alphabet expansion technology, the introduction of unnatural bases or base pairs into replicable DNA, has rapidly advanced as a new synthetic biology area. A hydrophobic unnatural base pair between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px) exhibited high fidelity as a third base pair in PCR. SELEX methods using the Ds– Px pair enabled high-affinity DNA aptamer generation, and introducing a few Ds bases into DNA aptamers extremely augmented their affinities and selectivities to target proteins. Here, to further scrutinize the functions of this highly hydrophobic Ds base, the thermal stabilities of double-stranded DNAs (dsDNA) containing a non-cognate Ds–Ds or G–Ds pair were examined. The thermal stability of the Ds– Ds self-pair was as high as that of the natural G–C pair, and apart from the generally higher stability of the G–C pair than that of the A–T pair, most of the 5′-pyrimidine-Ds-purine-3′ sequences, such as CDsA and TDsA, exhibited higher stability than the 5′-purine-Ds-pyrimidine-3′ sequences, such as GDsC and ADsC, in dsDNAs. This trait enabled the GC-content-independent control of the thermal stability of the designed dsDNA fragments. The melting temperatures of dsDNA fragments containing the Ds–Ds pair can be predicted from the nearest-neighbor parameters including the Ds base. In addition, the non-cognate G–Ds pair can efficiently distinguish its neighboring cognate natural base pairs from non-cognate pairs. We demonstrated that real-time PCR using primers containing Ds accurately detected a single-nucleotide mismatch in target DNAs. These unique properties of the Ds base that affects the stabilities of the neighboring base pairs could impart new functions to DNA molecules and technologies. KEYWORDS Synthetic biology, Unnatural base pair, Expansion of genetic alphabet, PCR, SNP 1

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detection INTRODUCTION Advancements in DNA technology have revolutionized a wide range of modern science and biotechnology. However, the existence of only four types of nucleotide components in DNA with hydrophilic sugars and phosphates has constrained the further advancement of the technology, especially when using DNA as functional materials. Thus, many researchers have been devoted to exploring novel DNA functions, by modifying the components or introducing non-natural components.1-7 Among them, studies on the genetic alphabet expansion of DNA by introducing unnatural base pairs (UBPs) have rapidly advanced.8-13 This is because UBPs can function in replication as a third base pair and are utilized in PCR-based biotechnologies13-17 or in vivo applications18,19 with artificial DNAs containing UBPs. Currently, two types of UBPs have been developed: one is natural-type hydrogen-bonded UBPs, such as Z–P20,21, and the other is hydrophobic UBPs, which lack clear hydrogen-bonded interactions between the pairing bases, such as Ds–Px22,23 and NaM–5SICS24,25. These UBPs were applied to DNA aptamer generation for SELEX (Systematic Evolution of Ligands by EXponential enrichment)16,17,26-28 and the creation of a semisynthetic organism18,19. The hydrophobic Ds–Px pair exhibits high fidelity in PCR amplification.23,29 We have developed a detection system for qPCR and a molecular beacon using the Ds–Px pair and its modified version.30,31 In addition, the Ds–Px pair was applied to SELEX (ExSELEX, genetic alphabet Expansion for SELEX) to generate high-affinity Ds-containing DNA aptamers that specifically bind to target proteins.16,26 Due to the high hydrophobicity of Ds, only a few Ds bases can significantly augment the affinities of DNA aptamers. Thus, hydrophobic UBs have the potential to confer novel and increased functionalities on nucleic acids. Besides the DNA aptamer application, hydrophobic UBs and UBPs have a wide range of uses due to their unique physical properties. A modified Ds base, 7-(2,2′-bithien-5-yl) imidazo[4,5-b]pyridine (Dss)32, is strongly fluorescent, and the 2-nitropyrrole moiety of Px acts as a quencher.31 Other hydrophobic self-pairs, such as PICS–PICS33 and 34DMPy–34DMPy34, developed by Romesberg’s group, exhibit high thermal stability in duplex DNAs. Furthermore, the 34DMPy–34DMPy and 34DMPy– natural-base pairs destabilize the duplex DNAs containing natural-base mispairs.34 However, as for the Ds base, very limited information has been available about how the Ds base affects and increases DNA functions and properties. To explore the further potentials of the Ds base, we examined the thermal stabilities of double-stranded DNA (dsDNA) fragments containing Ds bases. Here, we 2


