Keywords

1 Introduction

Inter-strand photo-cross-linking of nucleic acids is one of the most useful technologies for regulating nucleic acid functions, such as transcription and replication of the genome, gene silencing activity of microRNAs, and translational activity of mRNAs. The sequence specific and thermally irreversible covalent bond formation between the nucleic acid strand and artificial photo-cross-linkable oligonucelotide caused by the photo-cross-linking reaction enables us to regulate sequence-specifically and spatiotemporally the target nucleic acid function.

As the pioneering works in this field, in the late 1980s, Miller and co-workers reported psoralen-modified oligonucleotide (Fig. 1, left) as the first photo-cross-linkable synthetic oligonucleotide [1, 15, 24]. They introduced trioxalen, a natural DNA photo-cross-linker that can be obtained from plants, at the 5′ end of synthetic oligodeoxyribonucleotides (ODN(s)), and clearly demonstrated that the ODNs cross-linked with single-stranded DNA and double-stranded DNA [14] under UVA (365 nm) irradiation. The findings led to the study on photoresponsive synthetic oligonucleotides. In the late 2000s, advanced-type ODNs having psoralen-modified adenosine were reported by Murakami and co-workers (Fig. 1, center) [11, 13]. Due to the restricted and favorable position of psoralen moiety in double-stranded nucleic acid consisting of the ODN and the complementary nucleic acid, these ODNs have higher photoreactivity compared to 5′ psoralen-modified ODNs. The psoralen-modified ODNs can photo-cross-link with not only single-stranded DNA, but also with single-stranded RNA and double-stranded DNA. The wide photoreactivity toward nucleic acid strands enables us to photoregulate various nucleic acid functions, such as transcription, [2, 7, 8, 18, 19, 29, 31] translation [3, 16] and microRNA activity [17].

Fig. 1
figure 1

Psoralen-modified photo-cross-linkable oligonucleotides

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In our group, as the next generation of photo-cross-linkers, 3-cyanovinylcarbazole-modified oligonucleotides (Fig. 2) have been developed [26, 30]. Since it has extremely high photoreactivity compared to other artificial photo-cross-linkable ODNs, the 3-cyanovinylcarbazole-modified ODNs can photo-cross-link with complementary DNA or RNA within a few seconds of UVA irradiation. Currently, the 3-cyanovinylcarbazole-modified photo-cross-linkable ODNs are applied to various fields of scientific study, such as the construction of DNA-based nanomaterials [23], SELEX-based aptamer selection [21], site-specific nucleic acid editing [4, 5], and gene regulation in cells [25].

Fig. 2
figure 2

3-cyanovinylcarbazole-modified photo-cross-linkable oligonucleotides

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In this chapter, the synthetic methods and properties of the 3-cyanovinylcarbazole-modified ODNs, and the applications for gene silencing are described.

2 Synthesis of 3-Cyanovinylcarbazole-Based Photo-Cross-Linker

Currently, 3-cyanovinylcarbazole-modified ODN is the most reactive photo-cross-linkable ODN in the world. However, at this time, the reagents for the modification of ODNs are not commercially available. Therefore, in this section, a practical method to synthesize the 3-cyanovinylcarbazole-modified ODNs is described.

The phosphoramidite monomer of CNVK can be obtained by multi-step organic synthesis as shown in Scheme 1 from commercially available carbazole (1) as a starting material. At first, the electron rich C3 carbon of carbazole is iodized by a general electrophilic halogenation procedure using NaIO4 as a catalyst. In this reaction, 3,6-diiodocarbazole by-product is sometimes generated because the electron density of C6 carbon of the target product, e.g. 3-iodocarbazole (2), is larger than that of the starting material. To obtain the target within a high yield, the homogeneity and the molar equivalent of all the reagents should be carefully checked before starting the reaction. 3-Cyanovinylcarbazole (3) can be obtained easily by the Mizoroki-Heck reaction [10, 20] using acrylonitrile and Pd(OAc)2 as a vinyl donor and palladium catalyst, respectively. In this reaction, a microwave synthesizer can be used for quick and high-yield preparation of the target compound. By adopting this equipment, the reaction is finished within 10 min. To couple 3-cyanovinylcarbazole (3) and the deoxyribose ring, 1-chloro-2-deoxy-3,5-di-O-p-toluoyl-D-ribofuranose (chlorosugar) can be used as a commercially-available reagent. The chlorosugar can also be obtained by the literature method [9]. Briefly, the methoxy substitution of 1 OH of 2-deoxy-D-ribofuranose is performed with methanol solution of acetyl chloride, the toluoyl protection of 5 and 3 OH groups of 1′-methoxy-2′-deoxy-D-ribofuranose is carried out using p-toluoyl chloride, and then the chlorination of 1 carbon of 1-methoxy-2-deoxy-3,5-di-O-p-toluoyl-D-ribofuranose is performed with HCl generated from acetyl chloride. According to this procedure, chlorosugar can be obtained as white precipitate generated in the reaction mixture; therefore, we can obtain chlorosugar just by using a simple filtration procedure.

