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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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Enrichment effects on positively selected sequences.(A) Enrichment level plotted against Z-scores. Sequences selected by Genomic SELEX for Hfq were clustered if the best alignment shows mutual identity ≥85%. The cluster size is shown along the x-axis, and the Z-score of the sequence along the y-axis. The sequences were binned into cluster size ranges of 10, and the boxes represent the distribution of Z-scores within the range of cluster sizes. The boxes cover the 25%–75% range of the data, the line within the box is the median and the whiskers indicate 1.5× the interquartile range. If enrichment were dependent on a high Z-score, we would expect to see an increase in the median Z-score as cluster sizes increase, however this is not the case. In fact, the Z-score of any given sequence appears to vary nearly as much with enriched sequences as with unenriched sequences. (B) As a control, we plotted the same analysis with round 9 of Neutral SELEX, showing that enrichment is a signal of the positive selection of sequences.
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pone-0009169-g004: Enrichment effects on positively selected sequences.(A) Enrichment level plotted against Z-scores. Sequences selected by Genomic SELEX for Hfq were clustered if the best alignment shows mutual identity ≥85%. The cluster size is shown along the x-axis, and the Z-score of the sequence along the y-axis. The sequences were binned into cluster size ranges of 10, and the boxes represent the distribution of Z-scores within the range of cluster sizes. The boxes cover the 25%–75% range of the data, the line within the box is the median and the whiskers indicate 1.5× the interquartile range. If enrichment were dependent on a high Z-score, we would expect to see an increase in the median Z-score as cluster sizes increase, however this is not the case. In fact, the Z-score of any given sequence appears to vary nearly as much with enriched sequences as with unenriched sequences. (B) As a control, we plotted the same analysis with round 9 of Neutral SELEX, showing that enrichment is a signal of the positive selection of sequences.

Mentions: This might also indicate that a sequence must be structurally unstable in order to be enriched by SELEX. In order to determine if this is the case, we estimated the enrichment level of each sequence by clustering the pool of positively selected Hfq sequences based on mutual sequence identity. Each sequence's enrichment was measured as the number of sequences in the cluster to which it belonged. We visualized the relationship between enrichment and stability in a box plot shown in Figure 4A. The Z-scores of the sequences did not substantially increase with enrichment level, and furthermore the variation of Z-score is similar between the enriched and non-enriched sequences. Therefore, it seems that SELEX favors structurally unstable sequences in general, independent of the positive selection.


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

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

Enrichment effects on positively selected sequences.(A) Enrichment level plotted against Z-scores. Sequences selected by Genomic SELEX for Hfq were clustered if the best alignment shows mutual identity ≥85%. The cluster size is shown along the x-axis, and the Z-score of the sequence along the y-axis. The sequences were binned into cluster size ranges of 10, and the boxes represent the distribution of Z-scores within the range of cluster sizes. The boxes cover the 25%–75% range of the data, the line within the box is the median and the whiskers indicate 1.5× the interquartile range. If enrichment were dependent on a high Z-score, we would expect to see an increase in the median Z-score as cluster sizes increase, however this is not the case. In fact, the Z-score of any given sequence appears to vary nearly as much with enriched sequences as with unenriched sequences. (B) As a control, we plotted the same analysis with round 9 of Neutral SELEX, showing that enrichment is a signal of the positive selection of sequences.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2820082&req=5

pone-0009169-g004: Enrichment effects on positively selected sequences.(A) Enrichment level plotted against Z-scores. Sequences selected by Genomic SELEX for Hfq were clustered if the best alignment shows mutual identity ≥85%. The cluster size is shown along the x-axis, and the Z-score of the sequence along the y-axis. The sequences were binned into cluster size ranges of 10, and the boxes represent the distribution of Z-scores within the range of cluster sizes. The boxes cover the 25%–75% range of the data, the line within the box is the median and the whiskers indicate 1.5× the interquartile range. If enrichment were dependent on a high Z-score, we would expect to see an increase in the median Z-score as cluster sizes increase, however this is not the case. In fact, the Z-score of any given sequence appears to vary nearly as much with enriched sequences as with unenriched sequences. (B) As a control, we plotted the same analysis with round 9 of Neutral SELEX, showing that enrichment is a signal of the positive selection of sequences.
Mentions: This might also indicate that a sequence must be structurally unstable in order to be enriched by SELEX. In order to determine if this is the case, we estimated the enrichment level of each sequence by clustering the pool of positively selected Hfq sequences based on mutual sequence identity. Each sequence's enrichment was measured as the number of sequences in the cluster to which it belonged. We visualized the relationship between enrichment and stability in a box plot shown in Figure 4A. The Z-scores of the sequences did not substantially increase with enrichment level, and furthermore the variation of Z-score is similar between the enriched and non-enriched sequences. Therefore, it seems that SELEX favors structurally unstable sequences in general, independent of the positive selection.

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