Hypersensitive substrate for ribonucleases

Abstract

A substrate for a hypersensitive assay of ribonucleo-lytic activity was developed in a systematic manner. This substrate is based on the fluorescence quenching of fluorescein held in proximity to rhodamine by a single ribonucleotide embedded within a series of deoxynucleotides. When the substrate is cleaved, the fluorescence of fluorescein is manifested. The optimal substrate is a tetranucleotide with a 5′,6-carboxyf luorescein label (6-FAM) and a 3′,6-carboxy-tetramethylrhodamine (6-TAMRA) label: 6-FAM-dArUdAdA-6-TAMRA. The fluorescence of this substrate increases 180-fold upon cleavage. Bovine pancreatic ribonuclease A (RNase A) cleaves this substrate with a k_cat/K_m of 3.6 × 10⁷ M^-1 s^-1. Human angiogenin, which is a homolog of RNase A that promotes neovascularization, cleaves this substrate with a k_cat/K_m of 3.3 × 10⁷ M^-1 s^-1. This value is >10-fold larger than that for other known substrates of angiogenin. With these attributes, 6-FAM dArUdAdA-6-TAMRA is the most sensitive known substrate for detecting ribo-nucleolytic activity. This high sensitivity enables a simple protocol for the rapid determination of the inhibition constant (K_i) for competitive inhibitors such as uridine 3′-phosphate and adenosine 5′-diphosphate.