DOI: 10.1002/cbic.201402346
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Controlling the Fluorescence of Benzofuran-Modified Uracil Residues in Oligonucleotides by Triple-Helix Formation Takashi Kanamori,[a] Hiroki Ohzeki,[b] Yoshiaki Masaki,[b] Akihiro Ohkubo,[b] Mari Takahashi,[c, d] Kengo Tsuda,[c, d] Takuhiro Ito,[c, d] Mikako Shirouzu,[c, d] Kanako Kuwasako,[c, d, e] Yutaka Muto,[c, d, e] Mitsuo Sekine,*[b] and Kohji Seio*[b] We developed fluorescent turn-on probes containing a fluorescent nucleoside, 5-(benzofuran-2-yl)deoxyuridine (dUBF) or 5-(3methylbenzofuran-2-yl)deoxyuridine (dUMBF), for the detection of single-stranded DNA or RNA by utilizing DNA triplex formation. Fluorescence measurements revealed that the probe containing dUMBF achieved superior fluorescence enhancement than that containing dUBF. NMR and fluorescence analyses indicated that the fluorescence intensity increased upon triplex
formation partly as a consequence of a conformational change at the bond between the 3-methylbenzofuran and uracil rings. In addition, it is suggested that the microenvironment around the 3-methylbenzofuran ring contributed to the fluorescence enhancement. Further, we developed a method for detecting RNA by rolling circular amplification in combination with triplex-induced fluorescence enhancement of the oligonucleotide probe containing dUMBF.
Introduction their fluorescence in the absence of targets;[11] Seitz and coworkers developed forced intercalation probes by controlling the conformation of the excited state of thiazole dyes upon intercalation between the base pairs of the probe–target duplexes;[12–15] Pedersen and Filichev have reported a series of the fluorescent probes bearing pyrene or thiazole dyes in triplexforming oligodeoxynucleotides (TFOs) to enabled the detection of duplex DNAs.[16–17] In contrast to these dye-based fluorescence probes, fluorescent pyrimidine nucleosides with a small substituent at position 5 have been reported.[18–37] Such fluorescent modified pyrimidine nucleosides incorporated into oligonucleotides serve as fluorescent dyes, while retaining Watson–Crick base pairing ability. One advantage of these fluorescent pyrimidines is that they cause minimal conformational disturbance to the native structures of nucleic acids and the complexes incorporating them. Thus, attempts have been made to use oligonucleotides containing fluorescent-modified pyrimidines as fluorescence probes that change their fluorescence upon binding to the targets; here, the substituent at position 5 becomes partly sandwiched between two bases. This molecular interaction changes the microenvironment of the substituents and their conformation, thus increasing or decreasing fluorescence intensities. However, in many cases, the magnitude of the fluorescence change is small.[28–29, 31, 34–35, 37] This is probably because the substituents at position 5 of the pyrimidine base extrude into major groove and are not completely covered by the upper and lower bases. To significantly enhance the on/off ratio of fluorescence signals, we studied the triple-helix formation of oligodeoxynucleotides containing 5-arylpyrimidines, based on our previous
Fluorescent oligonucleotides are promising tools for the detection of functional nucleic acids in vivo and in vitro and for studying the interactions of nucleic acids with proteins or small molecules.[1–8] Fluorescent oligonucleotides that emit fluorescence only in the presence of targets have been used as fluorescent probes. To enable target-dependent fluorescence emission, various mechanisms have been proposed: molecular beacons use the interactions of chromophores with quenching dyes in the absence of targets;[9–10] Okamoto reported a mechanism that uses H-aggregation of two thiazole dyes to quench
[a] Dr. T. Kanamori Education Academy of Computational Life Sciences Tokyo Institute of Technology 4259 Nagatsuta, Midoriku, Yokohama 226-8501 (Japan) [b] H. Ohzeki, Dr. Y. Masaki, Prof. A. Ohkubo, Prof. M. Sekine, Prof. K. Seio Department of Life Science, Tokyo Institute of Technology 4259 Nagatsuta, Midoriku, Yokohama 226-8501 (Japan) E-mail: [email protected] [email protected] [c] M. Takahashi, K. Tsuda, Dr. T. Ito, Prof. M. Shirouzu, Dr. K. Kuwasako, Prof. Y. Muto RIKEN Systems and Structural Biology Center 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045 (Japan) [d] M. Takahashi, K. Tsuda, Dr. T. Ito, Prof. M. Shirouzu, Dr. K. Kuwasako, Prof. Y. Muto RIKEN Center for Life Science Technologies 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045 (Japan) [e] Dr. K. Kuwasako, Prof. Y. Muto Faculty of Pharmacy Department of Pharmaceutical Sciences, Musashino University 1-1-20, Shinmachi Nishitokyo-city, Tokyo 202-8585 (Japan) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbic.201402346.
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Full Papers studies.[38–39] As the 5-arylpyrimidine residue, we selected fluorescent 5-(benzofuran-2-yl)uracil (UraBF) because of its high fluorescence quantum yield (F) and fluorescence increase dependence on DNA–DNA hybridization.[35] We further designed its derivative 5-(3-methylbenzofuran-2-yl)uracil (UraMBF). As shown in Figure 1 A, the benzofuran moieties of UraBF and UraMBF when located in the major groove can rotate when the
Results and Discussion Ground-state conformations of UraBF and UraMBF First, we calculated the electronic energies of UraBF and UraMBF to clarify the conformational differences in these nucleobase analogues. In these calculations, the CC bond between the benzofuran and uracil rings was rotated in a stepwise manner, and the energy of each conformer was calculated with the B3LYP/6-31G (d) model in Gaussian 09[43] in gas phase and in a water model, generated with the polarizable continuum model (PCM).[44] In the gas phase (dashed line in Figure 2 A),
Figure 1. Microenvironment and conformations of 5-(benzofuran-2-yl)uracil (UraBF) and 5-(3-methylbenzofuran-2-yl)uracil (UraMBF). A) Duplex with the target nucleic acid, and B) triplex with the target nucleic acid and triplexforming oligonucleotide incorporating propylene linker (C3).
oligodeoxynucleotide containing them forms a duplex with target nucleic acids. However, upon binding to triplex-forming oligonucleotides (TFOs) with a propylene linker (C3)[38–39] at the counter position of the UraBF or UraMBF, the benzofuran ring becomes sandwiched between two bases. These flanking bases make the benzofuran and uracil rings coplanar (Figure 1 B), thus inducing fluorescence. It has been reported that insertion of an alkyl group into the ortho position of fluorescent biaryls induced the large twist between two aryl rings of biaryls and thus affected their photophysical properties, such as fluorescence emission wavelength.[40–42] Furthermore, the fluorescence intensity increases upon changes to the conformations of the biaryls from the twisted form to coplanar.[42] Based on this, we designed UraMBF in addition to the previously reported UraBF. It was expected that the introduction of a methyl group would increase the population of the twisted-form UraMBF in the duplex state and induce a larger fluorescence enhancement in the triplex state. At the same time, the binding of UraBF or UraMBF to TFO (Figure 1 B) makes the microenvironment of the benzofuran ring more hydrophobic. This change in the microenvironment can also enhance the fluorescence intensity. Although the nucleoside UraBF and oligonucleotides containing UraBF have been reported,[31, 35] the fluorescence properties in triplex DNA have not been reported. We observed a large fluorescence enhancement of UraBF and UraMBF upon the triplex formation. Our triplex-induced fluorescence probes could be used to detect single-stranded nucleic acids such as micro RNAs.
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Figure 2. Potential energy vs. torsion angle (q; C4-C5-CB2-CB3) of A) 1-methyl-5-(benzofuran-2-yl)uracil and B) 1-methyl-5-(3-methylbenzofuran-2-yl)uracil. Relaxed potential energy scan was performed with B3LYP/6-31G (d) (Gaussian 09). a: gas, c: water; for the calculation in the water model, a PCM solvation model was used.
UraBF had an energy minimum at q = 08 (conformation where the benzofuran and uracil rings are coplanar). In contrast, UraMBF had an energy minimum at q = 338 in the gas phase with a twisted conformation (Figure 2 B). This clearly indicates that the incorporation of a methyl group stabilizes the twisted conformation to relieve the steric hindrance caused by uracil. The solvent effects for PCM (solid line) lowered the energy barrier for the rotation; however, the energy168
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Full Papers minimized conformation did not change. These results clearly support our hypothesis that incorporation of a methyl group stabilizes the twisted conformation. Synthesis and photophysical properties of dUBF and dUMBF Next, the corresponding nucleosides were synthesized to elucidate the fluorescence properties. Benzofuran-modified deoxyuridine dUBF (3 a; aglycon UraBF) and dUMBF (3 b; aglycon UraMBF) were synthesized by using Suzuki–Miyaura cross-coupling reactions (Scheme 1). Several 5-aryl-modified pyrimidines[22–23, 45]
Figure 3. Absorption (50 mm, c) and fluorescence (10 mm, a) spectra of 3 b in various solvents (lex = 300 nm). For measurement of absorption and fluorescence spectra, samples contained 5 and 1 % DMSO, respectively, for solubility.
ing phosphoramidites 5 a and 5 b for oligonucleotide synthesis. To study the photophysical properties of 3 b, the UV absorption and steady-state and time-resolved fluorescence were measured in various solvents (Figure 3, Table 1). Because the photophysical properties of 3 a[35] and related nucleosides[21, 29, 31] have already been reported, the properties of 3 b could be compared with these. Compound 3 b showed an absorption peak at ~ 300 nm in every solvent tested, and at ~ 260 nm in the case of water (Figure 3). The absorption at ~ 300 nm was strongest when 3 b was dissolved in ethyl acetate (the least polar solvent) and weakest in water (the most polar solvent). The acetonitrile and methanol solutions showed absorption peaks between these. The maximum-absorption wavelength (lmax) of 3 b ranged from 293 to 306 nm in different solvents (Table 1). Upon irradiation at 300 nm, the fluorescence emitted by 3 b was weakest in water and stronger in organic solvents such as methanol, acetonitrile, and ethyl acetate (Figure 3). The maximum-fluorescence emission wavelength (lem) changed significantly in different solvents (from 432 to 484 nm). To analyze the effect of solvent on the absorption and emission wavelengths, the Stokes shifts (difference between the emission and absorption maxima) were plotted against the microscopic solvent polarity parameter, ET(30).[47] The plot (Figure 4) shows a positive correlation, thus indicating stabilization of the excit-
Scheme 1. Synthesis of fluorescent nucleosides, dUBF (3 a) and dUMBF (3 b), and their phosphoramidites, 5 a and 5 b. a) 0.03 equiv Pd(OAc)2, 0.08 equiv TPPTS, 2.2 equiv Na2CO3, H2O/CH3CN (2:1, v/v), 45 8C, overnight (62 % for 3 a (R = H), 97 % for 3 b (R = Me)); b) 1.1 equiv DMTrCl, pyridine, RT, 3 h for 4 a and 6 h for 4 b (83 % for 4 a, 81 % for 4 b); c) 1.5 equiv [(iPr)2N]2P(OCE), 0.8 equiv HN(iPr)2, 0.8 equiv 1H-tetrazole, CH2Cl2, RT, overnight (59 % for 5 a (R = H), 84 % for 5 b (R = Me)).
