Template Directed Reversible Photochemical Ligation of Oligodeoxynucleotides

We demonstrated that 5-vinyldeoxyuridine (VU) and 5-carboxyvinyldeoxyuridine (CVU) can be used to photoligate a longer oligonucleotide (ODN) from smaller ODNs on a template. By performing irradiation at 366 nm, these artificial nucleotides make photoligated ODNs with high efficiency without any side reactions. Moreover, by performing irradiation at 312 nm, these photoligated ODNs were reversed to the original ODN. VU needs to be irradiated 366 nm for 6 h, but CVU needs to be irradiated at 366 nm for 15 min. Finally, we made a self-assembled structure with an ODN containing CVU and observed the photoligated ODN by photoirradiation.


Introduction
There are many methods of template-directed chemical ligation of oligonucleotides (ODNs) via a native phosphodiester bond [1] or non-native linking [2]. Template-directed chemical ligation can ligate not only DNA, but also other biomolecules such as protein-like molecules [3]. Template directed OPEN ACCESS synthesis has been used for DNA nanotechnology [4], the selection of amplifiable small-molecule libraries [5], the release of drugs [6], and as a diagnostic means of detecting the presence of the nucleic acid template [7]. One method of template-directed chemical ligation is non-enzymatic chemical ligation [8,9]. This method was researched for new gene manipulation and as a design method for nanostructures, because it does not have restrictions of a substrate and reaction conditions are suitable for enzymatic reactions. In particular, photochemical ligation has many useful characteristics. For example, there is no need to add other reagents and the ligation reaction is easily regulated by irradiation wavelength and intensity. However, there are only a few methods of performing photochemical reactions [10][11][12]. Previously reported methods for DNA photochemical ligation are thymine dimer formation [10], photoreactions of DNA containing appended stilbenes [11] and ligated DNA using anthracene [12]. However, these methods have a serious problem for practical utilization, such as the low yields of photochemical ligation products and the use of short wavelengths that injure other biological components. We reported that 5-vinyldeoxyuridine (VU) can be used to photolink a longer ODN from five smaller identical ODNs on a template with high efficiency without any side reactions by photoirradiation at 366 nm [13]. This reaction ligated T and VU via [2+2] photocyclization [14]. Moreover, we have reported 5-carboxyvinyldeoxyuridine (CVU) [15] an artificial nucleotide which is photoresponsive. The photochemical reaction of VU and CVU is shown in Scheme 1. In this study, we synthesized a longer ODN from four smaller different ODNs by photochemical ligation with VU and 5-vinlycytosine (VC) [16]. Next, we conducted the same experiment using CVU and confirmed the template dependence. Finally, we researched photochemical ligation activity using CVU.

