SELEX or SELEX-seq / HT-SELEX

Systematic Evolution of Ligands by Exponential Enrichment / High-Throughput Systematic Evolution of Ligands by Exponential Enrichment

From the time that the first SELEX experiments were described by 3 independent groups in 1990 (Ellington et al., 1990) (Tuerk et al., 1990) (Sullenger et al., 1990), the method has been adapted to a wide range of technologies (Chen et al., 2016) (Takahashi et al., 2016). A highly multiplexed, parallel HT-SELEX method was developed for NGS (Jolma et al., 2010). A variation of SELEX-seq (Slattery et al., 2011) uses Nextera adapter sequences for efficient library preparation (Zhang et al., 2016).

In this method, proteins are expressed as fusions with streptavidin-binding peptide (SBP), conjugated to Gaussia luciferase, in the pD40htSELEX expression vector. Each DNA ligand contains a 14 bp randomized region (14N), and a 5 bp barcode that uniquely identifies the individual SELEX sample. Partially nested primers are used in successive SELEX rounds. A double-stranded DNA mixture containing all possible 14 bp sequences is incubated with a DNA-binding protein immobilized into a well of a 96-well plate, resulting in binding of DNA to the protein. After washing and elution, the resulting population of more specific sequences is amplified by PCR and sequenced (Caroli et al., 2016)

Advantages:

Disadvantages:

  • Could contain sequence bias


Reagents:

Illumina Library prep and Array Kit Selector



Reviews:

Anzalone A. V., Lin A. J., Zairis S., Rabadan R. and Cornish V. W. Reprogramming eukaryotic translation with ligand-responsive synthetic RNA switches. Nat Methods. 2016;13:453-458

Jijakli K., Khraiwesh B., Fu W., et al. The in vitro selection world. Methods. 2016;106:3-13



References:

Urak K. T., Shore S., Rockey W. M., Chen S. J., McCaffrey A. P. and Giangrande P. H. in vitro RNA SELEX for the generation of chemically-optimized therapeutic RNA drugs. Methods. 2016;103:167-174

Ahirwar R., Vellarikkal S. K., Sett A., et al. Aptamer-Assisted Detection of the Altered Expression of Estrogen Receptor Alpha in Human Breast Cancer. PLoS One. 2016;11:e0153001

Iaboni M., Fontanella R., Rienzo A., et al. Targeting Insulin Receptor with a Novel Internalizing Aptamer. Mol Ther Nucleic Acids. 2016;5:e365

Long Y., Qin Z., Duan M., et al. Screening and identification of DNA aptamers toward Schistosoma japonicum eggs via SELEX. Sci Rep. 2016;6:24986

Janowski R., Heinz G. A., Schlundt A., et al. Roquin recognizes a non-canonical hexaloop structure in the 3′-UTR of Ox40. Nat Commun. 2016;7:11032

Schneider T., Hung L. H., Schreiner S., et al. CircRNA-protein complexes: IMP3 protein component defines subfamily of circRNPs. Sci Rep. 2016;6:31313

Stewart H., Bingham R. J., White S. J., et al. Identification of novel RNA secondary structures within the hepatitis C virus genome reveals a cooperative involvement in genome packaging. Sci Rep. 2016;6:22952

Oakes B. L., Xia D. F., Rowland E. F., et al. Multi-reporter selection for the design of active and more specific zinc-finger nucleases for genome editing. Nat Commun. 2016;7:10194