Non-Enzymatic DNA Cleavage Reaction Induced by 5Ethynyluracil in Methylamine Aqueous Solution andApplication to DNA Concatenation
Shuji Ikeda1, Kazuki Tainaka3, Katsuhiko Matsumoto1, Yuta Shinohara5, Koji L. Ode3, Etsuo A. Susaki3,
Hiroki R. Ueda1,2,3,4,5*
1 Laboratory for Synthetic Biology, Quantitative Biology Center, RIKEN, Chuo-ku, Kobe, Japan, 2 Laboratory for Systems Biology, Center for Developmental Biology, RIKEN,
Chuo-ku, Kobe, Japan, 3 Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan, 4 Department of
Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka, Japan, 5 Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan

Abstract
DNA can be concatenated by hybridization of DNA fragments with protruding single-stranded termini. DNA cleavage
occurring at a nucleotide containing a DNA base analogue is a useful method to obtain DNA with designed protruding
termini. Here, we report a novel non-enzymatic DNA cleavage reaction for DNA concatenation. We found that DNA iscleaved at a nucleotide containing 5-ethynyluracil in a methylamine aqueous solution to generate 59-phosphorylated DNA
fragment as a cleavage product. We demonstrated that the reaction can be applied to DNA concatenation of PCR-amplified
DNA fragments. This novel non-enzymatic DNA cleavage reaction is a simple practical approach for DNA concatenation.
Citation: Ikeda S, Tainaka K, Matsumoto K, Shinohara Y, Ode KL, et al. (2014) Non-Enzymatic DNA Cleavage Reaction Induced by 5-Ethynyluracil in Methylamine
Aqueous Solution and Application to DNA Concatenation. PLoS ONE 9(3): e92369. doi:10.1371/journal.pone.0092369
Editor: Giovanni Maga, Institute of Molecular Genetics IMG-CNR, Italy
Received December 22, 2013; Accepted February 21, 2014; Published March 19, 2014
Copyright: ß 2014 Ikeda et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Program for Innovative Cell Biology by Innovative Technology from the Ministry of Education, Culture, Sports, Scienceand Technology (MEXT) of Japan (to HRU), a Grant-in-Aid for Scientific Research (S) (Grant No. 25221004) (to HRU), a Grant-in-Aid for Scientific Research on
Innovative Areas (Grant No. 23115006) from MEXT/Japan Society for the Promotion of Science (JSPS) (to HRU), a grant for the strategic programs for R&D(President’s discretionary fund) of RIKEN (to HRU), an intramural Grant-in-Aid from the RIKEN Center for Developmental Biology and RIKEN Quantitative Biology
Center (to HRU), a grant from Core Research for Evolutional Science and Technology (CREST) (to HRU), the RIKEN Special Postdoctoral Research Program (to EAS),
a Grant-in-Aid from Japan Foundation for Applied Enzymology (to EAS), and a Grant-in-Aid for JSPS Fellows A2559890 (to YS). The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: HRU, SI, and KT are listed on the patent describing a process for the production of a 59-phosphorylated oligodeoxynucleotide comprisingcleavage of an oligodeoxynucleotide including a non-native base. Patent Co-operation Treaty (PCT) Patent No. JP2013/058349. This does not alter our adherence
to PLOS ONE policies on sharing data and materials.
* E-mail: uedah-tky@umin.ac.jp

ity of 8-oxoguanine and the requirement of O2 bubbling for the
degradation of DNA containing 8-oxoguanine.
Here, we report a novel DNA cleavage reaction induced by 5ethynyluracil. The reaction occurs in a methylamine aqueous
solution to cause DNA cleavage at a nucleotide containing 5ethynyluracil. One of the cleavage products is a 59-phosphorylated
DNA fragment which is favourable for enzymatic ligation. We
applied the reaction to the cleavage of PCR-amplified DNA
fragments and showed the resulting DNA fragments can beconcatenated. The DNA cleavage requires only the addition andremoval of methylamine enabling a simple procedure for DNA
concatenation. Sequencing results also indicate that the mutagenicity of 5-ethynyluracil might be low as would be expected given
its structural similarity to thymine (Figure 1).

