molecules

Review
G-Quadruplexes as An Alternative Recognition
Element in Disease-Related Target Sensing
Jeunice Ida 1 , Soo Khim Chan 1 , Jörn Glökler 2 , Yee Ying Lim 1 , Yee Siew Choong 1 and
Theam Soon Lim 1,3, *
1 Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Penang 11800, Malaysia;
[email protected] (J.I.); [email protected] (S.K.C.); [email protected] (Y.Y.L.);
[email protected] (Y.S.C.)
2 Division of Molecular Biotechnology and Functional Genomics, Technical University of Applied Sciences
Wildau, Hochschulring 1, 15745 Wildau, Germany; [email protected]
3 Analytical Biochemistry Research Centre, Universiti Sains Malaysia, Penang 11800, Malaysia
* Correspondence: [email protected]; Tel.: +604-653-4852; Fax: +604-653-4803




Received: 11 February 2019; Accepted: 16 March 2019; Published: 19 March 2019


Abstract: G-quadruplexes are made up of guanine-rich RNA and DNA sequences capable of
forming noncanonical nucleic acid secondary structures. The base-specific sterical configuration
of G-quadruplexes allows the stacked G-tetrads to bind certain planar molecules like hemin (iron
(III)-protoporphyrin IX) to regulate enzymatic-like functions such as peroxidase-mimicking activity,
hence the use of the term DNAzyme/RNAzyme. This ability has been widely touted as a suitable
substitute to conventional enzymatic reporter systems in diagnostics. This review will provide a brief
overview of the G-quadruplex architecture as well as the many forms of reporter systems ranging
from absorbance to luminescence readouts in various platforms. Furthermore, some challenges and
improvements that have been introduced to improve the application of G-quadruplex in diagnostics
will be highlighted. As the field of diagnostics has evolved to apply different detection systems, the
need for alternative reporter systems such as G-quadruplexes is also paramount.

Keywords: G-quadruplex; DNAzyme; colorimetric; fluorescence; luminescence; diagnostics

1. Introduction
Just over 60 years ago, Watson and Crick published their seminal paper on the DNA structure [1].
In it, they emphasized two principle features of the DNA molecule that are the complementary nature
of the base sequences on the two antiparallel oriented strands and the double-helical nature of the
polymer. This has helped shaped the way we perceive DNA structure today and allow researchers
to design complex nanostructures according to this rule. DNA is a promising engineering material,
not just for its outstanding data storage capacity, but for its flexibility, design and efficient chemical
synthesis. A deeper understanding of DNA has allowed DNA to expand its presumed main role in
genetic-information storage to a more versatile function due to the robustness of the structures it may
form. Apart from the Watson-Crick double helix structure, DNA can also adopt alternative structures
ranging from disordered single strands to higher-order structures such as G-quadruplex (see Figure 1)
as a consequence of dynamic molecular events.
A DNA G-quadruplex (G4) structure can arise from specific G-rich sequence(s) forming G-tracts.
Guanine bases associate with guanines from neighboring G-tracts through Hoogsteen hydrogen
bonding to form a square planar called guanine tetrad (G-tetrad) or guanine quartet (G-quartet).
The G-tetrad would then stack with one another within the G-tetrad core resulting in a G4 structure.

Molecules 2019, 24, 1079; doi:10.3390/molecules24061079 www.mdpi.com/journal/molecules

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Molecules 2019, 24, 1079 2 of 30

(a) (b)
(a) (b)
Figure
Figure 1. Comparison
1. Comparison of of
(a)(a) DNAdouble
DNA doublehelix
helix(PDB:
(PDB:1BNA)
1BNA)and
and(b)
(b) DNA
DNA G-quadruplex stabilized
stabilized by
Figure
K 1. Comparison
++ (PDB: 244D). of (a) DNA double helix (PDB: 1BNA) and (b) DNA G-quadruplex stabilized
by K
by K+ (PDB: 244D).
Hoogsteen
Hoogsteen hydrogen
hydrogen bonding,
bonding, a non-canonical/non-Watson-Crick
a non-canonical/non-Watson-Crick base
base configuration
configuration allows
allows forfor
Hoogsteenofhydrogen bonding, ain
non-canonical/non-Watson-Crick base configuration allows for
the formation of unusual base pairs in certain sequence contexts. G4 arrangements are found to to
the formation unusual base pairs certain sequence contexts. G4 arrangements are found bebe
the formation
immensely of unusual
polymorphicallowing base pairs
allowingitit toin certain
to represent sequence contexts. G4 arrangements are found to be
immensely polymorphic representaahugehugefamily
family of stable structures
of stable withwith
structures a typical overall
a typical
immensely
fold. The polymorphicpolymorphism
allowing it to motif
representthea G4
huge family of unique
stable structures with a typical
overall fold.rich
Thestructural
rich structural polymorphism of
motif as well
of the G4 asas its
well as its geometry are thought
unique geometry are to
overall
allow forfold. The
specific rich structural polymorphism motif of the G4 as well as its unique geometry are
thought to allow forrecognition and detection
specific recognition of metal ions,
and detection DNA,
of metal small
ions, molecules,
DNA, protein and
small molecules, enzyme
protein
thought
activity to allow
through for specific
a number recognition and detection of metal ions, DNA, small molecules, protein
and enzyme activity through of bindingof
a number modes in amodes
binding manner in analogous to that of double-helical
a manner analogous to that of double- DNA
and enzyme [2].
intercalators activity
The through a number
introduction of G4 of binding
structure modeswith
together in a its
manner
unique analogous
ability to to
bindthat of double-
with various
helical DNA intercalators [2]. The introduction of G4 structure together with its unique ability to bind
helical DNA
ligands intercalators
suchligands
as hemin [2]. The introduction
(see of G4 structure together withofitsnew unique abilityoftoDNA
bind
with various such asFigure
hemin2)(see
haveFigure
contributed
2) have to contributed
the identification functions
to the identification of new
with various
in biology. ligands such as hemin (see Figure 2) have contributed to the identification of new
functions of DNA in biology.
functions of DNA in biology.

(a)
(a)
Figure 2. Cont.
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(b)
(a) Chemical
Figure 2. (a) Chemical structure
structure of hemin (b) G-quadruplex-hemin complex.

Motivated by
Motivated this fact,
by this fact, the
the study
study onon DNA
DNA G4 G4 has
hasgained
gainedattention
attention from
from different
different research
research groups
groups
to apply for the design of selective probes in diagnostic assays. This review provides
to apply for the design of selective probes in diagnostic assays. This review provides an account an account of the
of
structural
the information
structural of G4
information ofbesides highlighting
G4 besides its diversity
highlighting as a reporter
its diversity in disease-related
as a reporter target
in disease-related
detection
target assays.assays.
detection The assays, specifically
The assays, targetingtargeting
specifically DNA, ATP, enzymes
DNA, and metaland
ATP, enzymes ions, are discussed
metal ions, are
with
discussed with a focus towards various distinct types of principles and mechanisms that performed
a focus towards various distinct types of principles and mechanisms that have been have been
and reported
performed and over the past
reported overfewthe
years.
past The
few claim
years.that
The G4-based
claim thatsensors
G4-basedcompare
sensorsfavorably
comparewith other
favorably
available detection strategies are discussed as well. It is therefore conceivable that the
with other available detection strategies are discussed as well. It is therefore conceivable that theimpact G4 could
have onG4
impact biomedical
could have diagnostic applications
on biomedical will increase
diagnostic applicationswithwill
newincrease
improvements
with new of the specific G4
improvements
design and implementation.
of the specific G4 design and implementation.