have been synthesized by the Suzuki–Miyaura cross-coupling reaction. One of the advantages over Stille coupling, which was previously used for the synthesis of 3 a and its derivatives, is avoidance of the harmful tributyltin reagent. In brief, the boronic acid esters of benzofurTable 1. Photophysical properties of 3 b in various solvents. kr and knr are radiative and nonradiative decay rate an (2 a) or 3-methylbenzofuran constants, respectively. (2 b), which was prepared by lem[a] [nm] Irel[b] F tave [ns] kr [ns1] knr [ns1] Solvent lmax [nm] using method of Miyaura,[46] were coupled to 5-iododeoxyuriwater 293 484 1.0 0.003 0.12 0.02 8.0 methanol 306 457 6.7 0.009 0.36 0.03 2.7 dine (1) to afford 3 a and 3 b, reacetonitrile 300 441 4.1 0.005 0.20 0.03 5.1 spectively (Scheme 1). The nuethyl acetate 301 432 5.0 0.007 0.15 0.05 6.5 cleosides were used for the fluo[a] Maximum fluorescence wavelength upon irradiation at 300 nm. [b] relative fluorescence intensity at each rescence measurements and lem. preparation of the correspondChemBioChem 2015, 16, 167 – 176
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Full Papers Dependence of fluorescence intensity on the solvent viscosity As previously reported for 3 a, the conformational change around the CC bond between the benzofuran and uracil rings can affect the fluorescence emission intensities.[35] To elucidate the effects of the conformational change, the fluorescence emission spectra of 3 b were measured at different solvent viscosities (Figure 5). Tor[29] and Srivatsan[31, 35] have report-
Figure 4. Relationship between Stokes shift and ET(30) for dUMBF (3 b).
ed state by polar solvent molecules. A similar dependence on solvent polarity was observed for 3 a.[35] In these studies of the nucleoside, very small shoulder bands were present in all cases (e.g., ~ 530 nm for methanol). Although this might indicate some aggregation of nucleosides, such bands were not observed in the later triplex cases and did not hamper the triplex studies. Next, the relative fluorescence intensities (Irel) were compared at the maximum-fluorescence wavelength: 6.7, 5.0, 4.1, and 1.0 in methanol, ethyl acetate, acetonitrile, and water, respectively. For 3 b, F was lowest in water (0.003), then 0.005, 0.007, and 0.009, in acetonitrile, ethyl acetate, and methanol, respectively. Interestingly, according to a previous report, the structurally similar 3 a showed the lowest F in acetonitrile (0.04) and the highest in water (0.19).[35] These results indicate that incorporation of a methyl group reverses the fluorescence quantum yield dependence on solvent polarity. In short, 3 a emits more intense fluorescence in more polar solvents such as water,[35] whereas the fluorescence of 3 b in water was the weakest among the solvents tested. The solvent-dependent photophysical properties of 3 b, which are different from those of 3 a (solvent dependence of the absorption maximum and the quantum yield), are probably due to the conformation change (twist angle between the uracil and the benzofuran ring) in 3 b in different solvents. We also measured the excited-state decay kinetics of 3 b by time-resolved fluorescence spectroscopy (Table 1, Table S1 and Figure S1 in the Supporting Information). The excited-state lifetime (tave) of 3 b in water was 0.12 ns, thus indicating that the incorporation of a methyl group reduces the excited-state lifetime (2.38 ns for 3 a;[35] 0.12 ns for 3 b) when dissolved in water. The lifetimes of 3 b in organic solvents ranged from 0.15 (ethyl acetate) to 0.36 ns (acetonitrile). We also calculated the radiative (kr) and the nonradiative (knr) decay constants of 3 b from the quantum yields and tave. The kr was lowest in water (0.02), among methanol, acetonitrile, and ethyl acetate. In contrast, the largest knr was in water. Thus, the weakest emission in water can be explained by the slowest radiative relaxation and the fastest nonradiative relaxation (Table 1). ChemBioChem 2015, 16, 167 – 176
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Figure 5. Fluorescence spectra at various solvent viscosities (lex = 350 nm, 2.0 mm 3 b, 25 8C).
ed the viscosity-dependent fluorescence increase of nucleosides containing 5-(furan-3-yl)uridine and 3 a. Similarly, the fluorescence emission of 3 b at 452 nm increased 14-fold in the viscous glycerol relative to that in the less viscous methanol (Figure 5). This can be attributed to restriction of the rotation of the CC bond between the 3-methylbenzofuran and uracil rings. Characterization of oligonucleotides containing dUBF or dUMBF Based on the above results, it was expected that 3 b (and similarly 3 a) would show a significant fluorescence increase when incorporated into DNA triplexes and forced into the coplanar conformation (Figure 1). Moreover, in the case of 3 b incorporated into triplexes, as UraMBF is surrounded by less-polar neighboring nucleobases, this might result in additional fluorescence enhancement. Thus, DNA triplexes were prepared by incorporating 3 a and 3 b, and their fluorescence properties were compared. Oligodeoxynucleotides were prepared (Figure 6) and their properties were studied. HP-UBF and HP-UMBF are hairpin (HP) duplexes incorporating adenine (Ade)-UraBF and Ade-UraMBF base pairs, respectively. TFO-1 with a propylene linker (C3) recognizes the HPs. To study the triplexes at pH 7.0, 2-aminopyridine nucleoside (Py)[48–50] was used instead of deoxycytidine in TFO-1 because the conditions must be acidic for the protonation of cytosine when a TFO containing deoxycytidine is used.[48] 170
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Figure 6. Sequences of hairpin oligonucleotides (HP-UBF and HP-UMBF) and TFO-1.
To confirm triplex formations, the melting temperatures (Tm ; transition from the triplex state to oligodeoxynucleotides) for HP-UBF or HP-UMBF with TFO-1 were measured: 33 8C for HP-UBF with TFO-1, and 28 8C for HP-UMBF with TFO-1 (Figure S2). These data indicate that both HP-UBF and HP-UMBF form triplexes with TFO-1. Next, the fluorescence spectra of HP-UBF and HP-UMBF were measured in the absence or presence of TFO-1. At 420 nm (lmax), the fluorescence intensity of HP-UBF increased 2.9-fold upon the addition of TFO-1, whereas at 420 nm, that of HPUMBF increased 16-fold. The greater enhancement of HP-UMBF can be attributed to the conformational effect (Figure 5) and the microenvironmental effect (Table 1). In terms of the conformational effects, it is expected that incorporation into DNA triplexes increases the fluorescence of both 3 a and 3 b by forcing planar conformations. In contrast, in terms of the microenvironmental effects, it is expected that incorporation into DNA triplex structures increases the fluorescence of 3 b, but reduces that of 3 a, because the fluorescence of 3 b increases in lesspolar organic solvents than in water, whereas that of 3 a decreases.[35] Thus, in terms of the fluorescence enhancement upon triplex formation, 3 b showed a larger enhancement than 3 a.
Figure 7. Fluorescence spectra of hairpin oligonucleotides: A) HP-UBF and B) HP-UMBF and their triplexes with TFO-1 (lex = 350 nm, 0.1 mm HP-UBF, HPUMBF, and TFO-1, 10 mm cacodylate buffer, 500 mm NaCl, 10 mm MgCl2, pH 7.0, 10 8C).
Structural evaluation of DNA triplex incorporating 3 b To investigate further the results based on the three-dimensional structure, we measured the 2D NMR data for ODN1, a DNA triplex incorporating 3 b based on a previously reported sequence (Figure 8).[51–52] The 3-methylbenzofuran of the UraMBF moiety is in a hydrophobic environment by stacking between T19 and T21, and the 3-methylbenzofuran and uracil rings of UMBF remain coplanar in this triplex (Figure 9). This indicates that both the solvatochromic effects and the conformational change of UraMBF contributed to the fluorescence enhancement. In addition to this structural consideration, the contribution of hydrophobic microenvironment was also indicated by the blue shift of the fluorescence of HP-UMBF (from 449 to 428 nm upon triplex formation; Figure 7).
Figure 8. Sequence of the DNA triplex (ODN1) used for the determination of the three-dimensional structure. EG: ethylene glycol linker, G + C: Hoogsteen base pair between guanine and protonated cytosine, A·T: Hoogsteen base pair between adenine and thymine.
Development of off/on fluorescence probes for singlestranded DNA or RNA Based on the above results, we used this triplex system as a fluorescence probe for single-stranded DNA or RNA. We used 3 b as a fluorescent nucleoside because its on/off ratio was higher than that of 3 a (Figure 7). We prepared target RNA, ChemBioChem 2015, 16, 167 – 176
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Figure 9. NMR-derived averaged structure of the DNA triplex (ODN1) incorporating UMBF (purple). For more details, see the Experimental Section, Table S2, and Figures S8, S9.
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Full Papers limit was estimated to be ~ 5.0 nm, which is comparable to those of other probes.[53] On the basis of the above results, a mixture of probes 1 and 2 can be used as a fluorescence probe for single-stranded DNA or RNA.
Application of off/on fluorescence probes to miRNA detection system by rolling-circle amplification
Figure 10. Sequences of oligonucleotides used for the fluorescence detection of single-stranded DNA or RNA.
By using probes 1 and 2, a smaller amount of RNA was detected along with a “padlock probe”, followed by RCA (Figure 12).[54] RCA is a convenient amplification method for the detection of nucleic acids because it can be performed under isothermal conditions.
target DNA, probe 1 incorporating 3 b, and probe 2, which was identical to TFO-1 (Figure 10). Target DNA and RNA oligonucleotides formed duplexes with probe 1 through Watson–Crick base pairing, and probe 2 formed Hoogsteen base pairs with probe 1 in the duplexes of probe 1 + target DNA, and probe 1 + target RNA. The fluorescence properties of this system are shown in Figure 11. The mixture of probes 1 and 2 emitted weak fluores-
Figure 12. Fluorescence detection of single-stranded RNA by using a padlock probe and RCA with probes 1 and 2.
We designed the following RCA systems. We chose an oncomiR,[55] miR-16 (5’-UAGCA GCACG UAAAU AUUGG CG-3’) as the target (Figure 12). We also prepared a padlock probe with 5’ and 3’ termini designed to bind to miR-16. A purine-rich 5’d(CAAAA AAGAT AGAAA C)-3’ sequence was also introduced into the padlock probe. After the binding of miR-16 to the padlock probe, the latter was ligated with T4 DNA ligase. The concatemeric DNA was synthesized from the 3’ terminus of miR-16 on the cyclized padlock probe by phi29 DNA polymerase. The concatemeric DNA contained multiple copies of a pyrimidine-rich triplex-forming site (3’-d(GTTTTTTCTATCTTTG)-5’), which is the target sequence of probes 1 and 2. After RCA, 1 and 2 were added, and the fluorescence was measured. As expected, a significant fluorescence enhancement was detected in the presence of 50 fmol of target RNA (Figure 13). This suggests that probes 1 and 2 formed triplexes with the pyrimidine-rich sites, and the triplex-induced fluorescence increase can be used as a fluorescence indicator of RCA.
