We report here the first synthesis of 5-phenyl-telluride-thymidine derivatives and the Te-phosphoramidite. We also report here the synthesis, structure and STM current-imaging studies of DNA oligonucleotides containing the nucleobases (thymine) derivatized with 5-phenyl-telluride functionality (5-Te). Our results show that the 5-Te-DNA is stable, and that the Te-DNA duplex has the thermo-stability similar to the corresponding native duplex. The crystal structure indicates that the 5-Te-DNA duplex structure is virtually identical to the native one, and that the Te-modified T and native A interact similarly to the native T and A pair. Furthermore, while the corresponding native showed weak signals, the DNA duplex modified with electron-rich tellurium functionality showed strong topographic and current peaks by STM imaging, suggesting a potential strategy to directly image DNA without structural perturbation.
We report here the first synthesis of 5-phenyl-telluride-thymidine derivatives and the Te-phosphoramidite. We also report here the synthesis, structure and STM current-imaging studies of DNA oligonucleotides containing the nucleobases (thymine) derivatized with 5-phenyl-telluride functionality (5-Te). Our results show that the 5-Te-DNA is stable, and that the Te-DNA duplex has the thermo-stability similar to the corresponding native duplex. The crystal structure indicates that the 5-Te-DNA duplex structure is virtually identical to the native one, and that the Te-modified T and native A interact similarly to the native T and A pair. Furthermore, while the corresponding native showed weak signals, the DNA duplex modified with electron-rich tellurium functionality showed strong topographic and current peaks by STM imaging, suggesting a potential strategy to directly image DNA without structural perturbation.
Well-behaved base pair recognitions and duplex structures of DNAs and RNAs have stimulated extensive research investigations, such as DNA nano-structure construction and self-assembling (1–4), disease and pathogen detection at single molecule level (5), oligonucleotide drug discovery (6) and nanoelectronic device design based on DNA conductivity and charge migration (7–12). Moreover, chemical modifications of nucleobases have been widely used to selectively tailor the chemical and biophysical properties of DNAs and RNAs and to probe their chemical and biochemical functions, including base pairing specificity, polymerase recognition, DNA damaging and repairing (13–19) and DNA current/conductivity imaging, including metallization and conjugating with conductive nanoparticles or polymers through scanning tunneling microscopy (STM) to image DNA (20–24).Tellurium, which was recently introduced to the sugar moiety of the nucleosides and nucleotides (25,26), is a non-metal element with a much larger atomic size (atomic radius: 1.40 Å) (27) in the same elemental family of oxygen (0.73 Å), sulfur (1.02 Å) and selenium (1.16 Å) (28). Tellurium has much higher metallic property and stronger electron delocalizability than the other ones in the same elemental family. Since DNA duplexes are relatively electron deficient, we hypothesize that an electron-rich tellurium atom will likely donate electron and facilitate electron delocalization and DNA imaging, when it is introduced into DNA duplexes via the nucleobases.Herein, we report the first synthesis of a tellurium-nucleobase-derivatized nucleoside (Te-thymidine or TeT), the Te-phosphoramidite and Te-DNAs. We found that the TeT-DNA duplex has the thermo-stability similar to the corresponding native duplex. Furthermore, our X-ray crystal structure studies of the Te-derivatized DNAs indicate that the Te-derivatized and native structures are virtually identical, and that TeT and A interact as well as the native T–A pair. Moreover, our STM imaging study reveals that the Te-DNAs show strong characteristic current peaks on graphite surface, suggesting a useful DNA imaging strategy without perturbation of the local and global structures.