2. The
2. The Basic
Basic Structure
Structure of of G-Quadruplex
G-Quadruplex
The ability
The ability of
of guanines
guaninestotoself-associate
self-associatetotoform formfour-stranded
four-stranded helical
helical structures,
structures, known
known as
as G-
G-quadruplex was first demonstrated in 1962 [3]. Even so, the fact that G-rich
quadruplex was first demonstrated in 1962 [3]. Even so, the fact that G-rich DNA solutions are able DNA solutions are able
to form
to form gelatinous
gelatinousaggregates
aggregateswas wasfirst noted
first notedin in
1910. However,
1910. However, their exact
their nature
exact was was
nature not noticed until
not noticed
these these
until gels were observed
gels were to form
observed to stacked
form stackedplanesplanes
of guanine tetramers
of guanine in a cyclic
tetramers arrangement.
in a cyclic Since
arrangement.
then, G4 have been extensively studied and were reported to be predominant
Since then, G4 have been extensively studied and were reported to be predominant in functional in functional regions of
the human
regions of genome
the human and genome
transcriptome. This also includes
and transcriptome. Thisreplication
also includesinitiation sites, human
replication telomeres,
initiation sites,
human telomeres, oncogene promoter regions and untranslated regions [4]. The formation of in
oncogene promoter regions and untranslated regions [4]. The formation of G4 at the telomeres G4the
at
macronuclei
the telomeresofinthe theciliate Stylonyvhia
macronuclei lemnae
of the wasStylonyvhia
ciliate first visualized
lemnae using
was afirstG4 selective
visualized antibody
using ainG4a
biologicalantibody
selective context [5].
in a It was suggested
biological contextbased[5]. It on
wasthe telomeric
suggested sequence
based on thethat two overhangs
telomeric sequencehave that
the propensity to associate end-to-end through the formation of four-stranded
two overhangs have the propensity to associate end-to-end through the formation of four-stranded conformations of two
fold-back hairpins
conformations of two which involves
fold-back a quadruplex
hairpins stem. aItquadruplex
which involves is further stabilized
stem. It is by stacked
further layers by
stabilized of
G-quartets
stacked thatofare
layers hydrogen-bonded
G-quartets in a cyclic mannerin[5].
that are hydrogen-bonded a cyclic manner [5].
The complementary C-rich
The complementary C-rich strand can strand can adopt
adopt another
another non-canonical
non-canonical structure
structure termed
termed as as i-motif.
i-motif.
It is
is based
based on onHoogsteen
HoogsteenC-C C-C + base-pairs, which
It + base-pairs, which waswas initially
initially deemed deemedto formto form
only only
underundernon-
non-physiological acidic conditions [6]. However, recently, Zeerati and co-workers
physiological acidic conditions [6]. However, recently, Zeerati and co-workers discovered that i-motif discovered that
i-motif structures are also able to form in the nuclei of human cells. This is mainly
structures are also able to form in the nuclei of human cells. This is mainly found in regulatory regions found in regulatory
regions
of of thegenome,
the human human genome, which includes
which includes the promoter
the promoter and telomeric
and telomeric regions. regions.
In genomic In genomic DNA,
DNA, i-motifs
i-motifs and its complementary G-rich sequences that can adopt G4
and its complementary G-rich sequences that can adopt G4 structures often coexist. ELISA resultsstructures often coexist. ELISA
results shown
shown by this by
samethisgroup
same proved
group proved
that iMab thatrecognizes
iMab recognizes
a widea range
wide range of i-motif
of i-motif DNA DNA structures
structures with
with
high high affinity
affinity but it but it does
does not not
bind bind
to toG4G4 structures,
structures, suggesting
suggesting thatG4
that G4and
andi-motif
i-motifcan
can bebe well
well
discriminated biochemically
discriminated biochemically [7]. [7].
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Molecules 2019, 24, x FOR PEER REVIEW 4 of 29

G4 could be formed from one, two, or four G-rich strands. Although the formation of G4 requires
G4 could be formed from one, two, or four G-rich strands. Although the formation of G4 requires
G-rich sequences, not all G-rich sequence can form G4 structures. G4s are composed of at least four G
G-rich sequences, not all G-rich sequence can form G4 structures. G4s are composed of at least four
residues as the fundamental building blocks, forming ring-like aromatic planar G-tetrad structures.
G residues as the fundamental building blocks, forming ring-like aromatic planar G-tetrad structures.
These structures
These areare
structures connected
connectedviavia
Hoogsteen
Hoogsteenhydrogen
hydrogenbonds,
bonds, involving the N1,
involving the N1,N7,
N7,06
06and
andN2
N2sites
sites
(see(see
Figure 3a) of the guanines [4]. Two or more G-quartets are able to stack on top of one
Figure 3a) of the guanines [4]. Two or more G-quartets are able to stack on top of one another, another,
resulting in the
resulting formation
in the formationofof
four-stranded
four-strandedhelical
helicalstructures
structures [8]
[8] (see Figure 3b).
(see Figure 3b).Loop
Loopsequences
sequenceslink
link
the the
Gs Gs
andand
determine the type of G4 structure to be formed.
determine the type of G4 structure to be formed.

(a) (b)
Figure 3. (a)
Figure Chemical
3. (a) Chemicalstructure ofofa G-quartet.
structure a G-quartet.Four
Fourguanines
guanines are
are bonded
bonded byby Hoogsteen
Hoogsteenhydrogen
hydrogen
bond
bond (dashes)
(dashes) andand themonovalent
the monovalentcation +
K acts
cation K +
actstotostabilize
stabilizethe structure.
the (b) (b)
structure. An Anintramolecular G-
intramolecular
quadruplex structure
G-quadruplex structure consisting
consistingofofthree
threeG-tetrads
G-tetrads and a G-quadruplex
and a G-quadruplex motif sequence
motif withwith
sequence four four
G-
tractsof
G-tracts ofthree
three guanines
guanines separated
separatedby byloops.
loops.

Originally,
Originally, it was
it was assumedthat
assumed thatG4s
G4soccur
occur only
only inin the
theform
formofofright-handed
right-handed helices. However,
helices. However,it
wasrecently
it was recentlydiscovered
discovered thatthat certain
certain G4s
G4s such
such asas the
the therapeutically
therapeutically active
activeG-rich
G-richoligonucleotide
oligonucleotide
AGRO100
AGRO100 (also
(also known
known as as AS1411)adopts
AS1411) adoptsa aleft-handed
left-handedhelical
helical conformation
conformation [9]. [9].Thus,
Thus,aside
asidefrom
from
the Z-form of double-stranded DNA (dsDNA), G4s can also adopt the unusual left-handedhelix.
the Z-form of double-stranded DNA (dsDNA), G4s can also adopt the unusual left-handed helix.
ThisThis property
property is critical
is critical if more
if more specificligands
specific ligandsarearetotobebedesigned
designed that
that can
can distinguish
distinguishbetween betweenthe the
helical conformations.
helical conformations.
Various
Various rules
rules governing
governing G4G4 foldinghave
folding havebeen
beenestablished
established with
with the
the ongoing
ongoingadvancement
advancementininthe the
structural studies of G4 [10-14]. The folding of G4 can occur through the folding of a single strand
structural studies of G4 [10–14]. The folding of G4 can occur through the folding of a single strand
(intramolecular) or the association of two (bimolecular) or four (tetramolecular) separate strands
(intramolecular) or the association of two (bimolecular) or four (tetramolecular) separate strands
(intermolecular) [12]. Intramolecular interactions in single-stranded DNA connects three loop regions
(intermolecular) [12]. Intramolecular interactions in single-stranded DNA connects three loop regions
linking four G-tracts of at least two consecutive Gs together [13].
linking four G-tracts of at least two consecutive Gs together [13].
Different sequences will evidently adopt alternative topologies, but in some cases various
Different sequences
conformations may also willbeevidently
derived adopt
from onealternative
particular topologies,
sequence but[14].in Depending
some caseson various
the
conformations may also be derived from one particular sequence [14]. Depending
environmental conditions, the conformations can interchange or are found to co-exist in different on the environmental
conditions, the conformations
states in parallel. Thus, it is notcan interchange
uncommon for or are foundstructures
quadruplex to co-exist in different
obtained states in parallel.
by crystallography to
Thus, it is not uncommon for quadruplex structures obtained by crystallography to
disagree with those obtained in solution [15]. Each of the four G-tracts can run in similar (parallel) disagree with those
or
obtained in solution
different [15]. Each
(anti-parallel) of thewith
direction fourrespect
G-tractsto can
eachrun in similar
other (parallel) or different
[4,16]. Anti-parallel conformation (anti-parallel)
may be
direction withinto
classified respect
twototypes
each other
as the[4,16]. Anti-parallel
guanines involved conformation may be syn-
have alternating classified
and into two types
anti-glycosyl
as the guanines involved
conformation along each have strand [17]. Insyn-
alternating and anti-glycosyl
addition, G4 have also conformation
been observedalongtoeach strand
adopt [17].
mixed
orientations
In addition, consisting
G4 have of parallel-antiparallel
also been observed to adopt strands to form a stable
mixed orientations structure
consisting [18]. This hybrid
of parallel-antiparallel
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orientation of G4 contains both consecutive guanines in the same (syn-syn or anti-anti) and different
strands to form a stable structure [18]. This hybrid orientation of G4 contains both consecutive
(syn-anti or anti-syn) conformations [17]. The four G-tracts are comprised of the same guanine rich
guanines in the same (syn-syn or anti-anti) and different (syn-anti or anti-syn) conformations [17].
oligonucleotides connected by loops of variable nucleotide lengths and sequences [16]. Figure 4
The four G-tracts are comprised of the same guanine rich oligonucleotides connected by loops of
highlights some of the basic types of G4 conformations: parallel, antiparallel and hybrid (see Figure
variable nucleotide lengths and sequences [16]. Figure 4 highlights some of the basic types of G4
4).
conformations: parallel, antiparallel and hybrid (see Figure 4).

