Review
The Structure and Function of DNA
G-Quadruplexes
Jochen Spiegel,1,4 Santosh Adhikari,2,4 and Shankar Balasubramanian1,2,3,*
Guanine-rich DNA sequences can fold into four-stranded, noncanonical secondary structures
called G-quadruplexes (G4s). G4s were initially considered a structural curiosity, but recent evidence
suggests their involvement in key genome functions such as transcription, replication,
genome stability, and epigenetic regulation, together with numerous connections to cancer
biology. Collectively, these advances have stimulated research probing G4 mechanisms and
consequent opportunities for therapeutic intervention. Here, we provide a perspective on the
structure and function of G4s with an emphasis on key molecules and methodological advances
that enable the study of G4 structures in human cells. We also critically examine recent mechanistic
insights into G4 biology and protein interaction partners and highlight opportunities for
drug discovery.
Beyond the DNA Double Helix
A chemist’s perspective on the function of a molecule, or a system of molecules, is typically led by a
consideration of how molecular structure dictates function. The most widely recognised DNA structure
is that of the classical DNA double helix [1], which defines a structural basis for the genetic code
via defined base-pairing. Yet, it is evident that DNA is structurally dynamic and capable of adopting
alternative secondary structures. One such class of DNA secondary structure is the four-stranded Gquadruplex
(G4). Herein, we discuss some of the key scientific history that has shaped our understanding
of this structural motif, its probable functions in biology, and major unanswered questions that
remain to be solved.
G4 Structure
The capacity for guanylic acid derivatives to self-aggregate was noted over a century ago [2].
Some 50 years later, fibre diffraction revealed that guanylic acids form four-stranded, righthanded
helices leading to a proposed model in which the strands are stabilised via Hoogsteen
hydrogen-bonded guanines to form co-planar G-quartets [3–5]. Subsequent biophysical studies
using DNA oligonucleotides with sequences from immunoglobulin switching regions or telomeres
(see Glossary) showed stable formation of G4 structures under near-physiological conditions
in vitro [6,7]. Stacks of G-quartets are stabilised by cations centrally coordinated to O6 of
the guanines with stabilising preference for monovalent cations in the order K+
> Na+
> Li+
(Figure
1A) [8]. G4s can be unimolecular or intermolecular and can adopt a wide diversity of topologies
arising from different combinations of strand direction (Figure 1D–F), as well as length and
loop composition [9,10]. Structural studies using X-ray crystallography and NMR spectroscopy
have provided detailed insights into the structure of DNA G4s primarily based on the human
telomeric repeat (Figure 1B,C) [11] or sequences derived from the promoter regions of certain
human genes such as MYC or KIT [12,13]. Based on biophysical studies on different G4 structures,
algorithms using sequence motifs such as GR3NxGR3NxGR3NxGR3 were developed and
deployed to predict putative G4 structures in genomic DNA [14,15]. Early models assumed
loop lengths no longer than seven and the requirement for four continuous stretches of Gs. Subsequently,
several G4 structures with longer loop lengths and discontinuities in G-stretches
causing bulges were observed [16,17], leading to alternative predictive models [18]. The recent
availability of large datasets on G4 formation has enabled the application of machine learning to
predict G4 forming propensity [19]. Further considerations include the effects of molecular
crowding [20] and DNA base modifications, such as cytosine methylation [21] and guanine oxidation
[22], on the stability of G4 structures.
1Cancer Research UK, Cambridge
Institute, Li Ka Shing Centre, Cambridge
CB2 0RE, UK
2Department of Chemistry, University of
Cambridge, Cambridge CB2 1EW, UK
3School of Clinical Medicine, University of
Cambridge, Cambridge CB2 0SP, UK
4These authors contributed equally to this
work.
*Correspondence: sb10031@cam.ac.uk
Highlights
Endogenous DNA G-quadruplex
(G4) structures have been detected
in human cells and mapped in
genomic DNA and in an endogenous
chromatin context by adapting
next-generation sequencing
approaches, to reveal cell typeand
cell state-specific G4 landscapes
and a strong link of G4s with
elevated transcription. Synthetic
small molecules and engineered
antibodies have been vital to probe
G4 existence and functions in cells.
Several endogenous proteins have
been found to interact with DNA
G4s, including helicases, transcription
factors, and epigenetic and
chromatin remodellers. Detailed
structural and functional studies
provided novel insight into G4–
protein interactions and revealed a
potential involvement of G4s in a
range of biological processes.
Multiple new lines of evidence
suggest that G4s play a role in
cancer growth and progression.
More G4s are detectable in cancer
cell states compared with normal
state, rendering G4s highly interesting
targets in drug discovery.
