Titlebook: Ribozyme Protocols; Philip C. Turner Book 1997 Springer Science+Business Media New York 1997

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Selection of Accessible Sites for Ribozymes on Large RNA Transcripts,However, the therapeutic target of antisense oligonucleotides and hammerhead ribozymes, large RNAs, are rich in structures, such as hairpins and pseudoknots, which may make certain target sequences inaccessible to the ribozyme (.–.). The methods described here are used in our laboratories to probe t

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Selection of Efficient Ribozyme Cleavage Sites in Target RNAs,ppointing failures. Even prediction of secondary structures of the target RNA by computer modeling with a more or less exact prediction of single-stranded or loop regions does not guarantee successful cleavage of the target RNA by a synthetic or an expressed ribozyme (.). Expensive screening tests w

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Chemical Synthesis, Analysis, and Purification of Ribozymes, group. Solid-phase chemistry currently provides the most effective means for the scale-up of RNA synthesis. The biological activity of chemically synthesized RNA (.–.), equivalent to that of RNA derived by transcription methods (.–.), is contingent on efficient synthesis and purification protocols.

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A Practical Method for the Production of RNA and Ribozymes,des. Although great progress has been made in improving the methods for DNA synthesis, such that production of large amounts has become commonplace, similar quantities of RNA are still difficult to obtain.

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Preparation of Templates for Production of Ribozymes and Substrates, by in vitro transcription can be used as substrates in a variety of experiments ranging from splicing and translation to structural studies. This chapter describes methods for the preparation of templates suitable for in vitro transcription.

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Enzymatic Synthesis and Characterization of Unmodified Ribozymes and Substrates,s can be end-labeled. Simple procedures generate a sequence ladder to confirm colinearity of template and transcript (.,.). This is not a “mere luxury,” since examples with significant template slippage have been reported (.). If a small labeled cleavage product is produced, also the exact cleavage

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