DNA photo-cross-linking using a pyranocarbazole-modified oligodeoxynucleotide with a d-threoninol linker

An alternative photo-cross-linker having a d-threoninol skeleton instead of the 2′-deoxyribose backbone in 3-cyanovinylcarbazole (CNVK) was investigated to improve the photoreactivity of photo-cross-linkers; the photo-cross-linking rate of 3-cyanovinylcarbazole with d-threoninol (CNVD) was found to be greater than that of CNVK. Therefore, in this study, a novel photo-cross-linker having pyranocarbazole (PCX) and d-threoninol instead of the 2′-deoxyribose backbone in PCX (PCXD) was developed. The PCXD in double-stranded DNA photo-cross-linked to a pyrimidine base at the −1 position of a complementary strand similar to PCX. Furthermore, the photoreactivity of PCXD was significantly higher than that of PCX. The introduction of d-threoninol improved the reactivity of pyranocarbazole to cytosine, the use of PCXD may extend the applicability of the photo-cross-linking reaction for DNA manipulation. In particular, this novel photo-cross-linker can contribute to the photochemical regulation of gene expression or biological events in a living cell.


Introduction
Photo-cross-linking reactions between biomolecules are used for various applications such as screening antigen interactions 1 and improving detection sensitivity 2 and the stability of biomolecular complexes. 3 In particular, the use of DNA photocross-linking in the formation of a thymine dimer induced by UV-irradiation 4 and interstrand photo-cross-linking with psoralen 5 has been reported. In recent years, with the development of nucleic acid medicine and DNA nanotechnology, these have been used for the photochemical regulation of an antisense effect, 6 improvement in stability of a DNA nanostructure, 7 and other applications. Many photo-cross-linkers such as bromouracil 8 and benzophenone 9 have also been reported. Among these, DNA photo-cross-linking via [2 + 2] photocycloaddition by psoralens 5 and coumarins 10 is useful for detection, manipulation, and regulation of nucleic acids because photoreversible manipulation is possible. Compared to the enzymatic method, photo-cross-linking can be used under a wide range of conditions without the addition of reagents. Although the intracellular usage of photo-cross-linking has already been reported, these require UV-irradiation, which pose a limitation for use in living cells because of phototoxicity. We report pyranocarbazole ( PC X) as a photo-cross-linker that can photo-crosslink to pyrimidine in complementary DNA or RNA strand under visible light. 11 It was anticipated that his photo-cross-linker would accelerate the intracellular application of nucleic acid photo-cross-linking such as photochemical regulation of gene expression 12 and detection of RNA strand; 13 however, photocross-linking using PC X to cytosine requires photoirradiation for 1 min, and it is necessary to speed up this process. Besides, the ribose backbone, D-threoninol backbone, 14 and serinol backbone 15 have been reported as the backbone of the articial nucleic acid. It was determined that 3-cyanovinylcarbazole modied D-threoninol ( CNV D) 16 considerably accelerated the photo-cross-linking reaction with cytosine using the D-threoninol backbone ( PCX D) (Fig. 1). Results and discussion PCX D was successfully synthesized according to the reaction scheme shown in Fig. 2. The PCX D was phosphoramidited using a general method 17 aer 5 0 DMTr protection to synthesize the oligodeoxynucleotide (ODN) containing PCX D. The 9 mer ODN which has A, T, G, C having deoxyribose and PCX D having Dthreoninol was synthesized with an automated DNA synthesis machine, and then, it was deprotected using 28% ammonium solution with a general method. Aer HPLC purication, it was analyzed using Matrix assisted Laser Desorption/Ionization (MALDI) analysis (Table 1).
First, we demonstrated the photo-cross-linking of PCX D in double-stranded DNA. The duplex containing PCX D was irradiated with 400 nm and analyzed using ultrahigh-performance liquid chromatography (UPLC). In the case of the pyrimidine base at the À1 position on the complementary ODN (cODN) strand, new peak identical to the photodimer appeared aer photoirradiation, although such new peak did not appear in the case of the purine base at the À1 position on the cODN strand. The new peak was identied to photoadduct of PCXD-ODN(C) and its complementary strand (found ¼ 5591.2, calcd [M + H] + ¼ 5591.0) by MALDI-TOF-MS analysis. This suggests that the photo-cross-linking reaction occurred only in the case of the pyrimidine base at the À1 position on the cODN strand.
To analyze the photoreactivity of PCX D, the time course of the photoreaction was monitored, and then, the reaction rate constant was analyzed with the assumption of rst-order reaction kinetics. As shown in Fig. 3, the reaction rate constant of PCX D with cytosine is 4.3-fold larger than that of PC X, suggesting that the relatively exible D-threoninol skeleton elevates the accessibility of the reactive double bond to the cytosine base on the complementary strand. The same effect was observed in the case of thymine as the target base of PCX D; the reaction rate constant was 1.1-folds larger than that of PC X. The difference in the reactivity between the thymine and the cytosine bases decreased and the photoreactivity enhanced compared with PC X, which indicates that the use of PCX D extends the applicability of the photo-cross-linking reaction for DNA manipulation.
To evaluate the duplex stability of ODN( PCX D), the melting temperature (T m ) of the duplex consisting of ODN(A PCX D) and ODN(GT) was evaluated and compared with the duplex including PC X. The sigmoidal melting curve of ODN( PCX D)/ ODN(T) duplex was observed, which suggests ODN( PCX D) and cODN(GT) formed a duplex structure. Then, we calculated the thermodynamic parameter from van't Hoff plots. 18 The DS through ODN( PCX D)/ODN(T) hybridization was smaller than that of ODN( PC X)/ODN(T), suggesting that a relatively exibility structure of PCX D inhibited the entropic loss via hybridization (Table 2). Therefore, PCX D has a higher exibility than PC X, and it induces a higher photo-cross-linking ability in cytosine than PC X. In addition, PC X and PCX D containing pyranocarbazole moieties have lower T m values compared to CNV K and CNV D containing 3-cyanovinylcarbazole moiety. When the polarity of pyranocarbazole (log P ¼ 1.12) and 3-cyanovinylcarbazole (log P ¼ 1.35) was examined, 19 it was found that pyranocarbazole has   a smaller log P and is hydrophilic. Therefore, it is considered that the T m value of the duplex containing 3-cyanovinylcarbazole is high and the entropy loss is large because of the stacking between bases and hydrophobic interactions.
To evaluate the effects of the surrounding bases on the photo-cross-linking of ODNs containing PCX D, double strand with all variations of base pairs at the À1, +1 position of PCX D and four different bases on the counter position to PCX D in cODN were prepared; the conversion aer 10 s of 400 nm irradiation was evaluated by UPLC. As shown in Fig. 4, in the case of T at the Z position in cODN, over 90% of the photo-cross-linking was obtained with 400 nm irradiation for 10 s. In the case of C at the Z position, photo-cross-linking was observed. However, in the case of A or G at the Z position, photo-cross-linking was not observed. These results indicate that the photo-cross-linking of PCX D has similar pyrimidine selectivity as those of CNV K, CNV D, and PC X. In addition, some differences in the reactivity were not observed among these duplexes, which indicated that the mechanism of effects such as local duplex stability and local electrostatic environment around the pyranocarbazole moiety remained does not affect the photo-cross-linking.

