G-quadruplex structural variations in human genome associated with single-nucleotide variations and their impact on gene activity.

G-quadruplexes (G4s) formed by guanine-rich nucleic acids play a role in essential biological processes such as transcription and replication. Besides the >1.5 million putative G-4-forming sequences (PQSs), the human genome features >640 million single-nucleotide variations (SNVs), the most common type of genetic variation among people or populations. An SNV may alter a G4 structure when it falls within a PQS motif. To date, genome-wide PQS-SNV interactions and their impact have not been investigated. Herein, we present a study on the PQS-SNV interactions and the impact they can bring to G4 structures and, subsequently, gene expressions. Based on build 154 of the Single Nucleotide Polymorphism Database (dbSNP), we identified 5 million gains/losses or structural conversions of G4s that can be caused by the SNVs. Of these G4 variations (G4Vs), 3.4 million are within genes, resulting in an average load of >120 G4Vs per gene, preferentially enriched near the transcription start site. Moreover, >80% of the G4Vs overlap with transcription factor-binding sites and >14% with enhancers, giving an average load of 3 and 7.5 for the two regulatory elements, respectively. Our experiments show that such G4Vs can significantly influence the expression of their host genes. These results reveal genome-wide G4Vs and their impact on gene activity, emphasizing an understanding of genetic variation, from a structural perspective, of their physiological function and pathological implications. The G4Vs may also provide a unique category of drug targets for individualized therapeutics, health risk assessment, and drug development.

Regulation of PDGFR-ß gene expression by targeting the G-vacancy bearing G-quadruplex in promoter.

G-quadruplex is an essential element in gene transcription that serves as a promising drug target. Guanine-vacancy-bearing G-quadruplex (GVBQ) is a newly identified G-quadruplex that has distinct structural features from the canonical G-quadruplex. Potential GVBQ-forming motifs are widely distributed in gene promoter regions. However, whether GVBQ can form in genomic DNA and be an effective target for manipulating gene expression is unknown. Using photo-crosslinking, dimethyl sulfate footprinting, exonuclease digestion and in vitro transcription, we demonstrated the formation of a GVBQ in the G-rich nuclease hypersensitivity element within the human PDGFR-ß gene promoter region in both single-stranded and double-stranded DNA. The formation of GVBQ in dsDNA could be induced by negative supercoiling created by downstream transcription. We also found that the PDGFR-ß GVBQ was specifically recognized and stabilized by a new synthetic porphyrin guanine conjugate (mPG). Targeting the PDGFR-ß GVBQ in human cancer cells using the mPG could specifically alter PDGFR-ß gene expression. Our work illustrates that targeting GVBQ with mPG in human cells can regulate the expression level of a specific gene, thus indicating a novel strategy for drug development.

Detection of genomic G-quadruplexes in living cells using a small artificial protein.

G-quadruplex (G4) structures formed by guanine-rich nucleic acids are implicated in essential physiological and pathological processes and serve as important drug targets. The genome-wide detection of G4s in living cells is important for exploring the functional role of G4s but has not yet been achieved due to the lack of a suitable G4 probe. Here we report an artificial 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 technique, 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 G4P 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.