Figure 4. Common conformations of G-quadruplex. (a) Parallel (b) Anti-parallel (c) Hybrid
(d) Anti-parallel
Figure 4. Common (dimolecular) (e) Parallel
conformations (tetramolecular).
of G-quadruplex. (a) Parallel (b) Anti-parallel (c) Hybrid (d) Anti-
parallel (dimolecular) (e) Parallel (tetramolecular).
The folding of G4 occurs rapidly and the obtained structures are complex, exhibiting great
conformational
The foldingdiversity
of G4 occurs [14]. This structural
rapidly and the polymorphism is mainly
obtained structures aredue to the properties
complex, exhibiting of the
great
loop, which includes
conformational diversity strand
[14].polarity, variations
This structural in strand stoichiometry
polymorphism is mainly due and to the
the sites on theofloop
properties the
that are connected to the G strands [4,19]. The structure of G4 may also
loop, which includes strand polarity, variations in strand stoichiometry and the sites on the loop that depend on the length and
composition
are connectedofto DNA the orG RNA,
strands the[4,19].
positions
The of the loops,
structure of the
G4orientation
may also depend of the chain on the andlength
the nature
and
of the cationsofinvolved
composition DNA or[16]. RNA, G-quartet regions,
the positions groove
of the dimensions,
loops, the orientation centralof channel
the chain ofand
the G4the as well
nature
as the
of the cations
negativeinvolved
electrostatic
[16]. potential
G-quartetofregions,
the anionic
groovebackbone are factors
dimensions, central that influences
channel of thethe G4binding
as well
selectivity
as the negativeof theelectrostatic
target molecules potential[4]. of the anionic backbone are factors that influences the binding
Otherofthan
selectivity the the sequence
target molecules requirements,
[4]. the integration of cations with the G4 is also vital in
determining the polymorphism
Other than the sequence requirements, the and stability of integration
the G4. The of ligands
cations actwithtothe coordinate
G4 is also with
vitalfour
in
electronegative
determining theO6 atoms from a and
polymorphism G-tetrad and of
stability another
the G4.fourTheof the adjacent
ligands act to stacked G-tetrad.
coordinate withInfourthe
presence of different
electronegative O6 atomscations, thea G-rich
from G-tetrad sequence
and anothermay adopt
four ofdifferent
the adjacentstructures
stacked [8,14]. This occurs
G-tetrad. In the
presence of different cations, the G-rich sequence may adopt different structures [8,14]. Thisquartet
as such ions contribute to variation in the interaction with the grooves and loops [20]. The occurs
arrangement
as such ions generates
contributea to central channel
variation thatinteraction
in the allows the cations
with the to settle
grooves whichandinfluences
loops [20]. theTheinteraction
quartet
arrangement generates a central channel that allows the cations to settle which influences the
of the G4 to the cations. Depending on the type of cations and the specific structure of the G4, the
positions
interactionofofthe thecations
G4 to the cancations.
be located along oronbetween
Depending the typethe quartetand
of cations plane the[20]. Important
specific structure features
of the
of the
G4, thecations
positionsand oftheir
thestructural
cations can influence
be locatedare their
alongradius and valency.
or between the quartetCations supporting
plane the G4
[20]. Important
structure are usually metal ions with the notable exception of the monovalent
features of the cations and their structural influence are their radius and valency. Cations supporting ammonium ion [21]
the G4 Generally,
structureG4 areformation
usually metal and ionsstabilization require exception
with the notable monovalent cations,
of the monovalentin particular,
ammonium Na+ and ion
more commonly, K + . K + is claimed to be more biologically relevant compared to that of Na + due
[21]
to itsGenerally,
higher intracellular
G4 formation concentration.
and stabilizationThe nature
requireof the ions affects
monovalent their exact
cations, location between
in particular, Na+ and
tetrads. +
K are observed be ad libitum +
more commonly, K+. K+ is to claimed to be moreequidistant
biologicallybetween
relevant each tetrad whereas
compared to that ofNaNaare located
+ due to its
within a range of geometries of a G4 [14]. Na + ions are slightly displaced from the G-quartet due to the
higher intracellular concentration. The nature of the ions affects their exact location between tetrads.
electrostatic
K + are observed repulsion
to be between
ad libitum adjacent Na+ , resulting
equidistant between in a slightly
each tetrad altered
whereas coordination
Na+ are located geometrywithin (see
a
Figure of 5) [22]. Not only +
range geometries of Ka G4 has [14].
a betterNacoordination with 06displaced
+ ions are slightly guanines,from this cation also possesses
the G-quartet due to lower
the
dehydration repulsion
electrostatic Na+ -containing
energy. Inbetween adjacentsolutions
+ , G4 formation can also occur but at a
devoidinofaKslightly
Na+, resulting altered coordination geometry
much
(see slower
Figure 5)rate
[22].[23].
NotTherefore,
only K+ has K+a in general
better is preferred
coordination over
with 06Na
+ in G4 formation. Another study
guanines, this cation also possesses
conducted also showed in the presence of KCl, the activity
lower dehydration energy. In Na -containing solutions devoid of K , G4 formation
+ doubled + that of NaCl [24].
can also occur but
at a much slower rate [23]. Therefore, K+ in general is preferred over Na+ in G4 formation. Another
study conducted also showed in the presence of KCl, the activity doubled that of NaCl [24].
Molecules 2019, 24, 1079 6 of 30
Molecules 2019, 24, x FOR PEER REVIEW 6 of 29

Figure 5. Location of K+ +(PDB: IJPG) and Na+ +(PDB: IJB7) in G-quadruplexes.

Figure 5. Location of K (PDB: IJPG) and Na (PDB: IJB7) in G-quadruplexes.
It has also been reported that other monovalent and divalent ions can in fact affect the stability
It has also been reported that other monovalent and divalent ions can in fact affect the stability
and structure of G4 [20]. It was suggested that the stability of G4 structures is influenced by ions in
and structure+of G42+ [20]. It was suggested that the stability of G4 structures is influenced by ions in
the order of K > Ca > Na+ > Mg2+ > Li+ and K+ > Rb+ > Cs+ [25]. It was also demonstrated that
the order of K > Ca > Na+ > Mg2+ > Li+ and K+ > Rb+ > Cs+ [25]. It was also demonstrated that interaction
+ 2+
interaction of G4 with type Ia and IIaa cations with respect to the stabilization of G4, follows the order
of G4 with type Ia and IIaa cations with respect to the stabilization of G4, follows the order of Sr2+ >
of Sr2+ > Ba2 + > Ca2+ > Mg2+ and K+ > Rb+ > Na+ > Li+ = Cs+ [26]. In another study, silver ions (Ag+ )
Ba2+ > Ca2+ > Mg2+ and K+ > Rb+ > Na+ > Li+ = Cs+ [26]. In another study, silver ions (Ag+) was shown to
was shown to also coordinate with guanine bases [27] and disrupt the structure of G4 significantly [28].
also coordinate with guanine bases [27] and disrupt the structure of G4 significantly [28]. This
This happens as Ag+ chelates guanines at the binding sites, N7 and C6 O [27], which are involved in G4
happens as Ag+ chelates guanines at the binding sites, N7 and C6O [27], which are involved in G4
formation. This results in the inhibition of the catalytic activity exhibited by G4-hemin DNAzyme [28].
yle size="10">[92]</style></DisplayText><record><rec-number>98</rec-number><foreign-keys><key app=
A further study has shown that no high order structural change was observe upon Ag+ addition,
[28]. A further study has shown that no high order structural change was observe upon Ag+ addition,
suggesting the destruction of G4 structure [29].
suggesting the destruction of G4 structure [29].
Disruption of G4 structures have also been observed with some divalent cations. Divalent cations
Disruption of G4 structures have also been observed with some divalent cations. Divalent
such as Mn2+ , Co2+ or Ni2+ may disrupt the structure of G4 even in the presence of K+ . Divalent cations
cations such as Mn2+, Co2+ or Ni2+ may disrupt the structure of G4 even in the presence of K+. Divalent
are capable of destabilizing G4 when the G4 and monovalent cation concentrations are sufficiently
cations are capable of destabilizing G4 when the G4 and monovalent cation concentrations are
low [30,31]. However, the effect of trivalent metal ions on G4 structures has not been well studied
sufficiently low [30,31]. However, the effect of trivalent metal ions on G4 structures has not been well
yet. Nevertheless, the trivalent ions were observed to destabilize G4 structures, as they were found
studied yet. Nevertheless, the trivalent ions were observed to destabilize G4 structures, as they were
to be remarkably reduced in the presence of metal chelators [32]. It was also found that the stacking
found to be remarkably reduced in the presence of metal chelators [32]. It was also found that the
of the quartets is better promoted in the presence of trivalent lanthanide metal ions such as La3+ ,
stacking of the quartets is better promoted in the presence of trivalent lanthanide metal ions such as
Eu3+ , Tb3+ , Dy3+ and Tm3+ [33]. This information is critical in the design of stable G4 structures for
La3+, Eu3+, Tb3+, Dy3+ and Tm3+ [33]. This information is critical in the design of stable G4 structures for
various applications.
various applications.
The effect of a number of G4-binding ligands on G4 structure and stability have been widely
The effect of a number of G4-binding ligands on G4 structure and stability have been widely
studied. Telomestatin, a natural product isolated from Streptomyces anulatus 3533-SV4 has been shown
studied. Telomestatin, a natural product isolated from Streptomyces anulatus 3533-SV4 has been
to be a very potent telomerase inhibitor. This is suggested by the similarity of the structure between
shown to be a very potent telomerase inhibitor. This is suggested by the similarity of the structure
telomestatin (see Figure 6a) and G-tetrad. The ability of telomestatin to either trap out pre-formed G4
between telomestatin (see Figure 6a) and G-tetrad. The ability of telomestatin to either trap out pre-
structures or facilitate the formation of G4 (see Figure 6b) can result in the sequestering of the telomeric
formed G4 structures or facilitate the formation of G4 (see Figure 6b) can result in the sequestering of
sequence (single-stranded d[T2 AG3 ]4 sequencecrucial for telomerase activity). Higher concentrations
the telomeric sequence (single-stranded d[T2AG3]4 sequencecrucial for telomerase activity). Higher
of telomestatin has been found to cause a significant increment of intramolecular basket-type
concentrations of telomestatin has been found to cause a significant increment of intramolecular
G4, suggesting that the telomestatin stabilizes or induces the intramolecular G4 formation [34].
basket-type G4, suggesting that the telomestatin stabilizes or induces the intramolecular G4
Interestingly, telomestatin can replace the need for monovalent cations, specifically K+ or Na+ to
formation [34]. Interestingly, telomestatin can replace the need for monovalent cations, specifically
stabilize intramolecular G4 structure, which is a unique trait among G4-interactive compounds.
K+ or Na+ to stabilize intramolecular G4 structure, which is a unique trait among G4-interactive
In contrast, a porphyrin compound, 5,10,15,20-tetrakis-(N-methyl-4-pyridyl)porphine (TMPyP4, see
compounds. In contrast, a porphyrin compound, 5,10,15,20-tetrakis-(N-methyl-4-pyridyl)porphine
Figure 7a) has been shown to preferentially facilitate the formation of intermolecular G4 structure (see
(TMPyP4, see Figure 7a) has been shown to preferentially facilitate the formation of intermolecular
Figure 7b). TMPyP4 is responsible to suppress the proliferation of alternative lengthening of telomeres
G4 structure (see Figure 7b). TMPyP4 is responsible to suppress the proliferation of alternative
in (ALT)-positive cells and inducing anaphase bridges in sea urchin embryos. However, telomestatin
lengthening of telomeres in (ALT)-positive cells and inducing anaphase bridges in sea urchin
embryos. However, telomestatin does not possess this effect suggesting the selectivity of telomestatin
 Molecules 2019, 24, 1079 7 of 30