Experimental
General 1 H NMR spectra were measured on a Bruker AVANCE III 400 system. Mass spectra were recorded on a Voyager PRO-SF, Applied Biosystems and HRMS were measured on a Solarix-JA (Bruker) HPLC was performed on a Chemcosorb 5-ODS-H column with JASCO PU-980, HG-980-31, DG-980-50 system equipped with a JASCO UV 970 detector at 260 nm. Reagents for synthesis for compound 1 to 5 were purchase from Aldrich, Wako, and TCI. Phosphoramidites of PCX D were synthesized by the procedure as follows. Other phosphoramidite reagents for the DNA sysnthesizer such as A, G, C, T-b-cyanoethyl phosphoramidite, and CPG support were purchased form Glen research. Other oligonucleotides were purchased from Fasmac (Japan) and used without farther purication. The synthesized ODN having deoxyribose backbone except PCX D.

Thermodynamic analysis of the hybridization
Thermodynamic parameters were obtained according to a method in the literature: where TM is the melting temperature of duplex, DS is entropy and DH is the entropy of duplex formation, respectively. R is the gas constant and CT is the total strand concentration. TM was measured at various concentrations of duplex in 50 mM Na-cacodylate buffer (pH 7.4) containing 100 mM NaCl by a spectrophotometer (V-630bio, Jasco) equipped with a temperature controller.

Conclusions
The PCX D in double-stranded DNA photo-cross-linked to the pyrimidine base at the À1 position of the complementary strand was similar to that for PC X, and the photoreactivity of PCX D was signicantly higher than that of PC X. The photo-crosslinking rate of PCX D having D-threoninol and cytosine was faster than that of PC X and cytosine, the applicability of the photocross-linking reaction for DNA manipulation may be expanded by the use of PCX D. We have already reported the importance of photo-cross-linking rate in photochemical antisense method in living cell and RNA FISH to E. coli 16S rRNA with a secondary structure. Therefore, this novel photo-crosslinker would contribute to the photoregulation of gene expression or biological events in living cells.

Conflicts of interest
There are no conicts to declare.