Figure 11. Fluorescence spectra for the detection of target DNA and RNA and quantum yields (lex = 350 nm, 0.2 mm probes 1, 2, target DNA, target RNA, 10 mm cacodylate buffer, 500 mm NaCl, 10 mm MgCl2, pH 7.0, 10 8C).
cence because they do not form Hoogsteen base pairs in the absence of target DNA or RNA, as revealed by the UV melting experiments (Figure S3). In contrast, in the presence of target DNA, the signal at 429 nm (lmax) increased 32-fold (from F = 0.055 to 0.39). Similarly, in the presence of target RNA, the fluorescence signal at 428 nm (lmax) increased 22-fold (from F = 0.055 to 0.29). It should be noted that formation of the triplexes at 10 8C was confirmed by Tm analysis (Figure S3). We also studied the effect of a mismatched base pair at the counter position of the UMBF residue (target DNA strand) on fluorescence intensity (Figure S4). Interestingly, similarly to the matched base pair (A–UraMBF), strong fluorescence enhancements were observed for every mismatch. These fluorescence enhancements also arose from triplex formation (Figure S5) and the induced coplanarity of the 3-methylbenzofuran ring and the uracil moiety. Finally, to determine the detection limit of our probes, we measured fluorescence in the presence of low concentration of target DNA (0 ~ 10 nm). Our probes showed liner fluorescence responses to target concentrations (Figure S6). The detection ChemBioChem 2015, 16, 167 – 176
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Conclusion We have developed new triplex-induced fluorescence probes that incorporate UraBF or UraMBF. The 3-methylbenzofuran and uracil rings of UraMBF in single- or double-stranded oligonucleotides are twisted with respect to each other before formation of triplexes with single-stranded DNA/RNA. The spectroscopic 172
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Full Papers [D6]DMSO (39.5 ppm) and CDCl3 (77.16 ppm) for 13C NMR, and 85 % phosphoric acid (0.0 ppm) for 31P NMR. 2-(3-Methylbenzofuran-2-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (2 b): 3-methylbenzofuran (1.30 g, 9.80 mmol), 4,4’-di-tertbutyl-2,2’-dipyridyl (dtbbp), (40 mg, 0.15 mmol), bis(1,5-cycloocta(49 mg, diene)di-m-methoxydiiridium(I) ([Ir(OMe)(1,5-cod)]2), 0.074 mmol), and bis(pinacolato)diboron (2.49 g, 9.80 mmol) were placed in a round-bottomed flask under argon. Then 1,2-dimethoxyethane (23 mL) was added, and the reaction mixture was stirred overnight at 80 8C. The crude product was purified by C-200 silica gel chromatography with n-hexane-ethyl acetate to give 2 b (2.38 g, 94 %); 1H NMR ([D6]DMSO): d = 7.65 (d, J = 7.8 Hz, 1 H), 7.55 (d, J = 8.3 Hz, 1 H), 7.39 (dd, J = 7.6, 7.8 Hz, 1 H), 7.26 (dd, J = 7.6, 7.3 Hz, 1 H), 2.40 (s, 3 H), 1.31 ppm (s, 12 H); 13C NMR ([D6]DMSO): d = 156.5, 130.0, 128.6, 126.3, 122.4, 120.5,111.4, 84.0, 24.6, 8.7 ppm; ESI-MS: calcd for C15H19BNaO3 + : 281.1319 [M+Na] + ; found 281.1321.
Figure 13. Fluorescence spectra of the reaction mixture after ligation and RCA in the presence or absence of miR-16 (lex = 350 nm, 0.2 mm probes 1 and 2, 10 8C). See the Experimental Section for details.
and NMR studies indicated that the significant increase in the fluorescence upon triplex formation can be attributed to changes in the microenvironment of the 3-methylbenzofuran moiety and increased coplanarity between the 3-methylbenzofuran and uracil rings. We also demonstrated the applicability of this triplex-induced fluorescence probe to miRNA detection in conjunction with RCA. Because our system involves only a simple interaction between the fluorescent 5-aryluridine and the abasic site in TFO, the system can be combined with other fluorescent 5-arylpyrimidines[20–23, 28–34, 36–37, 56–58] with different fluorescent colors. In addition, previously reported fluorescent probes such as thiazole-modified probes,[10–17] which have different fluorescent emission wavelengths from our probe (lem ~ 420 nm), can be used in combination with our probes. Furthermore, because of flexibly in the design of sequences flanking the 5-arylpyrimidines (as long as they can form triplexes), the triplex-induced fluorescence system can be applied to multicolor multiplex RNA detection. Notably, the triplex-forming sites are not restricted to homopurine sequences. Previously, we reported an artificial DNA triplex system incorporating modified pyrimidines with hydrogen-bonding sites at position 5.[39] By using such modified pyrimidines in probe 1, triplex-forming sites of any mixed sequence can be designed. Moreover, this strategy can be combined with isothermal nucleic acid amplification technologies[59–60] other than RCA, provided they generate single-stranded DNA or RNA. Studies are in progress in our laboratory to develop these applications and new fluorescent nucleosides based on this strategy.
5-(Benzofuran-2-yl)deoxyuridine (3 a): 5-iododeoxyuridine (800 mg, 2.26 mmol), palladium acetate (15 mg, 0.068 mmol), TPPTS (102 mg, 0.18 mmol), Na2CO3 (527 mg, 4.97 mmol) and 2(benzofuran-2-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (1.10 g, 4.51 mmol) were placed in a round-bottomed flask under argon. Degassed H2O/CH3CN (2:1, v/v, 20 mL) was added, and the mixture was stirred at 45 8C for 13 h. The mixture was evaporated under reduced pressure. The residue was diluted with small amount of H2O/CH3CN (2:1, v/v). The precipitate was filtered to give 3 a (486 mg, 62 %); 1H NMR ([D6]DMSO): d = 11.7 (br s, 1 H), 8.74 (s, 1 H), 7.61–7.53 (m, 2 H), 7.33–7.20 (m, 3 H), 6.23 (t, J = 6.1 Hz, 1 H), 5.31– 5.24 (m, 2 H) 4.33 (s, 1 H), 3.88 (s, 1 H), 3.73–3.65 (m, 2 H), 2.28– 2.23 ppm (m, 2 H); 13C NMR ([D6]DMSO): d = 160.3, 153.0, 149.4, 149.1, 137.1, 128.8, 124.3, 123.0, 121.0, 110.8, 104.7, 103.9, 87.7, 85.1, 70.1, 60.9, 40.5 ppm; ESI-MS: calcd for C17H16N2NaO6 + : 367.0901 [M+Na] + ; found 367.0903. 5-(3-Methylbenzofuran-2-yl)deoxyuridine (3 b): 5-iododeoxyuridine (1.39 g, 3.92 mmol), palladium acetate (27 mg, 0.12 mmol), TPPTS (176 mg, 0.31 mmol), Na2CO3 (0.913 g, 8.62 mmol) and 2-(3methylbenzofuran-2-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane 2 b (2.02 g, 7.84 mmol) were placed in a round-bottomed flask under argon. Degassed H2O/CH3CN (2:1, v/v, 43 mL) was added, and the mixture was stirred for 16 h at 45 8C. The mixture was evaporated under reduced pressure. The crude product was purified by C-200 silica gel chromatography with dichloromethane-methanol to give 3 b (1.36 g, 97 %); 1H NMR ([D6]DMSO): d = 11.66 (s, 1 H), 8.34 (1, 1 H), 7.57 (d, J = 7.6 Hz, 1 H), 7.50 (d, J = 8.1, 1 H), 7.31–7.23 (m, 2 H), 6.21 (t, J = 6.6 Hz, 1 H), 5.28 (m, 1 H), 5.07 (m, 1 H), 4.26 (m, 1 H), 3.81 (m, 1 H), 3.58–3.55 (m, 2 H), 2.19–2.17 ppm (m, 5 H); 13C NMR ([D6]DMSO): d = 161.0, 153.5, 149.9, 145.3, 141.5, 129.8, 124.4, 122.5, 119.5, 113.5, 110.9, 104.9, 87.6, 84.8, 70.3, 61.0, 40.4, 8.6 ppm; ESI-MS: calcd for C18H18N2NaO6 + : 381.1057 [M+Na] + ; found 381.1054.
Experimental Section
5-(Benzofuran-2-yl)-5’-O-(4,4’-dimethoxytrityl)deoxyuridine (4 a): 3 a (200 mg, 0.58 mmol) was coevaporated three times with pyridine and dissolved in pyridine (2.9 mL). 4, 4’-Dimethoxytrityl chloride (288 mg, 0.64 mmol) was added to the reaction mixture and stirred for 3 h at room temperature. After the completion of the reaction, methanol (1 mL) was added and the reaction mixture was evaporated. The crude product was extracted with water/dichloromethane and dichloromethane layer was collected and evaporated. Crude product was purified by C-200 silica gel chromatography with dichloromethane/methanol/0.5 % pyridine to give 4 a (310 mg, 83 %); 1H NMR ([D6]DMSO): d = 11.85 (br s, 1 H), 8.29 (s,
Materials: Reagents for DNA synthesis and unmodified DNA phosphoramidite units were purchased from Glen Research (Sterling, VA). Sep-Pak C18 cartridges for DNA purification were purchased from Waters (Milford, MA). Other reagents for the synthesis of phosphoramidite monomers were purchased from Tokyo Chemical Industry (Tokyo, Japan) and Sigma–Aldrich. DNA and RNA were purchased from Sigma–Aldrich. Dry solvents were purchased and stored over molecular sieves (4 ). 1H, 13C, and 31P NMR spectra were obtained at 500, 126, and 203 MHz, respectively. The chemical shifts were measured from [D6]DMSO (2.49 ppm) for 1H NMR, ChemBioChem 2015, 16, 167 – 176
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Full Papers 1 H), 7.55 (d, J = 7.6 Hz, 1 H), 7.44 (d, J = 7.6 Hz, 2 H), 7.31–7.29 (m, 5 H), 7.23–7.20 (m, 2 H), 7.14–7.09 (m, 2 H), 7.05–7.02 (m, 1 H), 6.77– 6.75 (m, 4 H), 6.51 (d, J = 8.3 Hz, 1 H), 6.21 (t, J = 6.5 Hz, 1 H), 5.35– 5.34 (m, 1 H), 4.21 (br s, 1 H), 3.99–3.97 (m, 1 H), 3.59–3.58 (m, 6 H), 3.36–3.34 (m, 1 H), 3.19–3.16 (m, 1 H), 2.30–2.28 ppm (m, 2 H); 13 C NMR (CDCl3): d = 160.6, 158.8, 153.8, 149.9, 147.8, 144.8, 136.0, 135.9, 135.4, 130.4, 130.3, 129.2, 128.5, 128.2, 127.2, 124.5, 123.0, 121.2, 113.5, 111.1, 107.3, 106.1, 87.1, 86.8, 86.0, 72.5, 63.7, 55.4, 41.7 ppm; ESI-MS: calcd for C38H34N2NaO8 + : 669.2207 [M+Na] + ; found 669.2208.
1.46 mmol) in dichloromethane (18 mL). The solution was stirred for 10 h at room temperature under argon. The reaction was diluted with dichloromethane and water, and extracted with dichloromethane. The combined organic layers were washed with water and brine, dried over MgSO4, and concentrated under reduced pressure. The crude product was purified by NH-silica gel chromatography with n-hexane/ethyl acetate to give 5 b (1.31 g, 84 %); 1 H NMR ([D6]DMSO): d = 11.75 (s, 1 H), 7.97–7.96 (m, 1 H), 7.52–7.50 (m, 1 H), 7.32–7.31 (m, 2 H), 7.21–7.16 (m, 8 H), 7.12–7.07 (m, 2 H), 6.72–6.71 (m, 4 H), 6.22–6.19 (m, 1 H), 4.50–4.44 (m, 1 H), 4.11–4.06 (m, 1 H), 3.76–3.44 (m, 10 H), 3.28–3.21 (m, 2 H), 2.76 (m, 1 H), 2.64 (m, 1 H), 2.47–2.36 (m, 2 H), 2.06 (s, 3 H), 1.17–1.09 (m, 9 H), 0.99– 0.97 ppm (m, 3 H); 13C NMR (CDCl3): d = 160.8, 158.7, 154.2, 150.0, 149.9, 144.5, 143.3, 139.9, 135.7, 135.6, 135.5, 130.8, 130.3, 130.2, 128.4, 128.3, 128.1, 127.1, 124.4, 122.3, 119.4, 117.9, 117.7, 115.3, 113.3, 111.2, 107.7, 87.0, 86.3, 86.1, 86.0, 74.2, 74.1, 73.8, 73.6, 63.4, 63.2, 58.7, 58.6, 58.4, 55.4, 43.6, 43.5, 43.4, 41.0, 25.0, 24.9, 24.8, 20.7, 20.5, 9.4 ppm; 31P NMR ([D6]DMSO): d = 148.6, 148.3 ppm; ESIMS: calcd for C48H53N4NaO9P + : 883.3442 [M+Na] + ; found 883.3434.
