DNA Oligonucleotide 3′-Phosphorylation by a DNA Enzyme

T4 polynucleotide kinase is widely used for 5'-phosphorylation of DNA and RNA oligonucleotide termini, but no natural protein enzyme is capable of 3'-phosphorylation. Here, we report the in vitro selection, of deoxyribozymes (DNA, enzymes) capable, of DNA oligonucleotide 3'-phosphorylation, using-a 5'-triphosphorylated RNA transcript (pppRNA) as the,phosphoryl donor. The basis of selection was the capture, during each selection round, of the 3'-phosphorylated DNA substrate terminus by 2-methylimidazole activation of-the 3'-phosphate (forming;31-MeImp)) and subsequent splint ligation with a 5'-amino DNA oligonucleotide, Competing and. precedented DNA-catalyzed reactions were-DNA phosphodiester hydrolysis Or deglycosylation, each also leading to a 3'-phosphate, but at a different nucleotide position within the DNA substrate. One oligonucleotide 3'-kinase deoxyribozyme, obtained from an N-40 random pool and named 3'Kin1, can 3'-phosphorylate nearly any DNA oligonucleotide substrate for which the 3'-terminus has the sequence motif 5'-NKR-3', where N denotes any oligonucleotide sequence, K = T or G, and R = A or G. These results establish the viabilty of in vitro selection for identifying DNA enzymes that 3'-phosphorylate DNA oligonucleotides.