Libraries nucleic acid

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

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. 

Dynamic combinatorial chemistry has recently been used to investigate the process of templating nucleic acid library members to form interesting structures. To date, the templating process has included the development... 

Aptamers are nucleic acids which exhibit a defined structure due to their nucleotide sequence and therefore, are able to specifically bind selected targets [1] (aptus [lat.] = fitting, sticking to). Aptamers and likewise, ribozymes [2] and deoxyribozymes [3] are selected in vitro by screening nucleic acid libraries. Here we describe in detail the selection of aptamers by a process called SELEX (Systematic Evolution of Ligands by Exponential enrichment) [4]. 

Usually, one starts with a nucleic acid library comprising 1014 to 1016 individual molecules [5]. This library size is assumed to be sufficient to contain sequences with the desired property [6]. Additional mutations may be introduced into selected nucleic acid variants by repeating the SELEX cycle, thereby increasing the number of screened nucleic acids with different sequences. 

The process of selecting an RNA catalyst with a particular function begins with a large pool of random sequences. This pool can be acquired by producing random single-stranded DNA on a nucleic acid synthesizer or by in vitro mutagenesis of an existing nucleic acid library [18,19]. Random sequence pools produced by chemical... 

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.

Famulok, M. (1998) in Combinatorial nucleic acid libraries the new world of aptamers and ribo-zymes., ed. Habenicht, U. (Springer Verlag, Heidelberg), pp. in press. 

Aptamers are short RNA or DNA molecules analogous to antibodies in their ability to bind tightly and specifically to protein antigens. Aptamers can be selected in vitro from large nucleic acid libraries. Because of their small size and the fact that they can be readily modified during chemical synthesis, aptamers have been adapted to a variety of diagnostic and therapeutic assays in the laboratory and clinic (1-5). 

In vitro selection (SELEX, Systematic Evolution of Ligands by Exponential Enrichment) has been employed for over a decade to evolve aptamers and artificial ribozymes with various catalytic functions [169, 170]. However, nucleic acids are, in contrast to proteins, limited to the four nucleobases, thus occupying a more narrow chemical space, which can limit successful SELEX experiments against particular targets. Modified nucleosides in nucleic acid libraries are useful tools in aptamer SELEX to expand the chemical space of DNA [171-174]. 

The introduction of functional groups on canonical nucleobases can be restrained, because these modified nucleobases need to be compatible with the enzymatic steps required in the SELEX process. A novel approach termed click-SELEX uses a modular strategy based on Cu -catalyzed azide-alkyne cycloaddition to generate modified nucleic acid libraries (Fig. 5) [175]. 

In future, this might be achieved by the introduction of photocleavable linker molecules to remove large residues prior to PCR amplification [175]. Thus, various chemical moieties such as aromatic residues, amino acids or lipid modifications could be introduced in nucleic acid libraries by this method, allowing rapid access to modified libraries for aptamer selection and the selection of novel DNA catalysts. 

Combinatorial Selections of Catalysts from Nucleic Acid Libraries 381... 

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