Molecules 2019, 24, x FOR PEER REVIEW 7 of 29

does not possess this effect suggesting the selectivity of telomestatin for intramolecular G4 structures
and TMPyP4 for intermolecular G4 structures [35]. TMPyP4, mainly known as DNA G4 stabilizer,
for intramolecular G4 structures and TMPyP4 for intermolecular G4 structures [35]. TMPyP4, mainly
was also reported to be able to unfold an extremely stable all purine RNA G4 (M3Q) and regulate the
known as DNA G4 stabilizer, was also reported to be able to unfold an extremely stable all purine
activity of a reporter gene through the disruption of G4 [36].
RNA G4 (M3Q) and regulate the activity of a reporter gene through the disruption of G4 [36].

(a)

(b)
Figure
Figure (a)(a)
6. 6. Chemical
Chemical structure
structure of of telomestatin.
telomestatin. (b)(b) Solution
Solution structure
structure of of
anan intramolecular
intramolecular (3 (3 + 1)
+ 1)
human
human telomeric
telomeric G-quadruplex
G-quadruplex bound
bound toto a telomestatin
a telomestatin derivative
derivative (PDB:
(PDB: 2MB3).
2MB3).

Pyridostatin (see Figure 8a) is a small molecule that is known to particularly stabilize G4
DNA complexes, triggers neurotoxicity and induce DNA double-strand breaks (DSBs) in cultured
neurons [37]. Also, zinc aminophthalocyanine (ZnAPC) (see Figure 8b) has been demonstrated to
bind to a G4-forming oligonucleotide derived from 50 -untranslated region of NRAS mRNA, causing a
selective cleavage of the target RNA G4 upon photo-irradiation [38].
 Molecules 2019, 24, x FOR PEER REVIEW 8 of 29

Molecules 2019, 24, 1079 8 of 30

Molecules 2019, 24, x FOR PEER REVIEW 8 of 29

(a)

(a)

(b)
Figure 7. (a) Chemical structure of TMPyP4 (b) A parallel stranded human telomeric quadruplex in
complex with the porphyrin TMPyP4 (PDB: 2HRI).

Pyridostatin (see Figure 8a) is a small molecule that is known to particularly stabilize G4 DNA
complexes, triggers neurotoxicity and induce DNA double-strand breaks (DSBs) in cultured neurons
[37]. Also, zinc aminophthalocyanine (ZnAPC)(b) (see Figure 8b) has been demonstrated to bind to a
G4-forming oligonucleotide derived from 5’-untranslated region of NRAS mRNA, causing a selective
Figure (a)Chemical
Figure7.7.(a) Chemicalstructure
structureof
ofTMPyP4
TMPyP4(b)
(b)AAparallel
parallelstranded
strandedhuman
humantelomeric
telomericquadruplex
quadruplexinin
cleavage of the target RNA G4 upon photo-irradiation [38].
complexwith
complex withthe
theporphyrin
porphyrinTMPyP4
TMPyP4(PDB:
(PDB:2HRI).
2HRI).

Pyridostatin (see Figure 8a) is a small molecule that is known to particularly stabilize G4 DNA
complexes, triggers neurotoxicity and induce DNA double-strand breaks (DSBs) in cultured neurons
[37]. Also, zinc aminophthalocyanine (ZnAPC) (see Figure 8b) has been demonstrated to bind to a
G4-forming oligonucleotide derived from 5’-untranslated region of NRAS mRNA, causing a selective
cleavage of the target RNA G4 upon photo-irradiation [38].

(a)
Figure 8. Cont.

(a)
Molecules 2019, 24, 1079 9 of 30
Molecules 2019, 24, x FOR PEER REVIEW 9 of 29

(b)
Figure
Figure 8. 8.Chemical
Chemicalstructure
structureof
of(a)
(a) pyridostatin
pyridostatin and
and (b)
(b) zinc
zincaminophthalocyanine
aminophthalocyanine(ZnAPC).
(ZnAPC).

Pressure
Pressure is is
a key
a keythermodynamic
thermodynamicfactor factor that
that can induce
inducestructural
structuralchanges
changesininbiomolecules
biomolecules duedue
to to
a volumetric
a volumetric decrease
decrease[39].
[39].Depending
Depending on on the specific
specific sequence,
sequence,G4 G4structures
structureshave
have been
been found
found to to
exhibit
exhibit exceptionallyhigh
exceptionally highthermodynamic
thermodynamic stability
stability [40].
[40]. This
Thissuperior
superiorproperty
propertyhas hasbeen
been exploited
exploited
in applications such as isothermal amplification in order to disrupt
in applications such as isothermal amplification order to disrupt the duplex structure for the duplex structure for primer
primer
binding
binding [41].However,
[41]. However,non-canonical
non-canonical DNA DNA structures
structuresare aremore
moresensitive
sensitivetotothetheeffects
effectsof of
pressure
pressure
compared
compared to to duplexeswhich
duplexes whichhave
havehigher
higherstability
stability under
under pressure
pressure[39].
[39].G4G4exhibits
exhibits positive
positive andandlarge
large
ΔV tr values, suggesting that thermal stability would decrease with increasing pressure resulting in
∆V tr values, suggesting that thermal stability would decrease with increasing pressure resulting in the
the unfolding
unfolding of G4.ofAG4. A study
study conducted
conducted using using
UV UV melting
melting underunder
highhigh pressure
pressure waswasableable
totomonitor
monitorthe
the unfolding process of human telomeric oligonucleotides. It was suggested
unfolding process of human telomeric oligonucleotides. It was suggested that the ∆Tm /∆P is less than that the ΔT m/ΔP is less

