Combinatorial RNA libraries

Fig. 3.4.3. Construction of a combinatorial RNA library. First, a DNA template having a random region of N positions between two fixed primer binding sites (primer A and B) is synthesized. AT7 promoter can be introduced...

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 formation of aptamers rests upon a technique termed SELEX (systematic evolution of ligands by exponential enrichment). It involves the synthesis of a DNA molecule with constant sections at both ends and a randomly variable segment of nucleotides in the middle. A variable segment as short as 10 nucleotides has a combinatorial library of 4 ° or about 10 different sequences a variable sequence of 30 nucleotides generates a combinatorial library of 4 or about 10 different sequences. The DNA mixture so synthesized is then enzymatically transcribed to a combinatorial RNA library. This mixture is applied to a chromatography column in which the substance of interest (e.g., amino acid, protein, drug) is bound. RNA molecules in the combinatorial library that do not bind with the target compound are simply washed out of the column. 

Diels-Alderase ribozymes (DAR), isolated from a combinatorial RNA library, cause a (2 X 10 )-fold acceleration of the Diels-Alder cycloaddition of anthracene covalently tethered to ribozyme and a biotinylated maleimide in aqueous-buffered medium (Scheme 5.15). Jaschke recently reported the action of Diels-Alderase ribozymes as true catalysts, in the sense that they catalyze the cycloaddition of anthracene that is not covalently tethered to RNA and biotin maleimide in aqueous-buffered medium. 

The SELEX technology (Systematic Evolution of Ligands by Exponential enrichment) was introduced by Larry Gold and Jack Szostak [1, 2] and provides a powerful tool for the in vitro selection of nucleic acids (aptamers) from combinatorial DNA or RNA libraries against a target molecule. 

When, as a synthetic chemist, you look at the structure of RNA, you may ask yourself how did this particular type of molecular structure enter the scene for the first time If you adhere to the principles of evolution, you are led to conclude that the structure is the result of a selection process. Selection from what An important possibility is that RNA had been selected (or had selected itself) from a combinatorially formed library of alternative structures. Again, if you, as a synthetic chemist, hypothesize about the type of chemistry that could have produced RNA for the first time, chemical reasoning leads you to consider that there are many chemically closely related alternative structures that could have had a comparable chance to be formed by the same kind of chemistry. In such a scenario, RNA was selected, or had selected itself, by functional criteria from a library of structurally related nucleic acid alternatives. Such a view defines a strategy for approaching the question experimentally you systematically make such potentially natural alternatives in the laboratory by chemical synthesis and compare them with RNA with respect to those chemical properties that are known today to be relevant for RNA s biological functions. Such comparisons may tell you. 

DNA libraries are obtained by a combination of the techniques of chemical solid-state synthesis and the combinatorial methods described later for peptides (see Sec. 9.3.2.2). DNA should, however, first be transformed to RNA libraries in order to yield useful recognition systems. Starting from a large pool of random-sequence DNA molecules, RNA sequences are obtained (see Fig. 8.4.3),... 

In vitro selection is a combinatorial approach in which functional molecules are selected from large libraries of randomized RNAs or DNAs by selection techniques that are suitable for the enrichment of a particular property such as the binding to a target molecule or a particular catalytic activity (Fig. 5). 

Fig. 10.6 Concept of multitarget affinity specificity screening (MASS). Macromolecular targets (typically structured RNA constructs or proteins) in nondenaturing buffers are mixed in solution with a collection of potential ligands derived from natural product fractions, combinatorial libraries, or other diverse compound collections. The...

McNaughton, B. R. Gareiss, P. C. Miller, B. L. Identification of a selective small-molecule ligand for HlV-l frameshift-inducing stem-loop RNA from an 11,325 member resin bound dynamoic combinatorial library. J. Am. Chem. Soc., 2007,129, 11306-11307. 

Figure 3.14 Top Design of the 11,325-member resin-bound dynamic combinatorial library (RBDCL). Bottom Left The HlVl frameshift-inducing stemloop RNA screened against the large RBDCL. Bottom Right Compound 3-3, the homodisulfide selected from the RBDCL that binds the HIV 1 frameshift-inducing stemloop with high affinity and selectivity.

Figure 3.17 Approach to the dynamic combinatorial modification of the TAR-binding aptamer. Left, italics The TAR RNA sequence. Left, bold The TAR-binding aptamer. Left, boxed The 2 -amino-2-deoxyuridine (U-NH ) for dynamic RNA modification. Left center Rb—Rd, the aldehyde library components. Right center Imino-linked DCL members. Right The selected nalidixic aldehyde appended to U-NH results in the TAR RNA-aptamer complex stabilization.

De Wildt, R.M.T., Finnem, R., Ouwehand, W.H., Griffiths, A.D., Van Venrooy, W.J., and Hoet, R.M.A. (1996). Characterization of human variable domain antibody fragments against the U1 RNA-associated A protein, selected from a synthetic and a patient derived combinatorial V gene library. Eur. J. Immunol., 26, 629-639. 

RNA catalysis has been proposed for use in preparing combinatorial libraries of organic structures for drug discovery [39]. As we learn more about the scope, reactivity, and specificity of RNA as a catalyst for organic reactions, it should be possible to use RNA to create new chemical diversity that parallels that found in biological systems, where proteins are the catalysts in the formation of natural products. 

Over the past several years, combinatorial libraries of biological molecules, such as peptides and nucleic acids, have proven invaluable as reagents with which to study molecular recognition of proteins and non-proteins. Such libraries have been used extensively to define the specificity of protein/protein, protein/RNA, protein/peptide, and RNA/small molecule interactions. 

Assuming that each chain library contains some 10s variants, the total number of combinations exceeds 1016 a figure which can only be achieved in the PCR amplified DNA/RNA-based combinatorial libraries [163-165],... 

An extension of this FTICR mass spectrometry-based screening technique has been to screen a combinatorial library for ligands to two receptors simultaneously (59, 60). In this example, the two receptors consisting of RNA constructs representing the prokaryotic (16s) rRNA and eukaryotic (18s) rRNA A-site were incubated simultaneously with an aminogly-... 

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