Recent studies have started to
explore the potential for synthetic
lethality and global modulation of
cancer gene transcription.
Trends in Chemistry, February 2020, Vol. 2, No. 2 https://doi.org/10.1016/j.trechm.2019.07.002
ª 2019 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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Small Molecules that Bind G4s
Replicative immortality is a hallmark of cancer and several studies have suggested that cancer cells
achieve unlimited proliferation by protecting ends of chromosomes [23]. Telomerase is a reverse transcriptase
enzyme that adds repeat segments to the 30
-end of telomeric DNA and is highly expressed
in most cancers [23,24]. One way to inhibit the action of telomerase was achieved using small molecules
that sequester telomeric ends as stable, liganded G4 structures, which are thought to render
telomeric ends inaccessible for telomerase-mediated extension [25]. An X-ray structure of the small
molecule daunomycin in complex with the G4 formed by four strands of d(TGGGGT) confirmed stacking
of small molecules on a terminal G-quartet [26]. Several structures based on NMR spectroscopy
and X-ray crystallography of small molecule-G4 complexes have since been reported [27]. The
concept of targeting telomeric G4 structures was extended to G4s located in gene promoters. A
cationic porphyrin TmPyP4, which binds to G4s (but does also bind duplex DNA) in vitro was shown
to inhibit transcription of oncogene MYC by a mechanism proposed to involve a G4 target in the
nuclease hypersensitivity element (NHE) in the MYC promoter [28]. Since then, a variety of different
G4-targeted ligands have been described to modulate the expression of genes carrying a sequence
capable of forming a G4 in their respective promoters. So far, few studies have investigated transcriptional
changes on a genome-wide level [29]. More carefully designed controls will be needed to
assess whether a particular G4 is in fact the main biological target or if changes in target gene expression
are a result of the ligand binding to other genomic (G4 or non-G4) targets. The central hypothesis
would be strengthened by more explicit evidence for G4 ligands actually engaging with G4 structures
in the promoters of affected genes in cells, for instance, by employing methods that enable the
genome-wide mapping of ligand binding sites in native chromatin [30,31].
To date, around 1000 small molecules targeting G4 structures have been reported in the G-Quadruplex
Ligands Database (http://www.g4ldb.org/) [32], with some examples of widely used ligands
shown in Figure 2B. Small molecule G4 binders generally have an aromatic surface for p-p stacking
Figure 1. G-Quadruplex (G4) Structures.
(A) Structure of a G-quartet formed by the Hoogsteen hydrogen-bonded guanines and central cation (coloured
green) coordinated to oxygen atoms. Crystal structure of human telomeric G4s (Protein Data Bank: 1KF1): (B)
top view and (C) side view; backbone is represented by grey tube and the structures are colour-coded by
atoms. Schematic representation of unimolecular G4s based on the strand direction: (D) parallel, (E) antiparallel,
and (F) hybrid with a bulge.
Glossary
ChIP-seq: chromatin immunoprecipitation
(ChIP) is a commonly
used method to detect interactions
between proteins and
DNA in a native chromatin context
that is based on the enrichment of
DNA associated with the protein
of interest. The combination with
high-throughput DNA
sequencing analysis (ChIP-seq)
allows for genome-wide detection
of protein binding sites. G4 ChIPseq
is a related technology
whereby an antibody directed
against the G4 structure rather
than a protein is used.
Chromatin: a complex of DNA
and proteins in eukaryotic cells. Its
main functions are the efficient
packaging of DNA to fit into the
volume of nucleus, the stabilisation
and further condensation of
DNA during cell division, and the
control of gene expression.
Euchromatin is a slightly packed
chromatin, which is usually transcriptionally
active, whereas heterochromatin
is more condensed,
inaccessible DNA that is usually
transcriptionally repressed.
DNA methyltransferases: a family
of enzymes that catalyse the
transfer of methyl groups to DNA.
They catalyse the formation of 5methylcytosine
at CpG dinucleotides
in mammalian cells.
fsp3
: number of sp3
hybridised
carbons/total carbon count.
Helicase: a class of enzymes that
can bind, unpack, and remodel
nucleic acids or nucleic acid protein
complexes. DNA helicases
play essential roles in vital cellular
processes. For instance, DNA
helicases unwind doublestranded
into single-stranded
DNA during replication or transcription
so that the strand information
can be copied.
Hypersensitivity site: (nuclease)
hypersensitivity sites are nucleosome-depleted
regions of chromatin
that are detected via their
very high sensitivity to cleavage
by nucleases. These regions are
less densely packed to allow the
binding of proteins, such as transcription
factors, and often mark
genomic regulatory elements.
