A NOVEL COMBINED CHEMICAL ENZYMATIC-SYNTHESIS OF CROSS-LINKED DNA USING A NUCLEOSIDE TRIPHOSPHATE ANALOG

A novel method using combined chemical and enzymatic reactions to allow the preparation of covalently cross-linked DNA duplexes has been described. The method can be used to specifically link two complementary bases of a DNA duplex containing all four natural bases. The modified nucleotide 9-(2-deoxy-5-O-triphospho-beta-d-ribofuranosyl)-N-6,N-6-ethano-2,6-diaminopurine (6edDTP) was prepared by total chemical synthesis and was found to be incorporated into DNA duplexes in the place of 2'-deoxyguanoisine 5'-O-triphosphate by the Klenow fragment of Escherichia coli DNA polymerase I, T4 and T7 DNA polymerases, avian myeloma virus reverse transcriptase, and rat DNA polymerase beta. Once incorporated, the aziridine of the nucleotide is rapidly opened by the N4 of the cytosine on the complementary strand to give cross-linked DNA, where the modified nucleotide is covalently joined to the complementary base by an ethano linkage. The duplexes produced were found to be recognized as substrates by various DNA polymerases. The K(m) for the incorporation of the 6edDTP into DNA catalyzed by the Klenow fragment of E. coli DNA polymerase I was found to be 29-mu-M, and the k(cat) was found to be 0.014 s-1. The modified nucleoside also served as a substrate for terminal deoxynucleotidyltransferase, where it was added to single-stranded DNA and then hybridized to a complementary strand, after which cross-linking of the two strands occurred within 1 min.