Results and Discussion
In a previous study, four of the same ODNs were ligated by photochemical ligation using V U [13].
In this study, we demonstrated the feasibility of ligating four different ODNs using V U and V C ( Figure 1A). After we elaborated the V U and V C (Scheme 2), we synthesized ODNs containing V U and V C by automated DNA synthesizer. Figure 1B shows the strategy of reversible photochemical ligation.
ODN 1 containing 32 P at the 5′ end (Experimental section 3.3) and the other ODNs were annealed and irradiated at 366 nm for 8 h and 312 nm for 1 h. Annealing and photoirradiation in detail is shown in experimental section 3.4. The result of denaturing PAGE analysis is shown in Figure 2.  Lane 1 and lane 2 are 12 mer control and 6 mer control respectively. Lane 3, which is ODN 1 and 2, was irradiated at 366 nm for 8 h in the presence of a template. We obtained the 12 mer band ligated ODN 1 and 2. Lane 4 is ODN 3 added to Lane 3. We obtained the 18 mer band ligated ODN 1, 2 and 3. Lane 5 is ODN 4 added to Lane 4. We obtained the 24 mer band ligated ODN 1, 2, 3 and 4. Lane 6 is lane 4 nonirradiated at 366 nm. We obtained the 6 mer band with only ODN 1 ligated. Lane 7, which is ODN 1, 3 and 4, was irradiated at 366 nm in the presence of the template. We obtained the 6 mer band, which is only ODN 1. Lane 8, which is ODN 1, 2 and 4, was irradiated at 366 nm in the presence of the template. We obtained the 12 mer band ligated ODN 1 and 2. Lane 9, which is ODN 1, 2, 3 and 4, was irradiated at 366 nm in the absence of the template. We obtained the 6 mer band, which is only ODN 1. Lane 10 is the same sample as that in lane 5. Lane 11 is lane 10 irradiated at 312 nm for 1 h. We obtained the 6 mer band, which is only ODN 1.
We confirmed that the initial domain becomes longer by comparison with the 2nd domain and 3rd domain. It showed the initial domain made photo cross linking by photoirradiation and this reaction advanced efficiently. This reaction did not proceed without the template and photoirradiation. The photoligated ODNs quantitatively reverted to the original ODNs by irradiation at 312 nm. V U and V C can be ligated in various pairs such as T♢ V U, T♢ V C and C♢ V U by photochemical ligation. So, we extended a ligation pair of only T♢ V U to four pairs. Next, we designed template directed synthetic DNA having branched DNA. Figure 3A shows the branched DNA. Branched DNA has various uses in signal amplification technology and in several types of nanotechnology, such as DNA computing, DNA nanostructures from self-assembled branched units, DNA sensors [17], and nano-electronic devices [18]. We previously reported Multiple-Branched DNA [15] and DNA computing [19] using branched DNA. We demonstrated the feasibility of reversible photoligation with CV U. The wavelengths used were 366 nm and 312 nm, the same wavelengths as when using V U. After elaborating the CV U, we synthesized ODNs containing CV U by automated DNA synthesizer. We synthesized ODNs containing CV U inside so we made branched DNA toward developing DNA nanotechnology ( Figure 3A). ODN 5 containing Cy3 fluorescence at the 5′ end, ODN 6, ODN 7, ODN 8 and template 2 was annealed and irradiated at 366 nm for 900 s. The result of denaturing PAGE analysis is shown in Figure 4. We obtained Cy3 fluorescence. Lane 1, which is ODN 5, was irradiated at 366 nm for 900 s in the presence of a template. We obtained the 10 mer band, which is only ODN 5. Lane 2 is ODN 6 added to Lane 1. We obtained the 20 mer band ligated ODN 5 and 6. Lane 3 is ODN 7 added to Lane 2. We obtained the 30 mer band ligated ODN 5, 6 and 7. Lane 4 is ODN 8 added to Lane 3. We obtained the 40 mer band ligated ODN 5, 6, 7 and 8. Lane 5 is lane 4 irradiated at 312 nm for 1800 s. Lane 6, which is ODN 5, 6, 7 and 8, was irradiated at 366 nm for 900 s in the absence template. Lane 7, which is ODN 5, 6, 7 and 8, was nonirradiated at 366 nm. We obtained the 10 mer band, which is only ODN 5 in Lane 5, 6 and 7.
The DNA becomes longer by adding DNA sequentially from the initial domain, which shows that CV U and T are connected by photochemical ligation. This reaction was completely finished for irradiation at 366 nm for 900 s as shown by the results of lane 4. Thus, 900 s is considered sufficient for utilization. And, this reaction did not advance in the absence of a template and nonirradiation at 366 nm. The photoligated ODNs were quantitatively reverted to the original ODNs by irradiation at 312 nm.
Next, we demonstrated photochemical ligation in other sequences ( Figure 5A). ODN 9 is ODN 7 linked Cy5 fluorescence at the 5′ end. The result of denaturing PAGE analysis is shown in Figure 5B. We obtained Cy5 fluorescence. Lane 1, which is ODN 9, was irradiated at 366 nm for 900 s in the presence of a template. We obtained the 10 mer band, which is only ODN 9. Lane 2 is ODN 10 added to Lane 1. We obtained the 20 mer band ligated with ODN 9 and 10. Lane 3 is ODN 6 added to Lane 2. We obtained the 30 mer band ligated with ODN 9, 10 and 6. Lane 4 is ODN 8 added to Lane 3. We obtained the 40 mer band ligated with ODN 9, 10, 6 and 8. Lane 5 is irradiated at 312 nm to Lane 4 for 1,800 s. Lane 6, which is ODN 9, 10, 6 and 8, was irradiated at 366 nm for 900 s in the absence of a template. Lane 7, which is ODN 9, 10, 6 and 8, was nonirradiated at 366 nm. We obtained the 10 mer band, which is only ODN 9 in Lane 5, 6 and 7. The same as the result above, we confirmed that the ligated product was produced by photochemical ligation, so we can use photochemical ligation regardless of the sequence. As photochemical ligation did not advance without a template, we can use this reaction for sensing the template. Additionally, these results show the difference of reactivity of V U and CV U on photochemical ligation so we can make the system only CV U ligated if photoirradiation was short and V U and CV U was ligated if the photoirradiation time was long by using these compounds respectively.
Next, we demonstrated the template dependence for changing the template in the same three domains ( Figure 6A). By using template 2, 3 or 4, we confirmed the size of the ligated product. In the presence of template 2, we should be able to confirm ligated ODN 5 and 6. In the presence of template 4, we should be able to confirm only ODN 5. In the presence of template 3, we should be able to confirm ligated ODN 5, 6 and 8.  Figure 6B. We observed Cy3 fluorescence and it showed the size of the ligation product of ODN 5.
We confirmed that ODN 5 and ODN 6 were ligated in the presence of template 1 and only ODN 5 was ligated in the presence of template 2. In the presence of template 3, ODN 5, 6 and 7 were ligated. This result reflects the template dependence of photochemical ligation. Generally, template-directed DNA ligation can generate a related to the template DNA in a sequence-specific manner. However, this photochemical ligation method is a reversible ligation method, so we can rearrange ODNs using templates. Furthermore, this photochemical ligation is reversible so we can rearrange another sequence after ligated ODN is reverted to original ODNs by irradiation at 312 nm. In vivo, DNA works as an information carrier so it is suggested that we can re-edit the information of DNA.
Finally, we researched the activities of photochemical ligation with a self-assembled DNA structure (Figure 7). A self-assembled DNA structure is made by the characteristic of complementarity of DNA such as a holiday junction [20] and DNA origami [21]. Our construction of a self-assembled structure contributes toward developing DNA nanotechnology. We synthesized ODN containing CV U.  Figure 8A. We confirmed that ligation products become bigger according to photoirradiation time. Samples of 900 s and 1,800 s showed no change so this reaction was completely irradiated at 366 nm for 1,800 s. The size of the final structure became longer by optimization of the sequence. The average of nucleotide photoirradiation time was plotted ( Figure 8B). This figure shows that the nucleotide average increases very fast at first and does not change over 900 s. This photochemical ligation is not a bimolecular reaction such as enzyme-substrate [22] but all reactions advance in parallel simultaneously by photoirradiation over the whole reaction field. Moreover, this structure is not a normal double strand but a double strand containing branched DNA. This branched DNA is not made by a ligation enzyme. So, we can modify branched DNA such as biotin and nucleotide aptamer. In a previous study, a molecule was spotted onto DNA origami [23]. We can spot various molecules using this linear structure too. In this study, it was shown that DNA may be ligated or cut by photo efficiency. This means that it is possible to carry out reorganization collection on the information included in DNA. In our body, the reorganization collection of the DNA is not carried out, but after being transferred as RNA, the reorganization collection is carried out, such as by splicing. Without passing RNA, by carrying out the reorganization collection of the DNA, it might be possible to create the translation process to protein artificially.

