HRU, SI, and KT are listed on the patent describing a process for the production of a 5′-phosphorylated oligodeoxynucleotide comprising cleavage 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.
Conceived and designed the experiments: SI KT HRU. Performed the experiments: 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.
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 is cleaved at a nucleotide containing 5-ethynyluracil in a methylamine aqueous solution to generate 5′-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.
Genetic recombination is ubiquitous research tool in the biological sciences. Typically, restriction enzymes are used to cut and paste DNA fragments for genetic recombination
Site-specific DNA cleavage using a DNA base analogue can produce DNA with protruding termini which can be used for seamless DNA concatenation
Here, we report a novel DNA cleavage reaction induced by 5-ethynyluracil. The reaction occurs in a methylamine aqueous solution to cause DNA cleavage at a nucleotide containing 5-ethynyluracil. One of the cleavage products is a 5′-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 be concatenated. The DNA cleavage requires only the addition and removal 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 (
DNA 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 mm×150 mm) with a Gilson Chromatograph, Model 305. Flow rate of the solvent for HPLC was 3.0 mL•min−1. Detection wavelength of the UV detector for 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
Synthesis of DNA oligonucleotides containing 5-ethynyluracil is described in the Supporting Information (
PCR amplification of DNA fragments using primers containing 5-ethynyluracil is described in the Supporting Information (
The base induced-cleavage reaction of DNA containing 5-ethynyluracil has not previously been described in detail. Seela et al reported that DNA oligonucleotides containing 5-ethynyluracil are unstable at 55°C in aqueous ammonia, generating by-products
(A), (B) HPLC charts of T6(EU)T6 before (gray) and after (black) the reaction in 14% NH3aq (A) or 20% MeNH2aq (B) at 70°C for 2 hours. (C), (D) (EU)T2AT2GT2 (C) and T2AT2GT2(EU)T (D) before (gray) and after (black) the reaction in 20% MeNH2aq at 70°C for 2 hours.
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 (
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) (
R is expected to be an abasic sugar derivative.
DNA oligonucleotides, T5X(EU)XT5 (X = A, C, and G) and CGCA2T(EU)TA2CGC, were also cleaved to produce two products corresponding to P1 and P2 (
We applied the QBIC reaction to the concatenation of PCR-amplified DNA fragments. PCR amplification of a DNA fragment with primers containing 5-ethynyluracil generates DNA fragments containing 5-ethynyluracil in the primer-derived regions. Cleavage of the PCR-amplified DNA fragment would produce a gap. If the terminal regions of the two DNA fragments are complementary to each other, they should be able to hybridize by heating and cooling. To verify this, a simple plasmid construction was carried out (
(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) PCR-amplified 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 25°C for 48 h (lane 4,5), 37°C for 10 h (lane 6,7), and 70°C 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 25°C for 48 h (lane 3), 37°C for 10 h (lane 4), and 70°C for 0.5 h (lane 5). (F,G) Intact purified plasmids (F) and EcoRV-digested plasmids (G) derived from the DNA fragments cleaved at 25°C for 48 h (lane 2,3), 37°C for 10 h (lane 4–6), and 70°C 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.
Two pairs of primers shown in
The hybridized samples were diluted 20 times with H2O.
In this study, we report a novel DNA cleavage reaction occurring at a nucleotide containing 5-ethynyluracil in a methylamine aqueous solution. Although the reaction rate is faster at elevated temperatures, the reaction proceeded even at room temperature. One cleavage product is a 5′-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 be achieved in
(TIF)
(TIF)
(TIF)
(TIF)
(TIF)
(TIF)
(PDF)
(PDF)
(PDF)
(PDF)
(PDF)
We are grateful to Dr. Craig C. Jolley (Center for Developmental Biology, RIKEN) for his kind proofreading and improvement of the manuscript and Dr. Yayoi Hongo (Molecular Characterization Team, RIKEN) for measurement of ESI mass spectra. We thank Genome Resource and Analysis Unit for DNA sequencing.