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report the unique properties of non-cognate Ds–Ds and Ds–G pairs (Figure 1), besides the cognate UBPs,22,35 in DNA duplexes. These non-cognate UB pairs affected the stabilities of the neighboring natural base pairs: the Ds–Ds pair can cancel the general rule that the thermal stability of dsDNAs depends on their GC contents, and the Ds–G pair, like Romesberg’s 34DMPy–A pair,34 can enhance the recognition of a neighboring cognate natural base pair. These findings represent a novel use of the Ds base for genetic alphabet expansion technologies. RESULTS AND DISCUSSION Thermal Stabilities of dsDNA Containing Cognate and Non-cognate UBPs in Various Sequence Contexts. First, we examined the thermal stabilities of short dsDNA fragments (12-mer) containing cognate and non-cognate unnatural and natural base pairs (Table 1). The melting temperatures (Tm) of all of the fragments were measured in phosphate buffer (pH 7.0) containing 100 mM NaCl. DNA fragments containing Px cannot be chemically synthesized, because of its chemical instability under basic conditions. Thus, instead of Px, we used 2-nitropyrrole (Pn)36 and pyrrole-2-carbaldehyde (Pa)35 as the cognate pairing partners of Ds. The Ds–Pn and Ds– Pa pairs can also be used as third base pairs in replication and transcription,35-38 although their pairing selectivities are slightly lower than that of the Ds–Px pairing in PCR. As shown in Table 1, in contrast to the highly selective pairings of Ds–Pn and Ds–Pa as third pairs in polymerase reactions, their thermal stabilities were moderate, and lower than that of the A–T pair. However, the Ds–Ds self-pair exhibited high stability, similar to that of the G–C pair. In addition, the stability of the G–Ds pair was higher than that of the T–G wobble pair. Since the Ds base greatly affects DNA aptamer affinities to target proteins,16,26 we focused on the non-cognate UBPs involving the Ds base, such as Ds–Ds and G–Ds pairs, for subsequent thermal stability studies. Next, to determine the effects of the Ds–Ds and G–Ds pairs on the neighboring natural bases, we measured the Tm values of the dsDNA fragments in various sequence contexts (Table 2). Interestingly, the neighboring G–C pairs of these non-standard base pairs did not always increase the thermal stability of the dsDNA fragments. For example, the 5′-TDsA-3′/3′-ADsT-5′ (Tm = 48.1°C) and 5′-TGA-3′/3′-ADsT-5′ (Tm = 40.1°C) contexts are more stable than the 5′-GDsC-3′/3′-CDsG-5′ (Tm = 44.1°C) and 5′ -GGC-3′/3′-CDsG-5′ (Tm = 36.8°C) contexts, respectively. The data revealed that most of the dsDNAs with the natural pyrimidine bases on the 5′ side of Ds exhibited higher stability than those with the purine bases. Namely, the 5′-Py-Ds-Pu-3′ sequences (Py: pyrimidine, Pu: purine) tend to have higher stability than the 5′-Pu-Ds-Py-3′ 3



