Monitoring genomic sequences during SELEX using high-throughput sequencing: neutral SELEX.
BACKGROUND
SELEX is a well established in vitro selection tool to analyze the structure of ligand-binding nucleic acid sequences called aptamers. Genomic SELEX transforms SELEX into a tool to discover novel, genomically encoded RNA or DNA sequences binding a ligand of interest, called genomic aptamers. Concerns have been raised regarding requirements imposed on RNA sequences undergoing SELEX selection.


RESULTS
To evaluate SELEX and assess the extent of these effects, we designed and performed a Neutral SELEX experiment omitting the selection step, such that the sequences are under the sole selective pressure of SELEX's amplification steps. Using high-throughput sequencing, we obtained thousands of full-length sequences from the initial genomic library and the pools after each of the 10 rounds of Neutral SELEX. We compared these to sequences obtained from a Genomic SELEX experiment deriving from the same initial library, but screening for RNAs binding with high affinity to the E. coli regulator protein Hfq. With each round of Neutral SELEX, sequences became less stable and changed in nucleotide content, but no sequences were enriched. In contrast, we detected substantial enrichment in the Hfq-selected set with enriched sequences having structural stability similar to the neutral sequences but with significantly different nucleotide selection.


CONCLUSIONS
Our data indicate that positive selection in SELEX acts independently of the neutral selective requirements imposed on the sequences. We conclude that Genomic SELEX, when combined with high-throughput sequencing of positively and neutrally selected pools, as well as the gnomic library, is a powerful method to identify genomic aptamers.
Zimmermann
Bob
B
Max F Perutz Laboratories, Department of Biochemistry, University of Vienna, Vienna, Austria.
Gesell
Tanja
T
Chen
Doris
D
Lorenz
Christina
C
Schroeder
Renée
R
D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D016680:Genome, Bacterial / Q000235:genetics; D015698:Genomic Library; D023281:Genomics / Q000379:methods*; D035001:Host Factor 1 Protein / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D015203:Reproducibility of Results; D052156:SELEX Aptamer Technique / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C087373:Hfq protein, E coli; D035001:Host Factor 1 Protein