Applications of Modified Transcripts

This chapter provides protocols for two different methods that require modified transcripts, modification interference and phosphorothioate footprinting. Modification interference is used to identify positions (in substrate or ribozyme RNAs) where modified nucleotides interfere with ribozyme activity (1 –4 ), Since all noninterfering sites can be identified, it is safe to modify these sites without dramatic loss of activity. Thus, these results can be used for the rational design and synthesis of active, highly modified RNAs. In special cases, the enzymatic synthesis of highly modified ribozymes is possible. For example, in the synthesis of hammerhead ribozymes, CTP, GTP, and UTP can be substituted by the corresponding NTPαS (2 ). Furthermore, it should be possible to replace UTP by dTTPαS, since deoxyribose is acceptable at all uridine positions (5 ).