Identification of G-quadruplex clusters by high-throughput sequencing of whole-genome amplified products with a G-quadruplex ligand

Wataru Yoshida,Isao Karube,Kenichiro Hata,Keisuke Iida,Daniyah Habiballah Bay,Kazuhiko Nakabayashi,Hitomi Yoshioka,Kazuo Nagasawa,Hiroki Saikyo,Tomoko Kawai,Kazunori Ikebukuro

doi:10.1038/s41598-018-21514-7

Abstract

G-quadruplex (G4) is a DNA secondary structure that has been found to play regulatory roles in the genome. The identification of G4-forming sequences is important to study the specific structure-function relationships of such regions. In the present study, we developed a method for identification of G4 clusters on genomic DNA by high-throughput sequencing of genomic DNA amplified via whole-genome amplification (WGA) in the presence of a G4 ligand. The G4 ligand specifically bound to G4 structures on genomic DNA; thus, DNA polymerase was arrested on the G4 structures stabilised by G4 ligand. We utilised the telomestatin derivative L1H1-7OTD as a G4 ligand and demonstrated that the efficiency of amplification of the G4 cluster regions was lower than that of the non-G4-forming regions. By high-throughput sequencing of the WGA products, 9,651 G4 clusters were identified on human genomic DNA. Among these clusters, 3,766 G4 clusters contained at least one transcriptional start site, suggesting that genes are regulated by G4 clusters rather than by one G4 structure.

Highlights

G-quadruplex (G4) is a DNA secondary structure composed of two or more stacking G-quartets, a planar array of four guanine bases connected by a Hoogsteen hydrogen bond, and stabilised by a monovalent cation[1]
In the presence of 1 μM 7OTD, the non-G4-forming regions were amplified, whereas the G4-forming ones were not. These results demonstrate that 7OTD inhibits DNA polymerase extension on G4-forming regions in polymerase chain reaction (PCR)
The G4-seq analysis revealed that the human genome contains 716,310 G4-forming sequences stabilised by a G4 ligand[23]. These results suggest that Phi[29] DNA polymerase would be arrested on numerous G4-forming regions on genomic DNA and its inhibition would reduce the yield of total whole genome amplification (WGA) products

Summary

Introduction

G-quadruplex (G4) is a DNA secondary structure composed of two or more stacking G-quartets, a planar array of four guanine bases connected by a Hoogsteen hydrogen bond, and stabilised by a monovalent cation[1]. Clusters of G4-forming sequences that promote transcription and replication-dependent DNA damage induced by a G4 ligand have been identified in oncogenes and tumour-suppressor genes by ChIP-Seq of the DNA damage. We performed a whole genome amplification (WGA)[24] in the presence of a G4 ligand, followed by high-throughput sequencing of WGA products to identify G4 clusters in human genomic DNA. It has been reported that DNA polymerase was arrested on G4 structures stabilised by G4 ligand[25]. This led us to hypothesise that genomic DNA would be amplified by WGA, except for G4 clusters, in the presence of a G4 ligand. We could identify G4 clusters by analysing the WGA products using high-throughput sequencing technologies

Methods

Results

Conclusion