Data S1
MALDI TOF mass data of cleavage products
Products from T6(EU)T6; pT6 (P1), calcd C60H79N12O43P6 1842.2 [M−H]−; found
1841.8; T6pR (P2), calcd C66H90N13O45P6 1971.3 [M−H]−; found 1971.3; Productfrom (EU)T2AT2GT2; pT2AT2GT2 (P1), calcd for C80H103N22O54P8 2484.6 [M−H]−;found 2484.6; Product from T2AT2GT2(EU)T; T2AT2GT2pR (P2), calcd for
C86H114N23O56P8 2613.7 [M−H]−; found 2613.8; Products from T5A(EU)AT5; pAT5
(P1), calcd C60H78N15O41P6 1851.2 [M−H]−; found 1850.9; T5ApR (P2), calcd
C66H89N16O43P6 1980.3 [M−H]−; found 1980.2; Products from T5C(EU)CT5; pCT5
(P1), calcd C59H78N13O42P6 1827.2 [M−H]−; found 1827.0; T5CpR (P2), calcd
C65H89N14O44P6 1956.3 [M−H]−; found 1956.3; Products from T5G(EU)GT5; pGT5
(P1), calcd C60H78N15O42P6 1867.2 [M−H]−; found 1867.2; T5GpR (P2), calcd
C66H89N16O44P6 1996.3 [M−H]−; found 1996.2; Products from
CGCA2T(EU)TA2CGC; pTA2CGC (P1), calcd C58H74N23O36P6 1855.2 [M−H]−;found 1855.0; CGCA2TpR (P2), calcd C64H85N24O38P6 1984.3 [M−H]−; found
1984.3. R (C6H11NO2) was calculated on the supposition that the cleavage
reaction was accompanied by elimination of EU and addition of methylamineand OH−.

Method S1
Preparation of DNA oligonucleotides containing 5-ethynyluracil (EU)DNA oligonucleotides containing EU were synthesized on the DNA synthesizer
by using the standard phosphoramidite method. The phosphoramidite toincorporate EU into DNA oligonucleotides was synthesized according to theliterature [12,13]. Phosphoramidites of natural DNA nucleosides (BzdA, AcdC,
dmf
dG, and T) were purchased from Glen research. The coupling time of EU
phosphoramidite was extended to 30 minutes to ensure high coupling efficiency.
The last DMTr group was not deprotected on the DNA synthesizer (DMTr-ON).
After automated DNA synthesis, the CPG-support was put into a screw-cap tube
and 1 mL of 50 mM K2CO3/MeOH was added into the tube. The suspension wasleft at 25°C for 16 hours or 65°C for 4 hours. A small amount of H2O was added
to the suspension and MeOH in the suspension was removed by speed-vac.
H2O was added to the residue to reach a total volume of ≈500 μL and 28%
NH3aq (500 μL) was added to the suspension. The suspension was left at 25°C
for 16 hours. NH3 in the suspension was removed by speed-vac. The
suspension was passed through a 0.45 μm filter. The DMTr-ON DNA
oligonucleotides were purified by reversed-phase HPLC with a linear gradientover 20 minutes from 5 to 50% CH3CN in 50 mM ammonium formate (AF). After
speed-vac and freeze-drying to remove the solvent and AF, the DMTr group on
the DNA oligonucleotides was removed in 80% AcOH aqueous solution (100 μL)
at 25°C for 30 minutes. H2O was added to the solution to reach a total volume of≈1 mL. The DNA oligonucleotides were purified by reversed-phase HPLC with alinear gradient over 20 minutes from 5 to 20% CH3CN in 50 mM AF. The purified
DNA oligonucleotides were desalted and identified by MALDI TOF mass
spectrometry except for long primers (≥ 30 mer): T6(EU)T6, calcd for
C131H167N26O89P12 3901.5 [M−H]−; found 3901.8; (EU)T2AT2GT2, calcd for
C91H113N24O58P8 2718.8 [M−H]−; found 2718.9; T2AT2GT2(EU)T, calcd for
C101H126N26O65P9 3023.0 [M−H]−; found 3023.4; T5A(EU)AT5, calcd for
C131H165N32O85P12 3919.6 [M−H]−; found 3920.3; T5C(EU)CT5, calcd for
C129H165N28O87P12 3871.5 [M−H]−; found 3872.0; T5G(EU)GT5, calcd for
C131H165N32O87P12 3951.6 [M−H]−; found 3952.5; CGCA2T(EU)TA2CGC, calcd
for C127H157N48O75P12 3927.6 [M−H]−; found 3928.1; AG2TGCT3A(EU)GACTCTGC2G, calcd for C206H257N74O128P20 6437.1 [M−H]−; found 6437.8.

