Efficient Preparation of AppDNA/AppRNA by T4 DNA Ligase Aided by a DNA Involving Mismatched Mini-Hairpin Structure at Its 3 ' Side

Preparation of adenylated DNA, which has wide applications, is a great challenge because the obtained AppDNA is usually quickly ligated to the 3'-OH at the nick. Here, by using an Aid-DNA with a mini-hairpin structure at its 3' terminal, involving a 3-4-bp stem and a 3-nt loop, high adenylation yield (>90%) for most target sequences is obtained with no ligation by-products. The 5' side of the Aid-DNA is complementary to the DNA to be adenylated, and forms a pseudo-nick after hybridization. T4 DNA ligase, the most popular ligase, can complete the adenylation. By investigating the effects of stem lengths, types of mismatched base pairs, and reaction conditions, we found that both a short stem and the mismatched base pair at 3' end are essential to avoid ligation. More interestingly, the adenylation can be efficiently carried out even when another mismatched base pair flanks the 5' side of the nick, and the ligation is completely inhibited. Thus, adenylation of a DNA becomes simple just by changing the Aid-DNA sequence at the 5' side. This approach is also proved applicable for RNA adenylation, although the DNA ligase is used. Our finding is also helpful for understanding detailed mechanism of DNA ligation.