Synthesis of Oligonucleotides in This Experiment
Oligonucleotides were prepared by β-(cyanoethyl) phosphoramidite method on controlled pore glass supports (1 mmol) by using ABI3400 DNA synthesizer. Cyanoethylphosphoramidite of elaborated compounds was prepared as described above. After automated synthesis, the oligomer was detached from the support by soaking in conc. aqueous ammonia for 1 h at room temperature. Deprotection was conducted by heating the conc. aqueous for 10 h at 55 °C conc. aqueous ammonia was then removed by speedvac, and the crude oligomer was purified by reverse phase HPLC and lyophilized. The Purity and concentration of all oligonucleotides were determined by complete digestion with s.v. PDE, P-1 nuclease, and AP to 2′-deoxymononucleosides. DNA base sequence was shown in Table 1.

Photoirradiation of DNA Oligomer as Monitored by PAGE
In the V U and V C, to the reaction mixture (total volume 10 μL) 10 μM of loading buffer (a solution of 80% v/v formamide 1mM EDTA, 0.1% xylene cyanol, and 0.1% bromophenol blue) was added to quench the reaction and the samples (1-2 μL, ca 2-4 × 10 3 cpm) were loaded onto 15% (19:1) polyacrylamide 7M urea denaturing gel and electrophoresed at 700 V for 30 min. The gel was dried and exposed to X-ray film with an intensifying sheet at −80 °C. In the CV U, the reaction mixture was diluted with 8 M Urea in formamide 10 times. To this diluted solution (3 μL) containing initial ODN