5-(3-Methylbenzofuran-2-yl)-5’-O-(4,4’-dimethoxytrityl)deoxyuridine (4 b): 3 b (1.03 g, 2.87 mmol) was coevaporated three times with pyridine and dissolved in pyridine (29 mL). 4, 4’-Dimethoxytrityl chloride (1.07 g, 3.16 mmol) was added to the reaction mixture and stirred for 6 h at room temperature. After completion of the reaction, methanol (1 mL) was added, and the reaction mixture was evaporated. The crude product was extracted with water/dichloromethane, and the dichloromethane layer was collected and evaporated. The crude product was purified by C-200 silica gel chromatography with dichloromethane/methanol/pyridine (0.5 %) to give 4 b (1.54 g, 81 %); 1H NMR ([D6]DMSO): d = 11.7 (s, 1 H), 7.90 (s, 1 H), 7.51–7.49 (m, 1 H), 7.30–7.29 (m, 2 H), 7.22–7.05 (m, 9 H), 6.71–6.69 (m, 4 H), 6.19 (t, J = 6.6 Hz, 1 H), 5.36–5.34 (m, 1 H), 4.25 (m, 1 H), 3.97–3.94 (m, 1 H), 3.64–3.63 (m, 6 H), 3.18–3.13 (m, 2 H), 2.29–2.27 (m, 1 H), 2.03 ppm (s, 3 H); 13C NMR ([D6]DMSO): d = 160.8, 158.0, 157.9, 153.3, 149.8, 144.6, 144.5, 140.5, 135.5, 135.1, 129.8, 129.6, 129.5, 127.7, 127.5, 126.5, 124.2, 122.3, 119.3, 113.7, 113.0, 110.6, 105.2, 85.9, 85.8, 85.3, 70.5, 63.7, 54.9, 8.6 ppm; ESIMS: calcd for C39H36N2NaO8 + : 683.2364 [M+Na] + ; found 683.2378.
Synthesis and purification of oligonucleotides: DNA was synthesized in an ABI 392 DNA/RNA synthesizer (Applied Biosystems) with phosphoramidite chemistry. Oligonucleotides were purified in Sep-pak C18 cartridges with reverse-phase high performance liquid chromatography (HPLC) at 30 8C with a linear gradient of solvent I (NH4OAc (30 mm)) and solvent II (acetonitrile) at a flow rate of 1.0 mL min1 for 30 min. MALDI-TOF mass data of oligonucleotides: HP-UBF : [M+H] + Calcd 11134.89, found 11140.93; HP-UMBF : [M+H] + calcd 11148.90, found 11153.97; probe 1: [M+H] + calcd 5038.96, found 5039.94; probe 2 (TFO-Py): [M+H] + calcd 3965.68, found 3966.62; ODN-1: [M+H] + calcd 7918.48, found 7923.50.
5-(Benzofuran-2-yl)-5’-O-(4,4’-dimethoxytrityl)deoxyuridine 3’-(2cyanoethyl N,N-diisopropylphosphoramidite) (5 a): Compound 4 a (230 mg, 0.36 mmol) was coevaporated with anhydrous pyridine. 2-Cyanoethyl N,N,N’, N’-tetraisopropylphosphoramidite (160 mL, 0.50 mmol) was added to a stirred solution of 4 a, diisopropylamine (30 mL, 0.22 mmol), and 1H-tetrazole (15 mg, 0.22 mmol) in dichloromethane (3.6 mL). The solution was stirred overnight at room temperature under argon. The reaction was diluted with dichloromethane and water, and extracted with dichloromethane. The combined organic layers were washed with water and brine, dried over MgSO4, and concentrated under reduced pressure. The crude product was purified by C-200 silica gel chromatography with n-hexane/ethyl acetate/pyridine (0.5 %) to give 5 a (180 mg, 59 %); 1H NMR ([D6]DMSO): d = 11.88 (br s, 1 H), 8.35–8.33 (m, 1 H), 7.56–7.55 (m, 1 H), 7.46–7.44 (m, 2 H), 7.33–7.10 (m, 9 H), 7.05–7.03 (m, 1 H), 6.77–6.74 (m, 4 H), 6.55–6.48 (m, 1 H), 6.24–6.20 (m, 1 H), 4.47 (m, 1 H), 4.15–4.10 (m, 1 H), 3.75–3.68 (m, 1 H), , 3.60–3.57 (m, 6 H), 3.57–3.37 (m, 4 H), 3.22–3.19 (m, 1 H), 2.77–2.75 (m, 1 H), 2.65– 2.63 (m, 1 H), 2.49–2.40 (m, 2 H), 1.16–1.10 (m, 9 H), 0.98 ppm (m, 3 H); 13C NMR (CDCl3): d = 160.7, 160.6, 158.8, 153.7, 149.7, 149.6, 147.8, 144.8, 136.1, 136.0, 135.9, 135.8, 135.4, 130.4, 130.4, 129.2, 128.6, 128.5, 128.2, 127.2, 124.4, 122.9, 121.1, 117.9, 117.7, 113.4, 111.1, 107.2, 106.1, 87.0, 86.3, 86.0, 85.9, 73.9, 73.8, 73.5, 73.4, 63.3, 63.1, 58.7, 58.6, 58.5, 58.4, 55.4, 43.6, 43.5, 43.4, 40.9, 24.9, 24.8, 20.7, 20.5, 20.4 ppm. 31P NMR ([D6]DMSO): d = 148.6, 148.3 ppm; ESI-MS: calcd for C47H51N4NaO9P + : 869.3286 [M+Na] + ; found 869.3289.
Quantum yield measurement: Quantum yields were determined using 2-aminopurine riboside[4, 61] in water (lex = 303 nm, F = 0.68) or quinine[62] in H2SO4 (0.1 n; lex = 366 nm, F = 0.53) as reference. The quantum yields were calculated according to Equation (1): FFðSÞ AðRÞ F ðSÞ ¼ FFðRÞ AðSÞ F ðRÞ
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nðSÞ nðRÞ
2 ð1Þ
where FF(S) and FF(R) are the fluorescence quantum yields of the sample and the reference, respectively; F(S) and F(R) are the area under emission curve of the sample and the reference solution, respectively; A(S) and A(R) are the respective optical density of the sample and the reference solution at the wavelength of excitation, and n(S) and n(R) are the values of the refractive index for the respective solvents. 2-Aminopurine riboside was used as the reference for the quantum yield measurement of dUMBF monomer; quinine was used for single-strand or triplex DNA containing dUMBF (Table 1). Exited-state lifetime measurements: Excited-state lifetime measurements were performed with a FluoroCube spectrometer (HORIBA Jobin Yvon, Kyoto, Japan). For the measurements of nucleoside 3 b (Table 1), a 280 nm pulse laser was used. Emission signals were acquired at lmax. Obtained decay profiles were fitted to the theoretical equation to determine lifetimes by using DAS6 software (HORIBA). Radiative (kr) and nonradiative (knr) decay constants were determined from quantum yield (F) and average excitedstate lifetime (tave) with Equations (2) and (3):[63]
5-(3-Methylbenzofuran-2-yl)-5’-O-(4,4’-dimethoxytrityl)deoxyuridine 3’-(2-cyanoethyl N,N-diisopropylphosphoramidite) (5 b): Compound 4 b (1.20 g, 1.82 mmol) was coevaporated with anhydrous pyridine. 2-cyanoethyl N, N, N’, N’-tetraisopropylphosphoramidite (864 mL, 2.72 mmol) was added to a stirred solution of 4 b, diisopropylamine (206 mL, 1.46 mmol), and 1H-tetrazole (102 mg,
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k r ¼ F=tave
ð2Þ
1=tave ¼ kr þ knr
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Full Papers used for further analysis. MOLMOL[66] was used to visualize the final structures.
Enzymatic ligation of the padlock probe: miR-16 (0, 50, or 1000 fmol), padlock probe (5 mm), and ATP (10 mm) were dissolved in ligation buffer (5 mL, Tris·HCl (10 mm, pH 7.5), MgCl2 (10 mm)) and incubated at 65 8C for 3 min. Subsequently, the reaction tubes were cooled to room temperature (20 8C) and kept for 10 min. T4 DNA ligase (200 U) was added, and the mixture was incubated at 37 8C for 2 h. After the ligation, ligase was deactivated by heating at 65 8C for 10 min.
Acknowledgements We thank Prof. Masayuki Takeuchi (National Institute for Materials Science, NIMS) and Prof. Kazunori Sugiyasu (NIMS) for the excited-state lifetime measurements and valuable discussions. This work was supported by JSPS KAKENHI, and Adaptable and Seamless Technology Transfer Program through target-driven R&D, JST. This work was also supported by Strategic Young Researcher Overseas Visits Program for Accelerating Brain Circulation, JSPS.
Rolling circle amplification: After the ligation, the reaction mixtures (5 mL) were added to Tris·HCl (95 mL, 50 mm, pH 7.5) containing MgCl2 (10 mm), (NH4)2SO4 (10 mm), dithiothreitol (4 mm), phi29 DNA polymerase (10 U), and dNTPs (0.2 mm). The reaction mixtures were incubated at 30 8C for 8 h then heated to 65 8C for 10 min to inactivate the polymerase. Fluorescence measurements of RCA products: Reaction mixtures were diluted in cacodylate buffer (400 mL) containing probe 1 and 2; final concentrations: probe 1 and 2 (0.2 mm), cacodylate buffer (10 mm, pH 7.0), NaCl (500 mm), MgCl2 (10 mm). Before the fluorescence measurements, the reaction mixtures were heated at 85 8C for 5 min and then gradually cooled to 10 8C. The spectra were measured at 10 8C.
