XIE/2:2025/Dec

12.12

• Triplex DNA pH Test

Sample preparation

• ssDNA stocks: s1 (3'Cy5), S2, TFO (3'Cy3), S‑S1 (3'Cy5), S‑S2, and S‑TFO (3'Cy3) were each prepared at 1 mM in nuclease-free water.
• Buffers: PBS adjusted to pH 5.5, 6.5, 7.0, 7.5, and 8.5; prepare 50 mL of each pH condition.

Incubation

• For each pH condition, set up two groups (Group 1 and Group 2). To each reaction, first add 194 µL of the corresponding PBS buffer.

1. Group 1: add S1, S2, and TFO , 2 µL each.
2. Group 2: add S-S1, S-S2, and S-TFO , 2 µL each.

Name	Buffer	SsDNA
5.5-1	194 uL, pH 5.5	S1, S2, TFO, 2 uL each
5.5-2	149 uL, pH 5.5	S-S1, S-S2, S-TFO, 2 uL each
6.5-1	194 uL, pH 6.5	S1, S2, TFO, 2 uL each
6.5-2	194 uL, pH 6.5	S-S1, S-S2, S-TFO, 2 uL each
7.0-1	194 uL, pH 7.0	S1, S2, TFO, 2 uL each
7.0-2	194 uL, pH 7.0	S-S1, S-S2, S-TFO, 2 uL each
7.5-1	194 uL, pH 7.5	S1, S2, TFO, 2 uL each
7.5-2	194 uL, pH 7.5	S-S1, S-S2, S-TFO, 2 uL each
8.5-1	194 uL, pH 8.5	S1, S2, TFO, 2 uL each
8.5-2	194 uL, pH 8.5	S-S1, S-S2, S-TFO, 2 uL each

• PCR: 37℃ 3h

Fluorescence spectrophotometer

• Sample preparation: dilute each sample to 1%.
• Measurement channels

1. IDA channel: excitation 550 nm; emission 670 nm.
2. IDD channel: excitation 550 nm; emission 570 nm.
3. IAA channel: excitation 650 nm; emission 670 nm.

Conculution

• These ssDNAs predominantly form triplex structures at around pH 6.5.
• The S-ssDNAs only partially form triplex structures under similar conditions (≈ pH 6.5).

12. 20

• Formation of triplex S-DNA at different pH

Incubation

• Take 2 µL of the 1 mM stock and dilute with ultrapure water to a final volume of 200 µL (final concentration 10 µM). Use this as the working solution.

	S-TFO (Sy3)	S-S1 (Cy5)	S-S2
SS-DNA	2 uL	2 uL	2 uL
UP-water	198 uL	198 uL	198 uL

• Mix the ssDNAs and incubate at 37 °C for 1 h.

ssDNAs were mixed and incubated at 37 °C for 1 h in buffers of different pH.

Name	Buffer (uL)	S-TFO (uL)	S-S1 (uL)	S-S2 (uL)
6.5-1	188	4	4	4
6.5-2	188	4	4	4
6.5-3	188	4	4	4
6.5-D	196	4	0	0
6.5-A	196	0	4	0
6.7-1	188	4	4	4
6.7-2	188	4	4	4
6.7-3	188	4	4	4
6.7-D	196	4	0	0
6.7-A	196	0	4	0
6.8-1	188	4	4	4
6.8-2	188	4	4	4
6.8-3	188	4	4	4
6.8-D	196	4	0	0
6.8-A	196	0	4	0
6.9-1	188	4	4	4
6.9-2	188	4	4	4
6.9-3	188	4	4	4
6.9-D	196	4	0	0
6.9-A	196	0	4	0
7.0-1	188	4	4	4
7.0-2	188	4	4	4
7.0-3	188	4	4	4
7.0-D	196	4	0	0
7.0-A	196	0	4	0

Fluorescence spectrophotometer

• Sample preparation: dilute each sample to 400 ul.
• Measurement channels

1. IDA channel: excitation 550 nm; emission 670 nm.
2. IDD channel: excitation 550 nm; emission 570 nm.
3. IAA channel: excitation 650 nm; emission 670 nm.

12.23-12.25

Sample preparation

• ss DNA：take 2 ul of ssDNA(TFO、S1、S2、S-TFO、S-S1、S-S2) and dilute to 200 ul with UP water(10 uM).
• 1640 +++：pH 7.5

Incubation

• TFO、S1、S2

Name	1	2	3	4	5	6
Time	48 h	24 h	16 h	8 h	2 h	2 h
ssDNA (TFO, S1, S2)	each 1 uL	each 1 uL	each 1 uL	each 1 uL	each 1 uL	each 1 uL
1640+++	7 uL	7 uL	7 uL	7 uL	7 uL	PBS 7 uL

• S-TFO、S-S1、S-S2

Name	S-1	S-2	S-3	S-4	S-5	S-6
Time	48 h	24 h	16 h	8 h	2 h	2 h
ssDNA (S-TFO, S-S1, S-S2)	each 1 uL	each 1 uL	each 1 uL	each 1 uL	each 1 uL	each 1 uL
1640+++	7 uL	7 uL	7 uL	7 uL	7 uL	PBS 7 uL

Nativepage

• Page cncentration:15%(4 ml ACR) with goldview
• sample:10 ul sample mix with 2 ul louding buffer
• condition:gel runs at 120 v for 2 h in 1X TBE

12.29-12.31

Sample preparation

• ss DNA：take 2 ul of ssDNA(TFO、S1、S2、S-TFO、S-S1、S-S2) and dilute to 200 ul with UP water(10 uM).
• 1640 +++：pH 7.5

Incubation

• TFO、S1、S2

Name	1	2	3	4	5	6
Time	48 h	24 h	16 h	8 h	2 h	2 h
ssDNA (TFO, S1, S2)	each 3 uL	each 3 uL	each 3 uL	each 3 uL	each 3 uL	each 3 uL
1640+++	10 uL	10 uL	10 uL	10 uL	10 uL	PBS 7 uL

• S-TFO、S-S1、S-S2

Name	S-1	S-2	S-3	S-4	S-5	S-6
Time	48 h	24 h	16 h	8 h	2 h	2 h
ssDNA (S-TFO, S-S1, S-S2)	each 1 uL	each 1 uL	each 1 uL	each 1 uL	each 1 uL	each 1 uL
1640+++	7 uL	7 uL	7 uL	7 uL	7 uL	PBS 7 uL

Nativepage

• Page cncentration:15%(4 ml ACR) with goldview
• sample:10 ul sample mix with 2 ul louding buffer
• condition:gel runs at 120 v for 2 h in 1X TBE