NET-Seq

SELEX

From the time that the first SELEX experiments were described by 3 independent groups in 1990 the method has been adapted to a wide range of technologies^1-5. A highly multiplexed, parallel HT-SELEX method was developed for NGS.⁶ A variation of SELEX-seq ⁷ uses Nextera adapter sequences for efficient library preparation.⁸

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.

Pros:

• High-throughput and efficient
• Software pipelines are available^9,10

Cons:

• Could contain sequence bias¹¹

• 1. Ellington A. D. and Szostak J. W. In vitro selection of RNA molecules that bind specific ligands. Nature. 1990;346:818-822
• 2. Tuerk C. and Gold L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science. 1990;249:505-510
• 3. Sullenger B. A., Gallardo H. F., Ungers G. E. and Gilboa E. Overexpression of TAR sequences renders cells resistant to human immunodeficiency virus replication. Cell. 1990;63:601-608
• 4. Chen D., Orenstein Y., Golodnitsky R., et al. SELMAP - SELEX affinity landscape MAPping of transcription factor binding sites using integrated microfluidics. Sci Rep. 2016;6:33351
• 5. Takahashi M., Wu X., Ho M., et al. High throughput sequencing analysis of RNA libraries reveals the influences of initial library and PCR methods on SELEX efficiency. Sci Rep. 2016;6:33697
• 6. Jolma A., Kivioja T., Toivonen J., et al. Multiplexed massively parallel SELEX for characterization of human transcription factor binding specificities. Genome Res. 2010;20:861-873
• 7.Slattery M., Riley T., Liu P., et al. Cofactor binding evokes latent differences in DNA binding specificity between Hox proteins. Cell. 2011;147:1270-1282
• 8. Zhang Y., Lee J. K., Toso E. A., et al. DNA-binding sequence specificity of DUX4. Skelet Muscle. 2016;6:8
• 9. Hoinka J. and Przytycka T. AptaPLEX - A dedicated, multithreaded demultiplexer for HT-SELEX data. Methods. 2016;106:82-85
• 10. Caroli J., Taccioli C., De La Fuente A., Serafini P. and Bicciato S. APTANI: a computational tool to select aptamers through sequence-structure motif analysis of HT-SELEX data. Bioinformatics. 2016;32:161-164
• 11. Takahashi M., Wu X., Ho M., et al. High throughput sequencing analysis of RNA libraries reveals the influences of initial library and PCR methods on SELEX efficiency. Sci Rep. 2016;6:33697

1. 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
2. Jijakli K., Khraiwesh B., Fu W., et al. The in vitro selection world. Methods. 2016;106:3-13

1. 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
2. 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
3. Iaboni M., Fontanella R., Rienzo A., et al. Targeting Insulin Receptor with a Novel Internalizing Aptamer. Mol Ther Nucleic Acids. 2016;5:e365
4. 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
5. 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
6. Schneider T., Hung L. H., Schreiner S., et al. CircRNA-protein complexes: IMP3 protein component defines subfamily of circRNPs. Sci Rep. 2016;6:31313
7. 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
8. 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