Introduction
Genetic recombination is ubiquitous research tool in the
biological sciences. Typically, restriction enzymes are used to cut
and paste DNA fragments for genetic recombination [1]. These
enzymes cleave double-stranded DNA to produce sticky ends,
enabling sequence-specific DNA ligation. The recognition sequences for restriction enzymes, however, impose severe sequence
restrictions on the design of recombinant DNA sequences.
Recently, various seamless DNA concatenation methods havebeen reported, in which no specific sequence is required for DNA
joining [2–10].
Site-specific DNA cleavage using a DNA base analogue canproduce DNA with protruding termini which can be used forseamless DNA concatenation [8–10]. Giese et al. achieved nonenzymatic nucleotide-specific DNA cleavage by using 8-oxoguanine as a degradable DNA base to produce single-stranded
overhangs on PCR-amplified DNA [10]. Such non-enzymatic
DNA cleavage reactions have several advantages. 1) Reagents for
non-enzymatic reactions are generally less expensive than
enzymes. 2) Quality control of the reagents is easier than for
enzymes. 3) Non-enzymatic reactions are more often tolerent of
pH and ion concentrations than their enzymatic counterparts.
Their method does have two major limitations — the mutagenicPLOS ONE | www.plosone.org

Materials and Methods
GeneralDNA oligonucleotides were synthesized on an NTS H-6 DNA/RNA synthesizer. Analysis and purification of DNA oligonucleotides by reversed-phase HPLC was carried out on a CHEMCOBOND 5-ODS-H column (10 mm6150 mm) with a Gilson
Chromatograph, Model 305. Flow rate of the solvent for HPLC
was 3.0 mLNmin21. Detection wavelength of the UV detector for
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DNA Cleavage and Concatenation

Figure 1. Chemical structures of thymine (T) and 5-ethynyluracil (EU).
doi:10.1371/journal.pone.0092369.g001

HPLC analysis was 254 nm. MALDI TOF mass spectra were
measured with a Shimadzu AXIMA Assurance. DNA concentration was calculated from UV absorbance at 260 nm [11]. Molar
extinction coefficients of DNA oligonucleotides were calculated byregarding 5-ethynyluracil as thymine.

Cleavage of DNA oligonucleotides
Synthesis of DNA oligonucleotides containing 5-ethynyluracil is
described in the Supporting Information (Method S1) [12,13].
The same volume of 28% NH3aq or 40% MeNH2aq was added tothe aqueous solution of DNA oligonucleotides (100 mM) in ascrew-cap tube for the DNA cleavage. After incubation at 70, 37,
or 25 uC, methylamine in the reaction solutions was removed byspeed-vac. The residue was directly analysed by reversed-phase
HPLC with a linear gradient over 20 minutes from 5 to 20%
CH3CN in 50 mM ammonium formate (AF). The cleavage
products were purified by reversed-phase HPLC, desalted andidentified by MALDI TOF mass spectrometry (Data S1).

DNA concatenation
PCR amplification of DNA fragments using primers containing
5-ethynyluracil is described in the Supporting Information(Method S2) [12,13]. The PCR sample was transferred from the
PCR tube to a screw-cap tube. The same volume of 40%
MeNH2aq was added to the PCR sample to cleave DNA. The
solutions were incubated at 25uC for 48 hours, 37uC for 10 hours,
and 70uC for 0.5 hours, respectively. Methylamine in the sample
was removed by speed-vac. H2O was added to return the sample
to its post-PCR volume. The solutions of 1.5 and 2.2 kbp DNA
fragments were mixed in a 1:1 ratio and incubated at 40uC for 10
minutes for hybridization. The mixed solution was diluted 206
with H2O for the transformation of competent cells (TOYOBO,
Competent high DH5a). 1 mL of the diluted solution and 10 mL ofthawed competent cells were added to an ice-cold tube and the
mixture was left on ice for 30 minutes. After heat-shock at 42uC
for 45 seconds, SOC medium (200 mL) was added to the mixture.
The mixture was incubated at 37uC for 30 minutes and 50 mL ofthe mixture was plated on four LB agar plates containing
ampicillin (50 mgNmL21). After incubation at 37uC for 16 hours,
the numbers of the transformants on the plates were counted. The
transformants were picked up from the colonies and cultured in
3 mL LB medium containing ampicillin (50 mgNmL21) at 37uC for
16 hours. Plasmid was purified from the culture by using the
Wizard Plus minipreps DNA purification system (Promega).
Sequencing of the plasmids was carried out by using BigDye
Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) andan ABI3130 or 3170 automated sequencer (Applied Biosystems).