NaH 95% (6 mg, 0.25 mmol) was added to a solution of 4 (0.15 g, 0.25 mmol) in dry THF (1 ml) at room temperature in dry glove box. The mixture was stirred for 30 min until complete cease of hydrogen gas evolution, then cooled down to −78°C. n-BuLi (0.36 ml, 2.3 M solution in hexanes; 0.83 mmol) was added dropwise over 5 min. The mixture was stirred for 30 min, and then treated with (Ph)2Te2 (0.25 g, 0.75 mmol). The mixture was further stirred for 1 h at the same temperature (−78°C). NaCl (5 ml, saturated aqueous solution) was added, the reaction flask was placed at room temperature and allowed to warm up naturally to the room temperature. Water (10 ml) was added to the flask and EtOAc (3 × 20 ml) was used to extract the crude product. The organic phase was dried over MgSO4, and evaporated under reduce pressure. The residue was purified by flash silica gel chromatography (eluent: 40% EtOAc in hexane containing 1% Et3N) to give 5 (106 mg, 64%) as a colorless foam. Elution with 50% EtOAc in hexane gave 9 (33 mg, 29%) as a colorless foam. Spectral data for 7: 1H-NMR (CDCl3, δ): 8.64 (1H, br, NH, exchangeable with D2O), 7.82 (1H, s, H-6), 7.81–7.78 (2H, m, Ar), 7.33–7.24 (3H, m, Ar), 6.25 (1H, dd, H-1′, J = 5.6, 8.0 Hz), 4.31 (1H, m, H-3′), 3.92 (1H, m, H-4′), 3.63–3.54 (2H, m, H-5′a,b), 2.30 (1H, ddd, H-2′a, J = 2.4, 5.6, 13.2 Hz), 1.94 (1H, m, H-2′b), 0.90 (9 H, s, SiCMe3), 0.88 (9 H, s, SiCMe3), 0.09–0.07 (12 H, 4s, SiMe2); 13C-NMR (DMDO-d6) δ 162.65 (C4), 150.21 (C2), 149.77 (C-6), 138.62 (Ar), 129.56 (Ar), 128.41 (Ar), 113.08 (C-5), 89.12 (Ar), 88.48 (C4′), 85.85 (C-1′), 72.57 (C-3′), 63.16 (C-5′), 42.04 (C2′), 26.18 (CMe3), 26.08 (CMe3), 18.42 (CMe3), 18.42 (CMe3), −4.48 (SiMe2), −4.66 (SiMe2); HRMS (ESI-TOF): molecular formula: C27H43N2O5Si2Te; [M-H+]−: 661.1775 (Figure S6; calcd 661.1773).
NaH 95% (19 mg, 0.67 mmol) was added portion wise to a solution of 2 (0.516 g, 0.67 mmol) in dry THF (2 ml) at room temperature in dry glove box. The mixture was stirred for 30 min until complete cease of hydrogen gas evolution, then cooled down to −78°C and treated with n-BuLi (1.0 ml, 2.3 M solution in hexane; 2.3 mmol) dropwise over 10 min. The mixture was stirred for 30 min, and then treated with (Ph)2Te2 (0.75 g, 1.8 mmol). The mixture was further stirred for 1 h at the same temperature (−78°C). NaCl (10 ml, saturated aqueous solution) was added, the reaction flask was placed at room temperature and allowed to warm up naturally to the room temperature. Water (10 ml) was added to the flask and EtOAc (3 × 20 ml) was used to extract the crude product. The organic phase was dried over MgSO4 (s), and evaporated under reduce pressure. The residue was purified by flash silica gel chromatography (eluent: 40% EtOAc in hexanes containing 1% Et3N) to give 6 (0.39 g, 68%) as a colorless foam. Elution with 50% EtOAc in hexanes gave 8 (108 mg, 25%) as a colorless foam. Spectral data for 6: 1H-NMR (CDCl3, δ): 9.42 (1H, br, NH, exchangeable with D2O), 7.92 (1H, s, H-6), 7.70–6.86 (18H, m, Ar), 6.29 (1H, dd, H-1′, J = 6.4, 8.0 Hz), 4.27 (1H, m, H-3′), 3.99 (1H, m, H-4′), 3.81 (6H, 2 s, CH3O), 3.25 (2H, m, H-5′a,b), 2.37 (1H, ddd, H-2′a, J = 2.4, 5.6, 13.2 Hz), 2.08 (1H, m, H-2′b), 0.87 (9 H, 3 s, SiMe3), 0.09–0.05 (12 H, 4s, SiMe2); HRMS (ESI-TOF): molecular formula: C42H48N2O7TeSi; [M + H+]+: 851.2357 (Figure S1; calcd 851.2371).
Disopropylethylamine (44 µl, 0.25 mmol) and N,N-diisopropylamino cyanoethylphosphamidic chloride (57 mg, 0.24 mmol) were added to a solution of 7 (0.15 g, 0.2 mmol) in CH2Cl2 (2 ml) at 0°C. The mixture was stirred for 2 h at room temperature, and then was slowly poured into pentane (100 ml) under vigorous stirring. The produced white precipitate was filtered out, dissolved in CH2Cl2 (1 ml) and re-precipitated in pentane. The collected fine white powder was dried under reduced pressure to give 142 mg 10 (75% yield) as a mixture of two diastereomers, which was directly used for solid phase synthesis without further purification. 31P-NMR (Figure S8, CDCl3, δ): 149·3, 149·8; HRMS (ESI-TOF): molecular formula: C45H51N4O8PTe; [M-H+]−: 935.257 (Figure S3; calcd 935.242).