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sequences. The order of the sequence context stabilities around the G–Ds pair (Table 2) was very similar to that around the Ds–Ds pair. The stronger stability of the 5′-Py-Ds-Pu-3′ sequences might result from the enhanced stacking stability between the Ds and purine bases, as compared to that between the Ds and pyrimidine bases. First, since the Ds–Ds pair exhibits high stability, the large Ds bases might both adopt anti-conformations and become stacked upon each other in dsDNA, rather than forming an edge-to-edge planar structure with one of the bases adopting a syn-conformation (Figure 2). Second, in the stacked structures, there are two possible conformations of the Ds-stacking structures, in which the Ds base tilts toward the 3′ (5Ds3 packing) or 5′ (5Ds3 packing) side, as shown in Figure 2. Since the 5′-Py-Ds-Pu-3′ sequences have higher stability than the 5′-Pu-Ds-Py-3′ sequences, the Ds bases might stack with the 5′-neighboring purine base, adopting the 5Ds3 packing in the 5′-Py-Ds-Pu-3′ duplexes (5′-Py-5Ds3-Pu-3′). In contrast, the 5Ds3 packing in the 5′-Pu-Ds-Py-3′ sequences is less stable, because of the lower stability of the stacking between Ds and pyrimidine bases (5′-Pu-5Ds3-Py-3′). Other hydrophobic UBPs such as PICS–PICS, MMO2–5SICS, and NaM– 5SICS,33,39,40 reported by Romesberg’s group, also exhibit high thermal stabilities. Structural analyses revealed that these hydrophobic bases also adopt the anti-conformation and stack upon each other in the self-pair.12,39-42 Interestingly, each UBP adopts different conformations: MMO2–5SICS and NaM–5SICS take the same conformation with the suggested 5Ds3 packing of Ds–Ds, but PICS–PICS takes the 5Ds3 packing.12 Understanding the stabilization mechanism depending on the neighboring bases will require further considerations, including hydration and polarity. Ds Base-Mediated Thermal Stability Control of dsDNAs. Inspired by the unique stabilities of the G–Ds and Ds–Ds pairs, which do not always correlate with the neighboring G–C pair, we designed dsDNA fragments containing one Ds–G or Ds–Ds pair and different numbers (from six to nine) of G–C pairs, with similar thermal stabilities. As shown in Figure 3, the six dsDNA fragments consisting of only natural bases had different thermal stabilities (maximum ∆Tm value: 11.9°C) dependent on their GC contents. However, by replacing one G–C pair with the G–Ds pair, the thermal stabilities of these fragments became similar to each other (maximum ∆Tm value: 3.9°C). Furthermore, replacing the G–C pair with the Ds–Ds self-pair at the same position also closed the gap (maximum ∆Tm value: 2.8°C). The dependency of the dsDNA thermal stability on the GC content often makes it difficult to design DNA hybridization probes and PCR primers for multiplex analyses. Thus, the G–Ds and Ds–Ds pairs might be useful for further hybridization applications, such as multiplex detection probe design, DNA computing, and DNA nanostructure construction. 4


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To predict the Tm values of dsDNA fragments containing the Ds–Ds pair, we determined the nearest-neighbor parameters (∆H°, ∆S°, and ∆G°37), using the data shown in Table 2, by a hyperchromicity calculation method. The ∆S° and ∆G°37 values obtained from the melting curves were converted to those in a 1 M NaCl solution, to allow the calculation of the parameters (Table 3). We used the parameters for the natural base combinations reported by SantaLucia.43 With these parameters, we predicted the Tm values of several dsDNA fragments (Supplementary Table S1 and Figure 4). The calculated Tm values of the dsDNA fragments correlated relatively well with their observed Tm values, when the Ds–Ds pair was embedded near the center of the sequences. Non-Cognate Natural Base Pair Recognition by Neighboring G–Ds Pair in dsDNA. The G–Ds pair has a wide range of applications, relative to the Ds–Ds pair, because it can target natural DNA sequences by using a hybridization probe containing Ds located opposite the G site in the target. Although the G–Ds pair reduces the thermal stability of dsDNAs, most of the Tm values of 12-mers containing 5′-pyrimidine-Ds-3′ sequences are higher than room temperature (Table 2). Furthermore, we found that the G–Ds pair more efficiently distinguished the neighboring cognate natural base pairs from the non-cognate base pairs, relative to the G–C pair (Table 4). The non-cognate pairs with A located next to the G–Ds pair significantly reduced the thermal stability of the duplex (minimum ∆Tm value: 13.9°C), as compared to those next to the G–C pair (minimum ∆Tm value: 9.6°C). This Ds-containing hybridization probe was applied to real-time PCR to detect a single-nucleotide mismatch in target DNAs. We prepared two DNA templates: one (Temp-T60, 60-mer) is a matched target sequence and the other (Temp-C53, 53-mer) is a mismatched untargeted sequence with one base difference in the reverse primer region (Rev Primer in Figure 5A). To analyze both of the amplified products on a gel simultaneously, their lengths were varied relative to each other. To determine the optimal position of the embedded Ds base in the reverse primer, we prepared three different primer sets for each Ds- or C-containing sequence (20X-1, 20X-2, and 20X-3, X = Ds or C), in which each hybridization position was displaced by two bases on the templates. Using AccuPrime Pfx DNA polymerase, each of the reverse primers, and the mixed templates of Temp-T60 and Temp-C53, we performed PCR amplification with different temperatures for primer hybridization (Figure 5B). Besides the natural base substrates, an UB substrate (dPxTP) was added to be the pairing partner incorporated opposite Ds in the primer in PCR.23,30 The amplified products from both of the templates were analyzed by denaturing gel-electrophoresis (Figure 5C). All three Ds-containing primers recognized only the matched template 5



