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Int. J. Mol. Sci. 2013, 14(3), 5765-5774; doi:10.3390/ijms14035765
Abstract: Selective photocrosslinking reaction between 3-cyanovinylcarbazole nucleoside (CNVK) and 5-methylcytosine (mC), which is known as epigenetic modification in genomic DNA, was developed. The reaction was completely finished within 5 s of 366 nm irradiation, and the rate of this photocrosslinking reaction was ca. 30-fold higher than that in the case of unmodified normal cytosine. There were no significant differences in the thermodynamic parameters and the kinetics of hybrid formation of oligonucleotide (ODN) containing CNVK and its complementary ODN containing C or mC at the photocrosslinking site, and suggesting that the quick and selective photoreaction has potential for the selective detection of mC in the DNA strand via the photocrosslinking reaction.
5-Methylcytosine (mC) is the most abundant epigenetic modification in genomic DNA and plays a role in the epigenetic regulation of gene expression, genomic imprinting, cell differentiation, and tumorigenesis [1,2]. Therefore, the development of methodology for the detection of mC in genomic DNA is required. Various methods have been developed for detecting mC based on chemical and enzymatic concepts [3–5]. We previously reported on photoresponsive synthetic oligonucleotides (ODN(s)) containing 5-vinyl-2′-deoxyuridine (VU) derivatives [6–10] that can photoligate to target DNA via [2 + 2] photocycloaddition with a pyrimidine base in target DNA. In particular, VU derivatives having a hydrophobic group, such as cyano , cyclohexyl , cyclopentyl , and aromatic group  were selectively photoligated to ODNs containing mC because of the hydrophobic interaction between the 5-methyl group on target mC and hydrophobic moiety tethered to VU. However, the photoreactivity of these VU derivatives was low, and the photoreaction requires long photoirradiation time, approximately 10 min.
On the other hand, we previously reported that ODNs having 3-cyanovinylcarbazole nucleoside (CNVK) can photocrosslink to complementary DNA strand via [2 + 2] photocycloaddition between CNVK and pyrimidine base in complementary strand with 1 s of 366 nm irradiation [14–18]. If the rapid photocrosslinking reaction of CNVK had the selectivity for mC like VU derivatives having hydrophobic group, rapid and selective detection of mC in the target DNA sequence will be possible. In addition, such a quick and selective photocrosslinking reaction might be a basic reaction for in situmC mapping and imaging in genomic DNA.
In this manuscript, we describe the mC selective photocrosslinking reaction of ODNs containing CNVK and also describe what causes the selective photocrosslinking reaction from the viewpoint of the difference of thermodynamic, kinetic and electronic properties of the ODN duplex containing CNVK and mC or C.
2. Results and Discussion
2.2. Thermodynamics and Kinetics of the Hybridization between CNVK-ODN and ODN(mC)
To reveal the reason why the CNVK-ODN has selectivity for ODN(mC), at first, the melting profiles of the duplexes consisting of CNVK-ODN and ODN(mC) or ODN(C) were measured. As shown in Figure 3a and Table 2, no significant difference in the melting temperature (TM) of each duplexes was observed, although in the case of CNVK-ODN/ODN(C), sharper transition compared to CNVK-ODN/ODN(mC) was observed. This suggests that there are entropic differences between the hybridization of each duplex. To evaluate the thermodynamic parameters of these duplexes, van’t-Hoff experiments were performed according to a method in the literature  (Figure 3b, Table 2). The ΔG°37 of these duplexes were similar, indicating that the stability of these duplexes is not different from each other. In general, the C5 methylation of cytosine causes stabilization of DNA duplex (ΔΔG°37 = 0.5 kcal/mol) because of the increase of the hydrophobic stacking interaction with neighboring bases , however, in our case, such a stabilization effect was not observed. As the 3-cyanovinylcarbazole moiety in the CNVK-ODN/ODN(mC) duplex disturbs the stacking interaction around the mC (Figure 4), it seems that the stabilization effect caused by the hydrophobicity of the mC was cancelled and was not observed in our case. On the other hand, ΔH° and ΔS° of CNVK-ODN/ODN(C) were smaller than that in the case of CNVK-ODN/ODN(mC) (19% and 23%, respectively), however, the selective photoreaction of CNVK toward mC could not be explained clearly from the thermodynamic study. Kinetic experiments for the hybridization of CNVK-ODN/ODN(C) and CNVK-ODN/ODN(mC) were also conducted by surface plasmon resonance experiments. Unfortunately, no significant difference between CNVK-ODN/ODN(C) and CNVK-ODN/ODN(mC) was observed (Table 3). We concluded that the thermodynamic and also kinetic properties of the duplex formation were not major reasons for the selective photocrosslinking reaction for mC.