than −10
× 10−2× K 10−2 K·MPa −1. Hydration of biomolecules is largely affected by pressure, unlike folding of
−10 ·MPa−1 . Hydration of biomolecules is largely affected by pressure, unlike folding of a
a nucleic acid duplex which takes up water molecules during the folding process. Osmotic pressure
nucleic acid duplex which takes up water molecules during the folding process. Osmotic pressure
analysis showed that water molecules are released during the folding of G4 [39]. Therefore, there is
analysis showed that water molecules are released during the folding of G4 [39]. Therefore, there
a potential to develop nano-materials triggered by pressure effects, considering the difference in
is a potential to develop nano-materials triggered by pressure effects, considering the difference in
sensitivity of each DNA structure to pressure.
sensitivity of each DNA structure to pressure.
Previously available assay allows the identification of G4 formation with hyperchromism at 260
nmPreviously available at
and hypochromism assay allows
295 nm but the
it isidentification of G4 all
not able to identify formation with hyperchromism
three (parallel, antiparallel and at
260hybrid)
nm and G4 types [42]. A recent study investigated the relationship between thermalantiparallel
hypochromism at 295 nm but it is not able to identify all three (parallel, denaturation and
hybrid)
profilesG4andtypes
G4 [42]. A recent
structure basedstudy
on UV investigated
absorbance the [17].relationship
The meltingbetween
profiles ofthermal denaturation
three types of G4
profiles and G4 structure based on UV absorbance [17]. The melting profiles
structures (parallel, antiparallel and hybrid) at 243, 260, 275 and 295 nm suggest that the G4 topology of three types of G4
structures (parallel, antiparallel and hybrid) at 243, 260, 275 and 295 nm suggest
affect the denaturation profile of G4 (see Table 1). The work also developed an inexpensive assay that that the G4 topology
affect
not the
onlydenaturation
distinguishesprofileG4 andofDNAG4 (see Table 1).
duplexes The workbut
or triplexes, also developed
also an inexpensive
between parallel assayof
conformation that
notG4
only distinguishes
with the other G4 two andstructures
DNA duplexes or triplexes,
(antiparallel and but also between
hybrid). This was parallel conformation
achieved due to the of G4
hypochromism
with the other twoexhibited
structuresby parallel G4and
(antiparallel structures
hybrid). inThis contrast to the antiparallel
was achieved and hybrid
due to the hypochromism
structures
exhibited which shows
by parallel hyperchromism
G4 structures in contrastatto275 nm [17]. These
the antiparallel recent structures
and hybrid findings on whichthermal
shows
denaturation profiles
hyperchromism at 275 nmof G4 could
[17]. potentially
These boost further
recent findings development
on thermal of G4 in
denaturation sensingofsystems
profiles G4 could
and provide
potentially boostnewfurther
insightsdevelopment
for the establishment
of G4 inofsensing
novel sensing
systems strategies.
and provide new insights for the
establishment of novel sensing strategies.
Table 1. Thermal denaturation profile of different conformations of G4.
Table 1. Thermal denaturation profile of different
λ (nm) conformations of G4.
Conformation
243 260 275 295
λ (nm)
ConformationParallel + - - -
243 260 275 295
Antiparallel + -/+ + -
Parallel Hybrid + + −+ + −- −
Antiparallel + −/+ + −
(+) Hyperchromism and (−) hypochromism observed upon denaturing/heating the structure.
Hybrid + + + −
(+) Hyperchromism and (−) hypochromism observed upon denaturing/heating the structure.
Molecules 2019,24,
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3. G-Quadruplex-Based Detection System

3. G-Quadruplex-Based Detection System
3.1. DNAzyme-Based Colorimetric G-quadruplex/Hemin System
3.1. DNAzyme-Based Colorimetric G-Quadruplex/Hemin System

3.1.1.Conventional
3.1.1. ConventionalDNAzyme-Based
DNAzyme-BasedG-Quadruplex/Hemin
G-quadruplex/Hemin System
System
Horseradishperoxidase
Horseradish peroxidase(HRP)
(HRP)can canbe
beused
usedininananenzyme-conjugated
enzyme-conjugatedassay assayasasaalabel
labelmainly
mainlytoto
detect proteins in immunoassays and can also be adopted for DNA detection
detect proteins in immunoassays and can also be adopted for DNA detection too. As modifications cantoo. As modifications
can
be be expensive
expensive and cumbersome,
and cumbersome, the availability
the availability of a peroxidase-mimicking
of a peroxidase-mimicking DNAzymeDNAzyme formed
formed by
by the
the binding
binding of with
of a G4 a G4hemin
with is
hemin is an interesting
an interesting alternative alternative [43-45].
[43–45]. The The peroxidase
peroxidase mimickingmimicking
catalytic
catalytic activity of G4 DNAzyme catalyzes the oxidation
0 of 2,2′-azinobis(3-ethylbenzothiazoline-6-
activity of G4 DNAzyme catalyzes the oxidation of 2,2 -azinobis(3-ethylbenzothiazoline-6-sulfonic acid)
sulfonic
(ABTS 2− ) acid) (ABTSa2−colorimetric
to produce ) to producechange
a colorimetric changesolution
from a colorless from a colorless solution
to a visible blue-greento a product
visible blue-
(see
green product (see Figure 9). DNAzyme systems involving other substrates
0 0
Figure 9). DNAzyme systems involving other substrates like luminol, 3,3 ,5,5 -tetramethylbenzidinelike luminol, 3,3’,5,5’-
tetramethylbenzidine
sulfate sulfate (TMB),
(TMB), 4-chloro-1-naphthol 4-chloro-1-naphthol
(4-CN) (4-CN)
or scopoletin (Sc), or scopoletin
in the presence of (Sc), in the presence
hydrogen peroxideof
hydrogen
(H peroxide (H2O2) can also be used to produce colorimetric, electrochemical and fluorescence
2 O2 ) can also be used to produce colorimetric, electrochemical and fluorescence readouts [46–51].
readouts [46-51].
However, colorimetric However,
G4-hemincolorimetric G4-heminbiosensors
DNAzyme-based DNAzyme-based biosensors
are utilized are utilized
in diagnostics for itsin
diagnostics for its simplicity, fast analysis, low cost and most notably, the ability
simplicity, fast analysis, low cost and most notably, the ability to detect target molecules visually. to detect target
molecules
The ABTS2− visually. Thesupplemented
substrate ABTS substrate
2−
withsupplemented
H2 O2 DNAzyme with Hcan2O2 DNAzyme can be designed to
be designed to generate the
generate the colorimetric change as the outcome of the detection
colorimetric change as the outcome of the detection of a particular target. of a particular target.

Figure 9. A schematic illustration of simple colorimetric G-quadruplex DNAzyme system.

Figure 9. A schematic illustration of simple colorimetric G-quadruplex DNAzyme system.
Single nucleotide polymorphisms (SNPs) is a condition that may lead to the development of
numerousSinglehuman diseases
nucleotide which includes(SNPs)
polymorphisms complications such as that
is a condition Alzheimer’s,
may leadsickle celldevelopment
to the anemia, cysticof
fibrosis
numerous and human
certain cancers
diseases[52]. A series
which of sensors
includes utilizingsuch
complications proteinas enzymes havesickle
Alzheimer’s, been established
cell anemia,
for the detection
cystic fibrosis andof SNPs. Restriction
certain cancers enzymes can also
[52]. A series of be employed
sensors to detect
utilizing SNPs,
protein but are have
enzymes limited to
been
the recognition
established for sequence of the
the detection ofenzyme recognition
SNPs. Restriction domain.can
enzymes Ligase was
also be also reported
employed to beSNPs,
to detect capablebut
ofare
detecting
limited to SNPs in random DNA
the recognition sequences
sequence [53,54], but
of the enzyme this typedomain.
recognition of assayLigase
is relatively
was also expensive
reported
to be
and capable oftodetecting
vulnerable SNPs in random
the surrounding conditions DNA sequences
[55]. In view of [53,54], but this type
the limitations of assay methods,
of existing is relatively
a
expensive and vulnerable to the surrounding conditions [55]. In view of the limitations of existing
turn-off colorimetric sensor was developed utilizing the aforementioned DNAzyme system for the
methods, aofturn-off
recognition SNPs basedcolorimetric sensorDNAzyme
on G4-hemin was developed utilizingDNA
and Y-shaped the aforementioned
duplex. In this assayDNAzyme (see
system10),
Figure forOligo
the recognition of SNPsand
1 (G-rich sequence) based on 2G4-hemin
Oligo DNAzyme
were designed and Y-shaped
to be partially DNA duplex.
complementary to eachIn
this assay
other. Oligo (see Figure
1, Oligo 2 and10), Oligo
target DNA 1 (G-rich sequence)
will hybridize withandeachOligo
other 2towere
form designed
a Y-shapedtoDNA be partially
duplex
complementary
(3-way junction) into the
each other. Oligo
presence 1, Oligo
of target DNA. 2 and
Thistarget DNA will
will render hybridize with
the DNAzyme each other
sequence to form
unavailable
a Y-shaped
for DNA duplex
catalytic reaction, (3-way junction)
thus inactivating in the presence
the DNAzyme. Thereforeof thetarget DNA.
resulting This will
outcome render the
is a decrease of
DNAzyme
the absorbance sequence
readoutunavailable for catalytic
of the oxidation productreaction, 2 −
of ABTS thus inactivating the DNAzyme. Therefore
[56].
the resulting outcome is a decrease of the absorbance readout of the oxidation product of ABTS2− [56].
Molecules2019,
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11 of 29

