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Reversible photopadlocking on double‑stranded DNAAuthor(s)Fujimoto, Kenzo; Matsuda, Shigeo; Yoshimura,
Yoshinaga; Ami, Takehiro; Saito, Isao

Citation

Chemical Communications, 2007(28): 2698‑2970

Issue Date2007

TypeJournal Article

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authorURL

http://hdl.handle.net/10119/7903

RightsCopyright (C) 2007 Royal Society of Chemistry.
Kenzo Fujimoto, Shigeo Matsuda, Yoshinaga
Yoshimura, Takehiro Ami and Isao Saito, ChemicalCommunications, 2007(28), 2007, 2698‑2970.
http://dx.doi.org/10.1039/b707524c ‑ Reproducedby permission of The Royal Society of Chemistry

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Japan Advanced Institute of Science and Technology

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Reversible Photopadlocking on Double-Stranded DNA
Kenzo Fujimoto,*a Shigeo Matsuda,‡b Yoshinaga Yoshimura,a Takashi Matsumura,a and Isao Saito§bReceipt/Acceptance Data [DO NOT ALTER/DELETE THIS TEXT]
Publication data [DO NOT ALTER/DELETE THIS TEXT]
5 DOI: 10.1039/b000000x [DO NOT ALTER/DELETE THIS TEXT]

We describe a highly efficient method for reversible
photocircularization of oligonucleotide (ODN) on a doublestranded DNA template. 5-Carboxyvinyl-2’-deoxyuridinecontaining ODN was reversibly circularized around the target
10 sequence of the double-stranded plasmid DNA resulting information of a catenated plasmid.
As more of the sequence data for the human genome becomes
available, new approaches for drug discovery for the treatment anddiagnostics of human diseases caused by genetic mutations are
15 being developed. Previous studies have demonstrated that theexpression of specific genes at the level of transcription andtranslation can be controlled based on chemical approaches.1
Designed modified oligonucleotides (ODNs) including triple helixforming ODN2 and synthetic molecules such as polyamides3 and
420 PNA have been shown to bind sequence-specifically to doublestranded DNA owing to their ability to hybridize with theircomplementary sequences and to be useful in genetic regulations
because the resulting triple helix structures prevent DNA-binding
proteins from recognizing the target sequences and initiating the25 transcriptional process. Recently, circular ODNs as binding agents
have attracted considerable interest because of their unique binding
properties and high-affinity recognition of target sequences.5
Circular ODN probes are also promising reagents for genetic
analysis by forming a circle onto the target.6 Padlock probes can act
30 as inhibitors to block the DNA-protein interaction required for thetranscriptional initiation at sites where padlock probes bind to theplasmid DNA site-specifically. They also discriminate genesequences with single base mutations due to the high sequencespecificity of the padlock7 and serve as templates for rolling circle
835 replication.
Padlock probes catenated with the target sequences of the singlestranded form of the circular cloning vector M13mp18 have beensynthesized by both enzymatic and photochemical methods.6,9However, these methods are not suitable for a circular double40 stranded DNA target because enzymatic reactions are undesirable
under conditions where a triple helix is formed and because thephotochemical method is not efficient. Escudé et al. reported that alinear 89-mer ODN was circularized around a double-strandedaSchool of Materials Science, Japan Advanced Institute of Science andTechnology, Ishikawa, 923-1292, Japan. Fax: +81 761 51 1671; Tel: +81761 51 1671; E-mail: kenzo@jaist.ac.jpbDepartment of Synthetic Chemistry and Biological Chemistry, Faculty ofEngineering, Kyoto University, Kyoto, 606-8501, Japan† Electronic Supplementary Information (ESI) available: Experimental
details. See http://dx.doi.org/10.1039/b000000x/‡ Current address: Department of Chemistry, The Scripps ResearchInstitute, La Jolla, CA 92037, USA.
§ Current address: NEWCAT Institute, School of Engineering, Nihon
University, Koriyama, 963-8642, Japan.