Scheme 1
scheme 1

Synthesis of the phosphoramidite monomer of CNVK. a) I2, NaIO4, H2SO4, EtOH; b) acrylonitrile, Pd(OAc)2, tributylamine, DMF-H2O;c) KOH, TDA-1, chlorosugar, MeCN; d) MeONa, MeOH, CHCl3; e) DMTr-Cl, DMAP, Pyridine; g) (iPr2N)2POCE, Benzylthio-1H-tetrazole, MeCN

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After the deprotection of the p-toluoyl group at 5′ and 3′ OH groups of protected 3-cyanovinylcarbazole-1′-β-deoxyriboside using sodium methoxide, 3-cyanovinylcarbazole-1′-β-deoxyriboside (5, CNVK) can be obtained in good yield. Phosphoramidite monomer of CNVK can be obtained by 5′ OH DMTr protection and 3′ OH phosphoramidite modification with general procedures, respectively.

The phosphoramidite monomer of CNVD can be obtained by a multi-step organic synthesis (Scheme 2) from commercially available carbazole (1). After the synthesis of 3-cyanovinylcarbazole (3) using the same procedures of CNVK synthesis, an acetic acid derivative of 3-cyanovinylcarbazole, 3-cyanovinylcarbazol-9-yl-acetic acid (5), can be obtained easily by the coupling with ethyl bromoacetate, and hydrolysis of the ethyl ester. N-(3-cyanovinylcarbazol-9-yl-acetyl) D-threoninol (10, CNVD) can be obtained by a simple condensation reaction with carbodiimide. Phosphoramidite monomer of CNVD can be obtained by DMTr protection of primary alcohol and phosphoramidite modification of secondary alcohol with general procedures, respectively. CNVL can also be obtained with the same synthetic procedures using L-threoninol instead of D-threoninol in the case of CNVD.

Scheme 2
scheme 2

Synthesis of the phosphoramidite monomer of CNVD. a) I2, NaIO4, H2SO4, EtOH; b) acrylonitrile, Pd(OAc)2, tributylamine, DMF-H2O; c) K2CO3, BrCH2CO2Et; d) HCl; e) D-threoninol, WSC, HOBt, DMF; f) DMTr-Cl, DMAP, Pyridine; g) (iPr2N)2POCE, Benzylthio-1H-tetrazole, MeCN

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2.1 Synthetic Procedures of CNVK and Its Phosphoramidite Monomer

2.1.1 3-Iodocarbazole (2)

To a solution of carbazole (8.0 g, 47 mmol), iodine (6.0 g, 24 mmol) and NaIO4 (2.5 g, 12 mmol) in EtOH (400 mL) was added a solution of H2SO4 (5 mL) in EtOH (200 mL), and stirred at 65 °C for 1 h. After neutralization with NaOH, solvent was evaporated, redissolved in CH3Cl (700 mL) and washed with H2O and brine. The organic layer was condensed and recrystallized twice to give 2 as a white solid (4.9 g, 17 mmol, 36%).

2.1.2 3-Cyanovinylcarbazole (3)

To a suspension of 2 (1.5 g, 5.12 mmol) in 20% DMF/H2O (2.8 mL) in a pressure-resistant glass tube, palladium acetate (115 mg, 0.51 mmol), tributylamine (1.22 mL, 5.12 mmol) and acrylonitrile (0.84 mL, 12.8 mmol) were added, and microwave irradiated (60 W, 160 °C) by a microwave synthesizer (DISCOVER, CEM) for 10 min. The reaction was monitored by TLC (hexane/AcOEt, 4:1), which showed the absence of the starting material. After the reaction mixture was evaporated, the residue was chromatographed on a silica gel using hexane/AcOEt (3:1, v/v) as eluent to give 3 (0.85 g, 76%) as a white powder.