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2D NMR spectra and peak assignments: The NMR sample was prepared by dissolving ODN-1 in H2O (90 %)/D2O (10 %) with NaCl (100 mm) and MgCl2 (5 mm) at pH 5.2. The final oligonucleotide concentration in the NMR sample was 0.5 mm in a 300 mL Shigemi NMR tube. NMR experiments were performed at 288 K and 298 K for triplex DNA containing UMBF on Bruker 800 MHz spectrometers (Bruker AV800 equipped with cryoprobe or with a normal probe). The 1 H, 15N, and 13C chemical shifts were referenced to the frequency of the 2H lock resonance of water. The data were processes with Topspin 3.1 (Brucker). Exchangeable proton resonances were obtained with 2D NOESY experiments by using the jump-return method with a gradient pulse for water suppression. The imino proton spectra of the DNA triplex incorporating UMBF were assigned based on the 2D NOESY spectra observed in H2O. Assignment of the imino proton resonances proved the formation of the predicted base pairs in the constructs. 2D NOESY, 2D TOCSY, 2D DQF COSY and natural abundance 1H,13C HSQC spectra were measured for ODN-1. Assignment of the H7, H8, H11, and H12 protons of UMBF were confirmed by the 2D DQF-COSY and NOESY spectra. Assignments of H2 protons of adenine and of H6, H8, and H1’ protons of canonical deoxynucleotides and H7, H8, H11, and H12 proton of UMBF were confirmed by the 1H,13C HSQC spectrum. Assignments of the methyl group of thymine and H2’ and H2’’ protons of deoxynucleotides were confirmed by the 2D TOCSY and NOESY spectra. NOE distance restraints for exchangeable protons were obtained by using NOESY spectra with the jump-return scheme. In order to estimate the dihedral angle, sugar pucker was analyzed by using TOCSY and DQF COSY spectra. The NMR data were processed in NMRPipe.[64] Analyses of the processed data were performed with Sparky (T. D. Goddard and D. G. Kneller, SPARKY 3, University of California, San Francisco, CA). The three-dimensional structures of the DNA triplex incorporating UMBF were determined by combined automated NOESY cross-peak assignment and structure calculations with torsion angle dynamics implemented in the program CYANA 2.1.[65] 3D structure determination based on NMR data: The structure calculations started from 200 randomized conformers and used the standard CYANA[65] simulated annealing schedule, with 40 000 torsion angle dynamics steps per conformer. The 20 conformers that were most consistent with the experimental restraints were then
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Received: July 1, 2014 Published online on December 2, 2014
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Controlling the Fluorescence of Benzofuran-Modified Uracil Residues in Oligonucleotides by Triple-Helix Formation Takashi Kanamori,[a] Hiroki Ohzeki,[b] Yoshiaki Masaki,[b] Akihiro Ohkubo,[b] Mari Takahashi,[c, d] Kengo Tsuda,[c, d] Takuhiro Ito,[c, d] Mikako Shirouzu,[c, d] Kanako Kuwasako,[c, d, e] Yutaka Muto,[c, d, e] Mitsuo Sekine,*[b] and Kohji Seio*[b] We developed fluorescent turn-on probes containing a fluorescent nucleoside, 5-(benzofuran-2-yl)deoxyuridine (dUBF) or 5-(3methylbenzofuran-2-yl)deoxyuridine (dUMBF), for the detection of single-stranded DNA or RNA by utilizing DNA triplex formation. Fluorescence measurements revealed that the probe containing dUMBF achieved superior fluorescence enhancement than that containing dUBF. NMR and fluorescence analyses indicated that the fluorescence intensity increased upon triplex
formation partly as a consequence of a conformational change at the bond between the 3-methylbenzofuran and uracil rings. In addition, it is suggested that the microenvironment around the 3-methylbenzofuran ring contributed to the fluorescence enhancement. Further, we developed a method for detecting RNA by rolling circular amplification in combination with triplex-induced fluorescence enhancement of the oligonucleotide probe containing dUMBF.
Introduction their fluorescence in the absence of targets;[11] Seitz and coworkers developed forced intercalation probes by controlling the conformation of the excited state of thiazole dyes upon intercalation between the base pairs of the probe–target duplexes;[12–15] Pedersen and Filichev have reported a series of the fluorescent probes bearing pyrene or thiazole dyes in triplexforming oligodeoxynucleotides (TFOs) to enabled the detection of duplex DNAs.[16–17] In contrast to these dye-based fluorescence probes, fluorescent pyrimidine nucleosides with a small substituent at position 5 have been reported.[18–37] Such fluorescent modified pyrimidine nucleosides incorporated into oligonucleotides serve as fluorescent dyes, while retaining Watson–Crick base pairing ability. One advantage of these fluorescent pyrimidines is that they cause minimal conformational disturbance to the native structures of nucleic acids and the complexes incorporating them. Thus, attempts have been made to use oligonucleotides containing fluorescent-modified pyrimidines as fluorescence probes that change their fluorescence upon binding to the targets; here, the substituent at position 5 becomes partly sandwiched between two bases. This molecular interaction changes the microenvironment of the substituents and their conformation, thus increasing or decreasing fluorescence intensities. However, in many cases, the magnitude of the fluorescence change is small.[28–29, 31, 34–35, 37] This is probably because the substituents at position 5 of the pyrimidine base extrude into major groove and are not completely covered by the upper and lower bases. To significantly enhance the on/off ratio of fluorescence signals, we studied the triple-helix formation of oligodeoxynucleotides containing 5-arylpyrimidines, based on our previous
Fluorescent oligonucleotides are promising tools for the detection of functional nucleic acids in vivo and in vitro and for studying the interactions of nucleic acids with proteins or small molecules.[1–8] Fluorescent oligonucleotides that emit fluorescence only in the presence of targets have been used as fluorescent probes. To enable target-dependent fluorescence emission, various mechanisms have been proposed: molecular beacons use the interactions of chromophores with quenching dyes in the absence of targets;[9–10] Okamoto reported a mechanism that uses H-aggregation of two thiazole dyes to quench
[a] Dr. T. Kanamori Education Academy of Computational Life Sciences Tokyo Institute of Technology 4259 Nagatsuta, Midoriku, Yokohama 226-8501 (Japan) [b] H. Ohzeki, Dr. Y. Masaki, Prof. A. Ohkubo, Prof. M. Sekine, Prof. K. Seio Department of Life Science, Tokyo Institute of Technology 4259 Nagatsuta, Midoriku, Yokohama 226-8501 (Japan) E-mail: [email protected] [email protected] [c] M. Takahashi, K. Tsuda, Dr. T. Ito, Prof. M. Shirouzu, Dr. K. Kuwasako, Prof. Y. Muto RIKEN Systems and Structural Biology Center 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045 (Japan) [d] M. Takahashi, K. Tsuda, Dr. T. Ito, Prof. M. Shirouzu, Dr. K. Kuwasako, Prof. Y. Muto RIKEN Center for Life Science Technologies 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045 (Japan) [e] Dr. K. Kuwasako, Prof. Y. Muto Faculty of Pharmacy Department of Pharmaceutical Sciences, Musashino University 1-1-20, Shinmachi Nishitokyo-city, Tokyo 202-8585 (Japan) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbic.201402346.
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Full Papers studies.[38–39] As the 5-arylpyrimidine residue, we selected fluorescent 5-(benzofuran-2-yl)uracil (UraBF) because of its high fluorescence quantum yield (F) and fluorescence increase dependence on DNA–DNA hybridization.[35] We further designed its derivative 5-(3-methylbenzofuran-2-yl)uracil (UraMBF). As shown in Figure 1 A, the benzofuran moieties of UraBF and UraMBF when located in the major groove can rotate when the
Results and Discussion Ground-state conformations of UraBF and UraMBF First, we calculated the electronic energies of UraBF and UraMBF to clarify the conformational differences in these nucleobase analogues. In these calculations, the CC bond between the benzofuran and uracil rings was rotated in a stepwise manner, and the energy of each conformer was calculated with the B3LYP/6-31G (d) model in Gaussian 09[43] in gas phase and in a water model, generated with the polarizable continuum model (PCM).[44] In the gas phase (dashed line in Figure 2 A),
Figure 1. Microenvironment and conformations of 5-(benzofuran-2-yl)uracil (UraBF) and 5-(3-methylbenzofuran-2-yl)uracil (UraMBF). A) Duplex with the target nucleic acid, and B) triplex with the target nucleic acid and triplexforming oligonucleotide incorporating propylene linker (C3).
oligodeoxynucleotide containing them forms a duplex with target nucleic acids. However, upon binding to triplex-forming oligonucleotides (TFOs) with a propylene linker (C3)[38–39] at the counter position of the UraBF or UraMBF, the benzofuran ring becomes sandwiched between two bases. These flanking bases make the benzofuran and uracil rings coplanar (Figure 1 B), thus inducing fluorescence. It has been reported that insertion of an alkyl group into the ortho position of fluorescent biaryls induced the large twist between two aryl rings of biaryls and thus affected their photophysical properties, such as fluorescence emission wavelength.[40–42] Furthermore, the fluorescence intensity increases upon changes to the conformations of the biaryls from the twisted form to coplanar.[42] Based on this, we designed UraMBF in addition to the previously reported UraBF. It was expected that the introduction of a methyl group would increase the population of the twisted-form UraMBF in the duplex state and induce a larger fluorescence enhancement in the triplex state. At the same time, the binding of UraBF or UraMBF to TFO (Figure 1 B) makes the microenvironment of the benzofuran ring more hydrophobic. This change in the microenvironment can also enhance the fluorescence intensity. Although the nucleoside UraBF and oligonucleotides containing UraBF have been reported,[31, 35] the fluorescence properties in triplex DNA have not been reported. We observed a large fluorescence enhancement of UraBF and UraMBF upon the triplex formation. Our triplex-induced fluorescence probes could be used to detect single-stranded nucleic acids such as micro RNAs.
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Figure 2. Potential energy vs. torsion angle (q; C4-C5-CB2-CB3) of A) 1-methyl-5-(benzofuran-2-yl)uracil and B) 1-methyl-5-(3-methylbenzofuran-2-yl)uracil. Relaxed potential energy scan was performed with B3LYP/6-31G (d) (Gaussian 09). a: gas, c: water; for the calculation in the water model, a PCM solvation model was used.
UraBF had an energy minimum at q = 08 (conformation where the benzofuran and uracil rings are coplanar). In contrast, UraMBF had an energy minimum at q = 338 in the gas phase with a twisted conformation (Figure 2 B). This clearly indicates that the incorporation of a methyl group stabilizes the twisted conformation to relieve the steric hindrance caused by uracil. The solvent effects for PCM (solid line) lowered the energy barrier for the rotation; however, the energy168
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Full Papers minimized conformation did not change. These results clearly support our hypothesis that incorporation of a methyl group stabilizes the twisted conformation. Synthesis and photophysical properties of dUBF and dUMBF Next, the corresponding nucleosides were synthesized to elucidate the fluorescence properties. Benzofuran-modified deoxyuridine dUBF (3 a; aglycon UraBF) and dUMBF (3 b; aglycon UraMBF) were synthesized by using Suzuki–Miyaura cross-coupling reactions (Scheme 1). Several 5-aryl-modified pyrimidines[22–23, 45]
Figure 3. Absorption (50 mm, c) and fluorescence (10 mm, a) spectra of 3 b in various solvents (lex = 300 nm). For measurement of absorption and fluorescence spectra, samples contained 5 and 1 % DMSO, respectively, for solubility.
ing phosphoramidites 5 a and 5 b for oligonucleotide synthesis. To study the photophysical properties of 3 b, the UV absorption and steady-state and time-resolved fluorescence were measured in various solvents (Figure 3, Table 1). Because the photophysical properties of 3 a[35] and related nucleosides[21, 29, 31] have already been reported, the properties of 3 b could be compared with these. Compound 3 b showed an absorption peak at ~ 300 nm in every solvent tested, and at ~ 260 nm in the case of water (Figure 3). The absorption at ~ 300 nm was strongest when 3 b was dissolved in ethyl acetate (the least polar solvent) and weakest in water (the most polar solvent). The acetonitrile and methanol solutions showed absorption peaks between these. The maximum-absorption wavelength (lmax) of 3 b ranged from 293 to 306 nm in different solvents (Table 1). Upon irradiation at 300 nm, the fluorescence emitted by 3 b was weakest in water and stronger in organic solvents such as methanol, acetonitrile, and ethyl acetate (Figure 3). The maximum-fluorescence emission wavelength (lem) changed significantly in different solvents (from 432 to 484 nm). To analyze the effect of solvent on the absorption and emission wavelengths, the Stokes shifts (difference between the emission and absorption maxima) were plotted against the microscopic solvent polarity parameter, ET(30).[47] The plot (Figure 4) shows a positive correlation, thus indicating stabilization of the excit-
Scheme 1. Synthesis of fluorescent nucleosides, dUBF (3 a) and dUMBF (3 b), and their phosphoramidites, 5 a and 5 b. a) 0.03 equiv Pd(OAc)2, 0.08 equiv TPPTS, 2.2 equiv Na2CO3, H2O/CH3CN (2:1, v/v), 45 8C, overnight (62 % for 3 a (R = H), 97 % for 3 b (R = Me)); b) 1.1 equiv DMTrCl, pyridine, RT, 3 h for 4 a and 6 h for 4 b (83 % for 4 a, 81 % for 4 b); c) 1.5 equiv [(iPr)2N]2P(OCE), 0.8 equiv HN(iPr)2, 0.8 equiv 1H-tetrazole, CH2Cl2, RT, overnight (59 % for 5 a (R = H), 84 % for 5 b (R = Me)).
