Abstract
Conventional procedures to assay RNA degradation by a protein with ribonuclease (RNase) activity require a step to isolate intact RNA molecules, which are used as a substrate. Here, we established a novel “In-cell RNA hydrolysis assay” in which RNAs within cells are used as a substrate for the RNA-hydrolyzing protein, thereby avoiding the need to prepare intact RNA molecules. In this method, the degree of RNA degradation is indicated by the fluorescence intensity of RNA molecules released from fixed and permeabilized cells following treatment with the potential RNase. A catalytic 3D8 antibody capable of degrading RNAs and pancreatic RNase A were used as model RNases. Our data demonstrate that the novel In-cell RNA hydrolysis assay is a reliable and sensitive method to analyze the activities of potential RNA-hydrolyzing proteins such as catalytic antibodies.
Highlights
Some anti-DNA antibodies that are found at high titers in humans and mice with autoimmune diseases, such as systemic lupus erythematosus and multiple sclerosis [1, 2], are catalytic antibodies, called abzymes, capable of hydrolyzing DNA or RNA polymers [3,4,5]
When developing the novel In-cell RNA hydrolysis assay using RiboGreen, we postulated that if cellular RNAs are degraded by potential RNases in fixed and permeabilized cells, cleaved small RNA fragments would be released from the cells and could be detected using RiboGreen reagent (Fig. 1)
Fixed and permeabilized cells were incubated with 3D8 scFv antibody, HW6 scFv antibody, or RNase A, and the level of RNA in the conditioned medium was assessed in two ways (Fig. 2a): the fluorescence intensity of RiboGreen and at 260 nm (A260)
Summary
Some anti-DNA antibodies that are found at high titers in humans and mice with autoimmune diseases, such as systemic lupus erythematosus and multiple sclerosis [1, 2], are catalytic antibodies, called abzymes, capable of hydrolyzing DNA or RNA polymers [3,4,5]. Several methods have been developed to analyze the DNA-hydrolyzing activity of catalytic anti-DNA antibodies, in which plasmid DNA or synthetic nucleotides are used as a substrate [8,9,10,11,12]. These methods can be used to analyze the RNA-hydrolyzing capacity of proteins with RNase activity, such as anti-DNA antibodies, by substituting the DNA substrates (plasmid DNA or synthetic deoxyribonucleotides) for RNA substrates RNA molecules are extremely vulnerable to degradation by RNases, and RNA samples are often contaminated with these enzymes during their preparation and storage, resulting in high background in RNA hydrolysis assays
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