Figure 10. Schematic illustration for sequence-specific recognition of single-stranded DNA based upon
Figure 10.
Y-shaped Schematic
DNA duplex illustration
and G4-hemin for DNAzyme.
sequence-specific recognition of single-stranded DNA based
upon Y-shaped DNA duplex and G4-hemin DNAzyme.
An abnormal concentration of sodium (Na+ ) will affect both cellular and electrical functions
An abnormal
in biological systems.concentration
Hence, it isofassociated
sodium (Na with+) will affect both cellular and electrical functions in
many diseases such as heart failure and kidney
biologicalQuantitative
diseases. systems. Hence,detection +
it isofassociated
Na is required with for many diseases such
the diagnosis of suchas diseases.
heart failure and akidney
However, large
amount K+ can interfere
diseases.ofQuantitative detection
with Na of +Na + is required for the diagnosis of such diseases. However, a
sensing in physiological systems making the development of
Na + detection
large amount of K+ can ainterfere
systems with Na+As
huge challenge. sensing
G4s have in physiological systems towards
a higher sensitivity making the K+ compared
development to
Na +
of Na + detection
, this makes G4 systems
basedasensor
huge challenge.
development As G4s
for Na +
haveeven a higher
moresensitivity
challenging. towards
A study K reported
+ comparedthe to
Na+, this makes
development of aG4
Na + ion sensor
based sensor development
guided by thefor Nathat
fact + even more challenging. A study reported the
there are specific G4s possessing different
development of
conformations a Na+ ionon
depending sensor guided by
the presence the fact
of either K+thator Na + [57].
there areInspecific G4s possessing
this study, a known G4different
named
conformations
p25 was utilized depending
due to itsonability
the presence
to exhibit of either K or Na conformation
+
an antiparallel + [57]. In this study, withaNa + butG4
known named
a hybrid
p25 was utilized
conformation upondue to its ability
the presence of K+to. With
exhibit
increasing Na+ concentration,
an antiparallel conformation even with Napresence
in the of K+ ,
+ but a hybrid

conformation
the p25 G4 undergoes upon theapresence of K+. With increasing
specific conformational transition.Na+ concentration,
Due to the difference even inin thebinding
presence of K+,
affinity
theG4
of p25 G4 undergoes
various conformationa specific
towardsconformational
hemin (discrepancytransition. inDue to theactivity),
catalytic difference and inby
binding affinity
applying the
of G4 various
ABTS-H O conformation
system, a towards
colorimetric probehemin
for Na + was successfully
(discrepancy in catalytic activity),
established and
[58]. by applying the
2 2
ABTS-HFurther2O2 system,
research a colorimetric
on G4-hemin probe for Na was
DNAzyme + successfully
reported established [58].
that terbium/G4-hemin DNAzyme is
able to Further
exhibit research on G4-hemin
a significantly higher DNAzyme
catalytic reported
activity in that
comparison with K+ -induced
terbium/G4-hemin DNAzyme is able to
G4-hemin
exhibit a significantly higher catalytic activity in comparison
DNAzyme [51]. The quantitative data obtained from this study showed that the activity factor with K +-induced G4-hemin DNAzyme

of Tb3+
[51]. The quantitative
-induced G4-hemindata obtained
DNAzyme from K+study
andthis -induced showed G4-heminthat the activity factor
DNAzyme were of Tb3+and
11.55 -induced
2.02
G4-hemin DNAzyme
respectively [51], clearly and K+-induced
indicating thatG4-hemin
the former DNAzyme
has a higher were 11.55 andactivity
peroxidase 2.02 respectively [51],
than the latter.
clearly
This wouldindicating
allow forthata the former
wider has a higher
application of theperoxidase
sensing platform activityasthan thepreviously
it was latter. Thisthought
would allow
to be
for a wider
restricted only K+ andofNa
to application + as
the sensing platform
the inducer of G4 asstructure
it was previously
formation. thought to be restricted to only
K+ and Na+mentioned
It was as the inducer of that
earlier G4 structure
G4-heminformation.
DNAzyme would be significantly inhibited in the presence
It was
of silver ions mentioned
[28]. However, earlier
the that G4-hemin
addition DNAzyme
of hydrogen sulfidewould(H2 S) is beable
significantly
to remove inhibited in the
the coordinated
Ag + through
presence of silver ions [28].
competitive However,
binding owing thetoaddition
its strong ofaffinity
hydrogen to Ag + [59].
sulfide (HThis
2S) is able tothen
would remove
remove the
coordinated Ag +
+ through competitive binding owing to its strong affinity
the inhibitory effect by Ag , thus by reforming the G4 as well as its peroxidase-like activity. CD to Ag + [59]. This would then

removeanalysis
spectra the inhibitory effect byG4-hemin
of Tb-induced Ag+, thusDNAzyme
by reforming showedthe G4 as well as change
a structural its peroxidase-like
to the originalactivity.
state
CD spectra
upon addition analysis
of H2 SofinTb-induced
the presence of Ag+ ions.
G4-hemin DNAzyme The ability showedof Tba3+structural change to the
-induced G4-hemin original
DNAzyme
state
to upon
exhibit addition
a higher of H2Sactivity
catalytic in the compared
presence of to K Ag+ -induced
+ ions. The ability of Tb3+-induced G4-hemin
G4-hemin DNAzyme, a colorimetric
DNAzyme to exhibit a higher catalytic activity compared to K+-induced G4-hemin DNAzyme, a
Molecules 2019, 24, 1079 12 of 30

detection system for H2 S was developed [29]. This is important as H2 S is found to take part in various
physiological processes and its abnormal level has been associated with symptoms of various diseases
such as Down syndrome, Alzheimer disease and diabetes. In this setup, Tb/G4-hemin DNAzyme
was first prepared before introducing Ag+ to suppress the peroxidase-like activity. The addition of
Ag+ causes the green color of the solution to change to colorless and the absorbance of ABTS-H2 O2 to
decrease, reflecting the loss in catalytic activity. A green color was found to reappear upon the addition
of H2 S indicating the reformation of the G4 structure and the recovery of the peroxidase-like activity.
With the enhancement of H2 S concentration, the absorbance of ABTS-H2 O2 increases gradually which
forms the basis of the system. The selectivity of this detection system was examined by introducing
various competing anions. Even at concentrations of 100-fold higher, the tested anions were not
able to recover the catalytic activity of Tb/G4-hemin DNAzyme. This implies the sensitivity of the
detection system towards H2 S [29]. The claim that Tb3+ -induced G4-hemin DNAzyme exhibits a
higher peroxidase-like activity than the G4-hemin DNAzyme induced by K+ and Na+ was further
examined in this study by adding K+ and Na+ into the system instead of Tb3+ . The result was found to
be consistent in which the Tb3+ -induced system was able to exhibit higher peroxidase-like activity [51].
Not only did this system exhibited a higher catalytic activity, it also displayed the ability to be applied
on serum samples with no retardation of activity [29].

3.1.2. DNAzyme-Based Colorimetric Split G-Quadruplex System

It was found that the structure of G-rich nucleic acid sequences can be split to two parts and act
as a binary probe, which are reported to exhibit extraordinary selectivity [60]. In a split system, two
separate short G-rich oligonucleotides can be combined to form a common G4 wherein the guanines
of the G4 are usually distributed on two different strands allowing the target strand to bring them
together through hybridization. Formation of a G4 structure can thus turn on the catalytic property for
signal readout [61]. The number of bases of the whole split G4 is often twelve. In has been reported
that the separation of split G4 designs was best implemented in the loop region of the G4, thus the
twelve G bases of the G4 are always divided in ratios of either 2:2 or 1:3. However, a study reported
that the assembly of two equally-split parts could easily form an active hemin aptamer even in the
absence of target DNA producing a background signal [62].
Hence, a number of studies resorted to splitting the unabridged G-rich strand into an asymmetric
ratio instead [63,64]. In a system involving the asymmetrical split DNAzyme strategy to rapidly and
visibly detect rifampin-resistant M.tb with mutations in the rpoB gene, unequal ratio (3:1) of probe
A and probe B were assembled through hybridization with the target ssDNA [64]. Since the target
DNA was originally in dsDNA form, asymmetric PCR was applied to produce ssDNA. To ensure
sufficient quantity of target DNA was available and to increase the sensitivity of the assay, nested
PCR was conducted. Interestingly, the PCR product was added into the detection system without
the need for purification prior to the assay. A new strategy was designed to minimize the adverse
effects of complicated secondary structures of the target DNA to further improve the efficiency of the
probes. The 50 -end of Probe A was designed to match the 30 -end of the target ssDNA with three GGG
repeats. The 30 -end of Probe B contained one GGG repeat was designed to match the 50 -end of the
target DNA. Therefore, two dsDNA were formed at the two ends of the target DNA when the target
DNA was added to the mixture of probes A and B. A G4 structure was formed in the middle due to the
folding of the overhanging strands from Probe A and Probe B. The formed DNA G4 is then converted
into a DNAzyme that will subsequently catalyze the H2 O2 -mediated oxidation of ABTS2− through
peroxidation-like activity when bound with hemin. As a result, an oxidation product, ABTS− was
formed and a visible green color was observed [64].
Further studies on split G4 have been done to elucidate the effects of split G4 guanine ratio. The
effects of separation between the guanine bases with either more or less guanine bases in different
strands of the split G4 was studied to determine the optimal ratio to split the G4. Six split modes
were tested and it was shown that the performances of the modes 3:9 and 6:6 were both moderate
 Molecules 2019, 24, 1079 13 of 30