have been synthesized by the Suzuki–Miyaura cross-coupling reaction. One of the advantages over Stille coupling, which was previously used for the synthesis of 3 a and its derivatives, is avoidance of the harmful tributyltin reagent. In brief, the boronic acid esters of benzofurTable 1. Photophysical properties of 3 b in various solvents. kr and knr are radiative and nonradiative decay rate an (2 a) or 3-methylbenzofuran constants, respectively. (2 b), which was prepared by lem[a] [nm] Irel[b] F tave [ns] kr [ns1] knr [ns1] Solvent lmax [nm] using method of Miyaura,[46] were coupled to 5-iododeoxyuriwater 293 484 1.0 0.003 0.12 0.02 8.0 methanol 306 457 6.7 0.009 0.36 0.03 2.7 dine (1) to afford 3 a and 3 b, reacetonitrile 300 441 4.1 0.005 0.20 0.03 5.1 spectively (Scheme 1). The nuethyl acetate 301 432 5.0 0.007 0.15 0.05 6.5 cleosides were used for the fluo[a] Maximum fluorescence wavelength upon irradiation at 300 nm. [b] relative fluorescence intensity at each rescence measurements and lem. preparation of the correspondChemBioChem 2015, 16, 167 – 176
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Full Papers Dependence of fluorescence intensity on the solvent viscosity As previously reported for 3 a, the conformational change around the CC bond between the benzofuran and uracil rings can affect the fluorescence emission intensities.[35] To elucidate the effects of the conformational change, the fluorescence emission spectra of 3 b were measured at different solvent viscosities (Figure 5). Tor[29] and Srivatsan[31, 35] have report-
Figure 4. Relationship between Stokes shift and ET(30) for dUMBF (3 b).
ed state by polar solvent molecules. A similar dependence on solvent polarity was observed for 3 a.[35] In these studies of the nucleoside, very small shoulder bands were present in all cases (e.g., ~ 530 nm for methanol). Although this might indicate some aggregation of nucleosides, such bands were not observed in the later triplex cases and did not hamper the triplex studies. Next, the relative fluorescence intensities (Irel) were compared at the maximum-fluorescence wavelength: 6.7, 5.0, 4.1, and 1.0 in methanol, ethyl acetate, acetonitrile, and water, respectively. For 3 b, F was lowest in water (0.003), then 0.005, 0.007, and 0.009, in acetonitrile, ethyl acetate, and methanol, respectively. Interestingly, according to a previous report, the structurally similar 3 a showed the lowest F in acetonitrile (0.04) and the highest in water (0.19).[35] These results indicate that incorporation of a methyl group reverses the fluorescence quantum yield dependence on solvent polarity. In short, 3 a emits more intense fluorescence in more polar solvents such as water,[35] whereas the fluorescence of 3 b in water was the weakest among the solvents tested. The solvent-dependent photophysical properties of 3 b, which are different from those of 3 a (solvent dependence of the absorption maximum and the quantum yield), are probably due to the conformation change (twist angle between the uracil and the benzofuran ring) in 3 b in different solvents. We also measured the excited-state decay kinetics of 3 b by time-resolved fluorescence spectroscopy (Table 1, Table S1 and Figure S1 in the Supporting Information). The excited-state lifetime (tave) of 3 b in water was 0.12 ns, thus indicating that the incorporation of a methyl group reduces the excited-state lifetime (2.38 ns for 3 a;[35] 0.12 ns for 3 b) when dissolved in water. The lifetimes of 3 b in organic solvents ranged from 0.15 (ethyl acetate) to 0.36 ns (acetonitrile). We also calculated the radiative (kr) and the nonradiative (knr) decay constants of 3 b from the quantum yields and tave. The kr was lowest in water (0.02), among methanol, acetonitrile, and ethyl acetate. In contrast, the largest knr was in water. Thus, the weakest emission in water can be explained by the slowest radiative relaxation and the fastest nonradiative relaxation (Table 1). ChemBioChem 2015, 16, 167 – 176
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Figure 5. Fluorescence spectra at various solvent viscosities (lex = 350 nm, 2.0 mm 3 b, 25 8C).
ed the viscosity-dependent fluorescence increase of nucleosides containing 5-(furan-3-yl)uridine and 3 a. Similarly, the fluorescence emission of 3 b at 452 nm increased 14-fold in the viscous glycerol relative to that in the less viscous methanol (Figure 5). This can be attributed to restriction of the rotation of the CC bond between the 3-methylbenzofuran and uracil rings. Characterization of oligonucleotides containing dUBF or dUMBF Based on the above results, it was expected that 3 b (and similarly 3 a) would show a significant fluorescence increase when incorporated into DNA triplexes and forced into the coplanar conformation (Figure 1). Moreover, in the case of 3 b incorporated into triplexes, as UraMBF is surrounded by less-polar neighboring nucleobases, this might result in additional fluorescence enhancement. Thus, DNA triplexes were prepared by incorporating 3 a and 3 b, and their fluorescence properties were compared. Oligodeoxynucleotides were prepared (Figure 6) and their properties were studied. HP-UBF and HP-UMBF are hairpin (HP) duplexes incorporating adenine (Ade)-UraBF and Ade-UraMBF base pairs, respectively. TFO-1 with a propylene linker (C3) recognizes the HPs. To study the triplexes at pH 7.0, 2-aminopyridine nucleoside (Py)[48–50] was used instead of deoxycytidine in TFO-1 because the conditions must be acidic for the protonation of cytosine when a TFO containing deoxycytidine is used.[48] 170
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Figure 6. Sequences of hairpin oligonucleotides (HP-UBF and HP-UMBF) and TFO-1.
To confirm triplex formations, the melting temperatures (Tm ; transition from the triplex state to oligodeoxynucleotides) for HP-UBF or HP-UMBF with TFO-1 were measured: 33 8C for HP-UBF with TFO-1, and 28 8C for HP-UMBF with TFO-1 (Figure S2). These data indicate that both HP-UBF and HP-UMBF form triplexes with TFO-1. Next, the fluorescence spectra of HP-UBF and HP-UMBF were measured in the absence or presence of TFO-1. At 420 nm (lmax), the fluorescence intensity of HP-UBF increased 2.9-fold upon the addition of TFO-1, whereas at 420 nm, that of HPUMBF increased 16-fold. The greater enhancement of HP-UMBF can be attributed to the conformational effect (Figure 5) and the microenvironmental effect (Table 1). In terms of the conformational effects, it is expected that incorporation into DNA triplexes increases the fluorescence of both 3 a and 3 b by forcing planar conformations. In contrast, in terms of the microenvironmental effects, it is expected that incorporation into DNA triplex structures increases the fluorescence of 3 b, but reduces that of 3 a, because the fluorescence of 3 b increases in lesspolar organic solvents than in water, whereas that of 3 a decreases.[35] Thus, in terms of the fluorescence enhancement upon triplex formation, 3 b showed a larger enhancement than 3 a.
Figure 7. Fluorescence spectra of hairpin oligonucleotides: A) HP-UBF and B) HP-UMBF and their triplexes with TFO-1 (lex = 350 nm, 0.1 mm HP-UBF, HPUMBF, and TFO-1, 10 mm cacodylate buffer, 500 mm NaCl, 10 mm MgCl2, pH 7.0, 10 8C).
Structural evaluation of DNA triplex incorporating 3 b To investigate further the results based on the three-dimensional structure, we measured the 2D NMR data for ODN1, a DNA triplex incorporating 3 b based on a previously reported sequence (Figure 8).[51–52] The 3-methylbenzofuran of the UraMBF moiety is in a hydrophobic environment by stacking between T19 and T21, and the 3-methylbenzofuran and uracil rings of UMBF remain coplanar in this triplex (Figure 9). This indicates that both the solvatochromic effects and the conformational change of UraMBF contributed to the fluorescence enhancement. In addition to this structural consideration, the contribution of hydrophobic microenvironment was also indicated by the blue shift of the fluorescence of HP-UMBF (from 449 to 428 nm upon triplex formation; Figure 7).
Figure 8. Sequence of the DNA triplex (ODN1) used for the determination of the three-dimensional structure. EG: ethylene glycol linker, G + C: Hoogsteen base pair between guanine and protonated cytosine, A·T: Hoogsteen base pair between adenine and thymine.
Development of off/on fluorescence probes for singlestranded DNA or RNA Based on the above results, we used this triplex system as a fluorescence probe for single-stranded DNA or RNA. We used 3 b as a fluorescent nucleoside because its on/off ratio was higher than that of 3 a (Figure 7). We prepared target RNA, ChemBioChem 2015, 16, 167 – 176
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Figure 9. NMR-derived averaged structure of the DNA triplex (ODN1) incorporating UMBF (purple). For more details, see the Experimental Section, Table S2, and Figures S8, S9.
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Full Papers limit was estimated to be ~ 5.0 nm, which is comparable to those of other probes.[53] On the basis of the above results, a mixture of probes 1 and 2 can be used as a fluorescence probe for single-stranded DNA or RNA.
Application of off/on fluorescence probes to miRNA detection system by rolling-circle amplification
Figure 10. Sequences of oligonucleotides used for the fluorescence detection of single-stranded DNA or RNA.
By using probes 1 and 2, a smaller amount of RNA was detected along with a “padlock probe”, followed by RCA (Figure 12).[54] RCA is a convenient amplification method for the detection of nucleic acids because it can be performed under isothermal conditions.
target DNA, probe 1 incorporating 3 b, and probe 2, which was identical to TFO-1 (Figure 10). Target DNA and RNA oligonucleotides formed duplexes with probe 1 through Watson–Crick base pairing, and probe 2 formed Hoogsteen base pairs with probe 1 in the duplexes of probe 1 + target DNA, and probe 1 + target RNA. The fluorescence properties of this system are shown in Figure 11. The mixture of probes 1 and 2 emitted weak fluores-
Figure 12. Fluorescence detection of single-stranded RNA by using a padlock probe and RCA with probes 1 and 2.
We designed the following RCA systems. We chose an oncomiR,[55] miR-16 (5’-UAGCA GCACG UAAAU AUUGG CG-3’) as the target (Figure 12). We also prepared a padlock probe with 5’ and 3’ termini designed to bind to miR-16. A purine-rich 5’d(CAAAA AAGAT AGAAA C)-3’ sequence was also introduced into the padlock probe. After the binding of miR-16 to the padlock probe, the latter was ligated with T4 DNA ligase. The concatemeric DNA was synthesized from the 3’ terminus of miR-16 on the cyclized padlock probe by phi29 DNA polymerase. The concatemeric DNA contained multiple copies of a pyrimidine-rich triplex-forming site (3’-d(GTTTTTTCTATCTTTG)-5’), which is the target sequence of probes 1 and 2. After RCA, 1 and 2 were added, and the fluorescence was measured. As expected, a significant fluorescence enhancement was detected in the presence of 50 fmol of target RNA (Figure 13). This suggests that probes 1 and 2 formed triplexes with the pyrimidine-rich sites, and the triplex-induced fluorescence increase can be used as a fluorescence indicator of RCA.