Synthesis of the 5-Te-functionalized DNA
All the DNA oligonucleotides were chemically synthesized in a 1.0 μmol scale using an ABI392 or ABI3400 DNA/RNA Synthesizer. The ultra-mild nucleoside phosphoramidite reagents were used in this work (Glen Research). The concentration of the Te-uridine phosphoramidite was identical to that of the conventional ones (0.1 M in acetonitrile). Coupling was carried out using a 5-(benzylmercapto)-1H-tetrazole (5-BMT) solution (0.25 M) in acetonitrile. The coupling time was 25 s for both native and modified samples. Trichloroacetic acid (3%) in methylene chloride was used for the 5′-detritylation. Synthesis were performed on control pore glass (CPG-500) immobilized with the appropriate nucleoside through a succinate linker. All the oligonucleotides were prepared with DMTr-on form. After synthesis, the DNA oligonucleotides were cleaved from the solid support and fully deprotected by the treatment of 0.05 M K2CO3 solution in methanol for 8 h at room temperature. The 5′-DMTr deprotection was performed in a 3% trichloroacetic acid solution for 2 min, followed by neutralization to pH 7.0 with a freshly made aqueous solution of triethylamine (1.1 M) and extraction by petroleum ether to remove DMTr-OH.
HPLC analysis and purification
The Te-DNA oligonucleotides were analyzed and purified by reverse phase high performance liquid chromatography (RP-HPLC) both DMTr-on and DMTr-off. Purification was carried out using a 21.2 × 250 mm Zorbax, RX-C8 column at a flow rate of 10 ml/min. Buffer A consisted of 20 mM triethylammonium acetate (TEAAc, pH 7.2), while buffer B contained 50% aqueous acetonitrile and 10 mM TEAAc, pH 7.1. Similarly, analysis was performed on a Zorbax SB-C18 column (4.6 × 250 mm) at a flow of 1.0 ml/min using the same buffer system. The DMTr-on oligonucleotides were eluded with up to 100% buffer B in 20 min in a linear gradient, while the DMTr-off oligonucleotides were eluted with up to 70% of buffer B in a linear gradient in the same period of time. The collected fractions were lyophilized; the purified compounds were re-dissolved in water. The pH was adjusted to 7.0 after the final purification of the Te-oligonucleotides without the DMTr group.
Thermodenaturization of duplex DNAs
Solutions of the duplex DNAs (2 μM) were prepared by dissolving the DNAs in a buffer containing NaCl (50 mM), sodium phosphate (10 mM, pH 7.2), EDTA (0.1 mM) and MgCl2 (2 mM). The solutions were then heated to 90°C for 3 min, cooled slowly to room temperature and stored at 4°C overnight before measurement. Prior to thermal denaturation, argon was bubbled through the samples. Each denaturizing curves were acquired at 260 nm by heating and cooling for four times in a rate of 0.5°C/min using Cary-300 UV–visible spectrometer equipped with temperature controller system.
Crystallization
The purified oligonucleotides (1 mM) were heated to 70°C for 2 min, and cooled slowly to room temperature. The 2′-Se--dU was incorporated into the Te-DNA for the crystallization facilitation. Both native buffer and Nucleic Acid Mini Screen Kit (Hampton Research) were applied to screen the crystallization conditions with l mM of DNA sample at different temperatures using the hanging drop method by vapor diffusion.
Data collection
Glycerol (30%), PEG 400 or the perfluoropolyether was used as a cryoprotectant during the crystal mounting, and data collection was taken under nitrogen gas at 99°K. The Te-DNA-8-mer crystal data were collected at beam line X12C in NSLS of Brookhaven National Laboratory. A number of crystals were scanned to find the one with strong anomalous scattering at the K-edge absorption of selenium. The distance of the detector to the crystals was set to 150 mm. The wavelengths of 0.9795 Å was chosen for data collection. The crystals were exposed for 10–15 s per image with 1° oscillation, and a total of 180 images were taken for each data set. All the data were processed using HKL2000 and DENZO/SCALEPACK (29).