Methods S2
PCR amplification by using primers containing 5-ethynyluracil (EU)KOD Fx neo (TOYOBO) was used for PCR. Before PCR, CGCA2T(EU)TA2CGC
in ×1 buffer for KOD Fx neo was heated with a temperature program, 94°C, 2
min → [98°C, 10 sec → 60°C, 30 sec → 68°C, 90 sec] ×30 → 4°C. After heating,the sample was directly analyzed by reversed-phase HPLC with a linear gradientover 20 minutes from 5 to 20% CH3CN in 50 mM AF (Fig. S5). PCR solution (50μL) contained primers (0.4 μM, each), dNTPs (400 μM, each), buffer (×1), KOD
Fx neo (1 U), and template DNA, EcoRI-digested pBluescript-sk(–) (200 ng) or
linear λ phage DNA (1 ng). The linear λ phage DNA (N3011) was purchasedfrom New England Biolabs. DNA fragments for the construction of the 3.7 kbp
plasmid were amplified using a temperature program, 94°C, 2 min → [98°C, 10
sec → 60°C, 30 sec → 68°C, 90 sec] ×30 → 4°C. The PCR-amplified DNA
fragments were analyzed on a 1% agarose gel containing ethidium bromide in
TAE buffer by electrophoresis.

Methods S3
DNA base analogues for DNA cleavage
We found that the DNA base analogues described below can cause the same
DNA cleavage as that induced by EU. Their synthetic schemes are shown
following their respective synthetic procedures.

Preparation of T6(T1)T6
T6(T1)T6 was postsynthetically prepared from the DNA oligonucleotide
containing 5-ethynyluracil (PU) [17]. AgNO3aq (100 mM, 200 μL) was added toT6(PU)T6 aqueous solution (1 mM strand concentration, 200 μL) [18]. The
solution was mixed and left at 25°C for 16 hours. After addition of NaOHaq (10 M,
20 μL) [19], the solution was mixed and left at 25°C for 2 hours. H2O (600 μL)
containing acetic acid (20 μL) was added to the solution. The solution was
filtered with 0.45 nm filter. The product was purified by reversed-phase HPLC
with a linear gradient over 20 minutes from 8 to 20% CH3CN/50 mM AF. The
retention time of the product was 12.5 minutes. After desalination, the productwas identified by MALDI TOF mass spectrometry: T6(T1)T6, calcd for
C137H173N26O90P12 3995.6 [M−H]−; found 3995.6.

6-N-(3-propynyl)-5-methyl-2'-deoxycytidine (1)Thymidine (1.21 g, 5.00 mmol) was suspended in CH3CN (20 mL). Triethylamine(5.58 mL, 40.0 mmol) and acetic anhydride (1.89 mL, 20.0 mmol) were added tothe suspension. After stirring at 25°C for 21 hours, methanol (10 mL) was added
to the solution and stirred for 2 hours. 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 waswashed 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), CH3CN (30 mL), andtriethylamine (7.67 mL, 55.0 mmol) were added to the residue. POCl3 (932 μL,
10.0 mmol) was slowly added to the solution under constant stirring. After stirringfor 1 hour, the solvent was removed by evaporation. AcOEt (100 mmL) andsaturated 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.
Propargylamine (961 μL, 15.0 mmol) was added to the solution. After stirring at
25°C for 2 hours, methanol (30 mL) and 28% NH3aq (30 mL) were added to thesolution. The solution was stirred at 25°C for 24 hours. The solvent was removedby evaporation and the product 1 was purified by silica gel column
chromatography (10–15% MeOH/CH2Cl2). The product 1 was obtained as awhite powder (873 mg, 3.13 mmol, 63%): 1H NMR (DMSO-d6) δ 7.65 (d, J = 0.9,
1H), 7.57 (t, J = 5.7, 1H), 5.19 (d, J = 4.6, 1H), 5.01 (t, J = 5.3, 1H), 4.23-4.18 (m,1H), 4.14-4.01 (m, 2H),3.76-3.74 (m, 1H),3.62-3.50 (m, 2H), 3.04 (t, J = 2.5 ,1H),2.12-2.05 (m, 1H),1.99-1.92 (m, 1H),1.84 (s, 3H); 13C NMR (DMSO-d6) δ
162.4, 154.9, 138.0, 101.7, 87.2, 84.8, 81.5, 72.6, 70.4, 61.4, 40.3, 29.4, 13.0;
HRMS (ESI) calcd for C13H17N3O4Na ([M+Na]+) 302.1116, found 302.1117.
