Capillary electrophoresis of oligonucleotides in sieving liquid polymers in isoelectric buffers

Abstract

Analysis of oligonucleotides (especially in regard to assessing the purity of antisense compounds) is typically performed in 18%T sieving liquid polyacrylamide, in 30% formamide and 7 M urea. Up to 600 V/cm have been reported, with transit times, for 20 to 25 oligomers, of 15–20 min. We show that the same analysis can be performed in isoelectric buffers, typically histidine (His), and in more dilute linear polyacrylamides, e.g. 10%T (at 0%C), with much reduced analysis times. A series of His concentrations has been explored, ranging from 25 to 150 mM. Best performance is obtained in 100 mM His (at pH = pI, i.e., 7.47 at 25°C), dissolved in 7 M urea, in presence of 10% sieving liquid linear polyacrylamide. Such a buffer allows delivering 800 V/cm without any loss of resolution due to Joule heating, with retention of very high resolving power down to fragments as short as tetranucleotides. Under these conditions, the analysis time for an antisense oligonucleotide containing fragments from a 10-mer to 18-mer is in a time window of 4–5 min. It is shown that the smallest fragment (10-mer) migrates in the capillary at the remarkable speed of 5 cm/min.