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Nucleic Acids Research, 1994, Vol. 22, No. 14 2731-2739
© 1994

CHEMISTRY

A novel fluorogenic substrate for ribonucleases. Synthesis and enzymatic characterization

Ottilie Zelenko, Ulf Neumann, Wolfgang Brill1, Uwe Pieles1, Heinz E. Moser1 and Jan Hofsteenge*

Friedrich Miescher-Institut PO Box 2543, CH-4002 Basel 1Central Research Laboratories, Ciba Geigy Ltd CH-4002 Basel, Switzerland

*To whom correspondence should be addressed

Received May 3, 1994. Revised June 16, 1994. Accepted June 16, 1994.

The synthesis and enzymatic characterization of DUPAAA, a novel fluorogenic substrate for RNases of the pancreatic type is described. It consists of the dinucleotide uridylyl-3',5'-deoxyadenosine to which a fluorophore, o-aminobenzoic acid, and a quencher, 2,4-dinitroaniline, have been attached by means of phosphodiester linkages. Due to intramolecular quenching the intact substrate displayed very little fluorescence. Cleavage of the phosphodiester bond at the 3'-side of the uridylyl residue by RNase caused a 60-fold increase in fluorescence. This allowed the continuous and highly sensitive monitoring of enzyme activity. The substrate was turned over efficiently by RNases of the pancreatic type, but no cleavage was observed with the microbial RNase T1. Compared to the dinucleotide substrate UpA, the specificity constant with RNase A, RNase PL3 and RNase Us increased 6-, 18-, and 29-fold, respectively. These differences in increased catalytic efficiency most likely reflect differences in the importance of subsites on the enzyme in the binding of elongated substrates. Studies on the interactions of RNase inhibitor with RNase A using DUPAAA as a reporter substrate showed that it was well suited for monitoring this very tight protein - protein interaction using pre-steady-state kinetic methods.


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