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P2(T2AT2GT2(EU)T) was larger by 129 than that of T2AT2GT2p(2486) (Data S1). Heating at 120uC in a buffer degraded
P2(T2AT2GT2(EU)T) to produce a major product, namely, P2’(T2AT2GT2(EU)T) (Figure S2). The molecular mass of P2’(T2AT2GT2(EU)T) was indicated as 2486 by MALDI TOF mass
spectrometry (Data S1). Dephosphorylation of P2’ (T2AT2GT2(EU)T) by alkaline phosphatase generated T2AT2GT2, which
was confirmed by MALDI TOF mass spectrometry (Data S1) andcoinjection to reversed-phase HPLC with the authentic
T2AT2GT2. The results strongly suggest that P2’ (T2AT2GT2(EU)T) is T2AT2GT2p. It has been reported that 3’-phosphorylated DNA fragments can be generated by heat degradation of
DNA containing an abasic site [15], suggesting that the structureof P2(T2AT2GT2(EU)T)) might be similar to a cleavage productobtained by elimination of the DNA base [16]. We show thechemical formula of the DNA cleavage reaction in Figure 3.
DNA oligonucleotides, T5X(EU)XT5 (X = A, C, and G) andCGCA2T(EU)TA2CGC, were also cleaved to produce two
products corresponding to P1 and P2 (Figure S3). Here, we usedvolatile methylamine to facilitate removal from the samples. Other
primary amines such as ethylenediamine and 2-aminoethanol canbe also used for the DNA cleavage reaction (data not shown). We
have named the DNA cleavage reaction QBIC (Quantitative BaseInduced DNA Cleavage), as the reaction proceeded almost
quantitatively. We propose a cyclization-driven base eliminationinduced by nucleophilic attack by methylamine as the mechanismof the DNA cleavage reaction induced by 5-ethynyluracil (Method
S3). The DNA cleavage by other DNA base analogues is consistent
with the mechanism (Method S3, Figure S4) [17–26]. However,
the rate of the DNA cleavage induced by the DNA analogues was
slower than that using EU. Our preliminary results indicate thatthe reaction might be applicable to the preparation of 59phosphorylated DNA oligonucleotides by using the DMTr-ON
method on a DNA synthesizer (Method S4, Figure S5) [17].
We applied the QBIC reaction to the concatenation of PCRamplified DNA fragments. PCR amplification of a DNA fragmentwith primers containing 5-ethynyluracil generates DNA fragments
containing 5-ethynyluracil in the primer-derived regions. Cleavageof the PCR-amplified DNA fragment would produce a gap. If theterminal regions of the two DNA fragments are complementary toeach other, they should be able to hybridize by heating andcooling. To verify this, a simple plasmid construction was carriedout (Figure 4A). Before PCR, a DNA oligonucleotide containing
A, C, G, T, and EU bases was confirmed to be stable under PCR
conditions (Figure S6).
Two pairs of primers shown in Figure 4B were used to amplify
1.5 and 2.2 kbp DNA fragments from linear lambda phage DNA

Figure 2. Degradation of DNA oligonucleotides containing 5ethynyluracil. (A), (B) HPLC charts of T6(EU)T6 before (gray) and after(black) the reaction in 14% NH3aq (A) or 20% MeNH2aq (B) at 70uC for
2 hours. (C), (D) (EU)T2AT2GT2 (C) and T2AT2GT2(EU)T (D) before (gray)and after (black) the reaction in 20% MeNH2aq at 70uC for 2 hours.
doi:10.1371/journal.pone.0092369.g002

Results and Discussion
The base induced-cleavage reaction of DNA containing 5ethynyluracil has not previously been described in detail. Seela etal reported that DNA oligonucleotides containing 5-ethynyluracil
are unstable at 55uC in aqueous ammonia, generating by-products[12]. In order to investigate the degradation reaction, we prepared
DNA oligonucleotides containing 5-ethynyluracil (EU) according
to the literature [12,13]. As reported, a DNA oligonucleotide,
T6(EU)T6, generated the by-products under heating in aqueous
ammonia (Figure 2A). MALDI -TOF mass data indicate that themain peak in Figure 2A corresponds to T6(EU)T6. The molecularmass of the main by-product (MB), appearing just after the main
peak, is larger by 17 than that of T6(EU)T6.This indicates that MB
might be generated by addition of one molecule of ammonia to
T6(EU)T6.
To our surprise, we found that methylamine, which is a stronger
nucleophile than ammonia, caused nearly quantitative DNA
cleavage of T6(EU)T6 at the nucleotide containing 5-ethynyluracil.
Two products, P1(T6(EU)T6) and P2(T6(EU)T6), appeared as two
peaks on the reversed-phase HPLC chart after the reaction(Figure 2B). The molecular masses of P1(T6(EU)T6) andP2(T6(EU)T6) are indicated as 1843 and 1972 by MALDI TOF
mass spectrometry (Data S1). The results indicate that T6(EU)T6
was cleaved at the EU nucleotide. P1(T6(EU)T6) was expected tobe either pT6 or T6p because the molecular mass is identical tothose of pT6 and T6p. P2(T6(EU)T6) likely contains a residue
derived from the EU nucleotide because the molecular mass islarger than those of pT6 and T6p. Most of the T6(EU)T6 wascleaved after 30 minutes at 70uC, 10 hours at 37uC, and 48 hours
at 25uC in 20% MeNH2aq (Figure S1).
The cleavage reaction was examined in detail by using(EU)T2AT2GT2 and T2AT2GT2(EU)T. Treatment of (EU)T2AT2GT2 with methylamine generated a single main product,
namely, P1((EU)T2AT2GT2) (Figure 2C). P1((EU)T2AT2GT2) was
identified as pT2AT2GT2 by MALDI TOF mass spectrometry(Data S1) and coinjection with authentic pT2AT2GT2 [14] toreversed-phase HPLC. The methylamine treatment with
T2AT2GT2(EU)T generated a single main product, namely,
P2(T2AT2GT2(EU)T) (Figure 2D). The molecular mass of

