Non-Enzymatic DNA Cleavage Reaction Induced by 5-Ethynyluracil in Methylamine Aqueous Solution and Application to DNA Concatenation

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.

AcOEt (100 mmL) and saturated NaClaq (100 mL) were added to the residue and the product was extracted to the organic layer. The organic layer was washed with saturated NaClaq (100 mL) twice, dried over magnesium sulfate, and filtered. The solvent was removed by evaporation and the residue was dried under reduced pressure. 1,2,4-triazole (3.45 g, 50 mmol), CH 3 CN (30 mL), and triethylamine (7.67 mL, 55.0 mmol) were added to the residue. POCl 3 (932 μL, 10.0 mmol) was slowly added to the solution under constant stirring. After stirring for 1 hour, the solvent was removed by evaporation. AcOEt (100 mmL) and saturated NaClaq (100 mL) were added to the residue and the product was extracted to the organic layer. The organic layer was washed with saturated NaClaq (100 mL) twice, dried over magnesium sulfate, and filtered. The solvent was removed by evaporation and the residue was dried under reduced pressure [20]. 1,4-dioxane (30 mL) was added to the residue and the solution was stirred.
After dissolving the residue to CH 3 CN (10 mL), the phosphoramidite solution was used directly for automated DNA synthesis. T 6 (C1)T 6 was synthesized by standard phosphoramidite method on the DNA synthesizer. The protected oligonucleotide was cleaved by 28% NH 3 aq from the solid support and deprotected at 25°C for 16 hours in 28% NH 3 aq. After removal of ammonia by speed-vac, the aqueous solution was filtered through 0.45 μm filter. T 6 (C1)T 6 was purified by reversed-phase HPLC with a linear gradient over 20 minutes from 0 to 50% CH 3 CN/50 mM AF. The retention time of the product was 10.6 minutes. After the desalination, the product was identified by MALDI TOF mass spectrometry: T 6 (C1)T 6 , calcd for C 133 H 172 N 27 O 88 P 12 3928.6 [M−H] − ; found 3928.8.
After dissolving the residue to CH 3 CN (4.7 mL), the phosphoramidite solution was used directly for automated DNA synthesis. T 6 (C2)T 6 was synthesized by the standard phosphoramidite method on the DNA synthesizer. The last DMTr group was not deprotected on the DNA synthesizer. The protected oligonucleotide was cleaved by 28% NH 3 aq from the solid support and deprotected at 25°C for 16 hours in 28% NH 3 aq. After removal of ammonia by speed-vac, the aqueous solution was filtered through a 0.45 μm filter.
5'-DMTr-T 6 (C2)T 6 was purified by reversed-phase HPLC with a linear gradient over 20 minutes from 5 to 50% CH 3 CN/50 mM AF. The retention time of the 5'-DMTr-T 6 (C2)T 6 was 15.2 minutes. After speed-vac and freeze-drying to remove the solvent and AF, the DMTr group was removed at 25°C for 30 minutes in 80% AcOHaq (100 μL). The solution was diluted with H 2 O to be ≈1 mL and passed through a 0.45 m filter. T 6 (C2)T 6 was purified by reversed-phase HPLC with a linear gradient over 20 minutes from 8 to 20% CH 3 CN/50 mM AF.
After dissolving the residue in CH 3 CN (10 mL), the phosphoramidite solution was used directly for automated DNA synthesis. T 6 (A1)T 6 was synthesized by the standard phosphoramidite method on the DNA synthesizer. The last DMTr group was not deprotected on the DNA synthesizer. The protected oligonucleotide was cleaved from the solid support using 28% NH 3 aq and deprotected at 25°C for 16 hours in 28% NH 3 aq. After removal of ammonia by speed-vac, the aqueous solution was filtered through a 0.45 μm filter. 5'-DMTr-T 6 (C2)T 6 was purified by reversed-phase HPLC with a linear gradient over 20 minutes from 0 to 50% CH 3 CN/50 mM AF. The retention time of the 5'-DMTr-T 6 (A1)T 6 was 16.5 minutes.
After speed-vac and freeze-drying to remove the solvent and AF, the DMTr group was removed at 25°C for 30 minutes in 80% AcOHaq (100 μL). The solution was diluted with H 2 O to 1 mL and filtered through a 0.45 m filter.
The solution was stirred 25°C for 2 hours. The solvent was removed by evaporation. The product 9 was purified by silica gel column chromatography