2.1.3 3-Cyanovinylcarbazole-9-yl-1′-β-deoxyriboside-3′,5′-di-(P-toluoyl)ester (4)

To a solution of potassium hydroxide (2.29 g, 40.9 mmol) and tris[2-(2-methoxyethoxy)ethyl]amine (90.0 mg, 0.28 mmol) in CH3CN (360 mL), 3 (3.02 g, 13.9 mmol) was added and then stirred at room temperature for 30 min. To this reaction mixture chlorosugar (6.09 g, 14.3 mmol) was added and then stirred at room temperature for 60 min. The reaction was monitored by TLC (hexane/AcOEt, 4:1), which showed the absence of the starting material. After the reaction mixture was evaporated, the residue was chromatographed on a silica gel using CHCl3 as eluent to give 4 (7.89 g, 99%) as a yellow powder.

2.1.4 3-Cyanovinylcarbazole-9-yl-1′-β-deoxyriboside (5)

To a solution of 4 (7.89 g, 13.8 mmol) in MeOH (400 mL) was added 0.5 M methanolic MeONa (83.0 mL, 41.5 mmol) and CHCl3 (50 mL) and then stirred at room temperature for 1 h. The reaction was monitored by TLC (CHCl3/MeOH, 9:1), which showed the absence of starting material. After the reaction mixture was evaporated, the residue was chromatographed on a silica gel using CHCl3/MeOH (9:1, v/v) as eluent to give 5 (2.93 g, 63%) as a white powder.

2.1.5 5′-O-(4,4′-Dimethoxytrityl)-3-cyanovinylcarbazole-9-yl-1′-β -deoxyriboside (6)

To a solution of 5 (2.83 g, 8.47 mmol) in pyridine (12 mL) was added a solution of 4,4′-dimethoxytrityl chloride (3.45 g, 10.2 mmol) and 4-(dimethylamino)pyridine (0.21 g, 1.70 mmol) in pyridine (24 mL) on an ice bath and then stirred at room temperature for 23 h. After the reaction mixture was evaporated, the residue was chromatographed on a silica gel using CHCl3/MeOH (99:1, v/v) as the eluent to give 6 (4.20 g, 78%) as yellow foam.

2.1.6 5′-O-(4,4′-Dimethoxytrityl)-3-cyanovinylcarbazole-9-yl-1′-β -deoxyriboside-3′-O-(cyanoethoxy-N,N-diisopropylamino)phosphoramidite (7)

6 (0.32 g, 0.50 mmol) in a rubber sealed bottle was dissolved in CH3CN (1 mL) and co-evaporated three times in vacuo. A solution of 2-cyanoethyl N,N,N',N'-tetraisopropylphosphoramidite (160 μL, 0.50 mmol) in CH3CN (2.2 mL) and 0.45 M 1H-tetrazole in CH3CN (1.18 mL, 0.50 mmol) were added and then stirred at room temperature for 2 h. The reaction mixture was diluted with AcOEt (15 mL) and washed with a saturated aqueous solution of NaHCO3, and water. The organic layer was collected, dried over anhydrous magnesium sulfate, filtered, and evaporated in vaccuo to give the crude product of 7 (0.42 g, quant.) and was used as the automated DNA synthesizer without further purification.

2.2 Synthetic Procedures of CNVD and Its Phosphoramidite Monomer

2.2.1 Ethyl 3-cyanovinylcarbazol-9-yl-acetate (8)

3-Cyanovinylcarbazole (3) (1.1 g, 5 mmol) and NaH (60% oil suspension, 0.24 g, 6 mmol) were dissolved in dry DMF (20 mL) and stirred for 1 h at room temperature under N2 atmosphere. Ethyl bromoacetate (1.1 mL, 9.9 mmol) was added drop-wisely over 30 min. The reaction mixture was diluted with water (300 mL) and extracted with chloroform and then dried over sodium sulfate. After removal of the solvent, the residue was subjected to silica gel column chromatography (0–1% MeOH/CHCl3) to yield 4 as white solid (1.4 g, 4.6 mmol, 92%).

