In this study, we have demonstrated the viability of in vitro selection for identifying deoxyribozymes that 3′-phosphorylate DNA oligonucleotide substrates. Although the capture method is not strongly selective for the intended 3′-phosphorylated DNA terminus, one deoxyribozyme with 3′-kinase activity was identified. For this new deoxyribozyme, 3′Kin1, the same initially random (N40) region sequence can be used with many
different DNA substrate sequences by appropriate choice of the fixed-sequence segment on the 3′-side of the initially random region. At the first nucleotide position of the fixed-sequence segment, a T is favored, regardless of the DNA substrate sequence. At the remaining positions of the fixed-sequence segment, straightforward Watson−Crick base pairing should be included. With this arrangement, almost any DNA substrate that has sequence 5′-NKR-3′ can be 3′-phosphorylated, where N denotes any oligonucleotide sequence, K = T or G, and R =A or G. Certain other substrate sequences may also be 3′-phosphorylated.