5'-O-dimetoxytrityl-6-N-(3-propynyl)-5-methyl-2'-deoxycytidine (2)Pyridine (10 mL) was added to 1 (312 mg, 1.12 mmol) and DMTrCl (454 mg,
1.34 mmol). The solution was stirred at 25°C for 1 hour. After addition of
methanol (10 mL), the solvent was removed by evaporation. The residual
pyridine was removed by coevaporation with CH2Cl2 and hexane. The product 2
was purified by silica gel column chromatography (2–5% MeOH, 2%
Et3N/CH2Cl2). The product 2 was obtained as a white powder (631 mg, 0.924
mmol, 83%): 1H NMR (DMSO-d6) δ 7.63 (t, J = 5.7, 1H), 7.51 (s, 1H), 7.39-7.35(m, 2H),7.32-7.19 (m, 7H), 6.90-6.86 (m, 4H),6.21 (t, J = 6.6, 1H), 5.34 (d, J =4.1, 1H), 4.31-4.25 (m, 1H), 4.14-4.02 (m, 2H),3.90-3.86 (m, 1H),3.72 (s, 6H),
3.22-3.16 (m, 2H),3.09-3.00 (m, 6H),2.21-2.06 (m, 2H),1.50 (s, 3H), 1.23-1.15(m, 9H); 13C NMR (DMSO-d6) δ 162.3, 158.1, 157.9, 154.8, 144.7, 137.4, 135.5,
135.3, 130.0, 127.9, 127.7, 126.8, 113.2, 113.0, 101.9, 85.8, 85.4, 84.6, 81.4,
72.6, 70.5, 63.6, 55.0, 45.3, 29.4, 8.4; HRMS (ESI): calcd for C34H35N3O6Na([M+Na]+) 604.2525, found 604.2424.
Preparation of T6(C1)T6
2 (291 mg, 0.426 mmol) was dissolved in CH3CN (5 mL). Disopropylethylamine(261 μL, 1.50 mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite(223 μL, 1.00 mmol) was added to the solution. The solution was stirred at 25°C
for 30 minutes. AcOEt (25 mL) and saturated NaClaq (25 mL) were added to thesuspension. The product was extracted to the organic layer. The organic layer
was washed with saturated NaClaq (25 mL) twice, dried over magnesium sulfate,and filtered. The solvent was removed by evaporation and the residue was driedby coevaporation with acetonitrile and further dried under reduced pressure.
After dissolving the residue to CH3CN (10 mL), the phosphoramidite solution
was used directly for automated DNA synthesis. T6(C1)T6 was synthesized bystandard phosphoramidite method on the DNA synthesizer. The protectedoligonucleotide was cleaved by 28% NH3aq from the solid support anddeprotected at 25°C for 16 hours in 28% NH3aq. After removal of ammonia byspeed-vac, the aqueous solution was filtered through 0.45 μm filter. T6(C1)T6
was purified by reversed-phase HPLC with a linear gradient over 20 minutes
from 0 to 50% CH3CN/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: T6(C1)T6, calcd for C133H172N27O88P12 3928.6 [M−H]−; found3928.8.

4-N-[N,N-di(n-butyl)aminomethylene]-5-phenylethynyl-2'-deoxycytidine (3)5-phenylethynyl-2'-deoxycytidine (708 mg, 2.16 mmol) [21] was dissolved inmethanol (10 mL). N,N-di(n-butyl)formamide dimethyl acetal (2.0 mL, 8.6 mmol)[22] was added to the solution. The solution was stirred at 25°C for 2 hours. The
solvent was removed by evaporation. The product 3 was purified by silica gelcolumn chromatography (0–10% MeOH/CH2Cl2). The product 3 was obtained asa pale brown oil (700 mg, 1.50 mmol, 69%): 1H NMR (DMSO-d6) δ 8.64 (s, 1H),
8.44 (s, 1H), 7.43-7.34 (m, 5H), 6.12 (t, J = 6.2, 1H), 5.23 (d, J = 4.1, 1H), 5.13 (t,J = 4.8, 1H), 4.26-4.21 (m, 1H), 3.84-3.81 (m, 1H), 3.70-3.44 (m, 6H), 2.26-2.20(m, 1H), 2.08-2.01 (m, 1H), 1.66-1.52 (m, 4H), 1.32-1.22 (m, 4H), 0.90 (t, J = 7.3,
3H), 0.82 (t, J = 7.3, 3H); 13C NMR (DMSO-d6) δ 169.7, 157.4, 153.4, 145.3,
130.7, 128.6, 128.1, 123.2, 97.5, 91.6, 87.6, 85.8, 84.5, 69.7, 60.7, 54.9, 51.4,
45.4, 41.0, 30.4, 28.5, 19.6, 19.1, 13.5; HRMS (ESI) calcd for C26H35N4O4([M+H]+) 467.2658, found 467.2653.