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DNA in the presence of a 20-mer template via a triple helix
formation by using T4 DNA ligase.10 The use of a photoreaction
instead of an enzymic reaction is more desirable for synthesizing
circular ODN around double-stranded DNA. Furthermore, thereversibility of the circularization11 is suitable for controlling gene
expression by a photochemical switch. We previously investigated
50 an efficient and reversible template-directed photoligation in a cissyn [2+2] manner via a vinyl group of modified ODN without
using chemical reagents.12 We report here the synthesis of circularODN onto a double-stranded template and the formation of padlock
probes onto plasmid DNAs by reversible photoligation via 5CV
55 carboxyvinyl-2’-deoxyuridine (U) (Fig. 1). We also demonstratethat padlock probes inhibit the access of a restriction enzyme forrecognizing the target sequences.
4560

Fig. 1 Schematic representation of reversible photopadlocking.

Fig. 2 Autoradiogram of a denaturing 20% polyacrylamide gelelectrophoresis of photoreaction of 32P-5'-end-labeled ODN 1 (2 µM) and65 ODN 2 and ODN 3 (10 µM) in a solution of NaCl (100 mM) and MgCl2 (20mM). Lane 1, before reaction; Lane 2, irradiation at 366 nm, 1 h (sodiumacetate 50 mM, pH 5.0); Lane 3, irradiation at 302 nm, 1 h of Lane 2; Lane4, irradiation at 366 nm, 1 h (sodium cacodylate 50 mM, pH 6.0); Lane 5,irradiation at 366 nm, 1 h (sodium cacodylate 50 mM, pH 7.0); Lane 6,70 irradiation at 366 nm, 1 h (Tris-HCl 50 mM, pH 8.0).

We synthesized ODN 1 (5’-d(CVUTTCCCCTCTT)-S10d(CCTCTTC)-3’; here S corresponds to a hexa(ethylene glycol)
linker fragment) containing CVU at its 5’-end and a triplex-forming
75 sequencewith a target duplex formed by ODN 2 (5’d(GAATTCGGAGAAGAAAGGGGAGAATTC)-3’) and ODN 3(5’-d(GAATTCTCCCCTTTCTTCTCCGAATTC)-3’) by standard
automated DNA synthesis using PEG building blocks andexamined an intrastranded circularization of ODN 1 on the duplex
80 by photoirradiation. When 5’-end-labeled ODN 1 was irradiated at366 nm for 1 hour at pH 5.0 in the presence of ODN 2 and ODN 3,ODN 1 disappeared quantitatively and a slowly migrating band wasobserved on a denaturing 20% polyacrylamide gel (Fig. 2, Lane 2).
The ratio of the migration of the slower band relative to that of the[journal], [year], [vol], 00–00 | 1

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original 34 mer ODN 1 (Lane 1) was ca. 0.9. This result indicatesthat the slower band in Lane 2 was a photocircularized product ofODN 1.12 This circular ODN 4 was converted reversibly to theoriginal linear ODN 1 by photoirradiation at 302 nm for 1 hour
(Lane 3). Although the circularization efficiency at pH 6.0 was
90 similar to that observed at pH 5.0, no circular product was observed
under the conditions under which the triple helix was not formed(Lane 5 and 6). The reactivity dependency on pH shown in Fig. 2
suggests that this photocircularization proceeded through a triple
helix formation.
95To examine the sensitivity of the photocircularization reaction tomismatches, photoreaction was carried out with various duplexes
that contained mismatches for ODN 1 at the ligation site as well as
at other sites (Fig. 3, and 4). In the case of one or two mismatchesat the ligation site, a dramatic decrease of the yield was observed (<100 2% yield, Lane 3, 4, and 5). When duplex 8 and duplex 9 whichwere matched at the ligation site and had several mismatches atother sites were used, circular ODN 4 was also not formed (Lane 6,and 7). These results clearly showed that the photocircularization
was highly sensitive to mismatched sequences.
85

130

target sequences was obtained in 47% yield (Fig. 6a, Lane 2). This
padlocked DNA 12 was quantitatively converted to linear ODN 1by photoirradiation at 302 nm (Lane 3). The padlock probes were
not detected in the use of pUC 18 which did not contain the target
sequences (Lane 4).

135

Fig. 5 Description of the plasmids and ODN 1 used in padlocking
experiments. Modified plasmids were constructed by insertion of duplex
consisting of ODN 2 and ODN 3 after treatment with EcoRI.
140105

Fig. 3 ODNs used in this study. Bold bases indicate the mismatched bases.

