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Photoinduced DNA end capping viaN^3‑methyl‑5‑
cyanovinyl‑2'‑deoxyuridine

Author(s)Fujimoto, Kenzo; Yoshimura, Yoshinaga; Ikemoto,Tadayoshi; Nakazawa, Akio; Hayashi, Masayuki;
Saito, Isao

Citation

Chemical communications, 2005(25): 3177‑3179

Issue Date2005

Type

Journal Article

Text version

author

URL

http://hdl.handle.net/10119/7902

RightsCopyright (C) 2005 Royal Society of Chemistry.
Kenzo Fujimoto, Yoshinaga Yoshimura, Tadayoshi
Ikemoto, Akio Nakazawa, Masayuki Hayashi and Isao
Saito, Chemical communications, 2005(25), 2005,3177‑3179. http://dx.doi.org/10.1039/b504162g ‑Reproduced by permission of The Royal Society ofChemistry

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Japan Advanced Institute of Science and Technology

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Photoinduced DNA end capping via N3-methyl-5-cyanovinyl-2’deoxyuridine
Kenzo Fujimoto,*ab Yoshinaga Yoshimura,b Tadayoshi Ikemoto,a Akio Nakazawa,c Masayuki Hayashic
and Isao SaitocReceipt/Acceptance Data [DO NOT ALTER/DELETE THIS TEXT]
Publication data [DO NOT ALTER/DELETE THIS TEXT]DOI: 10.1039/b000000x [DO NOT ALTER/DELETE THIS TEXT]

A modified oligodeoxynucleotide (ODN) containing N3-methyl-5cyanovinyl-2’-deoxyuridine reacts by photoirradiation at 366 nmwith an adenine residue of a complementary template ODN toyield an end-capped ODN in 87% yield.
Since the double helical structure of DNA was first described byWatson and Crick in 1953, a wide variability of DNA
conformations has been observed as non-ground state structures,such as hairpin-DNA, cruciform, Z-DNA and triple helix in nucleic
acid.1 It has been difficult to study such unusual DNA
conformations by biophysical analysis because of the narrow rangeof limited conditions under which they exist. Among thesestructures, the hairpin stem-loop structure has attracted interest
because of its generality in palindromic sequences associated withthe regulation of transcription and other biological functions.2 To
overcome these problems, chemical probes for the trapping andstabilization of such hairpin structures have been developed toexplore DNA conformations, dynamics and their biological roles.3
Recently, we have reported efficient and reversible templatedirected photoligations with ODNs containing 3’-terminal cytosineusing 5-vinyl-2’-deoxyuridine (VU) containing ODN at the 5’terminal.4 A remarkable stacking between a vinyl residue of VU and5’-pyrimidine within the same strand will be responsible for theefficient photoreaction in our template-directed DNA photoligation
system via VU. We have now examined photochemical end capping,using N3-methyl-5-cyanovinyl-2’-deoxyuridine (MCVU) instead ofVU, in which the more photoreactive vinyl group was incorporated.
The photoreactive cyanovinyl group in MCVU was designed to stackeffectively with a base in the opposite strand by an N3-methylgroup substitution that allows stabilization of the syn orientation ofMCV
U and release from the Watson−Crick base pair (Figure 1).
Herein we report the photochemical DNA end capping via MCVU
instead of VU to generate the stabilized hairpin analogue at its end.
MCV
U-containing ODN was synthesized according to thestandard phosphoramidite chemistry on a DNA synthesizer. The
phosphoramidite of MCVU was prepared in six steps from 5-iodo-2’deoxyuridine as shown in Scheme 1.5 Incorporation of MCVU into
ODN was confirmed byenzymatic digestion
andMALDI−TOF−MS.6
When 5’-d(MCVUGCGTG)-3’ ODN1(MCVU) was irradiated at
366 nm for 30 min in the presence of 5’-d(CACGCA)-3’
ODN1’(A) (Scheme 2), ODN1(MCVU-A) was produced in 87%
yield, as determined by HPLC analysis (Figure 2).7,8
MALDI−TOF−MS indicated that ODN1(MCVU-A) obtained by† Electronic Supplementary Information (ESI) available: Experimental
details. See http://www.rsc.org/suppdata/xx/b0/b000000x/* kenzo@jaist.ac.jp

This journal © Royal Society of ChemistryFig. 1 Proposed two conformer about the base pair between adenine andMCVU at the terminal site.

Scheme 1 Reagents and conditions: (a) TBDMSCl, imidazole, pyridine, 3 h,95%; (b) dimethylcarbonate, 18-crown-6, K2CO3, DMF, 3 h, 98%; (c)acrylonitrile, Pd(OAc)2, PPh3, 8 h, 70%; (d) TBAF, THF, 3 h, 85%; (e)DMTrCl, DMAP, pyridine, 75%; (f) P(N-iPr2)2O(CH2)2CN, tetrazole,
CH3CN, 2 h, 98%.