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(Temp-T60) well at hybridization temperatures of 63.2 and 68°C (Figure 5C), in which Temp-T60 was predominantly amplified. Among them, the better conditions were at 63.2°C using 20Ds-1 or 20Ds-2, and at 68°C using 20Ds-3. In contrast, the natural base primers (20C-1, -2, and -3) amplified both the targeted and untargeted templates similarly, and only at 68°C, Temp-T60 was more efficiently amplified as compared to Temp-C53. To confirm the ability of the Ds-containing probes to detect a mismatch, we demonstrated the single-nucleotide mismatch detection by real-time PCR using matched (Temp-T60) and mismatched DNA fragments (Temp-C60) with the same lengths (60-mer), in which only one base was altered. Based on the precedent results, we chose the 20Ds-1 and 20C-1 primers and two-step PCR cycle conditions: 15 sec at 94°C and 120 sec at 68°C, and two different template concentrations (25 and 2.5 pM). As shown in Figure 6, the Ds-containing primer (20Ds-1) predominantly recognized only the matched Temp-T60 fragment, and PCR using the natural-base primer (20C-1) equally amplified both of the DNA fragments. In addition to the effect of the G–Ds pair, the high discrimination between the matched and mismatched base pairs using the Ds-containing primers might partly result from the selectivity and stability of the Px–Ds pairing in PCR. The Ds base in the primer initially pairs with G in the template strand. However, in the following PCR cycles, the Ds base pairs with Px, because the Px substrate is incorporated opposite Ds in the primer strand during PCR amplification. Thus, like the G–Ds pair, the Px–Ds pair might also recognize the neighboring matched and mismatched pairs, allowing for the highly sensitive detection of a single-nucleotide mismatch in the target DNA through PCR amplification.

CONCLUSION We identified the unique thermal properties of the Ds–Ds and G–Ds pairs in dsDNA fragments. These non-cognate base pairs involving the Ds base affected the neighboring natural base pairs under a different rule from that of nature. The thermal stability of the Ds–Ds pair is as high as that of the G–C pair in the same sequence contexts, and the Ds– Ds and G–Ds pairs have unique neighboring effects in a duplex. In different sequence contexts containing the Ds–Ds and G–Ds pairs, the 5′-Py-Ds-Pu-3′ sequences (Py: pyrimidine, Pu: purine) tend to have higher stability than the 5′-Pu-Ds-Py-3′ sequences. Thus, the dsDNA fragments containing 5′-CDsA-3′ or 5′-TDsG-3′ are more stable than those containing 5′-GDsC-3′ or 5′-ADsC-3′, allowing the Ds–Ds and G–Ds pairs to counteract the effects of the GC content on dsDNA stabilities. So far, several self-pairs 6


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using hydrophobic UBPs and metal-chelated base analogues have been reported.33,34,44-50 However, the altered thermal properties and sequence context effects described herein have not been reported yet, to the best of our knowledge. The novel characteristics of the Ds–Ds and G–Ds pairs could expand the architecture of DNA hybridization applications in various areas, such as detection, diagnostics, and nanostructures. One example is the non-cognate G–Ds pair, which affects the thermal stability of its neighboring base pairs and recognizes cognate natural base pairs. This phenomena might be a characteristics of hydrophobic bases, as show in Romesberg’s 34DMPy–34DMPy and 34DMPy–natural-base pairs.34 As demonstrated here, the G–Ds pair could be applied to real-time PCR to efficiently detect a single-nucleotide mismatch. This is a starting point for the practical application to SNP assays. To increase the sensitivity, further extensive analyses, such as pursuing other combinations with different distances between the mismatch and the unnatural base positions, should also be performed. Our data could also provide new information to elucidate the tertiary structures of Ds-containing functional molecules. Our previous data indicated that Ds-containing DNA aptamers exhibit higher thermal stability and affinity to targets, as compared to Ds-to-A DNA aptamer variants.16,26 The incorporation of the Ds–Ds or G–Ds pair might allow the structural diversity of DNA molecules to be increased on demand. Thus, the combination of the unique stability of the Ds base with the genetic alphabet expansion and replication system using UBPs, such as Ds–Px and Ds–Pa,22,23,29,30,35,38,51 could provide further applications, which are now being explored. In addition, another Ds base analog linked to an additional thiophene residue (Dss), which exhibits strong fluorescence,31,32 might be useful for the same purposes. METHODS Oligonucleotides: DNA fragments (12-mers for Tm measurement, 20-mer primers, and 60-mer or 53-mer DNA templates for PCR) were chemically synthesized with an Applied Biosystems 392 DNA synthesizer, an Oligonucleotide Synthesizer nS-8 (Gene Design), or an H8 DNA synthesizer (K&A Laborgerate), using phosphoramidite reagents for the natural and Ds bases (Glen Research),35 or purchased from Invitrogen, Gene Design or Integrated DNA Technologies. The chemically-synthesized DNA fragments were purified by HPLC (for 12-mers) or denaturing polyacrylamide gel electrophoresis (for primers). Measurement of melting curves: The melting curves of the 12-bp DNA duplexes were recorded with a Shimadzu UV-2450 spectrometer equipped with a temperature controller (TMSPC-8). The absorbance of each sample (5 µM DNA duplex in 10 mM sodium 7