Next-generation sequencing:
(deep sequencing) modern highthroughput
sequencing technologies,
such as Illumina (Solexa),
Roche 454, and Ion Torrent
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with G-tetrads, a positive charge or basic groups to bind to loops or grooves of the G4, and steric bulk
to prevent intercalation with double-stranded DNA [33]. To improve G4 binding, the aromatic ring
count, positive charge, and number of hydrogen bond donors generally exceed what would be
Figure 2. G-Quadruplex (G4) Ligands.
(A) Crystal structure of a naphthalene diimide, MM41 bound to an intramolecular human telomeric DNA G4, colourcoded
by atoms; water molecules are shown as red spheres, MM41 carbon atoms are coloured green, surface of the
G4 is coloured light grey (Protein Data Bank: 3UYH). (B) Structures of selected widely used G4 ligands.
sequencing that produce data on
a genome scale in the form of
millions of short sequence reads.
8-oxoG: 8-oxoguanine is a common
DNA lesion resulting from
reactive oxygen species; incorporation
into the DNA sequence can
result in a mismatched pairing
with adenine, resulting in
mutations.
Promoter: region of DNA where
transcription initiates; typically
located directly upstream of the
transcription start site.
Telomere: region of repetitive
nucleotide sequence (TTAGGG)n
at the end of each chromosome.
Telomeres protect chromosomes
from end-to-end fusion and prevent
DNA damage and the loss of
genetic information. Telomeres
are typically shortened during cell
division. Telomerases are enzymes
that extend telomeres by
addition of repeat sequences to
the 30
-end, a process that is active
in stem cells and cancers, but
mostly absent in somatic cells.
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optimal for a small molecule with good pharmacokinetic properties [34,35]. Contrary to the classical
perspective, it is noteworthy that a G4 ligand that lacks traditional ‘drug-like’ properties has shown
significant accumulation and efficacy in tumour xenografts of human cancers [29]. The X-ray crystal
structure of the G4 ligand MM41 bound to a human telomeric G4 (Figure 2A) suggests that certain
structural features of G4 ligands can exploit additional interactions in the groove regions of the G4
structure [36]. Interactions with the groove and with the backbone phosphates do not require a flat
aromatic structure. Therefore, compounds with reduced planarity (or high fsp3
) and interactions
with the grooves and/or backbone phosphates may have merit for targeting G4s. Structure–activity
relationship studies on G4 ligands, controlling physicochemical properties such as planarity (fsp3
),
polarity (total polar surface area), lipophilicity (LogD), and rotatable bonds would enable an optimum
balance between G4 binding, solubility, and permeability. In the targeting of structured RNA elements,
it has previously been realised that increased planarity and strongly p-stacking compounds
leads to off-target activity and that these molecules are generally difficult to improve via further medicinal
chemistry [37].
G4 Detection and Mapping
Computational algorithms have predicted over 370 000 G4 sequence motifs in the human
genome, of which a general enrichment was observed in regions associated with genome regulation,
such as telomeres, promoters, and 50
untranslated regions [14,38]. G4s were first detected
in vivo using a G4 structure-specific antibody to stain G4s in the telomeres of ciliates, whereby
telomeric G4 structure formation was observed to be dynamically controlled through protein interactions
in a cell cycle-dependent manner [39,40]. Subsequently, G4s were visualised in human
cells [41–44] and cancer tissue [45]. Antibodies have been used to monitor the behaviour of G4
structures in human cell lines upon ligand treatment together with depletion of the G4 resolving
helicase FANCJ [41,42], to reveal increased numbers of G4 foci staining in nuclei after pyridostatin
[41] or telomestatin ligand treatment and FANCJ depletion in chicken DT40 cells [42]. Telomeric
BG4 foci colocalise with human telomerase, suggesting the enzyme is recruited to G4
structures (Figure 3A) [43].
Besides antibodies, small molecules have also been used to detect G4s in cells. Early studies with radiolabelled
G4 ligands also showed localisation at telomeres [46]. Subsequently, G4s were detected
in human cells by treatment with alkyne functionalised G4 ligands followed by cell fixation and
coupling to fluorophores via coper-catalysed azide-alkyne cycloaddition [47] or strain-promoted
azide-alkyne cycloaddition [48]. Intrinsically fluorescent molecules that display different fluorescent
emission or excitation maxima and fluorescent decay lifetimes upon binding to G4s have been developed
for imaging live cells. The uptake of these molecules to the nucleus and a displacement by the
G4 ligand pyridostatin have been monitored in living cells via fluorescence microscopy [49,50]. G4
specificity was further corroborated by colocalisation with the G4 structure-specific antibody BG4
in fixed cells [50].
