Protein SELEX

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)).

SELEX is a widely used technique for screening of aptamers which are nucleic acid ligands. According to this method, a pool of DNA with a random sequence region attached to a constant chain is constituted by amplification then transcribed to RNA. RNA pool is separated according to the affinity of RNA molecules to a target protein. DNA molecules obtained by reverse transcription from retarded RNA molecules are amplified and the cycle is repeated. 

Fig. 2.5 (a) An aptamer-target protein interaction (adapted from Tuerk and Gold 1990, p. 505). (b) Principle of the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process (adapted from Tombelli et al., 2005, p. 2424)... 

SELEX has also allowed the characterization of the RNA hairpin, which constitutes the iron responsive element (IRE) recognized by the iron regulatory factor (IRF) protein to post-transcriptionally regulate translatability and decay of mRNAs involved in iron import and storage in eukaryotic cells (Henderson et al., 1994). 

Anwar, A., Ali, N., Tanveer, R. and Siddiqui, A. (2000) Demonstration of functional requirement of polypyrimidine tract-binding protein by SELEX RNA during hepatitis C virus internal ribosome entry site-mediated translation initiation. J. Biol. Chem., 275, 34231-34235. 

Moine, H., Cachia, G., Westhof, E., Ehresmann, B. and Ehresmann, C. (1997) The RNA binding site of S8 ribosomal protein of Escherichia coli SELEX and hydroxyl radical probing studies. RNA, 3, 255-268. 

In their original publication about SELEX, Tuerk and Gold (1990) already speculated that a similar approach could be adapted to protein selection. They referred to experiments describing the isolation of particular mRNAs from a pool of variants by immunoprecipitation of the nascent polypeptides present in the mRNA-ribosome-polypeptide complexes (Korman et al., 1982 Kraus and Rosenberg, 1982). In fact, soon after the publication of SELEX (Tuerk and Gold, 1990) a patent application was filed (Kawasaki, 1991), proposing a similar approach to enrich peptides from libraries. 

Fourth, the selected (partitioned) molecules are reverse transcribed (if the library was RNA based) and then amplified by PCR using the primers fixed B and fixed A for DNA libraries PCR is sufficient. Thus, the subset of sequences that bound to the intended target becomes the pool for the second round of the SELEX process. Several more rounds of the SELEX process are done until the library complexity drops from 1015 sequences to (perhaps) a hundred, after which they are cloned and sequenced and tested for the desired properties. For protein targets, the SELEX process usually has taken from 7 to 15 rounds, although a robotic SELEX process will take fewer rounds (see Figure 20.2). The entire (front-loaded) SELEX process is diagrammed in Figure 20.2. 

FIGURE 20.3 Kd values from the first 100 SELEXs aimed at proteins. 

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