Reverse action of ribonuclease T1 in frozen aqueous systems

We have studied ribonuclease T1 (EC 3.1-27.3)-catalysed synthesis of guanylyl-(3'-->5')cytidine in frozen aqueous reaction mixtures at -10 degrees C and in solution at 0 degree C in order to investigate whether ribonuclease-catalysed synthetic reactions can take advantage of the yield-increasing effect of freezing as was reported for protease-catalysed peptide synthesis. Under frozen state conditions, substantially increased yields of GpC were obtained compared to the reactions in solution. From the fact that no irreversible hydrolysis of the 2'3'-cyclic donor was observed it can be concluded that transesterification of the newly formed phosphodiester bond is the most important yield-limiting factor in ribonuclease T1-catalysed dinucleoside phosphate synthesis.