Molecules
whereas2019,the
24, xbest
FOR performing
PEER REVIEWmode
was 4:8 [65]. The employment of split G4 has allowed13an of additional
29

dimension in sensor designs affording more flexibility. In comparison with G4 detection systems
using unabridged probe strands where the results are limited, this splitting strategy allows the G-
using unabridged probe strands where the results are limited, this splitting strategy allows the G-rich
rich segments to integrate into many patterns and the best ratio mode could be identified, leading to
segments to integrate into many patterns and the best ratio mode could be identified, leading to a
a reduced background signal.
reduced background signal.
3.1.3. DNAzyme-Based Colorimetric G-quadruplex/Hemin Molecular Beacon System
3.1.3. DNAzyme-Based Colorimetric G-Quadruplex/Hemin Molecular Beacon System
The split G4 design can also be applied to the molecular beacon (MB) concept devoid of the
The split G4 design can also be applied to the molecular beacon (MB) concept devoid of the
fluorophore-quencher complex whilst maintaining their specific catalytic activity. MB was first
fluorophore-quencher
reported in 1996 and has since complex
beenwhilst
one of maintaining
the most widely theirused
specific catalytic
systems activity.
for DNA MB was
biosensors first reported
[66,67].
in 1996 and has since been one of the most widely used systems for DNA
Originally, the MB system alone relies on the concept of a closed to opened conformational change biosensors [66,67]. Originally,
in the presence of a target DNA. The conformational change can be detected with simultaneouspresence
the MB system alone relies on the concept of a closed to opened conformational change in the
of a target
production of aDNA. The conformational
fluorescence signal. MBs arechange basically can be detected with
hairpin-shaped simultaneous
oligonucleotides thatproduction
contain of a
fluorescence
a stem-loop signal.
structure [68].MBs
TheareDNA basically
bases on hairpin-shaped
both sides of the oligonucleotides
stems are connected that contain
by hydrogen a stem-loop
structure
bonds whereas[68].the
The5’-DNA
and bases
3’-endonofboth the sides
MBs of arethe stems are connected
respectively labeled with by hydrogen bonds
a fluorophore andwhereas
a the
0
quencher 0
5 - and [69].
3 -end Theof construct
the MBs are willrespectively
then unfold labeled
in thewith a fluorophore
presence of the target and sequence
a quencher to [69]. The construct
hybridize,
resulting
will thenin unfold
the separation of the fluorophore
in the presence of the targetfrom the quencher
sequence to yield
to hybridize, a fluorescent
resulting output. of the
in the separation
However, besides
fluorophore frombeing
the costly
quencher it is to
known
yield that the utilization
a fluorescent output.of the fluorophore
However, besidesand quencher
being costlyalso
it is known
requires complicated modification and complex operation making
that the utilization of the fluorophore and quencher also requires complicated modificationit cumbersome [69]. Inand
thiscomplex
alternate rendition
operation makingof MB, G4 MB-based[69].
it cumbersome detection
In this of alternate
nucleic acids involves
rendition of no
MB,fluorophore-quencher
G4 MB-based detection of
complex
nucleic acids involves no fluorophore-quencher complex but incorporates thestem
but incorporates the guanines needed for the formation of the G4 into the formation
guanines needed of for the
theformation
MB (see Figure 11). Subsequent binding to the target will open the MB stem
of the G4 into the stem formation of the MB (see Figure 11). Subsequent binding to the target permitting the
guanines
will opento be made
the available
MB stem to engage
permitting theinguanines
G4 formation.
to be made available to engage in G4 formation.

Figure 11. Typical DNAzyme-based colorimetric G-quadruplex/hemin-molecular beacon system.

Figure 11. Typical DNAzyme-based colorimetric G-quadruplex/hemin-molecular beacon system.
Considering the advantages of MB and split G4 with the drawbacks of employing the
Consideringand
fluorophore thequencher,
advantages of MB
a study and split
combining theG4MBwith the drawbacks
technique of employing
and split G4 structure tothe
produce a
fluorophore and quencher, a study combining the MB technique and split G4 structure
label-free G-quadruplex DNAzyme MB oligonucleotide sensor for Pseudostellaria heterophylla (PH) to produce a was
label-free
developedG-quadruplex DNAzyme
[70]. In this system, aMB oligonucleotide
split mode G4 wassensor for in
utilized Pseudostellaria
place of theheterophylla (PH)
fluorophore/quencher
wascombination
developed [70]. In this
where onesystem, a splitserved
GGG repeat mode G4
as awas utilized
reporter andin tethered
place of the fluorophore/quencher
to both ends of the MB structure.
combination where one GGG repeat served as a reporter and tethered
A section of the oligonucleotide specific for the target sequence was made to bothto ends of the MBthe loop
complement
structure. A section of the oligonucleotide specific for the target sequence was made to complement
portion and act as the sensing element of the assay [70]. In the absence of target DNA, one pair of GGG
the loop portion and act as the sensing element of the assay [70]. In the absence of target DNA, one
repeat associates and forms an intermolecular G4 structure. A DNAzyme is generated through the
pair of GGG repeat associates and forms an intermolecular G4 structure. A DNAzyme is generated
high affinity binding of hemin to the formed G4. This complex results in a peroxidase-like activity to
through the high affinity binding of hemin to the formed G4. This complex results in a peroxidase-
like activity to produce a colored product with a strong UV-vis absorption signal. In the presence of
Molecules 2019, 24, 1079 14 of 30

produce a colored product with a strong UV-vis absorption signal. In the presence of the target DNA,
the target DNA will hybridize to the loop of the MB inhibiting the formation of intermolecular G4
Molecules 2019, 24, x FOR PEER REVIEW
DNAzyme due to the opening of the stem to form an inactive duplex DNA structure. This 14 of 29
results in a
catalytic activity loss with a weaker UV-vis absorption signal in the presence of
the target DNA, the target DNA will hybridize to the loop of the MB inhibiting the formation ofthe target DNA [70].
Not only can aG4DNAzyme
intermolecular DNAzyme MB due act as aopening
to the catalyticof enzyme
the stem and target
to form an recognition element,
inactive duplex DNA this
multifunctional
structure. This label-free
results inprobe can also
a catalytic function
activity as aa primer
loss with weaker for polymerization
UV-vis [71].in A
absorption signal thenovel
label-free DNAzyme
presence MBDNA
of the target strategy
[70]. was developed for the colorimetric amplification detection of p53
DNA. ForNot theonly can a DNAzyme
DNAzyme MB probe MBalone,
act as in
a catalytic enzyme
the presence of and target
target p53recognition
DNA the element,
catalyticthisactivity
of peroxidase-mimicking DNAzyme is locked in its hairpin structure resulting in anovel
multifunctional label-free probe can also function as a primer for polymerization [71]. A signal label-
readout.
free DNAzyme
Meanwhile, MB strategy was
the hybridization of developed for the colorimetric
the recognition element of amplification
MB with targetdetection
DNA of p53 DNA. the
promotes
For the DNAzyme MB probe alone, in the presence of target p53 DNA the catalytic activity of
formation of G4, triggering isothermal circular strand-displacement polymerization (ICSDP) reaction.
peroxidase-mimicking DNAzyme is locked in its hairpin structure resulting in a signal readout.
This provides a positive readout signal and dynamic response range of 7 orders of magnitude even
Meanwhile, the hybridization of the recognition element of MB with target DNA promotes the
without chemical
formation of modifications
G4, triggeringand additional
isothermal nucleic
circular acids [71].
strand-displacement polymerization (ICSDP)
reaction. This provides a positive readout signal and dynamic response range of 7 orders of
3.2. Non-Covalent Fluorescence-Based G-Quadruplex System
magnitude even without chemical modifications and additional nucleic acids [71].

3.2.1.3.2.
Conventional Non-Covalent Fluorescence-Based G-Quadruplex Sensing System
Non-Covalent Fluorescence-Based G-Quadruplex System
The early studies in the field of DNA-based sensing commonly used covalently-conjugated
3.2.1. Conventional Non-Covalent Fluorescence-Based G-quadruplex Sensing System
oligonucleotides which are labeled with fluorophore/quencher or donor/acceptor pairs. However,
The early studies
covalent conjugation of theinfluorophore
the field of hasDNA-based sensing commonly
some drawbacks as it may used covalently-conjugated
interfere with the operation of
oligonucleotides which are labeled with fluorophore/quencher or donor/acceptor
the assay, influencing the selectivity and binding affinity of the functional oligonucleotides. pairs. However,Another
limitation is the cost and time required to produce such conjugated oligonucleotides.theThus,
covalent conjugation of the fluorophore has some drawbacks as it may interfere with operation
label-free
of the assay, influencing the selectivity and binding affinity of the functional oligonucleotides.
approaches have emerged as a practical and cost-effective alternative to the fluorescently-labeled
Another limitation is the cost and time required to produce such conjugated oligonucleotides. Thus,
oligonucleotides in DNA-based sensing applications. This alternative approach allows for the probes to
label-free approaches have emerged as a practical and cost-effective alternative to the fluorescently-
interact non-covalently
labeled oligonucleotides withinDNA via different
DNA-based binding modes
sensing applications. Thisand eliminates
alternative the need
approach allowsfor
forcovalent
the
attachment to the nucleic acid backbone. The probes may bind either through
probes to interact non-covalently with DNA via different binding modes and eliminates the need for intercalation, groove
binding,
covalent attachment to the nucleic acid backbone. The probes may bind either through intercalation,of the
end stacking or electrostatic interactions that is designed not to affect the functionality
DNAgroove
[72,73].binding, end stacking or electrostatic interactions that is designed not to affect the
functionality of
Commonly the DNA
used [72,73]. G4 dyes includes derivatives of carbocyanine, phthalocyanines,
fluorescent
porphyrin, Commonly
carbazole, used fluorescent G4ethidium
anthracyclines, dyes includes derivatives
bromide of carbocyanine, phthalocyanines,
and triphenylmethane [73–75]. One of the
porphyrin, carbazole, anthracyclines, ethidium bromide and triphenylmethane
most popular and commonly studied G4 ligands which binds and stabilizes different [73-75]. Onetypes
of theof G4
most popular and commonly studied G4 ligands which binds and stabilizes different types of G4
structures is porphyrin [74]. Their binding to G4 can dramatically enhance the florescence intensity of
structures is porphyrin [74]. Their binding to G4 can dramatically enhance the florescence intensity
porphyrins. Protoporphyrin IX (PPIX) (see Figure 12), a ubiquitous heme precursor, has been shown
of porphyrins. Protoporphyrin IX (PPIX) (see Figure 12), a ubiquitous heme precursor, has been
to beshown
a G4-selective fluorescentfluorescent
to be a G4-selective probe in vitro. Fluorescence
probe in enhancement
vitro. Fluorescence was reported
enhancement to increase
was reported to
by 16-fold
increase by 16-fold with the addition of G4 to PPIX [76]. The preference of using PPIX as a reporter
with the addition of G4 to PPIX [76]. The preference of using PPIX as a signal signal is
mainly due to
reporter is the large
mainly duedependency of the affinity
to the large dependency between
of the affinityitbetween
and theit G4
andonthethe
G4 integrity of G4 [77].
on the integrity
It exhibits weaker
of G4 [77]. affinity
It exhibits for duplex
weaker affinityand
for anti-parallel G4 DNA, G4
duplex and anti-parallel butDNA,
selectively binds tobinds
but selectively parallel
to G4
parallel G4 DNA. In addition, the affinity can be controlled by the input DNA
DNA. In addition, the affinity can be controlled by the input DNA through hybridization [74,78]. through hybridization
[74,78].

