Ju Mei,†,‡,∥ Nelson L. C. Leung,†,‡,∥ Ryan T. K. Kwok,†,‡ Jacky W. Y. Lam,†,‡ and Ben Zhong Tang*,†,‡,§ †HKUST-Shenzhen Research Institute, Hi-Tech Park, Nanshan, Shenzhen 518057, China ‡Department of Chemistry, HKUST Jockey Club Institute for Advanced Study, Institute of Molecular Functional Materials, Division of Biomedical Engineering, State Key Laboratory of Molecular Neuroscience, Division of Life Science, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China Guangdong Innovative Research Team, SCUT-HKUST Joint Research Laboratory, State Key Laboratory of Luminescent Materials and Devices, South China University of Technology, Guangzhou 510640, China

Aggregation-Induced Emission: Together We Shine, United We Soar!

Telomeric ends of chromosomes, which comprise noncoding repeat sequences of guanine-rich DNA, are fundamental in protecting the cell from recombination and degradation. Disruption of telomere maintenance leads to eventual cell death, which can be exploited for therapeutic intervention in cancer. Telomeric DNA sequences can form four-stranded (quadruplex) structures, which may be involved in the structure of telomere ends. Here we describe the crystal structure of a quadruplex formed from four consecutive human telomeric DNA repeats and grown at a K(+) concentration that approximates its intracellular concentration. K(+) ions are observed in the structure. The folding and appearance of the DNA in this intramolecular quadruplex is fundamentally different from the published Na(+)-containing quadruplex structures. All four DNA strands are parallel, with the three linking trinucleotide loops positioned on the exterior of the quadruplex core, in a propeller-like arrangement. The adenine in each TTA linking trinucleotide loop is swung back so that it intercalates between the two thymines. This DNA structure suggests a straightforward path for telomere folding and unfolding, as well as ways in which it can recognize telomere-associated proteins.

Crystal structure of parallel quadruplexes from human telomeric DNA.

Four-stranded G-quadruplex nucleic acid structures are of great interest as their high thermodynamic stability under near-physiological conditions suggests that they could form in cells. Here we report the generation and application of an engineered, structure-specific antibody employed to quantitatively visualize DNA G-quadruplex structures in human cells. We show explicitly that G-quadruplex formation in DNA is modulated during cell-cycle progression and that endogenous G-quadruplex DNA structures can be stabilized by a small-molecule ligand. Together these findings provide substantive evidence for the formation of G-quadruplex structures in the genome of mammalian cells and corroborate the application of stabilizing ligands in a cellular context to target G-quadruplexes and intervene with their function.

/pdf/quantitative-visualization-of-dna-g-quadruplex-structures-in-11l455raiw.pdf

Quantitative visualization of DNA G-quadruplex structures in human cells

Guanine-rich DNA sequences of a particular form have the ability to fold into four-stranded structures called G-quadruplexes. In this paper, we present a working rule to predict which primary sequences can form this structure, and describe a search algorithm to identify such sequences in genomic DNA. We count the number of quadruplexes found in the human genome and compare that with the figure predicted by modelling DNA as a Bernoulli stream or as a Markov chain, using windows of various sizes. We demonstrate that the distribution of loop lengths is significantly different from what would be expected in a random case, providing an indication of the number of potentially relevant quadruplex-forming sequences. In particular, we show that there is a significant repression of quadruplexes in the coding strand of exonic regions, which suggests that quadruplex-forming patterns are disfavoured in sequences that will form RNA.

/pdf/prevalence-of-quadruplexes-in-the-human-genome-265uyv6k6o.pdf

Prevalence of quadruplexes in the human genome

G-quadruplexes are four-stranded DNA structures that are over-represented in gene promoter regions and are viewed as emerging therapeutic targets in oncology, as transcriptional repression of oncogenes through stabilization of these structures could be a novel anticancer strategy. Many gene promoter G-quadruplexes have physicochemical properties and structural characteristics that might make them druggable, and their structural diversity suggests that a high degree of selectivity might be possible. Here, we describe the evidence for G-quadruplexes in gene promoters and discuss their potential as therapeutic targets, as well as progress in the development of strategies to harness this potential through intervention with small-molecule ligands.

Targeting G-quadruplexes in gene promoters: a novel anticancer strategy?