5'-O-dimethoxytrityl-4-N-[N,N-di(n-butyl)aminomethylene]-5-phenylethynyl-2'-deoxycytidine (4)Pyridine (10 mL) was added to 3 (700 mg, 1.50 mmol) and DMTrCl (763 mg,
2.25 mmol). The solution was stirred at 25°C for 1 hour. After addition of
methanol (10 mL), the solvent was removed by evaporation. The residual
pyridine was removed by coevaporation with CH2Cl2 and hexane. The product 4
was purified by silica gel column chromatography (0–5% MeOH, 2%
Et3N/CH2Cl2). The product 4 was obtained as white powder (924 mg, 1.20 mmol,80%): 1H NMR (DMSO-d6) δ 8.65 (s, 1H), 8.17 (s, 1H), 7.44-6.81 (m, 18H), 6.14
(t, J = 6.6, 1H), 5.32 (d, J = 4.6, 1H), 4.31-4.26 (m, 1H), 4.02-3.97 (m, 1H), 3.644(s, 3H), 3.637 (s, 3H), 3.57-3.49 (m, 4H), 3.23-3.16 (m, 2H), 2.36-2.30 (m, 1H),
2.17-2.10 (m, 1H), 1.66-1.53 (m, 4H), 1.32-1.22 (m, 4H), 0.91 (t, J = 7.3, 3H),
0.82 (t, J = 7.3, 3H); 13C NMR(DMSO-d6) δ 169.7, 158.03, 158.01, 157.5, 153.4,
144.7, 144.3, 135.6, 135.4, 130.6, 129.7, 129.6, 128.3, 127.93, 127.86, 127.6,
126.6, 123.0, 113.2, 97.9, 91.7, 86.2, 85.9, 83.9, 70.6, 63.5, 54.9, 54.5, 45.4,
41.2, 30.4, 28.5, 19.6, 19.1, 13.5; HRMS (ESI): calcd for C47H53N4O6 ([M+H]+)
769.3965, found 769.3985.
Preparation of T6(C2)T6
4 (109 mg, 0.142 mmol) was dissolved in CH3CN (5 mL). Triethylamine (157 μL,
1.13 mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (126 μL,
0.565 mmol) was added to the solution. The solution was stirred at 25°C for 30
minutes. AcOEt (25 mL) and saturated NaClaq (25 mL) were added to thesuspension. The product was extracted to the organic layer. The organic layer
was washed with saturated NaClaq (25 mL) twice, dried over magnesium sulfate,
and filtered. The solvent was removed by evaporation and the residue was driedby coevaporation with acetonitrile and further dried under reduced pressure.

After dissolving the residue to CH3CN (4.7 mL), the phosphoramidite solution
was used directly for automated DNA synthesis. T6(C2)T6 was synthesized by
the standard phosphoramidite method on the DNA synthesizer. The last DMTrgroup was not deprotected on the DNA synthesizer. The protectedoligonucleotide was cleaved by 28% NH3aq from the solid support anddeprotected at 25°C for 16 hours in 28% NH3aq. After removal of ammonia byspeed-vac, the aqueous solution was filtered through a 0.45 μm filter.
5'-DMTr-T6(C2)T6 was purified by reversed-phase HPLC with a linear gradientover 20 minutes from 5 to 50% CH3CN/50 mM AF. The retention time of the
5'-DMTr-T6(C2)T6 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 H2O to be ≈1
mL and passed through a 0.45 m filter. T6(C2)T6 was purified by reversed-phase
HPLC with a linear gradient over 20 minutes from 8 to 20% CH3CN/50 mM AF.
The retention time of the product was 13.6 minutes. After the desalination, theproduct was identified by MALDI TOF mass spectrometry: T6(C2)T6, calcd for
C137H172N27O88P12 3976.6 [M−H]−; found 3977.1.

