Figure 6.

Conversion of a single deoxybonucleotide T (red, left) in a DNA strand to a deoxyribonucleotide C (green, right) using a combination of DNAzyme F-8 and natural enzymes. DNA substrate was cleaved by F-8 to generate both 3′- and 5′-phosphate termini. Then the 3′-phosphate group was removed by T4 polynucleotide kinase (PNK), and a right DNA splint was introduced. Subsequent extension and ligation restored the original DNA except for a substitution of T with C. Inset: PAGE analysis of the various stages of the single-nucleotide excision repair process. Lane 1, the original 43-nt 5′-³²P-labeled DNA substrate. Lane 2, the upstream cleavage product generated by F-8. The cleaved fragment migrates slightly faster than the corresponding 18-nt marker because of its 3′-phosphate group. The dotted box indicates the expected position of the downstream cleavage product, which was invisible on the autoradiogram since it contained no ³²P. Lane 3, the cleavage product after dephosphorylation with PNK. Lane 4, 5′-³²P-labeled marker that comigrates with the 17-nt upstream cleavage fragment. Lane 5, the reaction products obtained after purifying the cleavage fragments, annealing them with right DNA splint, extending them by a C (18 nt) using DNA polymerase, and finally ligating to generate full-length substrate (43 nt). Lane 6, synthetic DNA markers. Cleavage assays were under standard single-turnover conditions (about 1 μM deoxyribozyme combined with 10 nM substrate).