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phosphate, 100 mM NaCl, 0.1 mM EDTA, pH 7.0) was monitored at 260 nm from 15 to 95°C, at a heating rate of 0.5°C/min. Each melting temperature was determined by calculating the first derivative of the melting curve, using the IGOR Pro software (WaveMetrics, Inc.). Determination of thermodynamic parameters: The thermodynamic parameters, ∆H°, ∆S°, and ∆G°37, for duplex formation were obtained from the melting curve data by using a hyperchromicity calculation method with the Varian Cary WinUV software, similar to the previously reported method.52 The ∆S° and ∆G°37 values thus obtained were then converted to the values corresponding to 1 M NaCl, by using the previously reported equations.43 According to the reported procedure,52 the nearest-neighbor ∆H°, ∆S°, and ∆G°37 values for 5′-ADs-3′/5′-DsT-3′, 5′-TDs-3′/5′-DsA-3′, 5′-CDs-3′/5′-DsG-3′, and 5′-GDs-3′/5′-DsC-3′ were determined by a multiple regression analysis using Microsoft Excel, from the data of the eleven DNA duplexes shown in Table 2. The calculated parameters are summarized in Table 3. Prediction of melting temperatures: The predicted melting temperature of each 12-mer duplex (5 µM, in 100 mM NaCl) was calculated according to the literature.53-55 From each predicted Tm value in 1 M NaCl (Tm (K) = 1,000 × ∆H° (kcal/mol) / (∆S° (cal/(K·mol)) + R ln Ct), where R = 1.987 (cal/(K·mol)) and Ct = (0.000005+0.000005) /4), the predicted Tm value in 100 mM NaCl was calculated by using the equation reported by Owczarzy et al.53 PCR using Ds-containing and fluorescently-labeled primers: The PCR reaction (10 µl) was performed in 1× AccuPrime Pfx reaction mix (Invitrogen), supplemented with 0.1 mM dNTPs (final concentration: 0.4 mM each) and 0.5 mM MgSO4 (final concentration: 1.5 mM), in the presence of 50 µM Diol1-dPxTP, 500 nM each of a forward primer labeled with FAM at the 5ꞌ-end and a reverse Ds-containing primer as indicated, 0.25 nM each 60-mer matched and 53-mer mismatched DNA template (Temp-T60 or Temp-C53), and 0.05 U/µl AccuPrim Pfx DNA polymerase. PCR conditions were 94°C 1 min and then 15 cycles of 15 s at 94°C, 60 s at either 55, 63.2, or 68°C, and 60 s at 68°C. The PCR products were fractionated on a denaturing 15% polyacrylamide gel, and the band patterns generated from the matched and mismatched DNA templates were analyzed with an LAS4000 bioimager (GE Healthcare). Real-time PCR using Ds-containing primers: The PCR reaction (20 µl) was performed on a CFX96 real-time PCR system (BioRad), in 1× AccuPrime Pfx reaction mix (Invitrogen) supplemented with 0.1 mM dNTPs (final concentration: 0.4 mM each) and 0.5 mM MgSO4 (final concentration: 1.5 mM), in the presence of 50 µM Diol1-dPxTP, 500 nM each of a non-labeled forward primer and a reverse 8


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Ds-containing primer as indicated, 25 or 2.5 pM 60-mer matched or mismatched DNA template (Temp-T60 or Temp-C60), and 0.05 U/µl AccuPrim Pfx DNA polymerase. PCR conditions were at 94°C for 1 min and then 40 cycles of 15 s at 94°C and 120 s at 68°C with an optical read at the end of each step. ASSOCIATED CONTENT Supporting Information. Additional experimental data and results: DNA-duplex sequences used in Figure 4 and their observed and predicted Tm values. “This material is available free of charge via the Internet at http://pubs.acs.org.”