Structure determination and refinement
The crystal structure of Te-DNA-8-mer was solved by molecular replacement with Phaser (30) using the automated search mode, followed by the refinement of selenium and tellurium atom positions in both CNS (31) and Refmac 5.0 (32). The refinement protocol includes simulated annealing, positional refinement, restrained B-factor refinement and bulk solvent correction. The stereo-chemical topology and geometrical restrain parameters of DNA/RNA (33) have been applied. The topologies and parameters for modified dU with 2′-SeMe (UMS) and 5-tellurium (TTE) were constructed and applied. After several cycles of refinement, a number of highly ordered waters were added. Final, the occupancies of selenium and tellurium were adjusted. Cross-validation (34) with a 5–10% test set was monitored during the refinement. The σA-weighted maps (35) of the (2m|Fo| − D|Fc|) and the difference (m|Fo| − D|Fc|) density maps were computed and used throughout the model building.
Mass analysis of Te–C bond breaking caused by X-ray irradiation
In our work, a few crystals were analyzed by MALDI-TOF MS before X-ray irradiation. As a comparison, a few crystals were analyzed by MALDI-TOF MS after X-ray irradiation. This Te-functionality is relatively stable in the absence of X-ray irradiation, and no significant decomposition was observed in 2 months under air. The Te-DNA was also heated at 90°C over 1 h without significant decomposition.
STM experiment
In this work, highly oriented pyrolytic graphite (HOPG) was used as a support substrate to study DNA molecules (36). The HOPG was freshly cleaved with scotch tapes prior to each experiment, and treated with UV Surface Decontamination System (Novascan Technologies Inc.) for 20 min before modifying the surface of the hydrophobic HOPG to the hydrophilic surface. The bare HOPG surface did not show obvious structure changes even at the atomic level. Both the native and TeT-DNA samples (100 μl, 0.03 μM) were dissolved in PBS solution, and the sample solutions were then loaded on the HOPG surface. After 20 min, the DNA samples were scanned with STM in the PBS buffer. To reduce the influence of the leakage current, the newly cut AuSTM tip was coated with Apiezon W Wax (SPI Supplies Division of Structure Probe Inc.), and only the top of the tip was exposed as reported by Lindsay (37).
RESULTS AND DISCUSSION
Aryl-chalcogenides (S and Se), such as phenyl sulfenyl and phenyl selenyl moieties, have been tethered to the 5-position of pyrimidine nucleosides and been incorporated into DNA (38–41). Methods, which are available in the literature for the introduction of the alkyl or aryl chalcogenides (S and Se) at the 5-position of pyrimidines, include the electrophillic addition–elimination on the C5–C6 double bond (42), the palladium-catalyzed nucleophillic substitution of 5-mercururio derivatives (39,43), and the lithiation of 5-halo derivatives in the presence of the electrophilic species (44). However, no literature report can be found on the corresponding 5-aryl- or alkyl-tellurides. Incorporation of the tellurium functionality to the 5-position is a long-standing challenge. We have attempted, without success, to introduce an alkyltelluro or aryltelluro moiety at the 5-position of pyrimidines under several conditions. However, by applying the lithium–halogen exchange strategy (45) on a protected 5-iodo-2′-deoxyurdine, we have successfully incorporated the Te-functionality to the 5-position. We found that the key steps are the transient protection of 5-iodo-2′-deoxyuridine derivative 2 as a sodium salt, and the treatment with n-BuLi and (PhTe)2. To the best of our knowledge, we have established the first synthesis of the 5-Te-pyrimidine derivatives.The synthesis of the 5-TePh-deoxyuridine and 5-TePh-deoxyuridine phosphoramidite is showed in Scheme 1, starting from the silylation of commercially available 5-iodo-2′-deoxyuridine derivative 1. After purification, this intermediate (2) was treated with NaH/n-BuLi, followed by adding diphenylditelluride (PhTe)2. This reaction generated an inseparable mixture of the desired 5-PhTe derivative (6) and the tentative 6-PhTe derivative (in 10 : 1 ratio, with 72% yield), along with the reduced byproduct (8, in 25% yield). After several failed trials to separate the isomers, such as re-crystallization and very-slow column chromatography, we switched the starting compound to the 3′,5′-di-O-silyl derivatives. Unfortunately, this 5-isomer was still difficult to be purified from the 6-regio-isomer. Later, various concentrations of the reactant (3′-O-silyl-5′-DMTr derivative 2) were tested (Scheme 1). Finally, it was found that when the concentration of the reactant was increased to as high as 0.15 or 0.18 M in THF solution, the 6-TePh isomer could indeed be completely eliminated. Desired 5-Te-isomer 6 was formed as the major product, while 8 was formed as a minor byproduct. Then the desilylation of 6 with TBAF/THF was carried out, generating 7 quantitatively, which was finally converted into 5-phenyltelluro thymdine (TeT) phosphoramidite (10) for solid phase synthesis of the Te-DNA. The characteristic distributions of the tellurium isotopes in mass spectra of 6, 7 and 10 were observed and confirmed the incorporation of the Te-functionality.
