In-vitro selection strategies

In vitro selection strategies can be sub-divided into two types direct and indirect selections. These two types of selection experiments directed at the isolation of synthetic catalytic nucleic acids differ mainly by their technical concept, their design and their outcome. 

Tawfik and Griffith (1998) reported an in vitro selection strategy for catalytic activity using compartmentalization. Here, each member of the DNA library is encapsulated in an aqueous compartment in a water in oil emulsion. The compartments are generated from an in vitro transcription-translation system, and contain the components for protein synthesis. The dilution is chosen such that, on average, the water droplets contain less than one DNA molecule. The DNA is transcribed and translated in vitro in the presence of substrate, which is covalently attached to the DNA. Only translated proteins with catalytic activity convert the substrate to the product. Subsequently, all DNA molecules are recovered from the water droplets and the DNA linked to the product is separated from the unmodified DNA linked to the educt, which requires a method to discriminate between both. The modified DNA can then be amplified by PCR and used for a second selection cycle. The principle of this approach is depicted in Figure 6. 

At the same time, several laboratories have used in-vitro selection strategies to identify RNAs and DNAs that catalyze specific reactions [34-40]. The information from these and related studies now opens the possibility of expanding the molecular skeletons capable of storing sequence information into ones that can be read into complementary materials [41]. 

In a subsequent study, a similar in vitro selection strategy was applied towards Mg -, Mn -, and Zn -dependent DNAzymes with RNAse activity [32]. Although the reaction with the resultant DNA enzymes proceeded around 17 times slower than with the Pb -based system, focus remained on the Mg " -dependent systems, as they might eventually be active under intracellular conditions. Next, a transacting catalyst capable of 17 turnovers of a chimeric substrate within 5 h was developed. The most active clone was suggested to contain a three-stem junction, based on the sequence. This makes the structure more complex than that of its Pb " -dependent analog. 

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 in vitro selection/amplification strategy has also been applied to modified ONs, especially where the 2 -ribose position has been changed and where phosphorothioates or other phosphate replacements have been used (Fig. 10.25, top). Several strucmres of modified ON chains that have been synthetically produced to obtain constrained sequences or sequences with higher stability to nucleases have also been reported (Fig. 10.25, bottom). Examples of biosynthetic modified ON libraries are covered in the next section. 

Ribozyme-catalyzed reactions involving C-C bond formations have also been reported. Seelig and Jaschke (233) presented the in vitro selection of ribozyme catalysts for the Diels-Alder reaction between maleimide and anthracene, employing a 2 X lO -member library of 160-mer modified ONs (L28) with 120 randomized positions. The selection strategy used is shown in Fig. 10.40. Library L28 was prepared from the corresponding dsDNA sequences, and transcription initiation was performed in the presence of ternary complexes between guanosine monophosphate (10.57), PEG (10.58), and anthracene (10.59, step a. Fig. 10.40). The library obtained contained a 5 -anthracene-PEG appendage and was incubated with biotin-modified maleimide... 

Figure 9.1. An in-vitro selection experiment comprises various sequential steps, of which the first is the generation of a nucleic acid library of completely random sequences. This library is subjected to an appro-. priatc selection strategy which allows the separation of functional molecules from non-functional ones. The small proportion of nucleic acids with the desired activity is then amplified enzymatically and re-suh-jected to the selection procedure. This is necessary as the complexity of the library, which can contain up to 1016 different oligonucleotide sequences, makes it impossible to enrich for the active sequences in one single selection and amplification cycle. Therefore, a number of cycles are performed sequentially until the functional sequences are the majority species in the library mix, and these can be characterized by cloning and sequencing.

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