Template-directed reversible photocircularization of DNA via 5-vinyldeoxycytidine

Article abstract of DOI:10.1016/S0040-4039(00)01025-X

An efficient and reversible template-directed photoligation of oligodeoxynucleotides (ODNs) using 5-vinyldeoxycytidine ((v)c) was described. When ODN containing (v)C at the 5' end was photoirradiated with ODN containing thymine at the 3' end in the presence of a template ODN, an efficient photoligation was observed. Reversible photopadlocking of a circular DNA by the photocircularization using (v)C was demonstrated. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)01025-X

Tetrahedron Letters 41 (2000) 6451±6454
Template-directed reversible photocircularization of DNA
via 5-vinyldeoxycytidine
Kenzo Fujimoto, Shigeo Matsuda, Naoki Ogawa, Masayuki Hayashi and Isao Saito*
Department of Synthetic Chemistry and Biological Chemistry, Kyoto University, Kyoto 606-8501, Japan
Received 26 May 2000; revised 12 June 2000; accepted 16 June 2000
Abstract
An ecient and reversible template-directed photoligation of oligodeoxynucleotides (ODNs) using
5-vinyldeoxycytidine (VC) was described. When ODN containing VC at the 50 end was photoirradiated with
ODN containing thymine at the 30 end in the presence of a template ODN, an ecient photoligation was
observed. Reversible photopadlocking of a circular DNA by the photocircularization using VC was
demonstrated. # 2000 Elsevier Science Ltd. All rights reserved.
Although many methods are known for template-directed chemical ligation of ODN, a few
methods for photoinduced chemical ligation has been demonstrated.1 Recently, we disclosed an
ecient and reversible template-directed photoligation of oligodeoxynucleotides (ODNs) using
5-vinyldeoxyuridine (VU)-containing ODN at the 50 end.2 However, due to the limitation of U
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which is only applicable to adenine as a counter base in a duplex, a di€erent base that contains a
photoreactive vinyl group is required. We now report a reversible photoligation of 5-vinyl-
deoxycytidine (VC)-containing ODN at the 50 end with ODN containing thymine at the 30 end.3
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By the use of C-containing ODNs, we demonstrated a reversible formation of catenated DNA
via template-directed photocircularization.
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Phosphoroamidite building block 44 for the synthesis of C-containing ODNs was prepared
from 50-DMTr-deoxyvinyluridine 30-phosphoramidite 3.5 VC-containing ODNs were synthesized
according to the standard phosphoramidite chemistry using 4, coupled with a post-modi®cation
* Corresponding author.
0040-4039/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)01025-X

6452
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procedure of ammonia treatment as reported previously6 (Scheme 1). Incorporation of C into
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ODN was con®rmed by enzymatic digestion and ESI-TOF MS of the resulting C-containing
ODNs.7
Scheme 1. (a) DMTrCl, NEt3, pyridine, 18 h, 73%; (b) P(N-iPr)2O(CH)2)2CN, tetrazole, CH3CN, 1 h, 98%; (c) 1,2,4-
triazole, POCl3, NEt3, CH3CN, 3 h, 95%
When 32P-50-end-labeled d(TGTGCT) (ODN 5) and d(VCGCGTG) (ODN 6a) were irradiated
at 366 nm in the presence of template ODN 7a (Scheme 2), the expected ligated 12 mer ODN 8a
was produced in 93% yield as determined by densitometric assay of the PAGE (Fig. 1, lane 2).8
Quantitative formation of starting ODN 5 was observed by further irradiation of lane 2 at 302
nm (Fig. 1, lane 3). HPLC analysis of the photoirradiated mixture of ODN 5 and ODN 6a with
template ODN 7a con®rmed a clean and ecient formation of ligated ODN 8a with concomitant
disappearance of ODN 5 and ODN 6a. The ESI-TOF MS indicated that ODN 8a is a ligated
product between ODN 5 and ODN 6a.9 Enzymatic digestion of isolated ODN 8a indicated the
formation of dG, dT and dC in a ratio of 5:3:2 together with a dT-VC photoadduct.10 These
results indicated that ODN 8a is a photoligated product via the photolinking of 30-terminal
thymine and 50-terminal VC. The reversibility of the photoligation of ODN 8a (X=VC) and ODN
8b (X=VU) upon 302 nm irradiation was compared under identical conditions. As shown in
Fig. 1, VC-containing ODN is far superior to VU-containing ODN for the photosplitting of
ligated ODNs (lane 3 versus lane 6).
Scheme 2.

