Ribozymes, purification

As the fold of the RNA is critical, the folding step after purification of the RNA is important. The most commonly used folding protocols involve heat-cooling the RNA in a metal-free buffer such as Tris-EDTA and subsequently adding metals (Ke and Doudna, 2004) such as monovalent or divalent cations, most commonly Na/K+ and Mg2+. Slow renaturation of the folded RNA from denaturing conditions may also be effective, particularly in RNA-protein systems. Both the human 5 virus ribozyme... 

To measure the folding equilibrium of the Tetrahymena L-21Sca ribozyme in various counterions, 32P-labeled ribozyme is prepared by T7 in vitro transcription with a-[32P]-ATP, according to established protocols (Zaug et al, 1988). Free label is removed with a size-exclusion spin column (e.g., TE-10, BD Sciences), and the RNA used without further purification (Emerick and Woodson, 1993). If desired, the RNA can be end-labeled with 32P and purified by denaturing PAGE. As for all biochemical experiments on RNA, care must be taken to use water, reagents, and plastic consumables that are free of RNase. 

In this section is an overview of some historical aspects of enzyme studies with special emphasis on new methods of purification, structure determination, and research on the reaction mechanism of an enzyme. Enzyme application for medical and industrial use and development of novel enzymes such as catalytic RNA (ribozyme) and catalytic antibody (abzymes) are also briefly described. 

Wincott F, DiRenzo A, Shaffer C, Grimm S, Tracz D, Workman C, Sweedler D, Gonzalez C, Scaringe S, Usman N. Synthesis, deprotection, analysis and purification of RNA and ribozymes. Nucleic Acids Res 23 2677-2684, 1995. Sproat B, Coloima F, Mullah B, Tsou D, Andrus A, Hampel A, Vinayak R. An efficient method for the isolation and purification of oligoribonucleotides. Nucleosides Nucleotides 14 255-273, 1995. 

Enzymes, as you probably know, are proteins that can make chemical reactions happen in a more selective and faster way. At the end of each reaction cycle the enzymes remain unchanged so they act as catalysts. Since they occur in the living world we call them biocatalysts. Aside from proteins, ribonucleic acids and their fragments can act as catalysts and are called ribozymes, by analogy to enzymes. Enzymes are extracted from living tissues, for example, milk, saliva, liver, muscle have to be stored under carefully maintained conditions and, once outside living tissue, lose their activity fast. Isolation and purification of enzymes and assaying their activity have been major operations in biochemical and biomedical laboratories. Today,... 

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