The adaptation of next-generation sequencing has enabled the mapping of G4 structures in genomes.
An in vitro reference map of G4s in purified human genomic DNA was obtained using
differential G4-stabilising conditions (i.e., ligands or cations) to recognise G4-specific DNA polymerase
stalling sites during next-generation whole genome sequencing (G4-seq, see Figure 3B)
[51]. The G4-seq study identified more than 700 000 G4 sites in the human genome, exceeding
some earlier predictions. Many noncanonical G4s were identified that comprised longer loops,
as few as two G-tetrads or bulges caused by discontinuous G-tracts [16]. A recent study
extended G4-seq to a variety of other organisms to generate G4 maps and reveal a strong potential
for G4 formation in promoters that is particular to mammals (mouse, human), but mostly
absent in the other organisms studied [52].
It is important to consider the effects of chromatin and all its associated proteins on G4 stability and
formation, which are unaccounted for in maps generated by G4-seq or computational predictions.
Efforts have been made to probe the native G4 landscape in vivo. G4 formation has been inferred
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from chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq)
experiments that use antibodies against proteins that are known to bind G4s in vitro (Figure 3C).
Enrichment of genomic regions comprising computationally predicted G4 structures was observed
for a-thalassemia mental retardation X-linked (ATR-X) [53] and for the XPB and XPD helicases [54],
yeast telomere binding protein RAP1-interacting factor 1 (Rif) [55], and yeast PIF1 helicase [56].
Such studies are consistent with G4 formation in vivo and suggest that the function of the respective
proteins is linked to being physically associated with G4s.
The G4 stabilising ligand pyridostatin generates DNA double-strand breaks in cells. The sites of
pyridostatin-induced strand breaks were determined by ChIP-sequencing of gH2.AX, a phosphorylated
protein that marks stand break sites, and is found to occur predominantly at predicted G4
regions of the cellular genome (Figure 3D) [47]. In a similar approach, ChIP-seq of RAD51, which provides
a more narrow ChIP-seq signal at DNA damage sites, identified 3000 genomic targets of the
G4 ligand CX-5461 [57].
Figure 3. Detection and Mapping of DNA G-Quadruplexes (G4s).
(A) Visualisation of G4s in fixed or live cells using structure-specific antibodies as well as labelled or intrinsically fluorescent G4 ligands. The number of
detected G4 foci can be increased by small molecule treatment or helicase depletion. (B) High-throughput sequencing of G4s in human genomic DNA
(G4-Seq). Two consecutive sequencing runs, under normal and G4 stabilising conditions, provide a reference map and detect G4-dependent
polymerase stalling, respectively. (C) Chromatin immunoprecipitation employing antibodies against endogenous G4-binding proteins followed by nextgeneration
sequencing (ChIP-seq). Genomic occupancy of endogenous proteins is used to infer putative G4 sites. (D) Treatment with G4 ligands
induces G4-dependent DNA damage. ChIP-seq of DNA damage markers in combination with deep sequencing detects G4-associated regions. (E)
Permanganate oxidation of nucleotides in transiently unwound regions traps the unpaired state, resulting in sensitivity to a single-strand specific
nuclease. Computational prediction is then used to infer the type of underlying non-B-DNA structures based on sequence context. (F) G4-specific
chromatin immunoprecipitation and next-generation sequencing (G4 ChIP-seq). A G4-specific antibody (e.g., BG4) is used to precipitate G4 structures
directly from native chromatin and is identified by deep sequencing.
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Genome-wide potassium permanganate-dependent nuclease footprinting, which identifies singlestranded,
non-B DNA, was performed in mouse B cells and combined with computational analysis
to discern the type and enrichment of different non-B DNA structures (Figure 3E) [58]. This approach
revealed around 20 000 hypersensitivity sites featuring G4 motifs and suggested a transcriptiondependent
formation of the noncanonical DNA structures, when comparing resting and lipopolysaccharide-interleukin
4 activated B cells.
The BG4 antibody that binds a wide range of G4 structural types with broad selectivity and high affinity
was recently used to map endogenous G4 structures in fixed chromatin from human epidermal
keratinocytes (NHEKs) and from spontaneously immortalised HaCaT keratinocytes (G4 ChIP-seq, Figure
3F) [59]. In this study, 10 000 G4s were uncovered in precancerous HaCaT cells, while only 1000
were detected in the ‘normal’ counterpart NHEK cells. This represents only 1% of the sites with the
capability to form G4s as observed in G4-Seq [51], suggesting a strong influence of the local chromatin
context and other associated proteins on G4 formation in cells. The majority of G4s were found
in nucleosome-depleted chromatin regions and were enriched in regulatory regions. In addition, G4s
were particularly enriched at promoters of highly transcribed genes [59]. Mapping G4 landscapes in
three other cancer cell lines revealed only moderate overlap, indicating a strong cell-type specificity
[60].