Fig. 4 Autoradiogram of a denaturing 20% polyacrylamide gelelectrophoresis of photoreaction of 32P-5'-end-labeled ODN 1 (2 µM) andODN 2 (10 µM) and ODN 3 (10 µM) in sodium acetate buffer (50 mM, pH5.0) containing NaCl (100 mM) and MgCl2 (20 mM). Photoirradiation wasconducted in a quartz capillary cell with transilluminator at 366 nm for 1 h
115 at 0 °C. Lane 1, before reaction; Lane 2, ODN 1 + ODN 2 + ODN 3; Lane 3,ODN 1 + duplex 5; Lane 4, ODN 1 + duplex 6; Lane 5, ODN 1 + duplex 7;
Lane 6, ODN 1 + duplex 8; Lane 7, ODN 1 + duplex 9.
110

To investigate further the possibility of the formation ofpadlocked DNA on a circular double-stranded DNA target byphotocircularization of ODN 1, a duplex consisting of ODN 2 andODN 3 was treated with a restriction enzyme EcoRI and theresulting fragment was incorporated into pUC 18 plasmid DNA by
T4 DNA ligase. Modified plasmids 10 and 11 for photopadlocking
125 experiments were constructed that contained the target sequences
one and five times, respectively (Fig. 5).13 When ODN 1 and themodified plasmid 10 containing the target sequence at one site were
photoirradiated at 366 nm at pH 5.0 for 3 h, the band failed tomigrate on the gel and padlock probes catenated to the plasmid 10120

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Fig. 6 Autoradiogram of a denaturing 12% polyacrylamide gelelectrophoresis of photoreaction of 32P-5'-end-labeled ODN 1 (2 µM) andplasmid 10 (0.125 µg/µL) or plasmid 11 (0.125 µg/µL) in sodium acetate
145 buffer (50 mM, pH 5.0) containing NaCl (100 mM) and MgCl2 (70 mM).
(a) The formation of padlocked DNA on plasmid 10. Lane 1, before
reaction; Lane 2, ODN 1 + plasmid 10, irradiation at 366 nm, 3 h; Lane 3,irradiation at 302 nm, 1 h of Lane 2; Lane 4, ODN 1 + unmodified pUC 18,irradiation at 366 nm, 3 h; Lane 5, EcoRI treatment (37 °C, 1 h) of Lane 2;
150 Lane 6, HindIII treatment (37 °C, 1 h) of Lane 2. (b) The formation ofpadlocked DNA on plasmid 11. Lane 1, before reaction; Lane 2, ODN 1 +plasmid 11, irradiation at 366 nm, 3 h; Lane 3, irradiation at 302 nm, 1 h ofLane 2; Lane 4, EcoRI treatment (37 °C, 1 h) of Lane 2.

This result demonstrates that the photocircularization proceeded
when ODN 1 specifically recognized the target sequences via a
triple helix formation to form circular ODN 4, which was catenatedto plasmid 10. When the reaction mixture after photoirradiation at366 nm for 1 h was treated with EcoRI under appropriate
160 conditions, the plasmid was not cleaved because the circular ODN
4 bounded to the recognition sequence of EcoRI prevented theaccess of the restriction enzyme at the target site (Lane 5). Whenthe mixture was treated with restriction enzyme HindIII, which
recognizes ca. 50 bases upstream from the EcoRI recognition site,
165 the plasmid was then cleaved without inhibition by the padlock
probes (Lane 6). With the use of modified plasmid 11 containing
the five repeated target sequences, padlocked DNA was obtained in
20% yield (Fig. 6b, Lane 2). In this case, the plasmid was cleavedby EcoRI treatment because the enzyme recognized the sites where
170 circular ODN 4 did not bind (Lane 4). Based on these results, it wasconcluded that the padlock probes formed by photocircularization
actually inhibit the activity of the restriction enzyme. Reversible
photopadlocking as shown above may find applications forcontrolling DNA-protein interactions.
155

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CREATED USING THE RSC COMMUNICATION TEMPLATE (VER. 2.1) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILSIn this study, we demonstrated that CVU is very useful forreversible photocircularization on double-stranded DNA with highefficiency. We also showed that reversible photopadlocking on thetarget DNA duplex via photocircularization was realized to block
the access of the restriction enzyme.
180This paper is dedicated to the memory of the late Professor
Emeritus Yoshihiko Ito. This work was supported by a Grant-inAid for Scientific Research from the Ministry of Education, Culture,Sports, Science, and Technology, Japan.
175

240

13 J. Sambrook, E. F. Fritsch and T. Maniatis, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, New
York, 2nd edn, 1989.

245

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