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

• Combine Mal‑PEG4‑DBCO and SsDNA at a 1:2 molar ratio. Example preparation (nominal final concentration 100 μM for Mal‑PEG4‑DBCO):

Component	Volume
Mal-PEG4-DBCO (stock)	2 μL
SsDNA (stock)	4 μL
1× PBS (pH 6.5)	14 μL

• Incubate the mixture at room temperature for 4 h.

2. Antibody reduction

• Pre-dilute TCEP into citrate buffer (pH 5.5) before mixing with antibody to avoid antibody inactivation (TCEP stock is acidic).
• Example reduction mixture:

Component	Volume
Antibody (stock)	10 μL
TCEP (stock, pre-diluted)	5 μL
Citrate buffer (100 mM, pH 5.5)	85 μL

• 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 (centrifugal concentrator), performing four washes. Target final antibody volume: ~10–30 μL.

3. Antibody–DNA conjugation

• Because Mal‑DNA contains DMSO (~10%), dilute the Mal‑DNA in PBS prior to addition to antibody.
• Example conjugation setup:

Component	Volume / notes
Reduced antibody (from step 2)	variable (see note)
Mal-DNA (prepared in step 1)	5 μL
1× PBS (pH 6.5)	~50 μL (to reach desired antibody:Mal‑DNA ratio; example uses ~1:5)

• 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.

Notes

• Prepare TCEP dilutions fresh; avoid exposing antibodies to low pH or high DMSO concentrations.
• Adjust reagent volumes and molar ratios according to the actual antibody concentration and the desired degree of labeling.
• Verify conjugation by an appropriate analytical method (SDS‑PAGE, absorbance/fluorescence, or mass spectrometry).

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)