Protocol for Thiol-Modified Oligonucleotide Reduction

Thiol-modified oligonucleotides are supplied in the protected form with the disulfide linkage intact to minimize the potential for oxidation, which results in oligo dimer formation. To use the free thiol (– SH) in your application the disulfide linkage must be reduced with dithiothreitol (DTT). The following procedure is recommended.

Reactions:

For 5' Thiols:

R–S–S–(CH₂)₆–Oligo + 0.1 M DTT → HS–(CH₂)₆–Oligo

For 3' Thiols:

Oligo–(CH₂)₃–S–S–(CH₂)₃–OH + 0.1 M DTT → Oligo–(CH₂)₃–SH

Part 1: Oligo Sulfhydryl activation (Oligo-disulfide)

1. Prepare a 100mM DTT solution in sodium phosphate buffer, (pH 8.3 – 8.5). DTT is available as a solid from Sigma-Aldrich^® (Product No. D9779). For 5 mL of the 100 mM solution, add 77.13 mg of the DTT powder.
2. For 0–12.5 A₂₆₀ units, dissolve the non-reduced oligo in 125 µl of 100mM DTT. Incubate the solution at room temperature for one hour. For more than 12 A₂₆₀ units of oligo, maintain a 10:1 (µl DTT : A₂₆₀ unit) ratio.

Part 2: Removal of DTT and other reaction byproducts and elution in conjugation buffer

1. Equilibrate a NAP-10 column (e.g. Amersham Pharmacia Product No. 17-0854-01) with approximately 15 mL of the 50 mM sodium phosphate buffer, pH 6.0. Allow the equilibration buffer to completely enter the gel bed.
2. Add the sample to the NAP-10 column in a volume of 1.0 mL. Bring the total sample volume to 1.0 mL with sodium phosphate buffer, pH 6.0 as needed. Allow the sample to enter the gel bed completely.
3. Place a tube for sample collection under the column and elute with 1.0 mL of sodium phosphate pH 6.0.

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