Scheme 2 Photochemical end capping via ODN1(MCVU).

Fig. 2 HPLC profile of photoreaction of ODN1(MCVU) and ODN1’(A). (a)before photoirradiation; (b) irradiation at 366 nm for 30 min, 87% yield.

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Table 1 Melting temperature of end-capped ODN1(MCVU-A) incomparison with duplex ODN1(MCVU)/ODN1’(A) and T4 loop hairpin
ODN
Entry

OligomerTm/ °Ca123

ODN1(MCVU)/ODN1’(A)ODN1(MCVU-A)
5’-d(CACGCATTTTTGCGTG)-3’

28.1
74.5
42.6

a

UV melting curves were obtained in a 50 mM sodium cacodylate buffer(pH 7.0) containing 100 mM NaCl at a strand concentration of 5.0 µM.

structure with a minimum unit constructed from the base analogue.
We also investigated the resistance of the end-capped ODN tonucleolytic digestion by snake venom phosphodiesterase. Afterphotoirradiationofself-complementald(MCVUGCGCAATTGCGCA)2 ODN2(MCVU), doubly end-capped
ODN ODN2(MCVU-A) was isolated13 and used in nucleolytic
digestion for 30 min compared with quantitative degradation ofstarting ODN2(MCVU) (Figure 3, lane 4 and lane 7).14,15 No
degradation of ODN2(MCVU-A) was observed in phosphodiesterase
treatment for 24 h (Figure 3, lane 5). These results show that theend-capped ODN2(MCVU-A) increases significantly its stability inthe biological medium and its possibility as a decoy DNA for
directly targeting transcription factors and for globally controllingthe expression of genes.16
In conclusion, we have synthesized MCVU-containing ODN as aprobe for trapping and stabilizing the hairpin structure anddemonstrated the photochemical end capping of ODN via MCVU.
This MCVU-mediated photochemical end capping may findapplication in the investigation of nucleic acid structure andfunction.

Scheme 3 Photochemical end capping via ODN2(MCVU).

ODN2(MCV U-A)
ODN2(MCV U)
1234567Fig. 3 Time-dependent phosphodiesterase-mediated degradation of the endcapped ODN. Lane 1: ODN2(MCVU); lane 2: 366 nm irradiation of lane 1 for3 h; lane 3: isolated ODN2(MCVU-A); lane 4: phosphodiesterase treatment oflane 3 for 30 min; lane 5: phophodiesterase treatment of lane 3 for 24 h; lane
6: ODN2(MCVU); lane 7: phosphodiesterase treatment of lane 6 for 30 min.
Bands were visualized by silver staining method.

HPLC purification is a cross-adduct of ODN1(MCVU) andODN1’(A).9 Enzymatic digestion of isolated ODN1(MCVU-A)
showed the composition of dA, dG, dT and dC in a ratio of 1:4:1:4
together with dA-dMCVU photoadduct.10 These results clearly
indicate that ODN1(MCVU-A) was an end-capped ODN formed bycrosslinking between an adenine of ODN1’(A) and MCVU ofODN1(MCVU) at the strand end. Unfortunately, the dA-dMCVU
photoadduct derived from enzymatic digestion of ODN1(MCVU-A)
was too labile to be isolated because of its thermal instability inwater. However, its inability to be photoreversed by 254 nm
irradiation suggests that the dA-dMCVU photoadduct was the [2+2]cycloadduct between the vinyl group and 1,6-double bonds of an
adenine-like major photoadduct in the TpA sequence.11,12
To evaluate the stability of end-capped ODN, thermal
denaturation experiments were examined (Table 1). From entries 1
and 2, it can be seen that end capping of ODN produced asignificantly increased melting temperature (ΔTm = +46 °C),
indicating that this capped ODN traps the hairpin structure
photochemically. It is also observed that end capping of ODN
resulted in an increase in thermal stability by 32 °C as comparedwith the T4 loop hairpin ODN, reflecting the effect of the linker
conformationally restricting the hairpin conformation. Thus, thephotochemical end capping effectively stabilizes the hairpin
2 | [JOURNAL], 200X, 00, 0000

Kenzo Fujimoto,*ab Yoshinaga Yoshimura,b Tadayoshi Ikemoto,aAkio Nakazawa,c Masayuki Hayashic and Isao Saitoc
aThe School of Materials Science, Japan Advanced Institute of Scienceand Technology, Ishikawa 923-1292, Japan. Fax: +81 761 51 1671; Tel:+81 761 51 1671; E-mail: kenzo@jaist.ac.jp
bPRESTO, Japan Science and Technology Agency, Ishikawa 923-1292,
Japan.
cDepartment of Synthetic Chemistry and Biological Chemistry, Faculty ofEngineering, Kyoto University, Kyoto 606-8501, Japan.