In an alternative ChIP-seq approach, the G4 antibody, D1, was directly expressed in human cervical
carcinoma cells as a GFP-fusion protein. In agreement with the BG4 study, a majority of the
G4 signals were found at transcription start sites, in introns and intergenic regions [44]. Notably,
ca. 15% of G4s were observed in exons, which were not found by BG4 ChIP-seq [59]. This may
reflect different specificities of both G4 antibodies, differences in cell-type specific G4 landscapes,
or competition of the D1 antibody with endogenous G4-binding proteins to reveal
masked G4 structures.
Natural G4-Binding Proteins
The observation of G4s enriched at genome regulatory sites, and that G4 formation in cells is dynamic
and dependent on cell type and state, suggests that the G4 landscape can be regulated by cellular
proteins. Indeed, many natural proteins have been identified that interact with G4s (see G4 Interacting
Proteins Database, http://bsbe.iiti.ac.in/bsbe/ipdb/) [61]. The identification of G4 associated proteins
has mostly relied on affinity proteomics experiments employing RNA and DNA G4 oligomers as
baits to isolate G4 interactors from cellular extracts [62–65]. Other approaches involve computational
analysis of genomic protein binding sites to assess the enrichment of predicted G4 motifs within
these binding sites [54,66], or meta-analysis, in which shared structural features of G4-binding protein
domains were compared to predict new putative G4 interactors [67,68]. In a recent study, a series of
protein affinity pull-downs from nuclear lysates with oligonucleotide baits at different concentrations
was combined with isobaric tandem-mass-tag labelling and mass spectrometry. This revealed
apparent dissociation constants and binding profiles towards different G4 and transcription factor
consensus sequences for hundreds of nuclear proteins in parallel [69].
To date, over a dozen specialised helicases have been identified that target DNA G4s with up to picomolar
affinity (reviewed in [70]). Recent studies have provided structural and mechanistic insights
into G4 recognition and unwinding. Single-molecule imaging studies revealed a common repetitive
unfolding mechanism for the specialised helicases DHX36 (also known as RHAU), Blooms (BLM), and
Werner (WRN) helicases, albeit with different substrate specificity, and showed the capacity to
displace G4-stabilising ligands [71,72]. A cocrystal structure of bos taurus DHX36 helicase bound
to the MYC promoter G4 revealed a mode of binding in which the G4 makes contacts with a
DHX36-specific motif (DSM) and the C terminal OB-fold domain (Figure 4) [73]. Strikingly, the residues
of the a-helical DSM create a nonplanar hydrophobic surface that stacks on the top of a mixed quartet
of the quadruplex, which forms after partial unfolding of the original G4 by the protein. The binding
mode is somewhat reminiscent of that suggested for most small molecule G4 ligands, but suggesting
that absolute planarity of the ligand may not be needed [73]. These findings are complemented by
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the crystal structure of Drosophila melanogaster DHX36, revealing a positively charged pocket that
binds and destabilises the G4 [74].
In accordance with the G4 forming potential of the telomere repeat sequence, many telomere-associated
proteins have been shown to interact with G4 structures [63]. For instance, POT1 [75] and RPA
[76], two components of the shelterin complex, as well as the human CST complex [77], are able to
bind and unwind telomeric G4 structures and assist the action of telomerase.
As G4s are strongly enriched at promoters, it is unsurprising that several transcription factors and
transcriptional coactivators bind sites containing predicted G4 motifs, many of which show potential
to bind or unwind G4 structures in vitro. In particular, G4 structures at promoter regions of oncogenes
represent the most closely studied G4 sites. The nonmetastatic factor NM23-H2 has been reported to
Figure 4. Crystal Structure of DHX36 Bound to the c-Myc G4 (Protein Data Bank: 5VHE).
Overall structure is shown in cartoon representation with the domain organisation of DHX36 colour-coded (top).
The a-helical DHX36-specific motif (DSM) stacks on the 50
-quartet. The residues Ile65, Tyr69, and Ala70 form a
nonpolar surface similar to the proposed binding mode of most small molecule G-quadruplex (G4) ligands
(bottom).