AUTHOR INFORMATION Corresponding Author *Tel: +65-6824-7104. Fax: +65-6478-9083. E-mail: [email protected] ORCID Michiko Kimoto: 0000-0002-6261-7362 Ichiro Hirao: 0000-0002-1115-8079 Author Contributions M.K. performed the experiments. I.H. chemically synthesized the DNA fragments. M.K. and I.H. contributed to the experimental design, the data analysis, and the preparation of the manuscript.

Notes: Conflict of Interest There is potential competing interest. A patent application describing ideas presented in this article has been filed by TagCyx Biotechnologies and RIKEN. M.K. and I.H. own stock in TagCyx Biotechnologies. ACKNOWLEDGEMENTS: This work was supported by the Institute of Bioengineering and Nanotechnology (Biomedical Research Council, Agency for Science, Technology and Research, Singapore) (M.K., I.H.), a Grant-in-Aid for Scientific Research [KAKENHI 26248043] from the Ministry of Education, Culture, Sports, Science and Technology (I.H.), and by the Japan Science and Technology Agency (JST) Precursory Research for Embryonic 9



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Zhang, W., Gerloff, D. L., Huang, Z., Tan, W., and Benner, S. A. (2015) Evolution of functional six-nucleotide DNA. J. Am. Chem. Soc. 137, 6734-6737. (28) Zhang, L., Yang, Z., Le Trinh, T., Teng, I. T., Wang, S., Bradley, K. M., Hoshika, S., Wu, Q., Cansiz, S., Rowold, D. J., McLendon, C., Kim, M. S., Wu, Y., Cui, C., Liu, Y., Hou, W., Stewart, K., Wan, S., Liu, C., Benner, S. A., and Tan, W. (2016) Aptamers against Cells Overexpressing Glypican 3 from Expanded Genetic Systems Combined with Cell Engineering and Laboratory Evolution. Angew. Chem. Int. Ed. Engl. 55, 12372-12375. (29)

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Efficiency and Functionalization of Ds-Px Unnatural Base Pairs in PCR Amplification for a Genetic Alphabet Expansion System. ACS Synth. Biol. 5, 1220-1230. (30) Yamashige, R., Kimoto, M., Mitsui, T., Yokoyama, S., and Hirao, I. (2011) Monitoring the site-specific incorporation of dual fluorophore-quencher base analogues for target DNA detection by an unnatural base pair system. Org. Biomol. Chem. 9, 7504-7509. (31) Kimoto,

M., Mitsui, T., Yamashige, R., Sato, A., Yokoyama, S., and Hirao, I. (2010) A new unnatural base pair system between fluorophore and quencher base analogues for nucleic acid-based imaging technology. J. Am. Chem. Soc. 132, 15418-15426.

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FIGURE LEGENDS Figure 1. Chemical structures of UBPs involving Ds. Figure 2. Possible conformations of the Ds–Ds self-pair in a duplex DNA. These schematic structures were drawn based on the structural data of the MMO2–5SICS and NaM–5SICS base pairs.12,41,42 Figure 3. Design of probe sequences containing different GC contents with similar Tm values. Figure 4. Predicted Tm values plotted against the observed Tm values in 100 mM NaCl. Figure 5. PCR application of Ds-containing probes as primers for effective discrimination of a single-nucleotide mismatch in a DNA target. (A) Sequences used in the PCR analysis. (B) Experimental scheme of the PCR analysis using AccuPrime Pfx DNA polymerase. (C) Band patterns of full-length FAM-labeled PCR products under each condition, analyzed with an LAS4000 bio-imager. Figure 6. Application of a Ds-containing primer to real-time PCR, allowing the discrimination of a single-nucleotide mismatch.

15



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Figure 1.

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Figure 2.

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Figure 3.

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Figure 4.

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Figure 5.

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Figure 6.

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Table 1. Thermal stabilities of dsDNA fragments (12-mer) containing unnatural and natural bases.