8-(2-formylphenyl)-2'-deoxyadenosine (5)8-bromo-2'-deoxyadenosine (2.20 g, 6.66 mmol) [23], 2-formylbenzene boronic
acid (2.00 g, 13.3 mmol), palladium (II) acetate (150 mg, 0.666 mmol),
triphenylphosphine (525 mg, 2.00 mmol), and potassium carbonate (1.84 g, 13.3mmol) were stirred in acetonitrile (40 mL) and H2O (20 mL) at 80°C for 4 hours[24,25]. The solvent was removed by evaporation. Methanol (200 mL) was
added to the residue. The resulting solution was filtered. The solvent wasremoved by evaporation. The product 5 was purified by silica gel column
chromatography (10% MeOH/CH2Cl2). The product 5 was obtained as white
powder (1.81 g, 5.09 mmol, 76%): 1H NMR (DMSO-d6) δ 9.93 (s, 1H), 8.18 (s,1H), 8.09-8.07 (m, 1H), 7.90-7.80 (m, 2H), 7.74-7.72 (m, 1H), 7.46 (br, 2H), 5.90
(dd, J = 8.5, 6.2, 1H), 5.49 (dd, J = 8.3, 4.1, 1H), 5.17 (d, J = 4.1, 1H), 4.39-4.35(m, 1H), 3.82-3.79 (m, 1H), 3.65-3.59 (m, 1H), 3.50-3.44 (m, 1H), 3.19 (ddd, J =12.8, 8.7, 6.0, 1H), 2.06 (ddd, J = 13.0, 6.2, 1.8, 1H); 13C NMR (DMSO-d6) δ
191.7, 156.2, 152.3, 149.6, 147.4, 135.4, 133.8, 131.5, 131.4, 130.8, 129.5,
119.3, 88.3, 85.7, 71.3, 62.2, 37.4; HRMS (ESI) calcd for C17H18N5O4 ([M+H]+)
356.1359, found 356.1364.
6-N-[N,N-di(n-butyl)aminomethylene]-8-(2-formylphenyl)-2'-deoxyadenosine (6)5 (1.55 g, 4.36 mmol) was dissolved in methanol (20 mL). N,N-di(n-butyl)formamide dimethyl acetal (8.14 mL, 34.8 mmol) was added to the solution [22].
The solution was stirred at 25°C for 2 hours. The solvent was removed byevaporation. The product 6 was purified by silica gel column chromatography(2–5% MeOH/CH2Cl2). The product 6 was obtained as a pale brown oil (1.40 g,
2.83 mmol, 65%): 1H NMR (DMSO-d6) δ 9.92 (s, 1H), 8.91 (s, 1H), 8.45 (s, 1H),
8.09 (d, J = 7.8, 1H), 7.91-7.82 (m, 2H), 7.74 (d, J = 7.3, 1H), 5.94 (dd, J = 7.8,
6.8, 1H), 5.28 (dd, J = 7.6, 4.4, 1H), 5.18 (d, J = 4.1, 1H), 4.40-4.36 (m, 1H),
3.81-3.77 (m, 1H), 3.64-3.41 (m, 6H), 3.25-3.18(m, 1H), 2.09-2.04 (m, 1H),
1.62-1.52 (m, 4H), 1.33-1.22 (m, 4H), 0.94-0.83 (m, 6H); 13C NMR(DMSO-d6) δ
191.8, 159.5, 157.9, 151.7, 151.6, 149.3, 135.5, 133.8, 131.5, 131.3, 130.9,
129.7, 125.8, 88.2, 85.5, 71.2, 62.1, 50.9, 44.4, 37.2, 30.4, 28.7, 19.6, 19.1,13.7,
13.5; HRMS (ESI) calcd for C26H35N6O4 ([M+H]+) 495.2720, found 495.2718.
5'-O-dimethoxytrityl-6-N-[N,N-di(n-butyl)aminomethylene]-8-(2-formylphenyl)-2'-deoxyadenosine (7)Pyridine (10 mL) was added to 6 (1.31 g, 2.64 mmol) and DMTrCl (991 mg, 2.92mmol). The solution was stirred at 25°C for 1 hour. After addition of methanol (10mL), the solvent was removed by evaporation. The residual pyridine wasremoved by coevaporation with CH2Cl2 and hexane. The product 7 was purified
by silica gel column chromatography (2% MeOH, 2% Et3N/CH2Cl2). The product7 was obtained as a white powder (1.24 g, 1.56 mmol, 59%): 1H NMR(DMSO-d6) δ 9.89 (s, 1H), 8.88 (s, 1H), 8.25 (s, 1H), 8.06-8.04 (m, 1H),
7.88-7.78 (m, 3H), 7.33-7.31 (m, 2H), 7.21-7.15 (m, 7H), 6.81-6.73 (m, 4H), 5.98
(t, J = 6.9, 1H), 5.25 (d, J = 4.6, 1H), 4.57-4.53 (m, 1H), 3.94-3.90 (m, 1H), 3.71
(s, 3H), 3.69 (s, 3H), 3.59-3.53 (m, 2H), 3.44-3.32 (m, 3H), 3.20-3.12 (m, 2H),

2.11 (ddd, J = 13.0, 7.3, 4.3, 1H), 1.62-1.52 (m, 4H), 1.34-1.23 (m, 4H),
0.90-0.87 (m, 6H); 13C NMR(DMSO-d6) δ 191.6, 159.2, 157.9, 157.9, 157.7,
151.8, 149.5, 145.0, 135.7, 135.60, 135.55, 133.6, 131.7, 131.4, 130.7, 129.7,
129.5, 129.1, 127.6, 126.5, 125.6, 113.0, 112.9, 85.8, 85.2, 84.8, 71.0, 63.6,
54.94, 54.90, 50.9, 44.4, 36.3, 30.4, 28.7, 19.6, 19.1, 13.7, 13.5; HRMS (ESI):
calcd for C47H53N6O6 ([M+H]+) 797.4027, found 797.4046.