'If G-quadruplexes form so readily in vitro, Nature will have found a way of using them in vivo' (Statement by Aaron Klug over 30 years ago).During the last decade, four-stranded helical structures called G-quadruplex (or G4) have emerged from being a structural curiosity observed in vitro, to being recognized as a possible nucleic acid based mechanism for regulating multiple biological processes in vivo. The sequencing of many genomes has revealed that they are rich in sequence motifs that have the potential to form G-quadruplexes and that their location is non-random, correlating with functionally important genomic regions. In this short review, we summarize recent evidence for the in vivo presence and function of DNA and RNA G-quadruplexes in various cellular pathways including DNA replication, gene expression and telomere maintenance. We also highlight remaining open questions that will have to be addressed in the future.

G-quadruplexes and their regulatory roles in biology.

G-quadruplex structures: in vivo evidence and function

The potential dangers of using viruses to deliver and integrate DNA into host cells in gene therapy have been poignantly highlighted in recent clinical trials. Safer, non-viral gene delivery approaches have been largely ignored in the past because of their inefficient delivery and the resulting transient transgene expression. However, recent advances indicate that efficient, long-term gene expression can be achieved by non-viral means. In particular, integration of DNA can be targeted to specific genomic sites without deleterious consequences and it is possible to maintain transgenes as small episomal plasmids or artificial chromosomes. The application of these approaches to human gene therapy is gradually becoming a reality.

Towards safe, non-viral therapeutic gene expression in humans

Most eukaryotic telomeres contain a repeating motif with stretches of guanine residues that form a 3*-terminal overhang extending beyond the telomeric duplex region. The telomeric repeat of hypotrichous ciliates, d(T4G4), forms a 16-nucleotide 3*-overhang. Such sequences can adopt parallel-stranded as well as antiparallel-stranded quadruplex conformations in vitro. Although it has been proposed that guanine-quadruplex conformations may have important cellular roles including telomere function, recombination, and transcription, evidence for the existence of this DNA structure in vivo has been elusive to date. We have generated high-affinity single-chain antibody fragment (scFv) probes for the guanine-quadruplex formed by the Stylonychia telomeric repeat, by ribosome display from the Human Combinatorial Antibody Library. Of the scFvs selected, one (Sty3) had an affinity of Kd 5 125 pM for the parallel-stranded guanine-quadruplex and could discriminate with at least 1,000-fold specificity between parallel or antiparallel quadruplex conformations formed by the same sequence motif. A second scFv (Sty49) bound both the parallel and antiparallel quadruplex with similar (Kd 5 3‐5 nM) affinity. Indirect immunofluorescence studies show that Sty49 reacts specifically with the macronucleus but not the micronucleus of Stylonychia lemnae. The replication band, the region where replication and telomere elongation take place, was also not stained, suggesting that the guanine-quadruplex is resolved during replication. Our results provide experimental evidence that the telomeres of Stylonychia macronuclei adopt in vivo a guanine-quadruplex structure, indicating that this structure may have an important role for telomere functioning.

https://www.pnas.org/content/pnas/98/15/8572.full.pdf

In vitro generated antibodies specific for telomeric guanine-quadruplex DNA react with Stylonychia lemnae macronuclei.

Telomere end-binding proteins (TEBPs) bind to the guanine-rich overhang (G-overhang) of telomeres. Although the DNA binding properties of TEBPs have been investigated in vitro, little is known about their functions in vivo. Here we use RNA interference to explore in vivo functions of two ciliate TEBPs, TEBPα and TEBPβ. Silencing the expression of genes encoding both TEBPs shows that they cooperate to control the formation of an antiparallel guanine quadruplex (G-quadruplex) DNA structure at telomeres in vivo. This function seems to depend on the role of TEBPα in attaching telomeres in the nucleus and in recruiting TEBPβ to these sites. In vitro DNA binding and footprinting studies confirm the in vivo observations and highlight the role of the C terminus of TEBPβ in G-quadruplex formation. We have also found that G-quadruplex formation in vivo is regulated by the cell cycle–dependent phosphorylation of TEBPβ.

Hans J. Lipps

Papers

G-quadruplexes and their regulatory roles in biology.

G-quadruplex structures: in vivo evidence and function

Towards safe, non-viral therapeutic gene expression in humans

In vitro generated antibodies specific for telomeric guanine-quadruplex DNA react with Stylonychia lemnae macronuclei.

Telomere end-binding proteins control the formation of G-quadruplex DNA structures in vivo