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Monitoring genomic sequences during SELEX using high-throughput sequencing: neutral SELEX.

Zimmermann B, Gesell T, Chen D, Lorenz C, Schroeder R - PLoS ONE (2010)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Max F Perutz Laboratories, Department of Biochemistry, University of Vienna, Vienna, Austria.

ABSTRACT

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.

Methodology/principal findings: 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/significance: 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.

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Effects of SELEX on structural stability of RNA sequences.(A) Average Z-score of sequences in the initial library (brown bar), each pool of Neutral SELEX (green bars) and the sequences from the Hfq Genomic SELEX experiment after 9 rounds of selection (grey bar). Numbers indicate the SELEX cycle. (B) Comparison of the distributions of Z-scores of the 9th round of Neutral SELEX, the 9th round of Hfq Genomic SELEX and the genomic library. These are all plotted next to the normal distribution (expected from random sequence), shown with the black line.
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pone-0009169-g003: Effects of SELEX on structural stability of RNA sequences.(A) Average Z-score of sequences in the initial library (brown bar), each pool of Neutral SELEX (green bars) and the sequences from the Hfq Genomic SELEX experiment after 9 rounds of selection (grey bar). Numbers indicate the SELEX cycle. (B) Comparison of the distributions of Z-scores of the 9th round of Neutral SELEX, the 9th round of Hfq Genomic SELEX and the genomic library. These are all plotted next to the normal distribution (expected from random sequence), shown with the black line.

Mentions: As shown in Figure 3A, the initial library has a negative average Z-score, which differs from the Hfq average on the right. With each successive round of Neutral SELEX, the average MFE Z-score increases, and the average Z-score becomes positive after the 3rd round, and ultimately approaches the average of the Hfq pool. This is a clear indication that the SELEX process favors sequences that are less structurally stable.


Monitoring genomic sequences during SELEX using high-throughput sequencing: neutral SELEX.

Zimmermann B, Gesell T, Chen D, Lorenz C, Schroeder R - PLoS ONE (2010)

Effects of SELEX on structural stability of RNA sequences.(A) Average Z-score of sequences in the initial library (brown bar), each pool of Neutral SELEX (green bars) and the sequences from the Hfq Genomic SELEX experiment after 9 rounds of selection (grey bar). Numbers indicate the SELEX cycle. (B) Comparison of the distributions of Z-scores of the 9th round of Neutral SELEX, the 9th round of Hfq Genomic SELEX and the genomic library. These are all plotted next to the normal distribution (expected from random sequence), shown with the black line.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2820082&req=5

pone-0009169-g003: Effects of SELEX on structural stability of RNA sequences.(A) Average Z-score of sequences in the initial library (brown bar), each pool of Neutral SELEX (green bars) and the sequences from the Hfq Genomic SELEX experiment after 9 rounds of selection (grey bar). Numbers indicate the SELEX cycle. (B) Comparison of the distributions of Z-scores of the 9th round of Neutral SELEX, the 9th round of Hfq Genomic SELEX and the genomic library. These are all plotted next to the normal distribution (expected from random sequence), shown with the black line.
Mentions: As shown in Figure 3A, the initial library has a negative average Z-score, which differs from the Hfq average on the right. With each successive round of Neutral SELEX, the average MFE Z-score increases, and the average Z-score becomes positive after the 3rd round, and ultimately approaches the average of the Hfq pool. This is a clear indication that the SELEX process favors sequences that are less structurally stable.

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Max F Perutz Laboratories, Department of Biochemistry, University of Vienna, Vienna, Austria.

ABSTRACT

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.

Methodology/principal findings: 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/significance: 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.

Show MeSH