In-Cell RNA Hydrolysis Assay: A Method for the Determination of the RNase Activity of Potential RNases

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.

Fig. 1

Schematic representation of the concept of the In-cell RNA hydrolysis assay. a Cells containing intact RNAs. b Cells whose membranes have been permeabilized, allowing the release of small RNA fragments even in the absence of RNase treatment. c Cells release small RNA fragments via their membrane pores following treatment with an RNase. d Collection of degraded RNA fragments released from cells, which are labeled with RiboGreen and subjected to fluorescence intensity measurement

Fig. 2

The detection of RNAs released from fixed and permeabilized cells. a Experimental procedures for the data presented in (b) and (d). b A549 cells grown in a 48-well plate at a density of 5 × 10⁴ cells/well were fixed, permeabilized, and incubated with 100 μl of 10 μM RNase A or 10 μM 3D8 antibody for 2 h at 37 °C. An aliquot of the conditioned medium was added to RiboGreen, and the fluorescence intensity was analyzed. c Pure 16S and 23S rRNA from E. coli was incubated with RiboGreen in the presence or absence of RNase A prior to fluorescence intensity analysis. d A549 cells grown in a 6-well plate at a density of 5 × 10⁵ cells/well were fixed, permeabilized, and incubated with 10 μM RNase A or 3D8 antibody for 2 h at 37 °C. Proteins were removed from the conditioned medium by precipitation, and absorbance at 260 nm was measured. e Degradation of plasmid DNA (1 μg/ml) by DNase I (2 U) was tested using RiboGreen prepared in DNase I reaction buffer for 2 h at 37 °C. f Fixed and permeabilized cells in a 48-well plate were treated with RNase A or 3D8 antibody (10 μM) in the presence or absence of DNase I (2 U) for 2 h at 37 °C. The conditioned medium was mixed with RiboGreen prior to fluorescence intensity analysis. RFU relative fluorescence unit. Data represent the mean ± standard error of four independent experiments

Fig. 3

Quantitative analysis of RNA degradation in fixed and permeabilized cells treated with RNase A or 3D8 antibody. a Standard curve to determine the concentration of RNA. Pure rRNA at known concentrations was mixed with RiboGreen prior to fluorescence intensity analysis. b Dose-dependent RNA degradation. RNA degradation was quantitatively analyzed in fixed and permeabilized cells (5 × 10⁴ cells) treated with 1.25–10 μM RNase A or 3D8 antibody. c The sensitivity of this method. RNA degradation was quantitatively analyzed in fixed and permeabilized cells treated with various dilutions of RNase A generated from a stock solution of 1 μM. RFU, relative fluorescence unit. Data represent the mean ± standard error of four independent experiments

Fig. 4

Detection of RNA in fixed and permeabilized cells treated with RNase A or 3D8 antibody. a Schematic diagram of the experimental procedure to detect the level of RNA in cells according to the fluorescence intensity of RiboGreen. b, c A549 cells plated in a 24-well plate (b) or a 6-well plate (c) were incubated with 5-ethynyl uridine (EU) for 20 h, fixed, permeabilized, and incubated with 10 μM RNase A or 3D8 antibody for 2 h at 37 °C. Cells were applied to click chemistry for 30 min, extensively washed, and examined by confocal microscopy (b) and flow cytometry (c). Data are representative of three independent experiments. Scale bars represent 10 μm in (b)