Ribozyme artificial

Ribothymidylic acid residue 813 Ribozyme(s) 649 - 652, 239 artificial 652 hairpin 649,651s hammerhead 649, 651 leadzyme, 651s Ribulose 164s Ribulose bisphosphate 707s Ribulose bisphosphate carboxylase (Rubisco)... 

Silverman, S. K. (2009). Artificial functional nucleic acids Aptamers, ribozymes, and deoxyribozymes identified by in vitro selection. In Functional Nucleic Acids for Analytical Applications, (Y. Li and Y. Lu, eds.), pp. 47-108. Springer Science+Business Media, LLC, New York. 

By simulating evolution in vitro it has become possible to isolate artificial ribozymes from synthetic combinatorial RNA libraries [1, 2]. This approach has great potential for many reasons. First, this strategy enables generation of catalysts that accelerate a variety of chemical reactions, e.g. amide bond formation, N-glycosidic bond formation, or Michael reactions. This combinatorial approach is a powerful tool for catalysis research, because neither prior knowledge of structural prerequisites or reaction mechanisms nor laborious trial-and-error syntheses are necessary (also for non-enzymatic reactions, as discussed in Chapter 5.4). The iterative procedure of in-vitro selection enables handling of up to 1016 different compounds... 

The Houk group recently compared several artificial catalytic Diels-Alderase systems, including the ribozymes described here [11]. This study came to the conclusion that in none of these artificial systems there is a significant specific stabilization of the transition state. Acceleration arises predominantly from binding of the reactants, converting a second-order reaction of diene with dienophile into a first-order reaction of the termolecular complex of host, diene, and dienophile. The simultaneous presence of the two components of an intermolecular Diels-Alder reaction within the confined space of a cavity is the driving force that facilitates the reaction. 

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. 

Ribozymes acting on nucleotide sequences have often been selected from random libraries, and many artificial sequences have been obtained. Landweber and Porovskaya (212) recently reported the selection of a family of small ribozymes from a 1.6 X 10 -member, 132-mer hbrary L23 able to ligate multiple RNA substrates. In vitro selection was performed with three different RNA substrates for six cycles in the presence of Mg (step a, Fig. 10.33), and multiple substrates were used to select for... 

Figure 10.33 Selection of artificial ribozymes with dual, switchable RNA cleavage and ligation activity from the biosynthetic ON ribozyme library L23 the selection/amplification...

The existence of a coordination site for divalent cations and the possibility for the ribozyme to adopt two different conformations, one suited to ligation, the other to self-cleavage, were possible explanations for this dual activity. The occurrence of a parallel, catalytic activity that was selective for Mn , even though this cation was never used in the in vitro selection experiments, hinted at the flexibility of ribozymes able to adapt their properties to different conditions and that could allow the evolution of new catalytic activities. Other artificial ribozymes selected in vitro have been rejxMted by Vaish et al. (hammerhead-like) (215), Yu et al. (hairpin-like) (216), Robertson and Ellington (allosteric hgase activated by ON effectors) (217), and... 

Figure 10.35 Selection of artificial ribozymes with nucleotide-assembling properties from the biosynthetic ON ribozyme library L24 the selection/amplification process to 10.45 using the slection substrate 10.45.

Figure 10.37 Selection of artificial ribozymes with peptide-assembling properties from the biosynthetic ON riboyzyme labeled library L25 structure of the key intermediates 10.49 and 10.50.

Figure 10.39 Selection of artificial ribozymes with amino acidic cofactors from the biosynthetic modified on DNAzyme libraries L26 and L27 the selection/amplification process to the L-his dependent ribozymes 10.55 and 10.56.

Figure 10.40 Selection of artificial ribozymes with catalytic properties for a Diels-Alder reaction from the biosynthetic ON modified ribozyme library L28 library synthesis and the selection process using the biotinylated selection dienophile 10.60.

It is an impressive list since several hundred different natural group I and group II introns have been shown to exist. In addition, many artificial active ribozymes have been prepared. 

While enzymological research over several decades has provided a rather detailed understanding of how certain enzymes accelerate chemical reactions, knowledge about ribozyme mechanisms and catalytic strategies is scarce in comparison. For some natural ribozymes, mechanistic and structural studies were carried out, but artificial ribozymes remain largely uncharacterized. Therefore it is unclear in many cases whether these catalysts are passive containers working solely by substrate pre-organization, or whether there are specific effects on the activation parameters of the reaction. 

Table 14.2 Catalytic activities of artificial ribozymes (selected examples)...

An artificial ribozyme mimics this translation step of the ribosome. The spedlity of this selected ribozyme is based on the recognition of an adenosine moiety of the amino acid ester and allows the utilization of leucine- and phenylalanine- as weU as methionine-derivatized substrates. This tolerance for various amino acids indicates the possibility of selecting more general ribozymes for protein synthesis. Furthermore, a related ribozyme efficiently catalyses the synthesis of -30 different dipeptides from an aminoacyl-adenylate substrate. Ribozyme-mediated synthesis of uncoded peptides might have been an important step in the transition from a RNA to a peptide world before the anergence of the ribosome. ... 

Further artificial ribozymes are known to react with cosubstrates, for example acylating the thiol group of tethered Co-enzyme A with the AMP-activated biotin. These ribozymes also produce the crucial metabolic intermediates acetyl-CoA and butyryl-CoA at substantial reaction rates. For the selection of this ribozyme, the employed RNA pool had been coupled at its 5 -end to CoA by a previously isolated capping ribozyme. Ribozymes for a number of related reactions were isolated, and the reader is referred to an excellent review discussing in detail the current knowledge on ribozyme catalysis in the context of the RNA world hypothesis. ... 

In this last section, I will discnss stmcture and mechanism of one artificial ribozyme in more detail. This ribozyme, selected in my laboratory, is the only RNA catalyst for small-molecule chemistry with a known spatial stmcture, and due to extensive studies, it is arguably the best-characterized artificial ribozyme known to-date. These data provide for the first time an insight into how a small RNA can accelerate reactions different from phosphodiester chemistry, and what stractural prerequisites are required. 

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