Strand‐Biased Formation of G‐Quadruplexes in DNA Duplexes Transcribed with T7 RNA Polymerase

Liu, Jiaquan; Xiao, Shan; Hao, Yu-hua; Tan, Zheng

doi:10.1002/anie.201503648

Cited by 14 publications

(18 citation statements)

References 35 publications

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“…We further profiled G4P reads distribution in the RefSeq genes and found that G4s were concentrated at both sides of TSSs, with two peaks merging at the TSSs ( Figure 3F). The distribution largely correlated with the presence of 4G PQSs across the TSSs ( Figure 3G) and agreed with our previous in vitro studies in which transcription efficiently induced G4s at both sides of a TSS (18,(22)(23)(24)(28)(29)(30). The distribution of the two features between TSS and TES were distorted due to the scaling on the gene bodies of different sizes.…”

Section: G4p Recognizes G4s In Living Cellssupporting

confidence: 89%

“…The formation of the G4 in the plasmid was unlikely induced by the G4P because G4 was not detected when the CSTB motif was placed on the template strand, which we previously described as a strand-biased G4 formation in transcription (22). Furthermore, G4 was not detected by circular dichroism (CD) spectroscopy ( Figure 2A) and EMSA ( Figure 2B, lanes 3 and 5) when a CSTB-containing dsDNA ( Table S3) was incubated with G4P.…”

Section: Introductionmentioning

confidence: 85%

“…The plasmid accommodated a CSTB, c-MYC, or a mutated motif on the non-template or the template strand. Transcription with T7 RNA polymerase efficiently induces a formation of G4 on the non-template but marginally on the template strand (22). Accordingly, the G4s on the non-template strand led to a high enrichment of the corresponding plasmid.…”

Section: Introductionmentioning

confidence: 99%

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Detect Genomic G-Quadruplexes in Living Animal Cells with a Tiny Artificial Protein Probe

Zheng

Zhang

et al. 2020

Preprint

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G-quadruplex (G4) structures formed by guanine-rich nucleic acids are implicated in essential physiological processes and serve as important drug targets. The genome-wide detection of G4s in living cells is important for exploring the biological role of G4s but has not yet been achieved due to the lack of a suitable G4 probe. We engineered a 6.7 kDa G4 probe (G4P) protein that binds G4s with high affinity and specificity. We used it to capture G4s in living human, mouse, and chicken cells with the ChIP-Seq technology, yielding genome-wide landscape as well as details on the positions, frequencies, and sequence identities of G4 formation in these cells. Our results indicate that transcription is accompanied by a robust formation of G4s in genes. In human cells, we detected up to >123,000 G4 peaks, of which >1/3 had a fold increase of ≥5 and were present in >60% promoters and ~70% genes. Being much smaller than a scFv antibody (27 kDa) or even a nanobody (12-15 kDa), we expect that the G4P may find diverse applications in biology, medicine, and molecular devices as a G4 affinity agent.3 proteins. Moreover, the synergy between two binding domains dramatically improves affinity and selectivity towards G4s. Expression of the G4P in cells followed by chromatin immunoprecipitation (termed G4P-ChIP) allowed us to capture G4s in living human, mouse, and chicken cells through the ChIP-Seq technology, revealing genome-wide landscape and details on the locations, frequencies, and sequence identities of G4 formation in these cells. MATERIALS AND METHODSChIP-Seq data analysis. Clean paired-end sequencing data in fastq format were mapped to the human genome (hg19 or hg38 when comparing with downloaded hg38 data) using the Bowtie2 software (12) with the sensitive-local preset and --no-unal, --no-discordant, --no-mixed parameters. Mapped reads were written to bam files after being filtered by the samtools view (13) to remove low-quality alignments with the parameter -q 20 and by samtools rmdup to remove duplicates. Reads enrichment was calculated using the deeptools (14) plotEnrichment with the bam files. Reads bam files were also processed by the deeptools bamCompare to produce bigwig coverage files in subtract or ratio mode and normalized to RPKM. Profiles and heatmaps of reads were generated from the bigwig files using the deeptools computeMatrix followed by plotProfile and plotHeatmap, respectively, with region bed files derived from the NCBI RefSeq bed file downloaded from the UCSC website (http://genome.ucsc.edu/) unless otherwise indicated. Coordinate duplicates in the bed files were removed. Peaks of reads enrichment were identified with the macs2 software (15) using --qvalue 0.001, --keep-dup 1, and default values for the other parameters. ChIP-Seq data from public repositories were downloaded from the GEO (https://www.ncbi.nlm.nih.gov/geo/) or Encode (https://www.encodeproject.org/) database and processed as described above whenever applicable. Original sequencing (fastq) and processed (narrowPeak, bigwig) G4P-ChIP data...

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Section: G4p Recognizes G4s In Living Cellssupporting

confidence: 89%

Section: Introductionmentioning

confidence: 85%

Section: Introductionmentioning

confidence: 99%

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Detect Genomic G-Quadruplexes in Living Animal Cells with a Tiny Artificial Protein Probe

Zheng

Zhang

et al. 2020

Preprint

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“…Distinct effect on expression level when placing the GQ-forming sequence on antisense and sense strand has been observed before. [10,24,28,29] In contrast to the effect of GQ-and hairpin-forming sequences in the antisense strand, they caused a slight increase or no change in expression levels when located in the sense strand. Similarly, a study by the Hartig group showed that parallel GQ in the core promoter region of the sense strand has no effect on gene expression.…”

Section: Discussionmentioning

confidence: 99%

“…It has been shown earlier that effect on gene expression is strongly dependent on whether the GQforming sequence is located in the antisense or sense strand. [10,12,24] Therefore, we tested also the effect of sequences able to form a parallel GQ, an antiparallel GQ and a hairpin structure on expression levels when the sequences are inserted in the sense strand ( Figure 3A).…”

Section: Gene Expression Is Affected By the Sequence Inserted Into Thmentioning

confidence: 99%

Non-canonical Structures in Promoter Modulate Gene Expression in Escherichia coli

et al. 2018

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Herein we show how sequences that can form different non-canonical structures affect gene expression levels when inserted in the core of σ 70-dependent promoter, between the −35 and −10 elements recognized by RNA polymerase, in E. coli. We note that influence on level of GFP expression varies considerably depending on introduction of non-canonical structural elements in the antisense and sense strands as well as with their propensities to form G-triplex, G-hairpin, hairpin or G-quadruplex structures. Moreover, the extent of repression of expression does not relate to the in vitro thermal stability in a simple manner. Repression is most likely caused by steric interference rather than improper distance between the −35 and −10 elements. Although properties like thermal stability and topology can be somewhat different under in vivo and in vitro conditions, our results suggest that the extent of expression suppression cannot be dependent solely on thermal stabilities of Grich structures alone.

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Organische Chemie

Itzen¹,

Schaschke²,

Beifuß³

et al. 2016

Nachr Chem

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Strand‐Biased Formation of G‐Quadruplexes in DNA Duplexes Transcribed with T7 RNA Polymerase

Cited by 14 publications

References 35 publications

Detect Genomic G-Quadruplexes in Living Animal Cells with a Tiny Artificial Protein Probe

Detect Genomic G-Quadruplexes in Living Animal Cells with a Tiny Artificial Protein Probe

Non-canonical Structures in Promoter Modulate Gene Expression in Escherichia coli

Organische Chemie

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