Limits...
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.

Show MeSH
Trends in nucleotide frequencies of the Neutral SELEX and Hfq SELEX pools.Each group in the bar charts shows the difference in mononucleotide frequency (A) and dinucleotide frequency (B) from the averages of the E. coli K-12 genome. The groups begin on the left with the sequenced library in brown, then ten rounds of Neutral SELEX in green, and finally the Hfq Genomic SELEX content in grey.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2820082&req=5

pone-0009169-g005: Trends in nucleotide frequencies of the Neutral SELEX and Hfq SELEX pools.Each group in the bar charts shows the difference in mononucleotide frequency (A) and dinucleotide frequency (B) from the averages of the E. coli K-12 genome. The groups begin on the left with the sequenced library in brown, then ten rounds of Neutral SELEX in green, and finally the Hfq Genomic SELEX content in grey.

Mentions: We were also interested to see if SELEX amplification steps also influence the base composition. Since dinucleotide content influences the folding stability of genomic sequences [4], [15], [16], we were interested in both mono- and dinucleotide content. We measured the nucleotide content in each pool and compared this to the average content of the E. coli genome (Figure 5). In theory, the library content should be identical to that of the genome. However, the library content already shows a difference from the E. coli base composition, substantially contributing to the Neutral SELEX differences. Assuming the genomic sequence corresponds to the genome used experimentally, the library construction process must cause this difference. By visual comparison of Figure 5A along the ten rounds, we see a clear trend of increasing A and decreasing G content, and a weak trend of increasing U content. C content remains relatively stable. The chart in Figure 5B shows substantial trend of increasing AA, AU, GU, and UA dinucleotides and a substantial trend of decreasing in CG, GC and GG dinucleotides. Thus, the Neutral SELEX sequences show clear trends.


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

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

Trends in nucleotide frequencies of the Neutral SELEX and Hfq SELEX pools.Each group in the bar charts shows the difference in mononucleotide frequency (A) and dinucleotide frequency (B) from the averages of the E. coli K-12 genome. The groups begin on the left with the sequenced library in brown, then ten rounds of Neutral SELEX in green, and finally the Hfq Genomic SELEX content in grey.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0009169-g005: Trends in nucleotide frequencies of the Neutral SELEX and Hfq SELEX pools.Each group in the bar charts shows the difference in mononucleotide frequency (A) and dinucleotide frequency (B) from the averages of the E. coli K-12 genome. The groups begin on the left with the sequenced library in brown, then ten rounds of Neutral SELEX in green, and finally the Hfq Genomic SELEX content in grey.
Mentions: We were also interested to see if SELEX amplification steps also influence the base composition. Since dinucleotide content influences the folding stability of genomic sequences [4], [15], [16], we were interested in both mono- and dinucleotide content. We measured the nucleotide content in each pool and compared this to the average content of the E. coli genome (Figure 5). In theory, the library content should be identical to that of the genome. However, the library content already shows a difference from the E. coli base composition, substantially contributing to the Neutral SELEX differences. Assuming the genomic sequence corresponds to the genome used experimentally, the library construction process must cause this difference. By visual comparison of Figure 5A along the ten rounds, we see a clear trend of increasing A and decreasing G content, and a weak trend of increasing U content. C content remains relatively stable. The chart in Figure 5B shows substantial trend of increasing AA, AU, GU, and UA dinucleotides and a substantial trend of decreasing in CG, GC and GG dinucleotides. Thus, the Neutral SELEX sequences show clear trends.

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