G-quadruplexes are higher-order DNA and RNA structures formed from G-rich sequences that are built around tetrads of hydrogen-bonded guanine bases. Potential quadruplex sequences have been identified in G-rich eukaryotic telomeres, and more recently in non-telomeric genomic DNA, e.g. in nuclease-hypersensitive promoter regions. The natural role and biological validation of these structures is starting to be explored, and there is particular interest in them as targets for therapeutic intervention. This survey focuses on the folding and structural features on quadruplexes formed from telomeric and non-telomeric DNA sequences, and examines fundamental aspects of topology and the emerging relationships with sequence. Emphasis is placed on information from the high-resolution methods of X-ray crystallography and NMR, and their scope and current limitations are discussed. Such information, together with biological insights, will be important for the discovery of drugs targeting quadruplexes from particular genes.

Quadruplex DNA: sequence, topology and structure

Guanine-rich DNA sequences can form G-quadruplexes stabilized by stacked G–G–G–G tetrads in monovalent cation-containing solution. The length and number of individual G-tracts and the length and sequence context of linker residues define the diverse topologies adopted by G-quadruplexes. The review highlights recent solution NMR-based G-quadruplex structures formed by the four-repeat human telomere in K+ solution and the guanine-rich strands of c-myc, c-kit and variant bcl-2 oncogenic promoters, as well as a bimolecular G-quadruplex that targets HIV-1 integrase. Such structure determinations have helped to identify unanticipated scaffolds such as interlocked G-quadruplexes, as well as novel topologies represented by double-chain-reversal and V-shaped loops, triads, mixed tetrads, adenine-mediated pentads and hexads and snap-back G-tetrad alignments. The review also highlights the recent identification of guanine-rich sequences positioned adjacent to translation start sites in 5′-untranslated regions (5′-UTRs) of RNA oncogenic sequences. The activity of the enzyme telomerase, which maintains telomere length, can be negatively regulated through G-quadruplex formation at telomeric ends. The review evaluates progress related to ongoing efforts to identify small molecule drugs that bind and stabilize distinct G-quadruplex scaffolds associated with telomeric and oncogenic sequences, and outlines progress towards identifying recognition principles based on several X-ray-based structures of ligand–G-quadruplex complexes.

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Human telomere, oncogenic promoter and 5′-UTR G-quadruplexes: diverse higher order DNA and RNA targets for cancer therapeutics

The quadruplex structures formed by guanine-rich nucleic acid sequences have received significant attention recently because of growing evidence for their role in important biological processes and as therapeutic targets. G-quadruplex DNA has been suggested to regulate DNA replication and may control cellular proliferation. Sequences capable of forming G-quadruplexes in the RNA have been shown to play significant roles in regulation of polyadenylation and splicing events in mammalian transcripts. Whether quadruplex structure directly plays a role in regulating RNA processing requires investigation. Computational approaches to study G-quadruplexes allow detailed analysis of mammalian genomes. There are no known easily accessible user-friendly tools that can compute G-quadruplexes in the nucleotide sequences. We have developed a web-based server, QGRS Mapper, that predicts quadruplex forming G-rich sequences (QGRS) in nucleotide sequences. It is a user-friendly application that provides many options for defining and studying G-quadruplexes. It performs analysis of the user provided genomic sequences, e.g. promoter and telomeric regions, as well as RNA sequences. It is also useful for predicting G-quadruplex structures in oligonucleotides. The program provides options to search and retrieve desired gene/nucleotide sequence entries from NCBI databases for mapping G-quadruplexes in the context of RNA processing sites. This feature is very useful for investigating the functional relevance of G-quadruplex structure, in particular its role in regulating the gene expression by alternative processing. In addition to providing data on composition and locations of QGRS relative to the processing sites in the pre-mRNA sequence, QGRS Mapper features interactive graphic representation of the data. The user can also use the graphics module to visualize QGRS distribution patterns among all the alternative RNA products of a gene simultaneously on a single screen. QGRS Mapper can be accessed at http://bioinformatics.ramapo.edu/QGRS/.