Figure 11. Fluorescence spectra for the detection of target DNA and RNA and quantum yields (lex = 350 nm, 0.2 mm probes 1, 2, target DNA, target RNA, 10 mm cacodylate buffer, 500 mm NaCl, 10 mm MgCl2, pH 7.0, 10 8C).
cence because they do not form Hoogsteen base pairs in the absence of target DNA or RNA, as revealed by the UV melting experiments (Figure S3). In contrast, in the presence of target DNA, the signal at 429 nm (lmax) increased 32-fold (from F = 0.055 to 0.39). Similarly, in the presence of target RNA, the fluorescence signal at 428 nm (lmax) increased 22-fold (from F = 0.055 to 0.29). It should be noted that formation of the triplexes at 10 8C was confirmed by Tm analysis (Figure S3). We also studied the effect of a mismatched base pair at the counter position of the UMBF residue (target DNA strand) on fluorescence intensity (Figure S4). Interestingly, similarly to the matched base pair (A–UraMBF), strong fluorescence enhancements were observed for every mismatch. These fluorescence enhancements also arose from triplex formation (Figure S5) and the induced coplanarity of the 3-methylbenzofuran ring and the uracil moiety. Finally, to determine the detection limit of our probes, we measured fluorescence in the presence of low concentration of target DNA (0 ~ 10 nm). Our probes showed liner fluorescence responses to target concentrations (Figure S6). The detection ChemBioChem 2015, 16, 167 – 176
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Conclusion We have developed new triplex-induced fluorescence probes that incorporate UraBF or UraMBF. The 3-methylbenzofuran and uracil rings of UraMBF in single- or double-stranded oligonucleotides are twisted with respect to each other before formation of triplexes with single-stranded DNA/RNA. The spectroscopic 172
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Full Papers [D6]DMSO (39.5 ppm) and CDCl3 (77.16 ppm) for 13C NMR, and 85 % phosphoric acid (0.0 ppm) for 31P NMR. 2-(3-Methylbenzofuran-2-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (2 b): 3-methylbenzofuran (1.30 g, 9.80 mmol), 4,4’-di-tertbutyl-2,2’-dipyridyl (dtbbp), (40 mg, 0.15 mmol), bis(1,5-cycloocta(49 mg, diene)di-m-methoxydiiridium(I) ([Ir(OMe)(1,5-cod)]2), 0.074 mmol), and bis(pinacolato)diboron (2.49 g, 9.80 mmol) were placed in a round-bottomed flask under argon. Then 1,2-dimethoxyethane (23 mL) was added, and the reaction mixture was stirred overnight at 80 8C. The crude product was purified by C-200 silica gel chromatography with n-hexane-ethyl acetate to give 2 b (2.38 g, 94 %); 1H NMR ([D6]DMSO): d = 7.65 (d, J = 7.8 Hz, 1 H), 7.55 (d, J = 8.3 Hz, 1 H), 7.39 (dd, J = 7.6, 7.8 Hz, 1 H), 7.26 (dd, J = 7.6, 7.3 Hz, 1 H), 2.40 (s, 3 H), 1.31 ppm (s, 12 H); 13C NMR ([D6]DMSO): d = 156.5, 130.0, 128.6, 126.3, 122.4, 120.5,111.4, 84.0, 24.6, 8.7 ppm; ESI-MS: calcd for C15H19BNaO3 + : 281.1319 [M+Na] + ; found 281.1321.
Figure 13. Fluorescence spectra of the reaction mixture after ligation and RCA in the presence or absence of miR-16 (lex = 350 nm, 0.2 mm probes 1 and 2, 10 8C). See the Experimental Section for details.
and NMR studies indicated that the significant increase in the fluorescence upon triplex formation can be attributed to changes in the microenvironment of the 3-methylbenzofuran moiety and increased coplanarity between the 3-methylbenzofuran and uracil rings. We also demonstrated the applicability of this triplex-induced fluorescence probe to miRNA detection in conjunction with RCA. Because our system involves only a simple interaction between the fluorescent 5-aryluridine and the abasic site in TFO, the system can be combined with other fluorescent 5-arylpyrimidines[20–23, 28–34, 36–37, 56–58] with different fluorescent colors. In addition, previously reported fluorescent probes such as thiazole-modified probes,[10–17] which have different fluorescent emission wavelengths from our probe (lem ~ 420 nm), can be used in combination with our probes. Furthermore, because of flexibly in the design of sequences flanking the 5-arylpyrimidines (as long as they can form triplexes), the triplex-induced fluorescence system can be applied to multicolor multiplex RNA detection. Notably, the triplex-forming sites are not restricted to homopurine sequences. Previously, we reported an artificial DNA triplex system incorporating modified pyrimidines with hydrogen-bonding sites at position 5.[39] By using such modified pyrimidines in probe 1, triplex-forming sites of any mixed sequence can be designed. Moreover, this strategy can be combined with isothermal nucleic acid amplification technologies[59–60] other than RCA, provided they generate single-stranded DNA or RNA. Studies are in progress in our laboratory to develop these applications and new fluorescent nucleosides based on this strategy.
5-(Benzofuran-2-yl)deoxyuridine (3 a): 5-iododeoxyuridine (800 mg, 2.26 mmol), palladium acetate (15 mg, 0.068 mmol), TPPTS (102 mg, 0.18 mmol), Na2CO3 (527 mg, 4.97 mmol) and 2(benzofuran-2-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (1.10 g, 4.51 mmol) were placed in a round-bottomed flask under argon. Degassed H2O/CH3CN (2:1, v/v, 20 mL) was added, and the mixture was stirred at 45 8C for 13 h. The mixture was evaporated under reduced pressure. The residue was diluted with small amount of H2O/CH3CN (2:1, v/v). The precipitate was filtered to give 3 a (486 mg, 62 %); 1H NMR ([D6]DMSO): d = 11.7 (br s, 1 H), 8.74 (s, 1 H), 7.61–7.53 (m, 2 H), 7.33–7.20 (m, 3 H), 6.23 (t, J = 6.1 Hz, 1 H), 5.31– 5.24 (m, 2 H) 4.33 (s, 1 H), 3.88 (s, 1 H), 3.73–3.65 (m, 2 H), 2.28– 2.23 ppm (m, 2 H); 13C NMR ([D6]DMSO): d = 160.3, 153.0, 149.4, 149.1, 137.1, 128.8, 124.3, 123.0, 121.0, 110.8, 104.7, 103.9, 87.7, 85.1, 70.1, 60.9, 40.5 ppm; ESI-MS: calcd for C17H16N2NaO6 + : 367.0901 [M+Na] + ; found 367.0903. 5-(3-Methylbenzofuran-2-yl)deoxyuridine (3 b): 5-iododeoxyuridine (1.39 g, 3.92 mmol), palladium acetate (27 mg, 0.12 mmol), TPPTS (176 mg, 0.31 mmol), Na2CO3 (0.913 g, 8.62 mmol) and 2-(3methylbenzofuran-2-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane 2 b (2.02 g, 7.84 mmol) were placed in a round-bottomed flask under argon. Degassed H2O/CH3CN (2:1, v/v, 43 mL) was added, and the mixture was stirred for 16 h at 45 8C. The mixture was evaporated under reduced pressure. The crude product was purified by C-200 silica gel chromatography with dichloromethane-methanol to give 3 b (1.36 g, 97 %); 1H NMR ([D6]DMSO): d = 11.66 (s, 1 H), 8.34 (1, 1 H), 7.57 (d, J = 7.6 Hz, 1 H), 7.50 (d, J = 8.1, 1 H), 7.31–7.23 (m, 2 H), 6.21 (t, J = 6.6 Hz, 1 H), 5.28 (m, 1 H), 5.07 (m, 1 H), 4.26 (m, 1 H), 3.81 (m, 1 H), 3.58–3.55 (m, 2 H), 2.19–2.17 ppm (m, 5 H); 13C NMR ([D6]DMSO): d = 161.0, 153.5, 149.9, 145.3, 141.5, 129.8, 124.4, 122.5, 119.5, 113.5, 110.9, 104.9, 87.6, 84.8, 70.3, 61.0, 40.4, 8.6 ppm; ESI-MS: calcd for C18H18N2NaO6 + : 381.1057 [M+Na] + ; found 381.1054.
Experimental Section
5-(Benzofuran-2-yl)-5’-O-(4,4’-dimethoxytrityl)deoxyuridine (4 a): 3 a (200 mg, 0.58 mmol) was coevaporated three times with pyridine and dissolved in pyridine (2.9 mL). 4, 4’-Dimethoxytrityl chloride (288 mg, 0.64 mmol) was added to the reaction mixture and stirred for 3 h at room temperature. After the completion of the reaction, methanol (1 mL) was added and the reaction mixture was evaporated. The crude product was extracted with water/dichloromethane and dichloromethane layer was collected and evaporated. Crude product was purified by C-200 silica gel chromatography with dichloromethane/methanol/0.5 % pyridine to give 4 a (310 mg, 83 %); 1H NMR ([D6]DMSO): d = 11.85 (br s, 1 H), 8.29 (s,
Materials: Reagents for DNA synthesis and unmodified DNA phosphoramidite units were purchased from Glen Research (Sterling, VA). Sep-Pak C18 cartridges for DNA purification were purchased from Waters (Milford, MA). Other reagents for the synthesis of phosphoramidite monomers were purchased from Tokyo Chemical Industry (Tokyo, Japan) and Sigma–Aldrich. DNA and RNA were purchased from Sigma–Aldrich. Dry solvents were purchased and stored over molecular sieves (4 ). 1H, 13C, and 31P NMR spectra were obtained at 500, 126, and 203 MHz, respectively. The chemical shifts were measured from [D6]DMSO (2.49 ppm) for 1H NMR, ChemBioChem 2015, 16, 167 – 176
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Full Papers 1 H), 7.55 (d, J = 7.6 Hz, 1 H), 7.44 (d, J = 7.6 Hz, 2 H), 7.31–7.29 (m, 5 H), 7.23–7.20 (m, 2 H), 7.14–7.09 (m, 2 H), 7.05–7.02 (m, 1 H), 6.77– 6.75 (m, 4 H), 6.51 (d, J = 8.3 Hz, 1 H), 6.21 (t, J = 6.5 Hz, 1 H), 5.35– 5.34 (m, 1 H), 4.21 (br s, 1 H), 3.99–3.97 (m, 1 H), 3.59–3.58 (m, 6 H), 3.36–3.34 (m, 1 H), 3.19–3.16 (m, 1 H), 2.30–2.28 ppm (m, 2 H); 13 C NMR (CDCl3): d = 160.6, 158.8, 153.8, 149.9, 147.8, 144.8, 136.0, 135.9, 135.4, 130.4, 130.3, 129.2, 128.5, 128.2, 127.2, 124.5, 123.0, 121.2, 113.5, 111.1, 107.3, 106.1, 87.1, 86.8, 86.0, 72.5, 63.7, 55.4, 41.7 ppm; ESI-MS: calcd for C38H34N2NaO8 + : 669.2207 [M+Na] + ; found 669.2208.
