SELEX

The randomised nucleotide library that is used as the input in the initial selection procedure is conveniently generated on a DNA synthesiser and transcribed into an RNA pool by in vitro transcription. Designing the structure of the library is a crucial step which should be planned carefully. Three main types of libraries are in general use A fully randomised library a randomised region which is embedded in a natural RNA structure and a doped library where each position of a specific RNA sequence contains a 

ONA template synthesis In vitto transcription— l RNA poofl -Binding to ligand Separation 

An additional consideration is the required level of library complexity and RNA length. Due to experimental limitations the pool of RNAs has a practical limit of approximately 1015 molecules. This corresponds to approximately the number of sequence combinations in a randomised 25-nucleotide long oligonucleotide (425 — 1015). However, for some applications it is desirable to ran- 

8% denaturing polyacrylamide gel Nitrocellulose filter (BA85, Schleicher Schuell) 

2 x Renaturation buffer 20 mM HEPES-KOH, pH 7.0 2 mM MgCl2 200 mM KC1 Washing buffera Filter elution buffer 300 mM NaCl 1% SDS 

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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. 

Stoltenburg, R. Reinemann, C. Strehlitz, B. SELEX - A (r)evolutionary method to generate high-affmity nucleic acid ligands. Biomol. Eng. 2007, 24, 381 03. 

Transactivation-response element of HIV-1 [19] hnine exchange <230 SELEX, HPLC, and MALDI-TOFMS... 

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. 

Key words RNA aptamers, SELEX, Nicotinic acetylcholine receptors... 

A simple protocol detailed in this chapter was established to develop RNA aptamers that bind to the electric organ nAChR and that are displaced by cocaine (8) (see Fig. 1 for a scheme). This protocol can be easily transferred to SELEX applications with other receptors or cell-surface epitopes, given that these are enriched in membrane preparations. 

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). 

To perform SELEX experiment using nitrocellulose-filter binding, prepare filtration unit by preincubating a nitrocellulose sheet in incubation buffer. 

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

The RNA pool unlabeled or radiolabeled - as detailed later - is diluted in incubation buffer prior to the SELEX step, denatured and renatured as detailed in Subheading 3.7. 

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)).

For cloning and sequencing of individual RNA aptamers, nine SELEX cycles are necessary to obtain high-affinity RNA ligands for the nAChR. 

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). 

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). 

Alternative mRNA splicing detection and analysis of human exonic enhancer sequences using genomic SELEX... 

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 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. 

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.)...

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