Figure 3. Chemical formula of the DNA cleavage reaction. R is expected to be an abasic sugar derivative.
doi:10.1371/journal.pone.0092369.g003

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Figure 4. Construction of plasmid from two PCR-amplified DNA fragments. (A) Scheme of plasmid construction. (B) Primer sequences used
for PCR. The two sequences underlined in red and blue are complementary to each other. (C–G) Pictures of agarose gel electrophoresis. (C) PCRamplified DNA fragments 1.5 (lane 2) and 2.2 kbp (lane 3). (D) 1.5 and 2.2 kbp DNA fragments before (lane 2,3) and after DNA cleavage at 25uC for
48 h (lane 4,5), 37uC for 10 h (lane 6,7), and 70uC for 0.5 h (lane 8,9). MeNH2 was removed from the samples by speed-vac before electrophoresis. (E)Hybridized 1.5 and 2.2 kbp DNA fragments derived from those without cleavage reaction (lane 2) and cleaved at 25uC for 48 h (lane 3), 37uC for 10 h(lane 4), and 70uC for 0.5 h (lane 5). (F,G) Intact purified plasmids (F) and EcoRV-digested plasmids (G) derived from the DNA fragments cleaved at
25uC for 48 h (lane 2,3), 37uC for 10 h (lane 4–6), and 70uC for 0.5 h (lane 7–9). (H) Sequencing results of primer-derived regions of the plasmids.
Underlined letters correspond to EU in the primers.
doi:10.1371/journal.pone.0092369.g004
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DNA Cleavage and Concatenationand EcoRI-digested pBluescript-sk(–) [27], respectively. Successful
amplification of the fragments was indicated by agarose gelelectrophoresis (Figure 4C). The same volume of 40% methylamine aqueous solution was added to the PCR samples and the
mixtures were incubated at 70uC for 30 minutes, 37uC for
10 hours, and 25uC for 48 hours, respectively. After the cleavage
reaction, methylamine in the samples was removed by speed-vac
and the sample was diluted with H2O to restore the volume it had
after PCR. The cleaved DNA fragments were analyzed by agarose
gel electrophoresis (Figure 4D). The bands corresponding to the1.5
and 2.2 kbp DNA fragments were slightly weaker after cleavage at
higher temperature possibly due to alkali denaturation.of the DNA
fragments. The same volume solutions of the cleaved DNA
fragments were mixed and the resultant solution was incubated at
40uC for 10 minutes and cooled to 25uC for hybridization. The
hybridized samples were analyzed by agarose gel electrophoresis(Figure 4E). Weak bands with a size larger than those of the two
DNA fragments indicated that at least a part of them hybridized toform longer DNA fragments, although it was not clear that all the
hybridized structures of the two DNA fragments were stablethroughout the agarose gel electrophoresis.
The hybridized samples were diluted 20 times with H2O.
Escherichia coli (DH5a) was transformed using 1 mL of the diluted
samples. The transformants were plated on LB plates containing
ampicillin. The numbers of the transformed DH5a colonies were5968, 53617, and 1762 for samples with DNA cleavage
temperatures of 25uC, 37uC, and 70uC, respectively. Threeplasmids were purified from the transformants derived from eachDNA sample. The agarose gel electrophoresis of the intact
plasmids (Figure 4F) indicated that the size of one plasmid at lane
3 was larger than those of the other plasmids. Cleavage of theplasmids by EcoRV, which would cut one site of the 1.5 kbp
fragment, produced 3.7 kbp fragments (Figure 4G) as expected.
Those results indicated that the larger plasmid was the dimer ofthe 3.7 kbp DNA fragment. Sequencing of the primer-derived
region of the eight monomer plasmids made it clear that 5ethynyluracil was correctly replicated as T in the concatenatedDNA (Figure 4H). DNA concatenation experiments were carriedout for 14 other clones as well; mutations caused by 5ethynyluracilwere never observed (data not shown). One deletionof the plasmid derived from the sample cleaved at 70uC waspresumably caused by insufficient deprotection of the DMTrgroup during the automated DNA synthesis of the primer.