5′-CGCAT-X-GTTACC-3′ 3′-GCGTA-Y-CAATGG-5′

[a]

X–Y

Tm (℃)[a]

X–Y

Tm (℃)[a]

Ds–Ds

51.6

C–G

53.1

A–Ds

43.5

G–C

52.7

T–Ds

40.5

T–A

48.6

G–Ds

45.0

T–G

42.4

C–Ds

40.7

A–G

38.6

Pn–Ds

43.9

C–A

33.6

Pa–Ds

42.4

The absorbance at 260 nm of the DNA fragments (5 µM), in 10 mM sodium phosphate

(pH 7.0), 100 mM NaCl, and 0.1 mM EDTA, was monitored.

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Table 2. Thermal stabilities of DNA fragments (12-mer) containing the Ds–Ds or G–Ds pair in various sequence contexts.

5′-CGCAN1XN2TTACC-3′[a] 3′-GCGTN3DsN4AATGG-5′[a] Ds–Ds pair (X = Ds)

[a]

No

N1XN2

Tm (℃)[b]

1

CDsG

57.2

2

CDsA

53.1

3

TDsG

4

G–Ds pair (X = G) N1XN2

Tm (℃)[b]

CGG

49.9

–4.1

CGA

45.3

–4.6

51.6

–5.6

TGG

45.0

–4.9

GDsG

49.2

–8.0

GGG

41.1

–8.8

5

TDsA

48.1

–9.1

TGA

40.1

–9.8

6

ADsG

46.5

–10.7

AGG

39.4

–10.5

7

GDsA

44.6

–12.6

GGC

36.8

–13.1

8

GDsC

44.1

–13.1

GGA

36.4

–13.5

9

ADsA

42.5

–14.7

AGC

34.8

–15.1

10

ADsC

41.7

–15.5

AGA

34.7

–15.2

11

ADsT

37.7

–19.5

AGT

30.0

–19.9

∆Tm (℃)

∆Tm (℃)

N1–N3 and N2–N4 are any cognate natural base pairs. The absorbance at 260 nm of the DNA fragments (5 µM), in 10 mM sodium phosphate

[b]


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Page 24 of 26

Table 3. Nearest-neighbor parameters for DNA duplexes involving the Ds–Ds pair at 37°C. (5′-3′/5′-3′)

∆H° (kcal/mol)

∆S° (cal/(K·mol))

∆G°37 (kcal/mol)

CDs/DsG

-3.2 ± 1.3

-4.8 ± 3.8

-1.80 ± 0.11

TDs/DsA

-3.5 ± 1.3

-6.8 ± 3.8

-1.40 ± 0.11

GDs/DsC

-0.7 ± 1.3

-0.3 ± 4.0

-0.60 ± 0.12

ADs/DsT

0.5 ± 1.3

2.0 ± 4.0

-0.13 ± 0.12

AA/TT[a]

-7.9

-22.2

-1.00

AT/AT[a]

-7.2

-20.4

-0.88

TA/TA[a]

-7.2

-21.3

-0.58

CA/TG[a]

-8.5

-22.7

-1.45

[a]

-8.4

-22.4

-1.28

CT/AG[a]

-7.8

-21.0

-1.30

GA/TC[a]

-8.2

-22.2

-2.17

CG/CG[a]

-10.6

-27.2

-2.24

GC/GC[a]

-9.8

-24.4

-1.84

GT/AC

GG/CC[a]

-8.0

-19.9

-1.42

[a]

0.1

-2.8

0.98

Init. w/term. A/T[a]

2.3

4.1

1.03

Init. w/term. G/C

[a]

These parameters are listed according to the format reported in the literature.43

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Table 4. Thermal stabilities of dsDNA fragments containing matched or mismatched pairs in dsDNA fragments (12-mer) involving the Ds–G pair.

5′-CGCA-N-X-GTTACC-3′ 3′-GCGT-A-Y-CAATGG-5′ X–Y: G–Ds N–A

Tm

X–Y: G–C ∆Tm

[a]

(℃)

(℃)

T–A A–A G–A

45.5 26.9 29.3

– –18.7 –16.2

C–A

31.6

–13.9

[a]

N–A

Tm

∆Tm [a]

(℃)

(℃)

T–A A–A G–A

52.3 37.4 42.7

– –15.0 –9.6

C–A

39.8

–12.6

The absorbance at 260 nm of the DNA fragments (5 µM), in 10 mM sodium phosphate


25



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Abstract Graphic

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Unique Thermal Stability of Unnatural Hydrophobic Ds Bases in

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