Protocol 2

Antibody conjuagted DNA

Reagents and stock solutions

• Antibody (lexatumumab): 50 μM stock; prepare 10 μM working solution by dilution in 1× PBS.
• TCEP: 10 mM stock; prepare 100 μM working solution by dilution in nuclease-free water.
• Mal-PEG4-DBCO: 10 mM stock (DMSO); 1 mM working solution (DMSO).
• SsDNA-Azide: 1 mM stock (nuclease-free water).
• Citrate buffer: 100 mM, pH 5.5.

1. Preparation of Mal–DNA

Mal-PEG4-DBCO	SsDNA	Mal:DNA	1X PBS (pH 6.5)	Concentration
2 uL	4 uL	1:2	14 uL	100 uM

• Incubate the mixture at room temperature for 4 h.

2. Antibody reduction

Antibody	TCEP	AB:TCEP	Citrate buffer (pH 5.5)
10 uL	5 uL	1:5	85 uL

• Pre-dilute TCEP into citrate buffer (pH 5.5) before mixing with antibody to avoid antibody inactivation (TCEP stock is acidic).
• Incubate at 37 °C for 2 h to reduce disulfides and generate free thiols.
• Remove excess TCEP and exchange buffer to 1× PBS (pH 6.5) by ultrafiltration, performing four washes.

3. Antibody–DNA conjugation

Antibody (R)	Mal-DNA	AB:DNA	1X PBS (pH 6.5)
All	5 uL	1:5	50 uL

• Because Mal‑DNA contains DMSO (~10%), dilute the Mal‑DNA in PBS prior to addition to antibody.
• Incubate under appropriate conditions (e.g., room temperature for 1–4 h or 4 °C overnight) to allow maleimide–thiol coupling.
• Purify the conjugate by ultrafiltration/washing with 1× PBS (pH 7.4) four times to remove excess unreacted DNA.

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)