Ribozymes selection

The more successful strategy for the isolation of RNA- and DNA-based catalysts involves the direct screening of nucleic acids libraries for catalytic activity. This approach is called direct selection [6, 65, 77, 78, 86, 101-107]. In direct selections, nucleic acids that are capable of catalyzing a particular chemical transformation modify themselves with a tag or other characteristic that allows their preferential enrichment over those molecules which are catalytically inactive [108]. The design of ribozyme-selections involving reactions between two small substrates requires that one reactant be covalently attached to every individual member of the starting RNA pool. After the reaction with another substrate which usually carries the selection-tag has occurred, the self-modified RNA is immobilized on a solid support, separated from non-active molecules, and then cleaved off the support. 

Finally, novel nucleic acid catalysts have also been selected from random sequence pools (reviewed in Ref. 19). Joyce and co-workers have manipulated the function of the Group I self-splicing ribozyme, selecting variants that can utilize calcium or cleave DNA from partially randomized pools [20,21], Lorsch and Szostak [22] selected a polynucleotide kinase ribozyme from a completely random sequence pool that flanked a previously selected ATP binding site. Many of the novel ribozymes can catalyze reactions that are relevant to protein biosynthesis, bolstering arguments that translation may have arisen in a putative RNA world. For example, Lohse and Szostak [23] have selected ribozymes that can carry out an acyl transfer reaction, while Illangasekare et al. [24] have isolated a... 

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... 

Since it was first shown that functional nucleic acids could be selected from randomised libraries there have been many examples of aptamers and ribozymes selected for a variety of applications. There are a number of new reports on various aspects of aptamers, due in part to a special issue on this subject (Bioorganic and Medicinal Chemistry, 2001, volume 9, issue 10). Whilst most of the new aptamers that have been described are designed for binding to a given target, the number of aptamers that have catalytic activity is increasing. There are also examples now of allosteric aptamers, both for binding and with catalytic activity, that function only in the presence of a co-factor . 

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

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. 

Zhang, B., and Cecil, T. R., 1997. Peptide bond formation by in vitro selected ribozymes. Nature 59 99- 99. 

Two clinical studies with CD4+ lymphocytes expressing an antiviral were performed. Marking in both studies was low but there was some indication for a possible selective advantage conferred by the ribozyme (Buchschacher and Wong-Staal 2001 Macpherson et al. 2005). 

The catalytically active RNA species (ribozymes) have been shown in recent years to undergo an unbelievable range of reactions. This led to the question as to whether they are also involved in nucleotide syntheses. Unrau and Bartel (1998) have reported successful nucleotide syntheses carried out using ribozymes (see Sect. 6.5). It was possible to isolate in vitro selected RNA which acted catalytically in the synthesis of a pyrimidine nucleotide it remains unclear whether these results are important for biogenesis. 

Soon after this report, the group of M. Yaros, also working in Boulder, was able to demonstrate ribozyme activity with a much higher performance (Illangsekare, 1995). Using a random mixture of many billions of RNA sequences, they selected one species which was able to catalyse the aminoacyl synthesis. In other words, the selected ribozyme aminoacylated its 2 (3 ) end when offered phenylalanyl-AMP the addition of Mg2+ and Ca2+ was necessary. The catalysed reaction was about 105 times faster than in the absence of ribozyme. Thus the group was able to show that a fundamental reaction of contemporary protein biosynthesis can also be catalysed by a ribozyme (see Sect. 5.3.2). The assiduous search for further activities continues. 

The authors obtained an RNA ligase ribozyme using the method of in vitro evolution . Here, macromolecules are allowed to go through a series of synthetic cycles, which are followed by a proliferation phase, mutation and selection. As in Darwinian evolution, the goal is to carry out laboratory selection of molecules with certain required properties. 

Fig. 6.10 Schematic representation of the principle of the evolution of a ribozyme in a test tube. Several mutants are selected in each cycle and proliferate in the next step. Slightly modified after Culotta (1992)...

Agresti, J.J., Kelly, B.T., Jaschke, A., and Griffiths, A.D. (2005) Selection of ribozymes that catalyse multiple-turnover Diels-Alder cycloadditions by using in vitro compartmentalization. Proc. Natl. Acad. Sci. USA 102,16170-16175. 

RNAi and ribozymes represent two additional approaches to gene silencing/down-regulation with therapeutic potential. RNAi is an innate cellular process that achieves silencing of selected genes via an anti-sense mechanism. It shares many characteristics with the antisense-based approach described above, but also some important differences, e.g. in the exact mechanism by which the antisense effect is achieved. 

A rather new approach for detecting metal ions with very high sensitivity and selectivity utilizes DNAzymes. DNAzymes are a special class of enzymes formed from DNA nucleotides. Compared to proteins and ribozymes, they are more stable, structurally simpler, and therefore cheaper. As DNAzymes often require metal ion cofactors, they are interesting sensing platforms for these metal ions [149]. 

Keywords Ribozymes, In vitro selection. Nucleic acid libraries, Metallo enzymes, Aptamers. 

This is also true for a number of in vitro selected DNA enzymes which were selected under divalent metal-free buffer conditions [59,60]. These results contradict the common assumption that all ribozymes are metalloenzymes and provide a number of ribozymes for which it will be very interesting to determine their exact catalytic mechanisms at high resolution. 

While indirect selections work quite well for antibodies they have been less successful in the case of catalytic nucleic acids. There are only three examples which prove that it is possible in principle to obtain a ribo- or deoxyribozyme by selecting an aptamer that binds to a TSA A rotamase ribozyme [7], a ribozyme capable of catalyzing the metallation of a porphyrin derivative [92], and one catalytic DNA of the same function [93]. Another study reported the selection of a population of RNA-aptamers which bind to a TSA for a Diels-Alder reaction but the subsequent screen for catalytic activity was negative for all individual RNAs tested [94]. The attempt to isolate a transesterase ribozyme using the indirect approach also failed [95]. 

The only indirect selection that led to a catalytic DNA is a deoxyribozyme that catalyzes the same class of porphyrin metallation as the aforementioned ribozyme. The ssDNA oligonucleotide showed a k at of 13 h" for the insertion of into mesoporphyrin IX [93, 96-99]. This corresponds to a rate enhancement of 1400 compared to the uncatalyzed reaction which is as good as a catalytic antibody for the same reaction. 

A ribozyme activity that led to RNA-modifications that are analogous to the 5 -5 pyrophosphate caps of eukaryotic RNA transcripts was selected by Huang and Yarns [84]. Actually the author s intention was to isolate ribozymes which catalyze the formation of a mixed anhydride between an amino acid carboxylate and a 5 -terminal phosphate of an RNA, an activity that is chemically analogous to the activation of amino acids by ATP catalyzed by aminoacyl tRNA synthetases. However, while the selected ribozymes did... 

Many examples of catalytic nucleic acids obtained by in vitro selection demonstrate that reactions catalyzed by ribozymes are not restricted to phosphodiester chemistry. Some of these ribozymes have activities that are highly relevant for theories of the origin of life. Hager et al. have outlined five roles for RNA to be verified experimentally to show that this transition could have occurred during evolution [127]. Four of these RNA functionalities have already been proven Its ability to specifically complex amino acids [128-132], its ability to catalyze RNA aminoacylation [106, 123, 133], acyl-transfer reactions [76, 86], amide-bond formation [76,77], and peptidyl transfer [65,66]. The remaining reaction, amino acid activation has not been demonstrated so far. 

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