Scheme 1.
Synthesis of the 5-phenyltelluro-2′-deoxyuridine, its phosphoramidite, and the Te-DNAs. (A) TBDMS-Cl, Im., DMF, 4 h, room temperature (RT); (B) (i) NaH, THF, 15 min, RT; (ii) n-BuLi, THF, 10 min, −78°C; (iii) Ph2Te2, 1 h, −78°C; (C) TBAF, THF, 4 h, RT; (D) i-Pr2NP(Cl)CH2CH2CN, DIEA, CH2Cl2, 1 h, RT; (E) the solid-phase synthesis.
Synthesis of the 5-phenyltelluro-2′-deoxyuridine, its phosphoramidite, and the Te-DNAs. (A) TBDMS-Cl, Im., DMF, 4 h, room temperature (RT); (B) (i) NaH, THF, 15 min, RT; (ii) n-BuLi, THF, 10 min, −78°C; (iii) Ph2Te2, 1 h, −78°C; (C) TBAF, THF, 4 h, RT; (D) i-Pr2NP(Cl)CH2CH2CN, DIEA, CH2Cl2, 1 h, RT; (E) the solid-phase synthesis.Considering that the Te-functionality is electron rich, its oxidative removal might happen during solid-phase synthesis and purification. Fortunately, this Te-phosphoramidite is well compatible with the solid-phase synthesis conditions, thereby being incorporated into several designed DNA sequences. After K2CO3 (0.05 M in MeOH) cleavage and deprotection, the crude DNA solution was analyzed by analytical RP-HPLC. As showed in Figure 1, the coupling yield is over 95%. The synthesized Te-DNAs were then purified in a preparative RP-HPLC system in trityl-on form. After treated with 3% trichloroacedic acid for 2 min, the solution was neutralized with triethylamine and extracted with petroleum ether, followed by the second purification in trityl-off form. All the Te-modified DNA oligonucleotides were characterized by MALDI-TOF, and several Te-DNA oligonucleotides were listed in Table 1.
Figure 1.
HPLC analysis of 5-TePh-9-mer: [5′-DMTr-ATGG(5-TePh-T)GCTC-3′]. HPLC conditions: Welchrom C18-XB column (4.6 × 250 mm, 5 µ), 25°C, 1 ml/min, gradient from buffer A to 90% buffer B in 20 min. The retention time of the full-length sample is 15.3 min.
Table 1.
MALDI-TOF MS of the Te-derivatized DNA oligonucleotides
Entry
DNA sequences
Measured (calcd) m/z
1
ATGG(TeT)GCTC C93H114N32O54P8Te
[M]−: 2919.3 (2919.5)
2
CTCCCA(TeT)CC C89H113N27O53P8Te
[M]−: 2784.4 (2784.4)
3
CT(TeT)CTTGTCCG C111H140N32O69P10Te
[M]−: 3464.5 (3463.8)
4
GCG(TeT)ATACGC C102H125N38O58P9Te
[M+H+]+: 3218.0 (3218.7)
5
GTG(TeT)ACAC C83H101N30O46P7Te
[M]−: 2599.4 (2599.3)
6
G(2′-Se-dU)G(TeT)ACAC C83H101N30O46P7SeTe
[M+H+]+: 2679.2 (2679.2)
HPLC analysis of 5-TePh-9-mer: [5′-DMTr-ATGG(5-TePh-T)GCTC-3′]. HPLC conditions: Welchrom C18-XB column (4.6 × 250 mm, 5 µ), 25°C, 1 ml/min, gradient from buffer A to 90% buffer B in 20 min. The retention time of the full-length sample is 15.3 min.MALDI-TOF MS of the Te-derivatized DNA oligonucleotides
Thermodenaturization study of oligonucleotide duplexes containing the 5-Te derivatization
To evaluate the potential structure perturbation caused by this bulky tellurium moiety (Ph–Te), we conducted the UV–thermal denaturation studies. Our experimental results indicated that the Te-derivatized duplex structures are as stable as the corresponding natives (Table 2 and Figure 2). Clearly, the bulky tellurium substitution does not significantly change the melting temperatures and alter the duplex structure.