2.3. Extinction Coefficient of CNVK in DNA Duplex
To clear the reasons for the selective photocrosslinking reaction for mC, finally, we performed a UV titration experiment of the ODN(C) or ODN(mC) to the CNVK-ODN. Absorbance of 3-cyanovinylcarbazole moiety (366 nm) was monitored at various concentrations of ODN(C) or ODN(mC). As shown in Figure 5, in the case of ODN(C), absorbance at 366 nm was decreased with the increase of the complementary strand, however, absorbance at 366 nm was increased and the change was saturated at the equimolar concentration in the case of ODN(mC), suggesting that the electronic state of the 3-cyanovinylcarbazole moiety was different between CNVK-ODN/ODN(C) and CNVK-ODN/ODN(mC) duplexes. The difference in the extinction coefficient of CNVK in each duplex means that the excitation yield of the CNVK was different between the case of each duplex. Since the CNVK-ODN/ODN(mC) duplex has the higher extinction coefficient compared to CNVK-ODN/ODN(C) duplex, the selectivity of the photocrosslinking reaction for mC can clearly be explained by this phenomenon. Moreover, in the case of CNVK-ODN(A), the T50 of the photocrosslinking reaction with ODN(T) was ca. 40-fold lower than that of CNVK-ODN/ODN(C) (Table 1) and the absorbance at 366 nm was increased 20% by the addition of ODN(T), suggesting that the high reactivity observed in the photocrosslinking with T and mC can partly explained by the excitation yield of the CNVK in DNA double strand.
3. Experimental Section
3.1. Preparation of ODNs
ODN sequences were synthesized by the conventional phosphoramidite method by using an Applied Biosystems 3400 DNA synthesizer. The coupling efficiency was monitored with a trityl monitor. The coupling efficiency of crude mixture of CNVK was 97% yield. The coupling time for CNVK was 999 s. They were deprotected by incubation with 28% ammonia for 4 h at 65 °C and were purified on a InertSustain™ C18 column (GL Science, 5 μm, 10 × 150 mm) by reverse phase HPLC; elution was with 0.05 M ammonium formate containing 3%–20% CH3CN, linear gradient (30 min) at a flow rate of 3.0 mL/min. Preparation of oligonucleotides was confirmed by MALDI-TOF-MS analysis: CNVK-ODN [(M + H)+], calcd. 2812.53, found 2812.20, CNVK-ODN(A) [(M + H)+], calcd. 2796.53, found 2795.41, ODN(C) [(M + H)+], calcd. 2732.52, found 2732.93, ODN(mC) [(M + H)+], calcd. 2746.54, found 2746.93, ODN(U) [(M + H)+], calcd. 2733.51, found 2733.96, ODN(T) [(M + H)+], calcd. 2747.52, found 2747.94.