Notes and references
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R. R. Sinden, DNA Structure and Function, Academic Press: NewYork, 1994; N. R. Cozzarelli and J. C. Wang, DNA Topology and Its
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J. S. Nelson, L. Giver, A. D. Ellington and R. L. Letzinger,Biochemistry, 1996, 35, 5339; A. M. Pyle and J. B. Green, Curr.
Opin. Struct. Biol., 1995, 9, 303; P. P. Zarrinkar and J. R. Williamson,Science, 1995, 265, 918; J. R. Williamson, Nature Struct. Biol., 1994,1, 270; D. E. Draper, Acc. Chem. Res., 1992, 25, 201; B. C. F. Chuand L. E. Orgel, Nucleic Acids Res., 1991, 19, 6958; M. E. Bianchi,M. Beltrame and G. Paonessa, Science, 1989, 243, 1056.
S. E. Osborne, J. Volker, S. Y. Stevens, K. J. Bresleuer and G. D.
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K. Fujimoto, S. Matsuda, N. Takahashi and I. Saito, J. Am. Chem.
Soc., 2000, 122. 5646; K. Fujimoto, S. Matsuda, N. Ogawa, M.
Hayashi and I. Saito, Tetrahedron Lett., 2000, 41, 6451; K. Fujimoto,
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Tetrahedron Lett., 2000, 41, 9437.
MCV
U: λmax (water) 299 nm, ε 12,500 (ε at 366 nm, 85).
MALDI−TOF−MS: calcd. for ODN1( MCVU) [(M–H) –] 1873.30;
found 1873.47.
The yield was calculated based on ODN1’(A).
Each of the reaction mixtures containing ODN1(MCVU) (20 µM,strand concn) and ODN1’(A) (20 µM, strand concn) in 50 mM
sodium cacodylate buffer (pH 7.0) and 100 mM sodium chloride in aPyrex tube was irradiated with a 25 W transilluminator (366 nm,This journal © Royal Society of Chemistry

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910111213141516Communication5,700 µW/cm2) at 0 °C for 30 min. After irradiation, the progress ofphotoreaction was monitored by HPLC on a Chemcobond 5C18 ODS
column (4.6 × 150 mm, elution with a solvent mixture of 50 mM
ammonium formate, pH 7.0, linear gradient over 40 min from 3% to10% acetonitrile at a flow rate 1.0 mL/min).
MALDI−TOF−MS: calcd. for ODN1(MCVU-A) [(M–H) –] 3633.52;
found 3633.87.
MALDI−TOF−MS: calcd. for dA-dMCVU photoadduct [(M+H)+]
545.52; found 545.26.
Y. Wang, J. S. Taylor and M. L. Gross, Chem. Res. Toxicol., 2001,14, 738; Y. Wang, J. S. Taylor and M. L. Gross, Chem, Res. Toxicol.,1999, 12, 1077; X. Zhao and J. S. Taylor, Nucleic Acids Res., 1996,
24, 1561; S. N. Bose, R. J. H. Davies, S. K. Sethi and J. A.
McCloskey, Science, 1983, 723.
For reports on the formation of an aza-cyclobutane structure, see Y.
Yoshimura, Y. Ito and K. Fujimoto, Bioorg. Med. Chem. Lett., 2005,15, 1299; S. Paszyc, B. Skalski and G. Wenska, Tetrahedron Lett.,
1976, 6, 449.
The reaction mixture containing ODN2(MCVU) (20 µM, strand concn)
in 50 mM sodium cacodylate buffer (pH 7.0) and 100 mM sodium
chloride in a Pyrex tube was irradiated with a 25 W transilluminator(366 nm, 5,700 µW/cm2) at 0 °C for 3 h. Then, end-cappedODN2(MCVU-A) was obtained from the isolated peak at 13.5 minfrom HPLC analysis. The progress of photoreaction was monitoredby HPLC on a Chemcobond 5C18 ODS column (4.6 × 150 mm,elution with a solvent mixture of 50 mM ammonium formate, pH 7.0,linear gradient over 40 min from 3% to 12% acetonitrile at a flowrate 1.0 mL/min).
To a solution (0.5 mL) containing HPLC purified ODN2( MCVU) (40µM, strand concn) or ODN2(MCVU-A) (40 µM, strand concn), snake
venom phosphodiesterase (0.2 mL, 0.3 units/mL) was added andincubated at 37 °C.
PAGE analysis was carried out on 20% polyacrylamide gel andeletrophoresis at 280 V for 30 min.
M. A. Zanta, P. B. Valladier and J. P. Behr, Proc. Natl. Acad. Sci.,
1999, 96, 91.

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