Table 2.
UV-melting study of the Te-DNA duplexes
Entry
DNA duplex
Tm (°C)
1
5′-ATGG(TeT)GCTC-3′
40.3 ± 0.2
3′-TACCACGAG-5′
2
5′-ATGGTGCTC-3′
39.8 ± 0.3
3′-TACCACGAG-5′
3
5′-CT(TeT)CTTGTCCG-3′
44.1 ± 0.3
3′-GAAGAACAGGC-5′
4
5′-CTTCTTGTCCG-3′
44.0 ± 0.2
3′-GAAGAACAGGC-5′
Figure 2.
Normalized thermal–denaturation curves of the native and Te-modified DNA duplexes. The Te-DNA duplex: 5′-ATGG(5-TePh-T)GCTC-3′ and 5′-GAGCACCAT-3′. The solid and dashed curves represent the Te-modified and the corresponding native duplexes with 40.3 ± 0.2 and 39.8 ± 0.3°C as their melting temperatures, respectively.
Normalized thermal–denaturation curves of the native and Te-modified DNA duplexes. The Te-DNA duplex: 5′-ATGG(5-TePh-T)GCTC-3′ and 5′-GAGCACCAT-3′. The solid and dashed curves represent the Te-modified and the corresponding native duplexes with 40.3 ± 0.2 and 39.8 ± 0.3°C as their melting temperatures, respectively.UV-melting study of the Te-DNA duplexes
Crystallization and data collection of Te-DNA
To further confirm that this 5-TePh modification does not cause obvious structure perturbations, we have carried out the crystallization by using our 2′-Se modification strategy to facilitate the crystal growth (46–48). The modified DNA (5′-G-2′-SedU-G-TeT-ACAC-3′) was designed, synthesized and purified. Its crystallization conditions were screened with the Hampton Mini-Screening kit. As a result, the Te-DNA crystals were formed in all the 24 buffer conditions of the kit. Moreover, crystals with high-diffraction quality and reasonable sizes were generated in 2 days. Crystals, which formed in buffer #7 of the kit (10% v/v MPD, 40 mM sodium cacodylate pH 6.0, 12 mM sperminetetra–HCl, 80 mM potassium chloride and 20 mM magnesium chloride), were shown in Figure 3 as an example. By the crystal diffraction screening, the crystals formed in buffer #7 were identified to give the highest diffraction resolution (up to 1.50 Å). In addition, several data sets with high diffraction resolution were also collected for further refine purpose. The data collection and refine parameters are listed in Tables 3 and 4.
Figure 3.
Crystal photo of the Te-DNA octomer [5′-G(2′-SeMe-dU)G(TeT)ACAC-3]2 crystallized in buffer #7 with 0.2 × 0.1 × 0.1 mm in size.
Table 3.
X-ray data collection: the data collected at selenium K-edge
Data collection
λ = 0.9795 Å
Resolution range, Å (last shell)
18.89–1.50 (1.55–1.50)
Unique reflections
3659
Completeness (%)
94.4
Rmerge (%)
7.5 (42.7)
〈I/σ(I)〉
14.9 (2.4)
Redundancy
8.63 (5.30)
Reduced chi-squared
0.93 (0.42)
Rmerge = Σ|I − 〈I〉|/ΣI.
Table 4.