Preparation of T6(A1)T6
7 (159 mg, 0.200 mmol) was dissolved in CH3CN (5 mL). Triethylamine (223 μL,
1.60 mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (178 μL,
0.800 mmol) were added to the solution. The solution was stirred at 25°C for 30
minutes. AcOEt (25 mL) and saturated NaClaq (25 mL) were added to thesuspension. The product was extracted to the organic layer. The organic layer
was washed with saturated NaClaq (25 mL) twice, dried over magnesium sulfate,
and filtered. The solvent was removed by evaporation and the residue was driedby coevaporation with acetonitrile and further dried under reduced pressure.
After dissolving the residue in CH3CN (10 mL), the phosphoramidite solution wasused directly for automated DNA synthesis. T6(A1)T6 was synthesized by thestandard phosphoramidite method on the DNA synthesizer. The last DMTr group
was not deprotected on the DNA synthesizer. The protected oligonucleotide wascleaved from the solid support using 28% NH3aq and deprotected at 25°C for 16
hours in 28% NH3aq. After removal of ammonia by speed-vac, the aqueous
solution was filtered through a 0.45 μm filter. 5'-DMTr-T6(C2)T6 was purified byreversed-phase HPLC with a linear gradient over 20 minutes from 0 to 50%
CH3CN/50 mM AF. The retention time of the 5'-DMTr-T6(A1)T6 was 16.5 minutes.
After speed-vac and freeze-drying to remove the solvent and AF, the DMTrgroup was removed at 25°C for 30 minutes in 80% AcOHaq (100 μL). The
solution was diluted with H2O to 1 mL and filtered through a 0.45 m filter.
T6(C2)T6 was purified by reversed-phase HPLC with a linear gradient over 20
minutes from 5 to 20% CH3CN/50 mM AF. The retention time of the product was
14.9 minutes. After the desalination, the product was identified by MALDI TOF
mass spectrometry: T6(A1)T6, calcd for C137H172N29O88P12 4004.7 [M−H]−; found4005.2.

8-(2-formylphenyl)-2'-deoxyguanosine (8)8-bromo-2'-deoxyguanosine (3.46 g, 10.0 mmol) [26], 2-formylbenzene boronic
acid (3.00 g, 20.0 mmol), palladium (II) acetate (224 mg, 1.00 mmol),triphenylphosphine (787 mg, 3.00 mmol), and potassium carbonate (2.76 g, 20mmol) were stirred in acetonitrile (80 mL) and H2O (40 mL) at 80°C for 4 hours[24,25]. The solvent was removed by evaporation. Methanol (200 mL) was
added to the residue. The resultant solution was filtered. The solvent wasremoved by evaporation. The product 8 was purified by silica gel column
chromatography (10–20% MeOH/CH2Cl2). The product 8 was obtained as a paleyellow powder (2.97 g, 8.00 mmol, 80%): 1H NMR (DMSO-d6) δ 10.80 (br, 1H),
9.85 (s, 1H), 7.98 (dd, J = 7.8, 1.4, 1H),7.84-7.80 (m, 1H), 7.75-7.67 (m, 2H),
6.47 (br, 2H), 5.92 (dd, J = 8.2, 6.4, 1H), 5.10 (d, J = 4.1 Hz, 1H), 4.89-4.86 (m,1H), 4.21-4.18 (m, 1H), 3.72-3.69 (m, 1H), 3.49-3.38 (m, 2H), 2.98 (ddd, J = 13.2,
8.2, 5.9, 1H), 4.20 (ddd, J = 13.3, 6.4, 2.3, 1H); 13C NMR(DMSO-d6) δ 191.7,
156.5, 153.3, 151.8, 143.4, 135.5, 133.5, 132.5, 131.4, 130.1, 128.4, 117.5, 87.8,
84.6, 71.0, 61.9, 36.8; HRMS (ESI) calcd for C17H17N5O4Na ([M+Na]+) 394.1127,
found 394.1115.