2.2.2 3-Cyanovinylcarbazol-9-yl-acetic Acid (9)

4 (1.4 g, 4.6 mmol) and NaOH (0.18 g, 23 mmol) were dissolved in THF/MeOH/H2O (3:2:1, 30 mL) and stirred for 2 h at ambient temperature. After the addition of 1 N HCl (250 mL), the reaction mixture was extracted with EtOAc (300 mL) and washed with 1 N HCl. The organic layer was dried over MgSO4 and evaporated to yield 5 as white solid (1.2 g, 4.4 mmol, 96%).

2.2.3 N-(3-Cyanovinylcarbazol-9-yl-acetyl)-D-threoninol (10, CNVD)

5 (1.3 g, 4.5 mmol) and D-threoninol (0.47 g, 4.5 mmol) were added to dry DMF (20 mL) containing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC; 0.86 g, 4.5 mmol) and 1-hydroxybenzotriazole (HOBt; 0.69 g, 4.5 mmol), and stirred for 5 h at ambient temperature. Sat. NaCl aq. soln. was added and the obtained precipitate was collected and dried under vacuo to yield 6 as white solid (1.5 g, 4.1 mmol, 91%).

2.2.4 N-(3-Cyanovinylcarbazol-9-yl-acetyl)-1′-O-(4,4′-dimethoxytrityl)-D-threoninol (11)

A solution of 6 (0.40 g, 1.1 mmol), 4,4′-dimethoxytritylchloride (0.41 g, 1.2 mmol) and 4-dimethyl- aminopyridine (DMAP; 30 mg, 0.25 mmol) in dry pyridine (2 mL) were stirred at ambient temperature for 20 h. The reaction mixture was diluted with CHCl3, washed with H2O and the organic layer was dried over NaSO4. After the removal of the solvent, residue was subjected to silica gel column chromatography (CHCl3 with 0.2% TEA) to yield 7 (0.63 g, 86%).

2.2.5 N-(3-Cyanovinylcarbazol-9-yl-acetyl)-1′-O-(4,4′-dimethoxytrityl)-D-threoninol 3′-O-(Cyanoethoxy-N,N-diisopropylamino)phosphoramidite (12)

The residual trivial amount of water in 7 was removed by azeotropic distillation with dry acetonitrile (twice). Then, 7 (0.38 g, 0.57 mmol) was dissolved with 0.25 M solution of 5-benzylthio-1H-tetrazole in dry MeCN (2.3 mL, 0.57 mmol) and a solution of 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphoramidite (0.18 mL, 0.57 mmol) in dry acetonitrile (5 mL) was added and stirred under N2 atmosphere for 2 h. The crude mixture was dissolved in ethyl acetate. The solution containing 8 was washed with water, sat. NaHCO3 aq. soln. and brine. The organic layer was dried over MgSO4, and then filtered and the ethyl acetate was removed. The yellow foam (0.48 g, 0.55 mmol, 96%) was obtained and immediately used for DNA synthesis without further purification.

2.3 Synthesis of the Oligonucleotide Having CNVK or CNVD

ODNs having CNVK or CNVD can be obtained by the conventional cyano-ethylphosphoramidite chemistry using phosphoramidite monomer of CNVK or CNVD. If the automated DNA synthesizer has the extra phosphoramidite ports, ODNs having CNVK or CNVD can be synthesized easily by an automated DNA synthesizer. Due to the high stability of 3-cyanovinylcarbazole in cleavage and deprotection conditions, the general conditions (28% ammonium hydroxide, 55 °C, 8 h) are applicable. Crude ODN solution can be purified by reversed-phase HPLC in the same manner as general ODN purification.

2.4 Further Modification of CNVK- or CNVD-Modified ODNs

As the OH group on the 5′ and 3′ termini of the CNVK- or CNVD-modified ODNs is free for further modification, the general phosphoramidite-based modification method using 5′ or 3′ modifiers, such as FAM, biotin, amino, thiol, on the automated DNA synthesis is available. In the case of fluorescence labeling of the ODNs, a fluorophore whose stability is sufficiently high during the photo-cross-linking reaction, should be selected. In our cases, the Cy3 and Cy5 label is frequently used as the photo-stable fluorophore. To the best of our knowledge, fluorescein is not suitable for the fluorescent label of CNVK or CNVD-modified ODNs because of its lower photostability under UV irradiation.