Refinement statistics in Te-8-mer
Refinement
Resolution range, Å (last shell)
25–1.50 (1.55–1.5)
Number of reflections
6859 (424)
Rwork (%)
19.3
Rfree (%)
21.5
Number of atoms
Nucleic Acid (single)
162
Heavy atom
2 (Se, Te)
Water
28
RMSD
Bond length, Å
0.010
Bond angle
1.73
Average B-factors, Å2
All atoms
22.3
Wilson plot
19.6
Overall anisotropic B-values
B11/B22/B33
0.6/0.6/−1.2
Bulk solvent correction
Solvent density, e/Å3
0.44
B-factors, Å2
59.6
Coordinates error (c.-v.), 5 Å
Esd. from Luzzatt plot, Å
0.17
Esd. from SIGMAA, Å
0.15
Crystal photo of the Te-DNA octomer [5′-G(2′-SeMe-dU)G(TeT)ACAC-3]2 crystallized in buffer #7 with 0.2 × 0.1 × 0.1 mm in size.X-ray data collection: the data collected at selenium K-edgeRmerge = Σ|I − 〈I〉|/ΣI.Refinement statistics in Te-8-mer
Crystal structure of the Te-DNA 8-mer
This Te-DNA crystal has the same tetragonal space group P43212 as the corresponding native one (49). The occupancy of Se and Te are 1.0 and 0.35, respectively. The refined B-factor of Se and Te are 28.55 and 23.64, respectively, comparing with other atoms in the structure. The Te-DNA structure (1.50 Å resolution) revealed that both global and local structures of this Te-DNA are virtually identical to the native one (Figure 4A and B), indicating the Te-moiety does not cause significant perturbation. The structure result is also consistent with our biophysical study, suggesting that TeT and A (Figure 4B and C) pair as well as native T and A. Furthermore, the Te-DNA is stable under a higher temperature. As shown by Figure 5, the Te-DNA was heated at 90°C for 1 h without significant decomposition (<2%). However, since the Te–C bond is sensitive to X-ray irradiation, the occupancy of tellurium (∼40%) in the crystal structure is expected. The low electron density of the phenyl group (∼16% of expected density) is consistent with cleavage of these two Te–C bonds [Te–C5 and Te–C(Ph)] by the X-ray irradiation. To verify this, the mass analysis of the Te-DNA crystals was carried out. The Te-functionality is stable in the absence of X-ray irradiation, our MS analysis indicated no significant decomposition of the Te-DNA crystals in the crystallization droplet after 3 weeks (Figure 6A). However, after X-ray irradiation, the MALDI-TOF-MS spectra (Figure 6B) showed the loss of 78 (the phenyl group) and 203.9 mass units (the phenyltelluro group), indicating the partial cleavage of both Te–C bonds by X-ray irradiation.
Figure 4.
The global and local structures of the Te-DNA duplex [5′-G(2′-SeMe-dU)G(TeT)ACAC-3′]2. The red and yellow balls represent Te and Se atoms, respectively. (A) The Te-dsDNA structure (in cyan; 1.50 Å resolution; PDB ID: 3FA1) is superimposed over the native one (in magenta; PDB ID: 1DNS). (B) The local structure of the TeT/A base pair (in cyan) is superimposed over the native T/A base pair (in magenta). (C) The experimental electron density (2|Fo| − |Fc| map) of the TeT/A base pair (σ = 1.0). The green ball represents approximately one-sixth of phenyl group (one carbon).
Figure 5.
The Te-DNA thermal stability and HPLC analysis. Buffer A: 20 mM TEAAc; buffer B: 50% acetonitrile in buffer A. HPLC conditions: Welchrom C18-XB column (4.6 × 250 mm, 5 µ), 25°C, 1 ml/min, gradient from buffer A to 50% buffer B in 15 min. HPLC profiles of Te-DNA [5′-G(2′-SeMe-dU)G(TeT)ACAC-3′] thermal analysis: the solid line (without heating), the dashed line (heated in 10 mM Na-PO4 buffer, pH 7.3, at 90°C for 1 h).
Figure 6.
MALDI-TOF mass spectra of the Te-DNA [5′-G(2′-SeMe-dU)G(TeT)ACAC-3′; molecular formula: C83H101N30O46P7SeTe; F.W.: 2678.2]. (A) The crystal sample without X-ray irradiation; (B) the crystal sample with X-ray irradiation.
The global and local structures of the Te-DNA duplex [5′-G(2′-SeMe-dU)G(TeT)ACAC-3′]2. The red and yellow balls represent Te and Se atoms, respectively. (A) The Te-dsDNA structure (in cyan; 1.50 Å resolution; PDB ID: 3FA1) is superimposed over the native one (in magenta; PDB ID: 1DNS). (B) The local structure of the TeT/A base pair (in cyan) is superimposed over the native T/A base pair (in magenta). (C) The experimental electron density (2|Fo| − |Fc| map) of the TeT/A base pair (σ = 1.0). The green ball represents approximately one-sixth of phenyl group (one carbon).The Te-DNA thermal stability and HPLC analysis. Buffer A: 20 mM TEAAc; buffer B: 50% acetonitrile in buffer A. HPLC conditions: Welchrom C18-XB column (4.6 × 250 mm, 5 µ), 25°C, 1 ml/min, gradient from buffer A to 50% buffer B in 15 min. HPLC profiles of Te-DNA [5′-G(2′-SeMe-dU)G(TeT)ACAC-3′] thermal analysis: the solid line (without heating), the dashed line (heated in 10 mM Na-PO4 buffer, pH 7.3, at 90°C for 1 h).MALDI-TOF mass spectra of the Te-DNA [5′-G(2′-SeMe-dU)G(TeT)ACAC-3′; molecular formula: C83H101N30O46P7SeTe; F.W.: 2678.2]. (A) The crystal sample without X-ray irradiation; (B) the crystal sample with X-ray irradiation.