1.46 mmol) in dichloromethane (18 mL). The solution was stirred for 10 h at room temperature under argon. The reaction was diluted with dichloromethane and water, and extracted with dichloromethane. The combined organic layers were washed with water and brine, dried over MgSO4, and concentrated under reduced pressure. The crude product was purified by NH-silica gel chromatography with n-hexane/ethyl acetate to give 5 b (1.31 g, 84 %); 1 H NMR ([D6]DMSO): d = 11.75 (s, 1 H), 7.97–7.96 (m, 1 H), 7.52–7.50 (m, 1 H), 7.32–7.31 (m, 2 H), 7.21–7.16 (m, 8 H), 7.12–7.07 (m, 2 H), 6.72–6.71 (m, 4 H), 6.22–6.19 (m, 1 H), 4.50–4.44 (m, 1 H), 4.11–4.06 (m, 1 H), 3.76–3.44 (m, 10 H), 3.28–3.21 (m, 2 H), 2.76 (m, 1 H), 2.64 (m, 1 H), 2.47–2.36 (m, 2 H), 2.06 (s, 3 H), 1.17–1.09 (m, 9 H), 0.99– 0.97 ppm (m, 3 H); 13C NMR (CDCl3): d = 160.8, 158.7, 154.2, 150.0, 149.9, 144.5, 143.3, 139.9, 135.7, 135.6, 135.5, 130.8, 130.3, 130.2, 128.4, 128.3, 128.1, 127.1, 124.4, 122.3, 119.4, 117.9, 117.7, 115.3, 113.3, 111.2, 107.7, 87.0, 86.3, 86.1, 86.0, 74.2, 74.1, 73.8, 73.6, 63.4, 63.2, 58.7, 58.6, 58.4, 55.4, 43.6, 43.5, 43.4, 41.0, 25.0, 24.9, 24.8, 20.7, 20.5, 9.4 ppm; 31P NMR ([D6]DMSO): d = 148.6, 148.3 ppm; ESIMS: calcd for C48H53N4NaO9P + : 883.3442 [M+Na] + ; found 883.3434.
5-(3-Methylbenzofuran-2-yl)-5’-O-(4,4’-dimethoxytrityl)deoxyuridine (4 b): 3 b (1.03 g, 2.87 mmol) was coevaporated three times with pyridine and dissolved in pyridine (29 mL). 4, 4’-Dimethoxytrityl chloride (1.07 g, 3.16 mmol) was added to the reaction mixture and stirred for 6 h at room temperature. After completion of the reaction, methanol (1 mL) was added, and the reaction mixture was evaporated. The crude product was extracted with water/dichloromethane, and the dichloromethane layer was collected and evaporated. The crude product was purified by C-200 silica gel chromatography with dichloromethane/methanol/pyridine (0.5 %) to give 4 b (1.54 g, 81 %); 1H NMR ([D6]DMSO): d = 11.7 (s, 1 H), 7.90 (s, 1 H), 7.51–7.49 (m, 1 H), 7.30–7.29 (m, 2 H), 7.22–7.05 (m, 9 H), 6.71–6.69 (m, 4 H), 6.19 (t, J = 6.6 Hz, 1 H), 5.36–5.34 (m, 1 H), 4.25 (m, 1 H), 3.97–3.94 (m, 1 H), 3.64–3.63 (m, 6 H), 3.18–3.13 (m, 2 H), 2.29–2.27 (m, 1 H), 2.03 ppm (s, 3 H); 13C NMR ([D6]DMSO): d = 160.8, 158.0, 157.9, 153.3, 149.8, 144.6, 144.5, 140.5, 135.5, 135.1, 129.8, 129.6, 129.5, 127.7, 127.5, 126.5, 124.2, 122.3, 119.3, 113.7, 113.0, 110.6, 105.2, 85.9, 85.8, 85.3, 70.5, 63.7, 54.9, 8.6 ppm; ESIMS: calcd for C39H36N2NaO8 + : 683.2364 [M+Na] + ; found 683.2378.
Synthesis and purification of oligonucleotides: DNA was synthesized in an ABI 392 DNA/RNA synthesizer (Applied Biosystems) with phosphoramidite chemistry. Oligonucleotides were purified in Sep-pak C18 cartridges with reverse-phase high performance liquid chromatography (HPLC) at 30 8C with a linear gradient of solvent I (NH4OAc (30 mm)) and solvent II (acetonitrile) at a flow rate of 1.0 mL min1 for 30 min. MALDI-TOF mass data of oligonucleotides: HP-UBF : [M+H] + Calcd 11134.89, found 11140.93; HP-UMBF : [M+H] + calcd 11148.90, found 11153.97; probe 1: [M+H] + calcd 5038.96, found 5039.94; probe 2 (TFO-Py): [M+H] + calcd 3965.68, found 3966.62; ODN-1: [M+H] + calcd 7918.48, found 7923.50.
5-(Benzofuran-2-yl)-5’-O-(4,4’-dimethoxytrityl)deoxyuridine 3’-(2cyanoethyl N,N-diisopropylphosphoramidite) (5 a): Compound 4 a (230 mg, 0.36 mmol) was coevaporated with anhydrous pyridine. 2-Cyanoethyl N,N,N’, N’-tetraisopropylphosphoramidite (160 mL, 0.50 mmol) was added to a stirred solution of 4 a, diisopropylamine (30 mL, 0.22 mmol), and 1H-tetrazole (15 mg, 0.22 mmol) in dichloromethane (3.6 mL). The solution was stirred overnight at room temperature under argon. The reaction was diluted with dichloromethane and water, and extracted with dichloromethane. The combined organic layers were washed with water and brine, dried over MgSO4, and concentrated under reduced pressure. The crude product was purified by C-200 silica gel chromatography with n-hexane/ethyl acetate/pyridine (0.5 %) to give 5 a (180 mg, 59 %); 1H NMR ([D6]DMSO): d = 11.88 (br s, 1 H), 8.35–8.33 (m, 1 H), 7.56–7.55 (m, 1 H), 7.46–7.44 (m, 2 H), 7.33–7.10 (m, 9 H), 7.05–7.03 (m, 1 H), 6.77–6.74 (m, 4 H), 6.55–6.48 (m, 1 H), 6.24–6.20 (m, 1 H), 4.47 (m, 1 H), 4.15–4.10 (m, 1 H), 3.75–3.68 (m, 1 H), , 3.60–3.57 (m, 6 H), 3.57–3.37 (m, 4 H), 3.22–3.19 (m, 1 H), 2.77–2.75 (m, 1 H), 2.65– 2.63 (m, 1 H), 2.49–2.40 (m, 2 H), 1.16–1.10 (m, 9 H), 0.98 ppm (m, 3 H); 13C NMR (CDCl3): d = 160.7, 160.6, 158.8, 153.7, 149.7, 149.6, 147.8, 144.8, 136.1, 136.0, 135.9, 135.8, 135.4, 130.4, 130.4, 129.2, 128.6, 128.5, 128.2, 127.2, 124.4, 122.9, 121.1, 117.9, 117.7, 113.4, 111.1, 107.2, 106.1, 87.0, 86.3, 86.0, 85.9, 73.9, 73.8, 73.5, 73.4, 63.3, 63.1, 58.7, 58.6, 58.5, 58.4, 55.4, 43.6, 43.5, 43.4, 40.9, 24.9, 24.8, 20.7, 20.5, 20.4 ppm. 31P NMR ([D6]DMSO): d = 148.6, 148.3 ppm; ESI-MS: calcd for C47H51N4NaO9P + : 869.3286 [M+Na] + ; found 869.3289.
Quantum yield measurement: Quantum yields were determined using 2-aminopurine riboside[4, 61] in water (lex = 303 nm, F = 0.68) or quinine[62] in H2SO4 (0.1 n; lex = 366 nm, F = 0.53) as reference. The quantum yields were calculated according to Equation (1): FFðSÞ AðRÞ F ðSÞ ¼ FFðRÞ AðSÞ F ðRÞ
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nðSÞ nðRÞ
2 ð1Þ
where FF(S) and FF(R) are the fluorescence quantum yields of the sample and the reference, respectively; F(S) and F(R) are the area under emission curve of the sample and the reference solution, respectively; A(S) and A(R) are the respective optical density of the sample and the reference solution at the wavelength of excitation, and n(S) and n(R) are the values of the refractive index for the respective solvents. 2-Aminopurine riboside was used as the reference for the quantum yield measurement of dUMBF monomer; quinine was used for single-strand or triplex DNA containing dUMBF (Table 1). Exited-state lifetime measurements: Excited-state lifetime measurements were performed with a FluoroCube spectrometer (HORIBA Jobin Yvon, Kyoto, Japan). For the measurements of nucleoside 3 b (Table 1), a 280 nm pulse laser was used. Emission signals were acquired at lmax. Obtained decay profiles were fitted to the theoretical equation to determine lifetimes by using DAS6 software (HORIBA). Radiative (kr) and nonradiative (knr) decay constants were determined from quantum yield (F) and average excitedstate lifetime (tave) with Equations (2) and (3):[63]
5-(3-Methylbenzofuran-2-yl)-5’-O-(4,4’-dimethoxytrityl)deoxyuridine 3’-(2-cyanoethyl N,N-diisopropylphosphoramidite) (5 b): Compound 4 b (1.20 g, 1.82 mmol) was coevaporated with anhydrous pyridine. 2-cyanoethyl N, N, N’, N’-tetraisopropylphosphoramidite (864 mL, 2.72 mmol) was added to a stirred solution of 4 b, diisopropylamine (206 mL, 1.46 mmol), and 1H-tetrazole (102 mg,
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k r ¼ F=tave
ð2Þ
1=tave ¼ kr þ knr
ð3Þ
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Full Papers used for further analysis. MOLMOL[66] was used to visualize the final structures.
Enzymatic ligation of the padlock probe: miR-16 (0, 50, or 1000 fmol), padlock probe (5 mm), and ATP (10 mm) were dissolved in ligation buffer (5 mL, Tris·HCl (10 mm, pH 7.5), MgCl2 (10 mm)) and incubated at 65 8C for 3 min. Subsequently, the reaction tubes were cooled to room temperature (20 8C) and kept for 10 min. T4 DNA ligase (200 U) was added, and the mixture was incubated at 37 8C for 2 h. After the ligation, ligase was deactivated by heating at 65 8C for 10 min.
Acknowledgements We thank Prof. Masayuki Takeuchi (National Institute for Materials Science, NIMS) and Prof. Kazunori Sugiyasu (NIMS) for the excited-state lifetime measurements and valuable discussions. This work was supported by JSPS KAKENHI, and Adaptable and Seamless Technology Transfer Program through target-driven R&D, JST. This work was also supported by Strategic Young Researcher Overseas Visits Program for Accelerating Brain Circulation, JSPS.
Rolling circle amplification: After the ligation, the reaction mixtures (5 mL) were added to Tris·HCl (95 mL, 50 mm, pH 7.5) containing MgCl2 (10 mm), (NH4)2SO4 (10 mm), dithiothreitol (4 mm), phi29 DNA polymerase (10 U), and dNTPs (0.2 mm). The reaction mixtures were incubated at 30 8C for 8 h then heated to 65 8C for 10 min to inactivate the polymerase. Fluorescence measurements of RCA products: Reaction mixtures were diluted in cacodylate buffer (400 mL) containing probe 1 and 2; final concentrations: probe 1 and 2 (0.2 mm), cacodylate buffer (10 mm, pH 7.0), NaCl (500 mm), MgCl2 (10 mm). Before the fluorescence measurements, the reaction mixtures were heated at 85 8C for 5 min and then gradually cooled to 10 8C. The spectra were measured at 10 8C.
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Received: July 1, 2014 Published online on December 2, 2014
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