title: 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.
authors: Department of Biomedical Sciences, Graduate School, Ajou University, 206 World Cup-ro, Yeongtong-gu, Suwon, 443-749, South Korea.
authors: Youngsil
authors: Y
authors: Seo
authors: Hye-Ryeong
authors: HR
authors: Jun
authors: Joungmin
authors: J
authors: Lee
authors: Hyunjoon
authors: H
authors: Park
authors: Minjae
authors: M
authors: Kim
authors: Youngsoo
authors: Y
authors: Lee
authors: Myung-Hee
authors: MH
authors: Kwon
mesh_terms: D006868:Hydrolysis; D018613:Microscopy, Confocal; D012313:RNA / Q000378:metabolism*; D012260:Ribonucleases / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet
chemical_list: D012313:RNA; D012260:Ribonucleases
references: J Biol Chem. 2013 Dec 13;288(50):35877-85
references: 24155236
references: Mol Cell Biochem. 1974 May 30;3(3):179-86
references: 4209248
references: Biochemistry (Mosc). 2008 Nov;73(11):1242-53
references: 19120029
references: Biochim Biophys Acta. 2008 Dec;1778(12):2775-80
references: 18692020
references: J Mol Recognit. 2008 Sep-Oct;21(5):338-47
references: 18729241
references: PLoS One. 2014;9(4):e93001
references: 24736683
references: Clin Exp Immunol. 2015 Jan;179(1):5-10
references: 24533624
references: Proc Natl Acad Sci U S A. 2001 Feb 13;98(4):1793-8
references: 11172030
references: J Mol Recognit. 2010 Sep-Oct;23(5):486-94
references: 20119969
references: Mol Immunol. 1997 Oct;34(15):1083-93
references: 9519766
references: Proc Natl Acad Sci U S A. 1995 Jan 3;92(1):254-7
references: 7816827
references: J Biol Chem. 2006 Jun 2;281(22):15287-95
references: 16551636
references: Mol Biol Cell. 2002 Feb;13(2):425-34
references: 11854401
references: J Oral Maxillofac Pathol. 2012 Sep;16(3):400-5
references: 23248474
references: Autoimmune Dis. 2010 Jul 20;2011:462841
references: 21152217
references: Cell Mol Life Sci. 2009 Jun;66(11-12):1985-97
references: 19373434
references: J Immunol Methods. 2002 Nov 1;269(1-2):147-55
references: 12379359
references: FEMS Microbiol Ecol. 2010 Apr;72(1):143-51
references: 20059545