Probing the SELEX Process with Next-Generation Sequencing

Background SELEX is an iterative process in which highly diverse synthetic nucleic acid libraries are selected over many rounds to finally identify aptamers with desired properties. However, little is understood as how binders are enriched during the selection course. Next-generation sequencing offers the opportunity to open the black box and observe a large part of the population dynamics during the selection process. Methodology We have performed a semi-automated SELEX procedure on the model target streptavidin starting with a synthetic DNA oligonucleotide library and compared results obtained by the conventional analysis via cloning and Sanger sequencing with next-generation sequencing. In order to follow the population dynamics during the selection, pools from all selection rounds were barcoded and sequenced in parallel. Conclusions High affinity aptamers can be readily identified simply by copy number enrichment in the first selection rounds. Based on our results, we suggest a new selection scheme that avoids a high number of iterative selection rounds while reducing time, PCR bias, and artifacts.

[1]  Aniela Wochner,et al.  Semi-automated selection of DNA aptamers using magnetic particle handling. , 2007, BioTechniques.

[2]  Emmanuel Dias-Neto,et al.  Next-Generation Phage Display: Integrating and Comparing Available Molecular Tools to Enable Cost-Effective High-Throughput Analysis , 2009, PloS one.

[3]  Martin Vingron,et al.  A calibrated diversity assay for nucleic acid libraries using DiStRO—a Diversity Standard of Random Oligonucleotides , 2009, Nucleic acids research.

[4]  J C Cox,et al.  Automated selection of anti-protein aptamers. , 2001, Bioorganic & medicinal chemistry.

[5]  J. Kiel,et al.  Use of magnetic beads in selection and detection of biotoxin aptamers by electrochemiluminescence and enzymatic methods. , 2002, BioTechniques.

[6]  Zhaofeng Luo,et al.  In vitro selection of high-affinity DNA aptamers for streptavidin. , 2009, Acta biochimica et biophysica Sinica.

[7]  Seung Soo Oh,et al.  Quantitative selection of DNA aptamers through microfluidic selection and high-throughput sequencing , 2010, Proceedings of the National Academy of Sciences.

[8]  L. Gold,et al.  Interactions of Escherichia coli RNA with bacteriophage MS2 coat protein: genomic SELEX. , 2000, Nucleic acids research.

[9]  R. Britten,et al.  Repeated sequences in DNA. Hundreds of thousands of copies of DNA sequences have been incorporated into the genomes of higher organisms. , 1968, Science.

[10]  J. McIntosh,et al.  Single-strand DNA Aptamers as Probes for Protein Localization in Cells , 2003, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[11]  David R. Liu,et al.  Nucleic acid evolution and minimization by nonhomologous random recombination , 2002, Nature Biotechnology.

[12]  L. Gold,et al.  Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. , 1990, Science.

[13]  Markus Kalisch,et al.  Selection of Carbonic Anhydrase IX Inhibitors from One Million DNA-Encoded Compounds. , 2011, ACS chemical biology.

[14]  K. Reinert,et al.  RazerS--fast read mapping with sensitivity control. , 2009, Genome research.

[15]  D. Nieuwlandt.  In vitro selection of functional nucleic acid sequences. , 2000, Current issues in molecular biology.

[16]  Zoltán Konthur,et al.  Automation in the High-throughput Selection of Random Combinatorial Libraries—Different Approaches for Select Applications , 2010, Molecules.

[17]  R. Britten,et al.  Repeated Sequences in DNA , 1968 .

[18]  D. Shangguan,et al.  Conservative secondary structure motif of streptavidin-binding aptamers generated by different laboratories. , 2010, Bioorganic & medicinal chemistry.

[19]  Jörn Glökler,et al.  Nonradioactive fluorescence microtiter plate assay monitoring aptamer selections. , 2007, BioTechniques.

[20]  J. Szostak,et al.  In vitro selection of RNA molecules that bind specific ligands , 1990, Nature.

[21]  Juan M. Vaquerizas,et al.  Multiplexed massively parallel SELEX for characterization of human transcription factor binding specificities. , 2010, Genome research.

[22]  R. Stoltenburg,et al.  FluMag-SELEX as an advantageous method for DNA aptamer selection , 2005, Analytical and bioanalytical chemistry.

[23]  Charles Elkan,et al.  Fitting a Mixture Model By Expectation Maximization To Discover Motifs In Biopolymer , 1994, ISMB.