Several dinucleoside polyphosphates are acceptor substrates in the T4 RNA ligase catalyzed reaction

Several dinucleoside polyphosphates accept cytidine-3',5'-bisphosphate from the adenylylated donor 5'-adenylylated cytidine 5',3'-bisphosphate in the T4 RNA ligase catalyzed reaction. The 5'-adenylylated cytidine 5',3'-bisphosphate synthesized in a first step, from ATP and cytidine-3',5'-bisphosphate, is used as a substrate to transfer the cytidine-3',5'-bisphosphate residue to the 3'-OH group(s) of diguanosine tetraphosphate (Gp(4)G) giving rise to Gp(4)GpCp and pCpGp(4)GpCp in a ratio of approximate to 10 : 1, respectively. The synthesized Gp(4)GpCp was characterized by treatment with snake venom phosphodiesterase and alkaline phosphatase and analysis (chromatographic position and UV spectra) of the reaction products by HPLC. The apparent K-m values measured for Gp(4)G and 5'-adenylylated cytidine 5',3'-bisphosphate in this reaction were approximate to 4 mm and 0.4 mm, respectively. In the presence of 0.5 mm ATP and 0.5 mm cytidine-3',5'-bisphosphate, the relative efficiencies of the following nucleoside(5')oligophospho(5')nucleosides as acceptors of cytidine-3',5'-bisphosphate from 5'-adenylylated cytidine 5',3'-bisphosphate are indicated in parentheses: Gp(4)G (100); Gp(5)G (101); Ap(4)G (47); Ap(4)A (39). Gp(2)G, Gp(3)G and Xp(4)X were not substrates of the reaction. Dinucleotides containing two guanines and at least four inner phosphates were the preferred acceptors of cytidine-3',5'-bisphosphate at their 3'-OH group(s).