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recognise and unwind the MYC promoter G4 [78]. Conversely, binding by nucleolin was reported to
stabilise the G4 structure resulting in reduced transcription at this site [65]. Similarly, pull-down and
chromatin immunoprecipitation experiments showed that Myc-associated zinc finger (MAZ) and poly(ADP-ribose)
polymerase 1 (PARP-1) proteins bind the G4 structure found upstream of the KRAS
transcription start site [79]. However, hnRNP A1 and MAZ were shown to bind and unfold the KRAS
and HRAS promoter G4 in vitro, respectively [80,81]. Chromatin binding sites of the transcriptional
helicases XPB and XPD are enriched in predicted G4 motifs. Both helicases associate with G4s
in vitro, while XPD can also resolve G4 structures, suggesting that these proteins may affect transcription
via a G4-associated mechanism [54].
Furthermore, various epigenetic and chromatin remodelling enzymes selectively bind DNA G4
oligomers [69]. Genomic binding sites of the chromatin remodelling protein ATR-X are enriched
at GC-rich tandem repeats and CpG islands with the potential to fold G4 structures, and ATR-X
loss is implicated with G4-dependent replication stress, DNA damage, and copy number alterations
[53,82]. The DNA methyltransferase enzymes (DNMTs), which catalyse the formation of
5-methylcytosine at CpG dinucleotides in mammalian cells, bind to G4 structures with subnanomolar
affinity in vitro [83,84]. DNMT1 shows stronger interaction with G4s compared with duplex
DNA and loses enzyme activity upon G4 binding. Maps of DNMT1 chromatin binding sites and
endogenous G4s in human K562 leukaemia cells identified using ChIP-seq and G4 ChIP-seq,
respectively, revealed that at CpG islands most G4s occur where DNMT1 is bound, leading to
a proposal that G4s regulate DNA methylation by sequestering DNMTs [84]. Conversely, DNA
methylation can influence G4 topology [85] and may modulate binding by other G4-associated
proteins. For instance, CpG methylation at the hTERT gene promoter was suggested to induce
G4 formation, resulting in displacement of the CCCTC-binding factor (CTCF) and elevated transcription
[86].
G4-protein interactions may provide a means to recruit machinery to specific parts of the genome to
influence a wide range of cellular processes. Given that the G4 landscape is dynamic and dependent
on the functional state of cells, proteins may be responsible for regulating G4 structural dynamics
throughout the genome [60]. Approaches that can reveal the composition and dynamics of chromatin-associated
protein complexes [87,88] will be needed to uncover details of the proteins that
constitute the G4 interactome, which in turn may present opportunities for small molecule modulation
of these interactions.
Biological Role of G4s
The localisation of G4s at regions that regulate genome function have implicated G4s in a range of
biological processes. The finding that G4-rich telomeric repeats form G4 structures [6] suggested a
mechanistic link with telomerase-mediated extension of telomeres, prompting an exploration of
G4 stabilising ligands that may inhibit the growth of cancer cells by interfering with telomere maintenance
(reviewed in [89]). For instance, the mouse regulator of telomere elongation helicase 1
(RTEL1) was shown to maintain telomere integrity by unwinding telomeric G4s [90]. Telomeric G4s
had originally been suggested to impair telomerase function [91], but might also be important for
telomerase recruitment [43].
G4 structures are strongly associated with genomic and epigenetic instability, particularly when they
are not efficiently regulated. The yeast Pif1 helicase was shown to prevent G4-mediated genomic
instability and prevent DNA strand breaks [92,93]. Human Pif1 is recruited to DNA double-strand
breaks sites to promote homologous recombination (HR) at sequences predicted to form G4s. Treatment
with G4 stabilising ligands can impair Pif1 functionality, which can be rescued by PIF1 overexpression
[94]. G4s have also been suggested to function as potential sensor or trapping sites of
oxidative DNA damage caused by reactive oxygen species. Incorporation of 8-oxoG can affect stability
of promoter G4 structures, which resulted in altered expression levels in reporter gene assays
[95–97]. Moreover, 8-oxoG modification was shown to disrupt the formation of telomeric DNA G4s
[98] and to promote telomerase activity [99].
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Various models have been postulated for the biological role of G4 structures during DNA replication
[100,101]. G4 structures can act as an obstacle to replication fork progression when helicases are
disrupted [93,102]. Introduction of G4 forming sequences at the BU-1 locus of DT40 chicken cells,
resulted in pausing at the G4 sites (Figure 5). Additional replication stress due to nucleotide pool
depletion decoupled the replication machinery from restoration of the parental histones, ultimately
leading to altered gene expression [103]. Based on the same mechanism, small molecule stabilisation
of G4 structures induced replication-dependent loss of epigenetic information showing that G4s can
serve as obstacles to the replication machinery [104]. Conversely, mapping the genome-wide location
of replication origins using deep-sequencing of short nascent strands in four different human cell
lines uncovered enrichment of predicted G4s sequences, suggesting G4s may support initiation of
DNA replication [105].