2-N-[N,N-di(n-butyl)aminomethylene]-8-(2-formylphenyl)-2'-deoxyguanosine (9)8 (2.32 g, 6.25 mmol) was dissolved in methanol (20 mL). N,N-di(n-butyl)formamide dimethyl acetal (11.7 mL, 50.0 mmol) was added to the solution [22].
The solution was stirred 25°C for 2 hours. The solvent was removed byevaporation. The product 9 was purified by silica gel column chromatography(5–10% MeOH/CH2Cl2). The product 9 was obtained as a pale yellow powder(2.10 g, 4.11 mmol, 66%): 1H NMR (DMSO-d6) δ 11.51 (s, 1H), 9.89 (s, 1H),
8.53 (s, 1H), 8.02-8.00 (m, 1H), 7.87-7.83 (m, 1H), 7.77-7.73 (m, 1H), 7.69-7.67(m, 1H), 5.92 (dd, J = 7.8, 6.9, 1H), 5.16 (d, J = 4.2, 1H), 4.85-4.82 (m, 1H),
4.33-4.29 (m, 1H), 3.76-3.73 (m, 1H), 3.58-3.35 (m, 6H), 3.14 (ddd, J = 13.7, 7.3,
6.4, 1H), 2.05 (ddd, J = 13.3, 6.4, 2.8, 1H), 1.63-1.54 (m, 4H), 1.35-1.23 (m, 4H),
0.94-0.89 (m, 6H); 13C NMR(DMSO-d6) δ 191.7, 157.8, 157.4, 157.3, 150.5,
144.6, 135.5, 133.6, 132.1, 131.3, 130.2, 128.7, 120.5, 87.8, 85.0, 71.0, 62.0,
51.2, 44.9, 37.3, 30.3, 28.5, 19.6, 19.1, 13.7, 13.5; HRMS (ESI) calcd for
C26H34N6O5Na ([M+Na]+) 533.2488, found 533.2481.
5'-O-dimethoxytrityl-2-N-[N,N-di(n-butyl)aminomethylene]-8-(2-formylphenyl)-2'-deoxyguanosine (10)Pyridine (16 mL) was added to 9 (2.10 g, 4.11 mmol) and DMTrCl (1.53 g, 4.52mmol). The solution was stirred at 25°C for 1 hour. After addition of methanol (10mL), the solvent was removed by evaporation. The residual pyridine wasremoved by coevaporation with CH2Cl2 and hexane. The product 10 was purified
by silica gel column chromatography (80–98% AcOEt, 2% Et3N/n-hexane). The
product 10 was obtained as a pale yellow powder (2.20 g, 2.71 mmol, 66%): 1H
NMR (DMSO-d6) δ 11.52 (s, 1H), 9.88 (s, 1H), 8.38 (s, 1H), 7.98 (d, J = 7.8 Hz,
1H), 7.80-7.79 (m, 2H), 7.74-7.70 (m, 1H), 7.31-7.29 (m, 2H), 7.20-7.15 (m, 7H),
6.79-6.72 (m, 4H), 6.00 (dd, J = 8.0, 4.8 Hz, 1H), 5.19 (d, J = 5.1 Hz, 1H),
4.54-4.48 (m, 1H), 3.84-3.80 (m, 1H), 3.70 (s, 3H), 3.69 (s, 3H), 3.47-3.43 (m,2H), 3.31-3.26 (m, 1H), 3.21-3.13 (m, 3H), 3.07-3.04 (m, 1H), 2.17 (ddd, J = 13.7,
8.0, 5.8 Hz, 1H), 1.59-1.51 (m, 2H), 1.45-1.36 (m, 2H), 1.33-1.24 (m, 2H),
1.15-1.09 (m, 2H), 0.93-0.89 (m, 3H), 0.79-0.75 (m, 3H); 13C NMR(DMSO-d6) δ
191.6, 158.0, 157.9, 157.5, 157.3, 156.9, 150.3, 144.9, 144.8, 135.7, 135.6,
133.4, 132.5, 131.2, 130.1, 129.6, 129.4, 128.3, 127.6, 126.5, 120.4, 112.9, 85.4,
85.2, 84.0, 70.6, 63.6, 55.0, 54.9, 51.2, 45.0, 37.6, 30.2, 28.5, 19.6, 19.0, 13.7,
13.5; HRMS (ESI): calcd for C47H53N6O7 ([M+H]+) 813.3976, found 813.3976.
