Year: 1995
Type: book-chapter
Abstract: This chapter discusses the assessment of the RNA-binding activity of viral proteins by using the RNA-capture assay. The characterization of RNA-binding proteins can provide key insights into the mechanisms of viral replication as well as basic information on protein and RNA structure and function. Several methods have been used successfully to describe the interactions of protein and nucleic acids; these include gel mobility-shift electrophoresis, nucleic acid-conjugated column chromatography, filter binding assay, nucleic acid–protein photo-cross linking, nuclease protection (that is, footprinting), electron microscopy, and, recently, the crystallization of nucleic acid–protein complexes. The chapter describes an alternative technique, the RNA-capture assay, which is successfully used to detect and analyze the specificity of two rotavirus RNA-binding proteins. The RNA-capture assay employs immune-adsorbed protein as an affinity matrix to analyze RNA-binding activity. The RNA-capture assay depends primarily on the availability of a monospecific antibody that binds with reasonable affinity to the protein whose RNA-binding activity is to be assessed. Additionally, the assay requires that the selected antibody not perturb the RNA-binding activity of the protein. Some of the major advantages of the RNA-capture assay are as follows: (1) RNA probes of unlimited size can be used to examine RNA-binding activity; (3) the RNA-binding protein need not be purified a priori or subjected to harsh, possibly denaturing, conditions; and (3) the RNA-binding protein may be a component of large multimeric protein complexes.
Source: Methods in molecular genetics
Authors: John T. Patton, Jian Hua
Institution: University of Miami
Language: English
Topic: RNA Research and Splicing
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
Funder: National Institutes of Health
OpenAlex ID: W1505179576