(gray) and after (black) the reaction in 20% MeNH2aq at 70uC for
0.5 hours (A), 37uC for 10 hours (B), and 25uC for 48 hours (C).
(TIF)
Figure S2 Heat degradation of PB’. HPLC charts of
P2(T2AT2GT2(EU)T) before (gray) and after (black) the reaction
in sodium phosphate buffer (100 mM, pH 7.0 at 25uC) at 120uC
for 2 hours.
(TIF)
Figure S3 Reactivity of EU in various sequences of DNA
oligonucleotides. HPLC charts of T5A(EU)AT5 (A),T5C(EU)CT5 (B), T5G(EU)GT5 (C), and CGCA2T(EU)TA2CGC(D) before (gray) and after (black) reaction in 20% MeNH2aq at
70uC for 2 hours.
(TIF)
Figure S4 Reactivity of DNA oligonucleotides containing
designed DNA base analogues. (A–F) HPLC charts of
T6(EU)T6 (A), T6(T1)T6 (B), T6(C1)T6 (C), T6(C2)T6 (D),T6(A1)T6 (E), and T6(G1)T6 (F) before (gray) and after (black)
the reaction in 20% MeNH2aq at 70uC for 12 hours.
(TIF)
Figure S5 HPLC analysis of 59-phosphorylation of DNA
oligonucleotide. HPLC charts of crude DNA solution of
DMTr-(PU)T2AT2GT2 after NH3 treatment (gray) and crude
DNA solution of pT2AT2GT2 after ethylenediamine treatment(black). PU depicts 5-phenylethynyluracil.
(TIF)
Figure S6 Stability of DNA oligonucleotide containing A,
T, G, C, and EU under PCR condition. HPLC chart of
CGCA2T(EU)TA2CGC in PCR buffer (61) after a temperature
program, 94uC, 2 min R [98uC, 30 sec R 60uC, 30 sec R 68uC,
90 sec] 630 R 4uC.
(TIF)
Data S1

Method S1 Preparation of DNA oligonucleotides containing 5-ethynyluracil (EU).
(PDF)
Method S2 PCR amplification by using primers containing 5-ethynyluracil (EU).
(PDF)

Conclusions

Method S3 Preparation and cleavage of DNA oligonucleotides containing DNA base analogues.
(PDF)

In this study, we report a novel DNA cleavage reaction
occurring at a nucleotide containing 5-ethynyluracil in amethylamine aqueous solution. Although the reaction rate isfaster at elevated temperatures, the reaction proceeded even atroom temperature. One cleavage product is a 59-phosphorylated
DNA fragment, which is favourable for applications using
enzymatic DNA ligation. We applied the reaction to cleave
PCR-amplified DNA fragments, hybridized the DNA fragments,and showed that concatenation of the DNA fragments can beachieved in Escherichia coli. The sequencing data of the concatenated DNA indicats high fidelity of 5-ethynyluracil as a templatefor DNA replication. Because DNA cleavage requires only
addition and removal of methylamine, this procedure for DNA
concatenation is quite simple.

Method S4 59-phosphorylation of the DNA oligonucleotide using 5-phenylethynyluracil (PU).
(PDF)

Acknowledgments
We are grateful to Dr. Craig C. Jolley (Center for Developmental Biology,
RIKEN) for his kind proofreading and improvement of the manuscript andDr. Yayoi Hongo (Molecular Characterization Team, RIKEN) for
measurement of ESI mass spectra. We thank Genome Resource andAnalysis Unit for DNA sequencing.

Author Contributions

Supporting Information

Conceived and designed the experiments: SI KT HRU. Performed theexperiments: SI KT KM YS KLO EAS. Analyzed the data: SI KT KM
YS KLO EAS. Contributed reagents/materials/analysis tools: SI KT KM
YS KLO EAS. Wrote the paper: SI HRU.

Figure S1 DNA cleavage of T6(EU)T6 at 25, 37, and 706C
in 20% MeNH2aq. (A–C), HPLC charts of T6(EU)T6 before
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MALDI TOF mass data of cleavage products.

(PDF)

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