How do you purify oligonucleotide conjugates by HPLC?

Labeled oligonucleotides can be purified by reverse-phase HPLC using a standard analytical C8 or C18 column, and an analytical or semi-preparative HPLC instrument. The following protocol was optimized for the further purification of 0.1 – 1 mg labeled oligonucleotide (3 – 30 A₂₆₀ units).

1. Dissolve the pellet from the ethanol precipitation in 0.1 M triethylammonium acetate (TEAA).
2. Load the dissolved pellet onto the column in 0.1 M TEAA and run a linear 5 – 95% acetonitrile gradient over 30 minutes.

Note 1: There will be peaks that correspond to the unlabeled oligonucleotide, the labeled oligonucleotide, and the free dye. The actual order and number of these peaks depends on the length of the oligonucleotide and the purity of the sample.

Note 2: To determine the identity of the peaks, monitor the absorbance at both 260 nm and at the absorbance maxima (λ_max) for the dye. For instruments with only one detector, run two small samples, each monitored at a different wavelength. Unlabeled oligonucleotides will show an absorbance at 260 nm only. Free dye and labeled oligonucleotides will have absorbance at both 260 nm (A₂₆₀ for oligo) and at the absorbance maximum of the dye (λ_max). The dye-labeled oligonucleotides will have a higher A₂₆₀: λ_max ratio than the dye or hydrolyzed dye.