QGRS Mapper: a web-based server for predicting G-quadruplexes in nucleotide sequences

RNA structures in the untranslated regions (UTRs) of mRNAs influence post-transcriptional regulation of gene expression. Much of the knowledge in this area depends on canonical double-stranded RNA elements. There has been considerable recent advancement of our understanding of guanine(G)-rich nucleic acids sequences that form four-stranded structures, called G-quadruplexes. While much of the research has been focused on DNA G-quadruplexes, there has recently been a rapid emergence of interest in RNA G-quadruplexes, particularly in the 5'-UTRs of mRNAs. Collectively, these studies suggest that RNA G-quadruplexes exist in the 5'-UTRs of many genes, including genes of clinical interest, and that such structural elements can influence translation. This review features the progresses in the study of 5'-UTR RNA G-quadruplex-mediated translational control. It covers computational analysis, cell-free, cell-based and chemical biology studies that have sought to elucidate the roles of RNA G-quadruplexes in both cap-dependent and -independent regulation of mRNA translation. We also discuss protein trans-acting factors that have been implicated and the evidence that such RNA motifs have potential as small molecule target. Finally, we close the review with a perspective on the future challenges in the field of 5'-UTR RNA G-quadruplex-mediated translation regulation.

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5′-UTR RNA G-quadruplexes: translation regulation and targeting

G-quadruplexes are special secondary structures adopted in some guanine-rich DNA sequences. As guanine-rich sequences are present in important regions of the eukaryotic genome, such as telomeres and the regulatory regions of many genes, such structures may play important roles in the regulation of biological events in the body. G-quadruplexes have become valid targets for new anticancer drugs in the past few decades. Many leading compounds that target these structures have been reported, and a few of them have entered preclinical or clinical trials. Nonetheless, the selectivity of this kind of antitumor compound has yet to be improved in order to suppress the side effects caused by nonselective binding. As drug design targets, the topology and structural characteristics of quadruplexes, their possible biological roles, and the modes and sites of small-ligand binding to these structures should be understood clearly. Herein we provide a summary of published research that has set out to address the above problem to provide useful information on the design of small ligands that target G-quadruplexes. This review also covers research methodologies that have been developed to study the binding of ligands to G-quadruplexes.

G-Quadruplexes: Targets in Anticancer Drug Design

Guanine-rich nucleic acids are known to form highly stable G-quadruplex structures, also known as G-quartets. Recently, there has been a tremendous amount of interest in studying G-quadruplexes owing to the realization of their biological importance. G-rich sequences (GRSs) capable of forming G-quadruplexes are found in the vicinity of polyadenylation regions and are involved in regulating 3' end processing of mammalian pre-mRNAs. G-rich motifs are also known to play an important role in alternative, tissue-specific splicing by interacting with hnRNP H protein subfamily. Whether quadruplex structure directly plays a role in regulating RNA processing events requires further investigation. To date there has not been a comprehensive effort to study G-quadruplexes near RNA processing sites. We have applied a computational approach to map putative Quadruplex forming GRSs within the transcribed regions of a large number of alternatively processed human and mouse gene sequences that were obtained as fully annotated entries from GenBank and RefSeq. We have used the computed data to build the GRSDB database that provides a unique avenue for studying G-quadruplexes in the context of RNA processing sites. GRSDB website offers visual comparison of G-quadruplex distribution patterns among all the alternative RNA products of a gene with the help of dynamic graphics. At present, GRSDB contains data from 1310 human and mouse genes, of which 1188 are alternatively processed. It has a total of 379 223 predicted G-quadruplexes, of which 54 252 are near RNA processing sites. GRSDB is a good resource for researchers interested in investigating the functional relevance of G-quadruplexes, especially in the context of alternative RNA processing. It can be accessed at http://bioinformatics.ramapo.edu/grsdb/.

Manuel Viotti

Papers

GRSDB: a database of quadruplex forming G-rich sequences in alternatively processed mammalian pre-mRNA sequences