SELEX process

Aptamers are nucleic acid sequences that specifically bind proteins or low molecular-weight substrates. Aptamers are selected from a combinatorial library of lO -lO DNAs, using the Systematic Evolution of Ligands by Exponential Enrichment Process (SELEX). Numerous aptamers that specifically bind proteins or low molecular-weight substrates have been elicited in recent years. Also, their recognition properties have been used extensively to develop electrochemical [162,163] or optical... 

Aptamers are high-afFmity ligands selected from DNA or RNA libraries via SELEX process. SELEX is a process used for in vitro selection of aptamers, which are highly specific in binding as well as function due to the nucleotide sequence and shape. 

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.

Enrichment of high affinity candidates is usually achieved in 8 to 15 rounds of SELEX. Each rotmd takes approximately 2 days to perform. The process has been automated using robotic liquid handlers both for DNA (SomaLogic) and RNA aptamers (Cox, 2002). Next, the sequenced aptamer is prepared in bulk by conventional DNA synthesis chemistry and purified, then the aptamer arrayed onto a solid support. Thus, an aptamer is ready for application within 2 to 3 mo. Because the sequence is known, preparation of additional aptamer is easily accomplished using conventional oligonucleotide chemical synthesis. 

Use a word processing program to analyze random sequences for equal occurrence of AA, AC, AG, and AT motifs in the random regions (repeat for C, G, and T). If one or more of these motifs predominate or the base composition is not random in the random sequences, the pool may not contain enough different sequences for a successful SELEX experiment. 

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). 

Mount nitrocellulose sheet in autoclaved filtration unit. Assemble the following reaction mixtures for the SELEX process nAChR-enriched plasma membranes (800 pg/ml... 

The purified DNAs are used as templates for in vitro transcription reactions. Forward primer (P-40, same sequence as primer used during SELEX process) and reverse primer (p-22 pGEM). 

Figure 10.17 Schematic representation of the systematic evolution of ligands by exponential (SELEX) enrichment process. (See the color version of this figure in Color Plates Section.)...

Figure 10.17 Schematic representation of the systematic evolution of ligands by exponential (SELEX) enrichment process.

The search for RNAs with new catalytic functions has been aided by the development of a method that rapidly searches pools of random polymers of RNA and extracts those with particular activities SELEX is nothing less than accelerated evolution in a test tube (Box 26-3). It has been used to generate RNA molecules that bind to amino acids, organic dyes, nucleotides, cyano-cobalamin, and other molecules. Researchers have isolated ribozymes that catalyze ester and amide bond formation, Sn2 reactions, metallation of (addition of metal ions to) porphyrins, and carbon-carbon bond formation. The evolution of enzymatic cofactors with nucleotide handles that facilitate their binding to ribozymes might have further expanded the repertoire of chemical processes available to primitive metabolic systems. 

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. 

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]. 

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. 

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)... 

L Gold. The SELEX process a surprising source of therapeutic and diagnostic compounds. Harvey Lect 91 47-57, 1995. 

Unfortunately, enantiomeric or mirror-image nudeic adds cannot be used directly in the SELEX process because of the lack of (mirror-image) enzymes which would be needed to amplify them. 

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