Figure 3.

Sample data of R5 labeling and purification. (a) The sequence and secondary structure of the CS model DNA. The numbering of the phosphates is shown. (b) An example of anion-exchange HPLC results. The black trace is the 260-nm absorbance of a crude CS DNA strand with a phosphorothioate at position 4, and the red trace is the corresponding product after R5 labeling. The splitting in the unlabeled DNA is due to diastereomer separation. The labeled DNA elutes earlier than the unlabeled oligonucleotide due to the loss of a negative charge upon R5 attachment. The labeling efficiency is 94% based on the areas under respective peaks. The data shown were obtained using a PA-100 column with a flow rate of 2 ml min⁻¹, and the linear gradient used is shown in the inset table. Variation of this gradient has been used for DNA and RNA ranging from 12 to 44 nt long. For example, for a 44-nt DNA, the gradient is modified to Step 2: 0–20% B in 4 ml; Step 3: 20–30% B in 24 ml. (c) An example of a mass spectrum of a CS DNA strand with R5 attached at position 2. (d) An example of a 20% polyacrylamide denaturing gel for examining R5 labeling. The label is attached at position 3 of a CS DNA strand. Lane 1: DNA strand without modification; Lane 2: DNA strand containing one phosphorothioate; and Lane 3: DNA strand with one R5 attached.