STM imaging study
Since tellurium atom has the metallic property, we expect that the Te-modified DNA has higher visibility and conductivity under STM imaging than the corresponding native DNA. To investigate this, we synthesized the Te-DNA [9 nt, 5′-ATGG(TeT)-GCTC-3′, Figure 7], which is complementary to a native DNA [54 nt, 5′-(GAGCACCAT)6-3′] by six repeats, and conducted the STM studies. Interestingly, our experiments revealed that the Te-modified DNA duplex showed much stronger topographic and current peaks (Figure 7A and B) than the corresponding native duplex (Figure 7C and D). Clearly, both the topographic and current images of the Te-duplex showed that the Te-derivatization allowed better visualization of the 54-bp DNA duplex (calculated length: 18 nm) with ∼20 nm in measured length. Our result also suggested that the native and Te-modified duplexes can assemble with different orientations on the graphite surface, indicated by the topographic and current images of the native and Te-modified duplexes.
Figure 7.
The STM images of the Te-modified DNA duplex [5′-ATGG(TeT)-GCTC-3′ and 5′-(GAGCACCAT)6-3′] and its counterpart native duplex on HOPG. The arrows indicate the edges or current peaks of the measured molecules. (A) Topographic image of Te-duplex; (B) current image of Te-duplex; (C) topographic image of native duplex; (D) current image of native duplex. Comparing to the Te-modified DNA duplex, the native sample does not show obvious conductivity. The insets are 2D images corresponding to the 3D images. The sample bias: 0.50 V, the current set point: 100 pA.
The STM images of the Te-modified DNA duplex [5′-ATGG(TeT)-GCTC-3′ and 5′-(GAGCACCAT)6-3′] and its counterpart native duplex on HOPG. The arrows indicate the edges or current peaks of the measured molecules. (A) Topographic image of Te-duplex; (B) current image of Te-duplex; (C) topographic image of native duplex; (D) current image of native duplex. Comparing to the Te-modified DNA duplex, the native sample does not show obvious conductivity. The insets are 2D images corresponding to the 3D images. The sample bias: 0.50 V, the current set point: 100 pA.
CONCLUSIONS
In summary, we have first incorporated the Ph–Te-functionality (5-Te) to the 5-position of a pyrimidine, and successfully synthesized the Te-nucleobase-modified nucleoside, phosphoramidite and DNA oligonucleotides. The Te-modified DNA is stable, and the native and modified duplexes have the similar stability. Our biophysical and structural studies indicate that the Te-modified T and A interact as well as native T and A pair, and that the structures of the Te-derivatized and native duplexes are virtually identical. Furthermore, the Te-DNA duplex is readily visible under STM, suggesting a potential strategy to directly image DNA without the structural perturbation. This Te-modification will open a novel research avenue for nucleic acids, including mechanism and function studies, STM imaging and nano-electronic materials.
ACCESSION NUMBER
3FA1.
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online.
FUNDING
Georgia Cancer Coalition Distinguished Cancer Clinicians and Scientists Award and the US National Science Foundation (MCB-0824837). Funding for open access charge: US National Science Foundation (MCB-0824837).Conflict of interest statement. None declared.
Authors: Irena Kratochvílová; Karel Král; Martin Buncek; Alena Vísková; Stanislav Nespůrek; Anna Kochalska; Tatiana Todorciuc; Martin Weiter; Bohdan Schneider Journal: Biophys Chem Date: 2008-08-28 Impact factor: 2.352
Authors: Airlie J McCoy; Ralf W Grosse-Kunstleve; Paul D Adams; Martyn D Winn; Laurent C Storoni; Randy J Read Journal: J Appl Crystallogr Date: 2007-07-13 Impact factor: 3.304
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