Preparation of T6(G1)T6
10 (163 mg, 0.200 mmol) was dissolved in CH3CN (5 mL). Triethylamine (223 μL,
1.60 mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (178 μL,
0.800 mmol) were added to the solution. The solution was stirred at 25°C for 30
minutes. AcOEt (25 mL) and saturated NaClaq (25 mL) were added to thesuspension. The product was extracted to the organic layer. The organic layer
was washed with saturated NaClaq (25 mL) twice, dried over magnesium sulfate,
and filtered. The solvent was removed by evaporation and the residue was driedby coevaporation with acetonitrile and further dried under reduced pressure.
After dissolving the residue in CH3CN (10 mL), the phosphoramidite solution wasused directly for automated DNA synthesis. T6(G1)T6 was synthesized bystandard phosphoramidite method on the DNA synthesizer. The last DMTr group
was not deprotected on the DNA synthesizer. The protected oligonucleotide wascleaved by 28% NH3aq from the solid support and deprotected at 25°C for 16
hours in 28% NH3aq. After removal of ammonia by speed-vac, the aqueous
solution was filtered through a 0.45 μm filter. 5'-DMTr-T6(G1)T6 was purified byreversed-phase HPLC with a linear gradient over 20 minutes from 5 to 50%
CH3CN/50 mM AF. The retention time of the 5'-DMTr-T6(G1)T6 was 15.2
minutes. After speed-vac and freeze-drying to remove the solvent and AF, theDMTr group was removed at 25°C for 30 minutes in 80% AcOHaq (100 μL). The
solution was diluted with H2O to 1 mL and filtered through 0.45 m filter. T6(C2)T6

was purified by reversed-phase HPLC with a linear gradient over 20 minutes
from 5 to 20% CH3CN/50 mM AF. The retention time of the product was 14.6
minutes. After desalination, the product was identified by MALDI TOF mass
spectrometry: T6(G1)T6, calcd for C137H172N29O89P12 4020.7 [M−H]−; found4022.0.

DNA cleavage reaction using designed DNA base analogues
DNA cleavage was carried out using an aqueous solution of DNA
oligonucleotides (100 μM) in a screw-cap tube. The same volume of 40%
methylamine was added to the sample and the resulting solution was heated at
70°C in a water bath. After removal of methylamine by speed-vac, the sample
was directly analyzed by reversed-phase HPLC with a linear gradient over 20
min from 8% to 20% CH3CN in 50 mM AF. The cleavage products, P1 and P2
were purified, desalted by the HPLC and identified by MALDI TOF mass
spectrometry: P1 (pT6), calcd for C60H79N12O43P6 1843.2 [M−H]−; found 1841.6(P1(T6(T1)T6)), 1841.8 (P1(T6(C1)T6)), 1842.0 (P1(T6(C2)T6)), 1842.0(P1(T6(A1)T6)), and 1842.2 (P1(T6(G1)T6)); P2 (T6pR), calcd for C66H90N13O45P6
1971.3 [M−H]−; found 1971.0 (P2(T6(T1)T6)), 1971.4 (P2(T6(C1)T6)), 1971.1(P2(T6(C2)T6)), 1971.2 (P2(T6(A1)T6)), and 1971.0 (P2(T6(G1)T6)).

Methods S45'-phosphorylation of the DNA oligonucleotide using 5-phenylethynyluracil(PU)
Here, we used 5-phenylethynyluracil (PU) instead of 5-ethynyluracil because of
the more facile synthesis of the corresponding phosphoramidite [17].
DMTr-(PU)T2AT2GT2 was synthesized on the automated DNA synthesizer by the
DMTr ON method. DMTr-(PU)T2AT2GT2 was deprotected in 28% NH3aq at 25°C
for 16 hours. After removal of ammonia by speed-vac, the solution was passed
through a 0.45 μm filter and the solution was analyzed by reversed-phase HPLC
with a linear gradient over 20 min from 0% to 50% CH3CN in 50 mM AF. The
major product was purified, desalinated by reversed-phase HPLC, and identified
by MALDI TOF mass spectrometry: DMTr-(PU)T2AT2GT2, calcd for
C118H136N24O60P8 3099.3 [M−H]−; found 3097.7. The degradation of the
DMTr-containing moiety of DMTr-(PU)T2AT2GT2 was carried out by heating in 50%
ethylenediamine aqueous solution at 70°C for 8 hours. The solution was
neutralized by AcOH. The solution was passed through a 0.45 μm filter and thesolution was analyzed by reversed-phase HPLC. The major product was purified,desalinated by reversed-phase HPLC, and identified by MALDI TOF mass
spectrometry: pT2AT2GT2, calcd for C80H103N22O54P8 2484.6 [M−H]−; found2484.6.