Treatment with G4-stabilising small molecules resulted in reduced mRNA levels at genes that contain
G4 sequences in their respective promoters, such as the proto-oncogenes MYC [28] and KRAS [106],
supporting the hypothesis that G4s constitute an impediment to the progression of transcription machinery.
However, G4-stabilising ligands can also lead to DNA damage and recruit associated
response mechanisms [47,57]. Transcriptional changes at G4s may therefore be a result of either
direct G4 stabilisation or DNA damage-mediated transcriptional repression. Aberrant function of
the G4-resolving helicases WRN and BLM result in altered transcription of genes containing G4 motifs
in their promoter region, corroborating a link between G4s and transcription [107,108]. Moreover,
BLM-mutated cells derived from Bloom syndrome patients show high rates of sister chromatid exchange
at sites of G4 motifs in transcribed genes [109]. Notably, these helicases also process duplex
DNA, so not all the observed changes may be linked to G4 structures.
Where G4 structures form, the opposite DNA strand cannot form Watson-Crick base pairing. The Crich
opposite strand may be single stranded and potentially complexed with single-stranded binding
proteins [110,111]. It has also been postulated that secondary structures might form on the opposite
C-rich strand. Intercalated motif (i-motif) structures can be formed via stacks of intercalating hemiprotonated
C-neutral C base pairs (C+
:C), and are generally stabilised in slightly acidic pH [112]. Recent
experiments have used an i-motif-specific antibody to image i-motifs in the nuclei of fixed human cells
[113]. Furthermore, i-motif formation is cell cycle dependent, peaking at late G1 phase, whereas G4
formation was maximal during S phase [41], suggesting i-motifs and G4s may have different dependencies
or even be mutually exclusive [114]. Indeed, a previously proposed model for the MYC
Figure 5. G-Quadruplex (G4) Can Induce Epigenetic Reprogramming.
Unresolved G4 structures (e.g., G4 ligand treatment, impaired helicases) on the leading strand may promote uncoupling of DNA synthesis from opening of
the replication fork and disrupt histone recycling. Original histone modifications (blue) are lost and replaced with new histones (brown), resulting in
epigenetic reprogramming upstream of G4 sites.
Trends in Chemistry, February 2020, Vol. 2, No. 2 131
Trends in Chemistry
promoter region suggested that negative superhelicity initiated at the proximal promoter travels
upstream to mechanically perturb DNA structure [115] and it was suggested that a G4 and an i-motif
can each be bound and stabilised by different specialised proteins to play opposite roles in the control
of MYC gene transcription [116].
Approaches to map endogenous G4 structures such as permanganate footprinting, G4 ChIP-seq,
and immunofluorescence with G4 antibodies have been consistent with G4 structures marking
actively transcribing genes in human cells [58,59]. In addition, G4s can contribute to hypomethylation
of CpG islands in promoter regions, which also contributes to elevated gene expression [84]. Further
work is needed to unravel the mechanistic and molecular details of how G4s influence transcription.
Intervention and Therapeutics
G4s are associated with processes and control mechanisms that are important for the biology and
growth of cancer cells, including telomere biology, transcriptional regulation of cancer-related
genes, replication, and genome instability. Furthermore, G4 motifs are generally over-represented
in cancer-promoting genes [51,59] and experimental data has shown a higher presence of G4
structures in cancer states compared with normal states, which may favour G4s as molecular targets
for cancer. For example, antibody staining of stomach and liver cancer tissues showed higher levels of
G4 foci compared with normal tissue [45]. Also, G4 ChIP-seq detected tenfold more G4 sites in
cancer-like, immortalised HaCaT cells as compared with their normal human keratinocyte counterpart
[59,60].
G4 ligands such as pyridostatin and RHPS4 cause DNA double-strand breaks in cancer cells and activate
DNA repair pathways [47,117]. Cancer cells with impaired repair pathways are sensitive to G4
ligands. For example, cancer cells deficient in BRCA2, a vital component of the homologous recombination
(HR) repair pathway, are sensitive to pyridostatin derivatives and RHPS4 [118,119]. Similarly,
knockdown of PARP1, a key regulator of the nonhomologous end joining (NHEJ) repair pathway
resulted in cancer growth inhibition by RHPS4 both in cellular and xenograft models [117]. Notably,
pyridostatin was also toxic to HR-deficient cells resistant to olaparib, a PARP inhibitor [119]. This result
suggests that G4 ligands may be an effective therapeutic approach in HR-deficient cancers resistant
to PARP inhibitors. Indeed G4 ligand, CX-5461 is currently in human clinical trials for breast cancer
patients with BRCA1/2 germline aberrations [57]. Moreover, G4 ligands have also shown synergy
with DNA damaging therapies in ATR-X-deficient glioma cell models [82]. G4 ligands have also
been successfully used in combination with inhibitors of key proteins involved in the DNA repair
pathway. Pyridostatin synergises with NU7441, an inhibitor of DNA-PK, which is an important kinase
in the NHEJ pathway [118]. The combination of RHPS4 and PARP1 inhibitor GPI 15427 showed 50%
reduction in growth of HT29 colon tumour-bearing mice xenografts compared with 2% and 30%
with GPI 15427 and RHPS4 alone, respectively [117].
