Selection SELEX

In vitro selection (SELEX) (ligand binding or catalytic activity)... 

Degenerate Oligonucleotide Synthesis and In Vitro Selection (SELEX)... 

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]. 

In the DCC SELEX screen, aldehydes, the TAR RNA target, and a random library of 2 -amino RNAs were allowed to equilibrate. Next, the TAR RNA target and bound ligands were separated from the aptamer library. The selected 2 -amino RNAs that bound the TAR RNA target were then reverse transcribed into DNA and PCR amplified. These double-stranded... 

Figure 3.19 Schematic of the DCC SELEX system. Upper left A library of random 2 -amino RNAs are allowed to equilibrate via imine formation with aldehydes in the presence of target. Bottom left Modified RNAs are bound to the target. Bottom center Modified RNAs bound to the target are separated from unbound RNAs. Bottom right Selected RNAs are eluted and reverse transcribed and amplified to corresponding double-stranded DNA. Upper right The selected double-stranded DNA is transcribed to the 2 -amino RNAs. The selection process is repeated n-cycles and selected conjugated aptamers are identified.

The TAR RNA target sequence, the 2 -amino RNA library and the appended aldehydes were subjected to the DCC SELEX system. The screen selected a 19-nt sequence with U-NH appended at position 9 and unmodified at positions 6 and 7 (Eig. 3.20). Importantly, it was shown that different sequences were identified when control selections were carried out in the absence of aldehydes, proving that the imino-conjugated nucleic acids are being selected. 

Figure 3.20 TAR RNA DCC SELEX system, employing 2 -ammo-2-deoxyuri-dine (U-NH ) capable of reversible imine formation with the appended aldehydes Rb, Rc, and Re. Selected appended RNA aptamers and their corresponding dissociation constants are shown at the bottom.

Bugaut, A. Toulme, J-J. Rayner, B. SELEX and dynamic combinatorial chemistry interplay for the selection of conjugated RNA aptamers. Org. Bio-mol. Chem. 2006, 4, 4082 088. 

The first two rounds are performed under low stringency conditions to enhance RNA-protein binding and to avoid early depletion of sequences present in the SELEX RNA pool. For SELEX cycles 1-3 a nitrocellulose-filter binding assay is used to separate receptor-bound from free aptamers. Beginning from SELEX cycle 4, the nitrocellulose-filter binding and a gel-shifr selection step are employed as two consecutive selection processes (see Note 2). 

Fig. 2. Alternation ot gel-shitt and filter-binding selection steps Target-bound and unbound radiolabeled RNA aptamers are separated by polyacrylamide gel electrophoresis, visualized by autoradiography, purified from the gel, and used for the subsequent nitrocellulose-filter binding selection step. The experiments are earned out in the presence (-i-) and absence (-) of target protein using the SELEX cycles 0 (control), 3, and 7. The figure illustrates the increase of binding affinity of selected RNA pools, seen as augmented quantity of RNA retained together with the receptor protein at the top of the gel (modified from ref. (8)).

In order to avoid the selection and amplification of RNA molecules that do not bind to the target site, two assays for in vitro selection are both employed after SELEX cycle 3. In addition a negative preselection step can be used at which unspecific binders to nitrocellulose (used for separation of receptor-bound from unbound RNA molecules) are discarded (see Subheading 3.7). 

Although its use in ligand selection for large scale of affinity chromatography is not wide, ribosome display and systematic evolution of ligands by exponential enrichment (SELEX) may... 

SELEX (systematic evolution of ligands by exponential enrichment) is used to generate aptamers, oligonucleotides selected to tightly bind a specific molecular target. The process is generally automated to allow rapid identification of one or more aptamers with the desired binding specificity. 

Figure 1 illustrates how SELEX is used to select an RNA species that binds tightly to ATR In step (1), a random mixture of RNA polymers is subjected to unnatural selection by passing it through a resin to which ATP is attached. The practical limit for the complexity of an RNA mixture in SELEX is about 1015 different sequences, which allows for the complete randomization of 25 nucleotides (425 = 1015). When longer RNAs are used, the RNA pool used to initiate the search does not include all possible sequences. RNA polymers that pass through the column are discarded those that bind to ATP are washed from the column with salt solution and collected. The collected RNA polymers are amplified by reverse transcriptase to make many DNA complements to the selected RNAs then an RNA polymerase makes many RNA complements of the resulting DNA molecules. This new pool of RNA is subjected to the same selection procedure, and the cycle is repeated a dozen or more times. At the end, only a few aptamers, in this... 

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. 

Other RNA polymerases, like SP6 or T3, can also be used, provided that the corresponding promoter has been introduced. By using [o -32P] - nuc 1 eo tides the RNA can be labeled radioactively for easy determination of the amount of selected RNA. Thus, the progress of the SELEX process can be monitored. 

Figure 7. (continued) pre-selected molecules, (ii) creation of genetic diversity and (iii) selection of suitable candidates through intervention, for example by the SELEX technique (Figure 8). Molecular properties are tested after each selection phase and the cycles are terminated when either the desired result has been achieved or no further improvement of molecular properties has been observed.

Figure 8. A sketch of the SELEX technique used to select for molecules with optimal binding constants to predefined target molecules. The SELEX procedure selects for molecules with sufficiently high binding constants, so called aptamers, in two steps. Target molecules are attached to a chromatographic column which allows for selective retention of sufficiently strong binders. A different solvent is applied to release the binders and canalize them to the next selection round. Commonly some tens of selection cycles (Figure 7) are sufficient to isolate optimal binding RNA molecules.

In vitro selection is now routinely used in a number of laboratories. We discuss below the major features for running a SELEX experiment which typically involves three major steps (i) the synthesis of the library (ii) the selection of the oligomers of interest, and (iii) the amplification of the selected sequences (Figure 6.1). The use of RNA libraries makes it necessary to include a transcription step prior to selection and a reverse transcription step prior to amplification. More details can be found in recent reviews (Brody and Gold, 2000 Ellington, 1994 Famulok and Jenne, 1998 Famulok et al., 2000 Toulme, 2000). 

At first sight it may seem curious to use in vitro selection to identify RNA or DNA sequences that are able to interact with another nucleic strand. SELEX has been used in at least two cases first, in an attempt to extend the repertoire of triple-stranded structures, and second, to recognize folded RNA structures. Triple... 

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