Ribozymes engineering

Usman, N. Beigelman, L. McSwiggen, J.A. Hammerhead ribozyme engineering. Curr. Opin. Struct. Biol. 1996, 6 (4), 527-533. 

Cech T. Ribozyme engineering. Curr Opin Struct Biol 2 605-609, 1992. 

Ribozymes are enzymes that, in some cases, can cleave mRNA. A ribozyme is engineered so that it cleaves the mutant mRNA sequence, preventing its translation into a polypeptide. 

Fig. 3. The hepatitis delta virus ribozyme. A Secondary structure of the genomic HDV ribo-zyme RNA used for the determination of the crystal structure [37]. The color code is reflected In the three dimensional structure B of this ribozyme. PI to P4 indicate the base-paired regions. Nucleotides in small letters indicate the U1 A binding site that was engineered into the ribozyme without affecting the overall tertiary structure. The yellow region indicates close contacts between the RNA and the U1 A protein...

It has already been pointed out that a great deal of intracellular biochemistry is based on cofactors, with these cofactors, in turn, often being derived from nucleotides. However, while this indirectly implies the proficiency of ancient RNA catalysts, it does not prove that such catalysts could have existed. Although there are, for example, protein dehydrogenases and esterases, there are no modem ribozymes with similar activities. Just as engineering a ribozyme self-replicase will be an experimental demonstration that life could have arose via RNA, so the production of artificial ribozymes will be a demonstration that a metabolically complex RNA world may once have existed. 

The list of examples detailed above is by no means exhaustive. Several additional examples of the structure-based control of translation, such as repressor protein binding sequences on the mRNA transcripts and ribozymes [39], are among others that could be listed. The intentions of this chapter are not to enumerate examples of how structural characteristics of mRNA influence its translation. What should be evident from the preceding discussion is that mRNA structure is a critical determinant of translation, and synthetic DNA technology offers the possibility to alter and probe the effects of mRNA sequence on its structure and the resulting translation efficiency. Further developments in this area will be immensely valuable to metabolic engineering as it will enable practitioners to fine-tune the fluxes of desired metabolic pathways through modulation of protein levels. 

An important input for sensing applications of engineered nucleic acids has been their ability to show allosteric properties and thus perform as precision molecular switches. Allosteric ribozymes are frequently termed as aptazymes since they combine nucleic acid binding properties (aptamers) and catalytic activity like their protein enzyme counterparts. They possess an effector binding site that is distinct from the active site of the catalyst and it... 

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