Another approach described above is to target G4-containing genes involved in cancer progression
and manipulate their transcription. While the concept of regulating expression of individual genes
has been explored using several small molecules [28,106], it is rational to assume that most G4 ligands
will bind to other G4s and potentially regulate expression of many genes. This multigene targeting
approach can result in poly-pharmacology, affecting several pathways important for cancer
progression. In support of this view, global transcriptional profiling revealed that treatment of human
pancreatic ductal adenocarcinoma (PDAC) cell lines with the G4 ligand CM03 could induce global
downregulation of many G4-containing genes [29]. Analysis of the downregulated genes identified
G4-containing genes involved in pathways important for PDAC progression. CM03 reduced tumour
growth in PDAC xenografts as well as KPC mouse models at doses that do not cause any observable
toxicity. It is possible that a particular G4 ligand may result in a distinct profile of affected genes for
proteins involved in several pathways, suitable for targeting certain cancers. Global transcriptional
profiling of the cancer cells/tissues treated with G4 ligand may identify the affected pathways and
suggest cancers best suited for efficacy studies. A better understanding of the pathways affected
Outstanding Questions
G4 ligands: can we develop small
molecule G4 ligands with better
pharmacokinetic properties that
more effectively target G4s in cells?
Is it possible to selectively target individual
G4s or subclasses of G4s
based on the molecular topology
and sequence composition of the
loops of G4s? Is it necessary, or
desirable, to selectively target individual
G4s to achieve therapeutic
effects? How do we design suitable
functional assays to screen for and
validate direct G4 on-target effects
in cells?
Detection and mapping: can we
develop mapping approaches to
detect G4s genome-wide in native
chromatin with stranded information
and at single G4 resolution?
Do current antibody-based approaches
provide an incomplete
picture due to limited accessibility
and competition with endogenous
proteins? Can we develop detection
methods to monitor dynamic
G4-associated processes at highresolution
in living cells?
G4 landscape: under which conditions
do G4s form in cells? What is
the chromatin context? What are
the key protein regulators that
mediate G4 formation and depletion?
What is the crosstalk with
other epigenetic features? What
happens on the opposing strand
and what is the interplay with other
structural phenomena (e.g., i-motifs,
Z-DNA, R-loops)?
G4s and transcription: what is the
exact mechanism by which G4s affect
transcription? Can we control transcription
efficiently by manipulating
particular G4s or by interfering with
particular G4 interactors?
G4s and cancer: why are G4s enriched
at certain cancer genes?
Are G4 signatures suitable biomarkers
for diagnostic applications?
What pathways are G4s
involved in and are there phenotypes
that make patients more tractable
to G4 ligand therapies? What
synthetic lethalities can potentially
be exploited for combination
therapies?
132 Trends in Chemistry, February 2020, Vol. 2, No. 2
Trends in Chemistry
by G4 ligands may ultimately provide a rationale to consider combination therapy opportunities with
other drugs.
Concluding Remarks
During the past two to three decades, the study of DNA G4 structures has extended from biophysical
and structural work to studies in biological models and systems of increasing sophistication. Collectively,
these studies have advanced the hypothesis that the G4 DNA structure is intimately linked to a
number of biological functions. While there is more work to be done to unravel the mechanistic details
of how and why G4 structures influence biological function (see Outstanding Questions), there
appears to be rationale and merit in considering G4s as promising molecular targets for future
therapeutics.
Acknowledgements
We thank D. Tannahill for critical reading of the manuscript. The Balasubramanian laboratory is corefunded
by Cancer Research UK (C14303/A17197); Cancer Research UK programme (C9681/A18618);
S.B. is a Welcome Trust Senior Investigator (099232/Z/12/Z); J.S. gratefully acknowledges funding
from the EU H2020 Framework Programme (H2020-MSCA-IF-2016, ID: 747297-QAPs).
Disclaimer Statement
S.B. is a founder, shareholder, and scientific advisor to Cambridge Epigenetix Ltd.
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