Mechanistic analysis of the hepatitis delta virus (HDV) ribozyme: methods for RNA preparation, structure mapping, solvent isotope effects, and co-transcriptional cleavage.
Small ribozymes such as the hairpin, hammerhead, VS, glm S, and hepatitis delta virus (HDV) are self-cleaving RNAs that are typically characterized by kinetics and structural methods. Working with these RNAs requires attention to numerous experimental details. In this chapter we focus on four different experimental aspects of ribozyme studies: preparing the RNA, mapping its structure with reverse transcription and end-labeled techniques, solvent isotope experiments, and co-transcriptional cleavage assays. Although the focus of these methods is the HDV ribozyme, the methods should be applicable to other ribozymes.
Department of Chemistry, The Pennsylvania State University, University Park, PA, USA.
Durga M
DM
Chadalavada
Andrea L
AL
Cerrone-Szakal
Jennifer L
JL
Wilcox
Nathan A
NA
Siegfried
Philip C
PC
Bevilacqua
D002021:Buffers; D003001:Cloning, Molecular; D017666:Deuterium Oxide / Q000737:chemistry; D003698:Hepatitis Delta Virus / Q000201:enzymology / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D007554:Isotopes; D007700:Kinetics; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D048348:Reverse Transcription; D012997:Solvents / Q000737:chemistry*; D014158:Transcription, Genetic
D002021:Buffers; D007554:Isotopes; D016337:RNA, Catalytic; D012367:RNA, Viral; D012997:Solvents; D017666:Deuterium Oxide