3.2. Photoirradiation and UPLC Analysis
To avoid the absorption saturation, i.e., inner filter effect, the optical density of the sample at 366 nm was kept below 0.1. Photoirradiation was performed with an LED lamp (ZUV, 366 nm, 1600 mW/cm2, Omron Corporation, Kyoto, Japan) and a transilluminator (312 nm, Funakoshi) on an aluminum block incubator or water bath. The photoirradiated samples were analyzed with a UPLC system (Aquity, Waters, Milford, MA, USA) equipped with BEH Shield RP18 column (1.7 μm, 2.1 × 50 mm, elution was with 0.05 M ammonium formate containing 1%–10% CH3CN, linear gradient (10 min) at a flow rate of 0.4 mL/min, 60 °C).
3.3. Enzymatic Digestion, HPLC and MALDI-TOF-MS Analysis
The enzymatic digestion was carried out with the treatment of snake venom phosphodiesterase (0.2 U), P1 nuclease (1 U) and calf intestine alkaline phosphatase (20 U) in 50 mM Tris-HCl buffer (pH 9.0) containing 1 mM MgCl2, 0.1 mM ZnCl2 and 1 mM Spermidin at 37 °C for 5 h. After the purification of the photoadducts by reversed phase HPLC with InertSustain™ C18 (GL Science, 5 μm, 10 × 150 mm, elution was with 0.05 M ammonium formate containing 1%–50% CH3CN, linear gradient (50 min) at a flow rate of 3 mL/min), the molecular masses were analyzed with a MALDI-TOF-Mass spectrometer (Voyager DE-Pro-SF, Applied Biosystems, Foster City, CA, USA).
3.4. Thermodynamic and Kinetic Analysis of the Hybridization
Thermodynamic parameters were obtained by the following equations according to a method in the literature:
where TM is the melting temperature of duplex, ΔS° is entropy and ΔH° is the entharpy of duplex formation, respectively. R is the gas constant and CT is the total strand concentration. TM was measured at various concentrations of duplex (0.5–16 μM in 50 mM Na-cacodylate buffer (pH 7.4) containing 100 mM NaCl) by a spectrophotometer (V-630bio, Jasco, Tokyo, Japan) equipped with a temperature controller.
Kinetic parameters of the hybridization were collected by a surface plasmon resonance biosensor (BIAcore J, GE Healthcare, Buckinghamshire, UK) with avidine chips that having biotine-modified CNVK-ODN on the surface. After the injection of various concentrations of ODN(C) or ODN(mC), the sensorgrams were collected. (20 °C, 10 mM HEPES buffer (pH 7.4), 0.15 M NaCl, 3 mM EDTA, 0.005% Tween-20).
In this study, we found that ODN having CNVK can rapidly photocrosslink to mC in a complementary DNA strand and that the photocrosslinking reaction has a C5 methylation selective manner. As the photocrosslinking reaction was finished within 5 s of 366 nm irradiation, the reaction has the potential to become a key reaction for the selective detection of mC in the DNA strand and for the site specific induction of mC to T mutation. Thermodynamic and kinetic study revealed that the selectivity for mC of this photocrosslinking reaction is not caused by thermodynamics or kinetics of the hybridization. It seems that the conformational difference around the mC and C affects the selectivity of the quick, reversible photocrosslinking reaction. Based on the selective photoreaction toward mC, selective detection on genomic DNA would be possible with in situ hybridization technique using fluorescence- or biotin- labeled ODN having CNVK.
This study was partly supported by the Grant-in-Aid for Scientific Research (B) (90293894, K.F.) of The Ministry of Education, Science, Sports and Culture of Japan.
Conflict of Interest
The authors declare no conflict of interest.
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|Duplex||ΔG°37 kcal/mol||ΔH° kcal/mol||ΔS° cal/mol K||TM °C|
|CNVK-ODN/ODN(C)||−7.7||−49.8||−135||37.9 ± 0.9|
|CNVK-ODN/ODN(mC)||−7.6||−41.8||−110||37.4 ± 0.7|
|Duplex||KD (μM)||ka (×103 s−1 M−1)||kd (s−1)|
|CNVK-ODN/ODN(C)||2.4 ± 0.14||48||0.11|
|CNVK-ODN/ODN(mC)||2.2 ± 0.05||52||0.11|
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