3 Inter-Strand Photo-Cross-Linking Using ODNs Having CNVK or CNVD

In this section, the properties of the photo-cross-linking reaction of CNVK and CNVD, which currently are the most highly reactive photo-cross-linkers in the world, are described. Basic findings described here will enable the advanced application of the photo-cross-linking reaction of these photo-cross-linkable ODNs having CNVK or CNVD.

3.1 Properties of the Inter-Strand Photo-Cross-Linking Reaction of CNVK and CNVD in Nucleic Acid Double Strands

The inter-strand photo-cross-linking reaction of CNVK and CNVD occurs between the vinyl group of 3-cyanovinylcarbazole moiety and the C5-C6 double bond of the pyrimidine base situated at the –1 position of CNVK or CNVD in the complementary DNA or RNA strand with 366 nm UV irradiation (Fig. 3). Since the 312 nm irradiation under the denaturing condition causes reverse photoreaction and photo-splitting, the stability of the hybrid can be regulated freely by UV irradiation. As shown in Fig. 4, the local sequence around CNVK or CNVD largely affects the yield of the photo-cross-linking reaction. This indicates that the local duplex structure is important for the photo-cross-linking reaction. These reactions, photo-cross-linking and photo-splitting, can be monitored quantitatively by HPLC or denaturing PAGE analysis as the change in the retention time or mobility, respectively.

Fig. 3
figure 3

Schematic drawing of the photo-cross-linking and photo-splitting reaction of 3-cyanovinylcarbazole-modified ODNs

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Fig. 4
figure 4

Sequence dependency of the photo-cross-linking yields of CNVK- or CNVD-modified ODNs

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CNVK, which was first reported in 2008 [30], has great photoreactivity in the inter-strand photo-cross-linking reaction in the DNA double-strand. The reaction is finished within a few seconds of 366 nm irradiation using UV-LED. Although the pyrimidine bases, T and C, in the complementary strand can be cross-linked with CNVK, the reactivity depends on the bases, and the reactivity toward C is 27-fold lower than that toward T. This large difference of the photoreactivity dependent on the target pyrimidine base sometimes restricts the design of the ODN sequence for the photoreaction. In 2014, CNVD, which has a more flexible skeleton compared to CNVK, was developed [26] as the improved version of CNVK. The photoreactivity of CNVD is ca. 1.8-fold (toward T) and eightfold (toward C) greater than that of CNVK. In the case of CNVD, since the difference of the photoreactivity between T and C is closer than that of CNVK, the freedom for designing the sequences for the photo-cross-linking reaction is increased.

3.2 Light Source

For the photoreaction of CNVK and CNVD, a high-power UV-LED and a general transilluminator can be used depending on the experimental requirements. As the irradiation spotlight of UV-LED is small, ca. 100 mm2 at maximum, this light source is not suitable for the simultaneous irradiation of one lot of samples. If the experiment requires simultaneous irradiation of the samples, the transilluminator is suitable because the area that can be uniformly irradiated is larger, ca. 40,000 mm2, than that of UV-LED. In the case of the use of the transilluminator, the photoreaction proceeds more slowly compared to the case of UV-LED because of its lower irradiation energy compared to UV-LED.

3.3 Structural Insight of the DNA Duplex Including CNVK

As shown in Fig. 5, it was demonstrated that only the trans isomer of CNVK can react with the pyrimidine base on the complementary strand, and that reaction gives the photoadduct that was formed from the trans isomer of CNVK [6]. As the rate of the photo-isomerization of the cyanovinyl group is higher than that of the [2+2] photocycloaddition reaction, the rate determining step of the photo-cross-linking is the [2+2] photocycloaddition reaction. According to the structure of the photoadduct, the duplex structures consisting of CNVK-modified ODN and its complementary ODN before and after the photo-cross-linking reaction can be estimated by energy minimization calculation. As shown in Fig. 6, the structure of the duplex after the photo-cross-linking is slightly bent compared to that of before the photo-cross-linking. From the distance between the carbon atoms on certain nucleoside units, the major axis of the duplex is bent ca. 20° by the photo-cross-linking reaction. The degree of bend is comparable to the result that measured the bend in the CNVK photo-cross-linked DNA tile array [28].

