HPLC-UV Determination of a Mechlorethamine Crosslink in a DNA Duplex Containing a Cytosine-Cytosine Mismatch Pair

Abstract

Mechlorethamine is a nitrogen mustard that can form a crosslink at a C-C mismatch pair in a DNA duplex. These pairs may occur in DNA structures linked with Fragile X syndrome, a triplet repeat expansion disease, and mechlorethamine may be a useful probe for these structures. The molecular structure of the mechlorethamine C-C crosslink has not been determined, in part due to the poor recovery of the crosslinked duplex following gel electrophoretic separation. Here, we report the development of a simple high-performance liquid chromatographic (HPLC) method for isolation of the mechlorethamine crosslink in a DNA duplex containing a C-C mismatch pair. The mechlorethamine crosslink was prepared by incubating a DNA duplex d[CTCACACCGTGGTTC]•d[GAACCACCGTGTGAG] (underlined bases are a C-C mismatch pair) with mechlorethamine at room temperature for 2 hours. Isolation of the crosslinked duplex was most efficient using a Biobasic-C4 column with gradient elution of 5-15% acetonitrile in 100 mM TEAA and 0.1 mM EDTA over 60 min at a flow rate of 1 ml/min, a column temperature at 33ºC, and UV detection at 260 nm. The crosslinked duplex (25% of the total DNA) eluted at 40.54 min, while the unreacted DNA strands eluted at 28.02 and 25.81 min. Thus, the HPLC method gave good separation between the crosslink and the unreacted DNA. The percentage of crosslink out of the total DNA detected by this method was about 25%, in good agreement with our previous results for this reaction based on detection by gel electrophoresis. Therefore, we conclude that the HPLC method can be used for preparation of purified mechlorethamine-crosslinked C-C mismatch DNA for further structural characterization.