Detection of the sites of alkylation in DNA and polynucleotides by laser Raman spectroscopy

Abstract

A laser Raman study of the alkylation of calf thymus DNA, poly(dG).cntdot.poly(dC) and poly(dA).cntdot.(dT) was made using 2 water soluble alkylating agents: an antitumor drug, the difunctional methyl nitrogen mustard HN2 [N-methylbis(2-chloroethyl)amine hydrochloride], which forms interstrand cross-links, and the dimethyl nitrogen half mustard HN1 [N,N-dimethyl-2-chloroethylamine hydrochloride]. When an excess of the alkylating agent was used, the observed Raman frequencies due to the guanine ring modes in DNA and poly(dG).cntdot.poly(dC) changed virtually quantitatively to those of 7-methylguanosine, showing that essentially all of the guanine bases were alkylated in the N-7 position. Furthermore, this alkylated DNA formed a stable double helical complex at neutral pH in which the alkylated guanine residues are in the keto form. No changes in the Raman bands of any of the other bases were observed in alkylated DNA. The DNA double helix, completely alkylated in at the N-7 position of guanine, melts about 35.degree. C below that of the native DNA. Upon melting, the alkylated guanine changes from the keto to the zwitterionic form.