Benzimidazolium triflate-activated synthesis of (6-4) photoproduct-containing oligonucleotides and its application

In the solid-phase synthesis of oligonucleotides containing the pyrimidine( 6-4)pyrimidone photoproduct using a dinucleotide building block, considerab le amounts of by-products were found as the chain length increased. The by- products were the major product when a 49mer was synthesized on a 40 nmol s cale. It was assumed that these by-products were formed by the coupling of phosphoramidites with the N3 imino function of the 5' component of the (6-4 ) photoproduct. We examined imidazolium triflate and benzimidazolium trifla te to find an alternative activator for DNA synthesis. Imidazolium triflate prevented by-product formation to some extent, but the coupling yields wer e low. Benzimidazolium triflate was comparable to tetrazole in coupling eff iciency and reduced by-product formation to a great extent, without modific ation of the synthesizer program. The obtained 49mer was used to detect pro teins that recognize UV-damaged DNA in HeLa cell extracts. Two major protei n-DNA complexes were found when a 49mer duplex was used as probe, while a 3 0mer duplex failed to detect one of them. This application showed the usefu lness of long chain 'damaged' oligonucleotides in biochemical studies.