Fig. 5
figure 5

Photo-cross-linking reaction of CNVK in DNA duplex

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Fig. 6
figure 6

Predicted energy minimized structure of the duplexes containing CNVK before and after the photo-cross-linking

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4 Gene-Silencing Using CNVK Modified Antisense ODNs

Because of its ultra-fast photo-cross-linking properties, photo-cross-linkable ODNs having CNVK or CNVD might be applicable as the photoresponsive antisense ODNs for specific gene regulation through the photo-cross-linking with target mRNA. The ultra-fast photoresponse might enable us to regulate spatiotemporally the gene expression in cells without intense photo-damage to the cells and to investigate the function of the gene of interest at various places and timings in cells. There are some reports about the photo-cross-linkable antisense ODNs using psoralen as a photo-cross-linker [12, 22]; however, the lower photoreactivity of psoralen requires long-time UV irradiation that increases photo-cytotoxicity toward living cells. In this section, the practical design, experimental procedures and how to use CNVK-modified antisense ODNs for gene silencing in the cells are described.

4.1 Design of the Photoreactive Antisense ODNs Having CNVK

Similar to the general design method of antisense ODNs, the selectivity of the duplex formation with target mRNA and the stability of the hetero-duplex is crucial to obtain the effective sequence. As in the case of CNVK-modified ODNs, hybrid stability before the photo-cross-linking is lower than that of the unmodified duplex because the 3-cyanovinylcarbazole cannot pair with any canonical bases in the RNA strand, and the position of CNVK in the antisense ODNs is important to obtain the effective sequence. CNVK should be possessed at the termini of the antisense sequence to the greatest extent possible. In our cases, highly specific photoresponsive anti-sense ODNs for K-ras point-mutated mRNA [27], vascular endothelial growth factor mRNA [27], and for enhanced green fluorescent protein mRNA [25] were successfully obtained. These antisense ODNs can photo-cross-link completely with their target mRNA by 1 s of 366 nm irradiation in aqueous buffer solution.

4.2 Evaluation of the Photo-Cross-Linking Reaction with mRNA

In the antisense strategy for gene silencing, the binding between antisense ODNs and target mRNA is crucial for obtaining an effective silencing effect. To evaluate quantitatively the photo-cross-linking efficiency of the CNVK-modified ODNs and target mRNA, the quantification of the reverse-transcription inhibition is effective. As the reverse-transcription reaction is stopped around the position of the photo-cross-linked hetero-duplex caused by the photoreaction of CNVK, full-length cDNA derived from the reverse-transcription is decreased with the photo-cross-linking reaction (Fig. 7). Therefore, the photo-cross-linking efficiency of the photo-reactive antisense ODNs can be evaluated quantitatively by the decrease in the amount of full-length cDNA, which can be easily quantified by a real-time PCR method with an appropriate primer set.

Fig. 7
figure 7

Schematic drawing of the quantitative evaluation of the photo-cross-linking reaction between the CNVK-modified antisense ODNs and target mRNAs

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4.3 Photo-Induced Gene Silencing in Cells

Relying on the ultra-fast photo-cross-linking manner of the 3-cyanovinylcarbazole-modified photo-cross-linkable antisense ODNs, cellular mRNA might be photo-cross-linked and the gene expression might be down regulated with a few seconds of 366 nm irradiation toward antisense-treated cells. In our study, various photo-cross-linkable antisense ODNs having CNVK were designed for silencing the GFP gene in constitutively GFP expression cells (GFP–HeLa) [25]. Since a 60% decrease of target mRNA and 40% decrease of GFP protein were observed by 10 s irradiation of 366 nm UV-LED in the case of the most effective antisense ODN (Fig. 8), the applicability of the CNVK-based photo-cross-linkable ODNs was demonstrated. In particular, multiple-irradiation increases the gene-silencing effect of the antisense ODN, and the effect was comparable to that of siRNA, suggesting that further development of this strategy might provide more sophisticated and effective gene regulation tools that can regulate genes of interest in a spatiotemporal manner.

Fig. 8
figure 8

Antisense effect of photo-cross-linkable antisense ODN having CNVK toward GFP expression

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5 Summary

We described a practical method to synthesize and use photo-cross-linkable ODNs having CNVK and CNVD, which currently are the most highly reactive photo-cross-linkable ODNs in the world, toward gene silencing in the cells. We hope that further applications based on our photo-cross-linking technique will be developed by chemists, biologists and also medical researchers.