Protocol 2

Protocol

Triplex DNA pH Test

• Stock Solution: Dissolve in ultrapure water to 1 mM and store at −20 °C.

• Buffer: 0.5X TBE with 10 mM MgCl2 (pH: 5.5, 6.5, 7.0, 7.5, 8.5)

• Incubation: Dilute the stock with ultrapure water to 1% (10 µM). Add 4 µL each of TFO, S1, and S2 (or S‑TFO, S‑S1, S‑S2) to 388 ul buffers of different pH and incubate at 37 °C for 3 h.

pH (388 uL)	SsDNA 1 (Cy3) — 4 uL	SsDNA 2 (Cy5) — 4 uL	SsDNA 3 — 4 uL
5.5	TFO	S1	S2
5.5	S-TFO	S-S1	S-S2
6.5	TFO	S1	S2
6.5	S-TFO	S-S1	S-S2
7.0	TFO	S1	S2
7.0	S-TFO	S-S1	S-S2
7.5	TFO	S1	S2
7.5	S-TFO	S-S1	S-S2
8.5	TFO	S1	S2
8.5	S-TFO	S-S1	S-S2

Fluorometer

• Donor-only:Cy3-only
• Acceptor-only:Cy5-only
• 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.

Formuals

• α = mean(IDA_Donly / IDD_Donly)--(donor → acceptor leakage ratio)
• β = mean(IDA_Aonly / IAA_Aonly)--(acceptor direct‑excitation by the donor excitation ratio)
• IDA_corr = IDA_meas - α*IDD_meas − β*IAA_meas--(If the result < 0, set it to 0 .)
• Eapp = IDA_corr / (IDA_corr + γ * IDD_meas), here γ ≈ 1-- (approximate)