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Figure 1. Autoradiogram of a denaturing polyacrylamide gel electrophoresis of the photoreaction of ODN 6a,b and
32P-50-end-labeled ODN 5 in the presence of template ODN 7a,b. Lane 1: ODN 5+ODN 6a/ODN 7a, without photo-
irradiation; lane 2: irradiation of lane 1 at 366 nm, 3 h (93%); lane 3: irradiation of lane 2 at 302 nm, 1 h (quant.); lane
4: ODN 5+ODN 6b/ODN 7b, without photoirradiation; lane 5: irradiation of lane 4 at 366 nm, 3 h (98%); lane 6:
irradiation of lane 5 at 302 nm, 1 h (30%)
Reversible photopadlocking of circular plasmid DNA by the photocircularization using
VC-containing ODN was examined.1d A long 60 mer ODN 9 containing two sites that are
complementary to the 20 nucleotide section of plasmid M13mp18 DNA was synthesized by
standard automated DNA synthesis (Fig. 2). On the gel of the photoirradiated 32P-50-end-labeled
ODN 9 in the presence of M13mp18 DNA, a slower moving band which has the same mobility as
the band produced by T4 DNA ligase treatment appeared in 25% yield (Fig. 3, lane 3).11 Further
irradiation of lane 3 at 302 nm resulted in a complete reversion to original linear ODN 9 (lane 4).
As shown in lane 5, photopadlocked DNA was observed again by 366 nm irradiation, indicating
the reversibility of the photocircularization process. To our knowledge, this is the ®rst demonstration
of the reversible DNA photocircularization that produces DNA catenane, although irreversible
padlocking of plasmids has been reported in several systems.12,1d
In conclusion, we have demonstrated a reversible photoligation of ODNs containing thymine
at the 30 end with ODNs containing C at the 50 end. The C mediated reversible photoligation
was very useful for padlocking circular DNA to produce DNA catenane.
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Figure 2. Plasmid DNA and ODN used in this study. The target sequence was located from position 6054 to 6073 in
plasmid M13mp18. The sequence of 60 mer ODN was shown. 60 mer ODN 9 was hybridized to this plasmid at both 30
and 50 ends

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Figure 3. Autoradiogram of 12% denaturing gel electrophoresis for the photoligation of ODN 9 in the presence of
plasmid M13mp18. Lane 1: ODN 9; lane 2: ODN 9+M13mp18+T4 DNA ligase, rt, 3 h (20%); lane 3: ODN
9+M13mp18, irradiation at 366 nm, 3 h (25%); lane 4: irradiation of lane 3 at 302 nm, 30 min (quant); lane 5: irra-
diation of lane 4 at 366 nm 3 h (13%)
Acknowledgements
We thank CREST, Japan Science and Technology Corporation for funding.
References
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Nucleotides 1997, 16, 643. (c) Royer, G. P.; Cruickshank, K. A.; Morrison, L. E. European Patent 0324626A2.
(d) Liu, J.; Taylor, J.-S. Nucleic Acids Res. 1998, 26, 3300.
2. Fujimoto, K.; Matsuda, S.; Takahashi, N.; Saito, I. J. Am. Chem. Soc. 2000, 122, 5646.
3. VC: lmax (water) 289 nm, " 5,400 (" at 366 nm, 13).
4. 31P NMR (121 MHz, CDCl3): ꢀ 149.811, 150.386 (diastereomers).
5. Rahim, S. G.; Duggan, M. J. H.; Walker, R. T.; Jones, A. S.; Dyer, R. L.; Balzarini, J.; De Clercq, E. Nucleic
Acids Res. 1982, 10, 5285.
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S.; Nanbu, D.; Hari, Y.; Abdoh, J.; Morio, K.; Doi, T.; Imanishi, T. Tetrahedron Lett. 1998, 39, 5401
7. ESI-TOFF MS: calcd. for ODN 6a (C60H75N23O35P5) (M±H^): 1832.35; found: 1832.32.
8. The reaction mixture (total volume 10 mL) containing ODN 6 (7 mM, strand conc.), ODN 5 (ca. 2Â104 cpm, 0.1±
0.3 mM) and template ODN 7 (9 mM, strand conc.) in 50 mM sodium cacodylate bu€er (pH 7.0) was irradiated
with a transilluminator (366 nm or 302 nm) at 0ꢀC.
9. ESI-TOFF MS: calcd. for ODN 8a (C119H151N42O72P10) (M±H^): 3629.68; found: 3629.67.
10. ESI-TOFF MS: calcd. for dT-VC photoadduct (C20H25N4O10) (M±H^): 481.16; found: 481.15.
11. The reaction mixture (total volume 10 mL) containing M13mp18 DNA (90 nM, strand conc.) and ODN 9 (ca.
2Â104 cpm, 30 nM) in 50 mM sodium cacodylate bu€er (pH 7.0) was irradiated similarly.8
12. (a) Nisson, M.; Malmgren, H.; Samiotaki, M.; Kwiatkowski, M.; Chowdhary, B. P.; Landegren, U. Science 1994,
265, 2085. (b) Liu, D.; Daubendiek, S. L.; Zillman, M. A.; Ryan, K.; Kool, E. T. J. Am. Chem. Soc. 1996, 118,
1587. (c) Escude, C.; Garestier, T.; Helene, C. Proc. Natl. Acad. Sci. USA 1999, 96, 10603.