Figure Chemical
12.12.
Figure Chemicalstructure of protoporphyrin
structure of protoporphyrin IXIX (PPIX).
(PPIX).
Molecules 2019, 24, x FOR PEER REVIEW 15 of 29
Molecules 2019, 24, 1079 15 of 30
Molecules 2019, 24, x FOR PEER REVIEW 15 of 29
In a study conducted to measure the length of DNA based on the base number between two
selected
In aasequences,
In study PPIX was
study conducted
conducted to chosen the
to measure
measure as
thealength
probe of
length toDNA
of studybased
DNA the split
based on G4 base
on the
the formation
base number
number (see Figure two
between
between 13).
two
Therein,
selected two G-rich
sequences, strands
PPIX waswere hybridized
chosen as a to
probethe totarget
studystrand,
the driving
split G4
selected sequences, PPIX was chosen as a probe to study the split G4 formation (see Figure 13). the G-rich
formation strands
(see together
Figure 13).
in the middle
Therein,
Therein, two of the complex
two G-rich
G-rich strands molecule.
strands were
were PPIX in
hybridized
hybridized tosolution
to the
the target bound
target to the
strand,
strand, G4 and
driving
driving theits
the fluorescence
G-rich
G-rich strandsintensity
strands together
together
decreased
in the middle dramatically
of the in
complex the presence
molecule. PPIXof additional
in solution bases
bound intothe
the middle
in the middle of the complex molecule. PPIX in solution bound to the G4 and its fluorescenceG4 and of
its the target
fluorescence strand. The
intensity
addition
intensity of
decreased extra bases
dramatically
decreased in
in the
themiddle
dramatically in theofpresence
presence theadditional
of target of strand results
bases
additional inbases
theinmiddle
anthe
in unstable
of thestructure
middle target
of of split
strand.
the target G4
The
strand.
due to
addition theof increase
extra distance
bases in thebetween
middle them.
of the This
target in turn
strand decreases
results the
in an affinity
unstable
The addition of extra bases in the middle of the target strand results in an unstable structure of splitof the split
structure G4
of for PPIX
split G4
resulting
due to the
G4 due toin a decreased
increase
the increase fluorescence
distance between
distance betweenintensity
them. This[79].
them. in
Thisturnin decreases the affinity
turn decreases of theof
the affinity split
theG4 forG4
split PPIXfor
resulting in a decreased fluorescence intensity
PPIX resulting in a decreased fluorescence intensity [79]. [79].

Figure
Figure 13.
13. Activation
Activation of
of fluorescence
fluorescence of
of PPIX
PPIX triggered
triggered by
by distance
distance between
between two
two split
split G-quadruplex
G-quadruplex
strands
Figure (x:
13. number of
Activation bases
of added).
fluorescence
strands (x: number of bases added). of PPIX triggered by distance between two split G-quadruplex
strands (x: number of bases added).
Uniquely, G4 is able able to
to catalyze
catalyze Cu Cu2+2+ insertion into the PPIX (metalation) (see Figure 14a)
insertion into the PPIX (metalation) (see Figure 14a)
quenching
quenching the fluorescence of the responding ligand
the
Uniquely, fluorescence
G4 is able of
to the responding
catalyze Cu ligand to
2+ insertion produce
into
to aa decreased
the PPIX
produce fluorescence
(metalation)
decreased intensity.
(see Figure
fluorescence 14a)
intensity.
However,
However, aathe
quenching similar phenomenon
fluorescence
similar phenomenonof the is not reported
reported
responding
is not when
ligand
when using
to using
produce other toxic metal
a decreased
other toxic metal ions [80].
[80]. High
fluorescence
ions High Cu2+
intensity.
Cu 2+

levels
levels in
However, in vivo is claimed
a similar
vivo is claimed to be
phenomenon
to be toxic
toxic especially
is especially
not reported when
whenwhen theusing
the levelsother
levels are beyond
are beyond cellular
toxic metal needs.
ionsneeds.
cellular This
[80]. High
This Cuhigh
high2+

level
levels of
in Cu
level of Cu can be associated with some serious neurodegenerative medical conditions such as
vivo2+
2+ can
is be
claimed associated
to be with
toxic some
especiallyserious
when neurodegenerative
the levels are beyond medical
cellular conditions
needs. such
This high
as
Wilson
level
Wilson disease,
of disease, Menkes
Cu2+ canMenkes disease
be associated and
and Alzheimer’s
disease with some serious
Alzheimer’s disease [81].
[81]. Therefore,
neurodegenerative
disease Therefore, there
there is
medical aa need
need for
isconditions aa rapid
forsuch as
rapid
and
and sensitive
Wilson disease,
sensitive detection
Menkesmethod
detection disease of
method and
of such metal
metal ions.
Alzheimer’s
such ions. Taking
disease
Taking[81].advantage of
of this
Therefore,
advantage interaction
there
this is a needof
interaction forG4
of G4a in
in the
rapid
the
presence
and
presence of
sensitive Cu
of Cu 2+
2+ ,, aa sensitive
detection method
sensitive G4-based
of such probe
G4-based metal for
for Cu
probe ions. CuTaking
2+
2+ detection was
was developed
advantage
detection of [80].
[80]. PPIX
this interaction
developed of G4
PPIX actsinas
acts theaa
as
fluorescent
presence
fluorescentof probe
Cu2+, aand
probe and binds to
sensitive
binds to G4 in
in the
G4-based
G4 theprobe
absence
for of
absence of Cu
CuCu 2+ (see Figure
2+ detection
2+ was14b).
(see Figure developed [80]. PPIX acts as a
14b).
fluorescent probe and binds to G4 in the absence of Cu2+ (see Figure 14b).

Figure 14. (a) Insertion of Cu2+ into PPIX (metalation) catalyzed by G4. (b) A schematic illustration of
non-covalent
Figure 14. (a) fluorescence-based G-quadruplex
Insertion of Cu2+ into sensing
PPIX (metalation) system utilizing
catalyzed PPIX
by G4. (b) for Cu2+ illustration
A schematic detection. of
non-covalent
Figure fluorescence-based
14. (a) Insertion G-quadruplex
of Cu2+ into sensing
PPIX (metalation) system by
catalyzed utilizing
G4. (b)PPIX for Cu2+ detection.
A schematic illustration of
The intensity of fluorescence increases sharply due to the binding. In Cu
the2+ presence 2+
non-covalent fluorescence-based G-quadruplex sensing system utilizing PPIX for detection. of Cu ,
PPIX-Cu complex of
The intensity forms and G4 binds
fluorescence to this
increases complex.
sharply due In thisbinding.
to the case, although the G4 still
In the presence can2+bind
of Cu to
, PPIX-
Cu complex formsofand
The intensity G4 binds to
fluorescence this complex.
increases Indue
sharply thisto
case,
the although
binding. Inthethe
G4presence
still can of
bind
Cu to, PPIX-
2+ PPIX-
Cu complex forms and G4 binds to this complex. In this case, although the G4 still can bind to PPIX-
Molecules 2019, 24, 1079 16 of 30
Molecules 2019,
Molecules 2019, 24,
24, xx FOR
FOR PEER
PEER REVIEW
REVIEW 16 of
16 of 29
29

Cu complex,
Cu complex,
PPIX-Cu the fluorescence
the
complex, fluorescence intensity
intensity
the fluorescence is much
is
intensity much
is muchlower.
lower. This
This
lower. Thisassay
assay
assayprovides
providesaaareliable
provides reliable platform
reliableplatform
platform forfor
effective detection with a low detection limit
effective detection with a low detection limit of 3.0 nM.of 3.0 nM.
In aa later
In later study,
study, aa detection
detection system
detection system waswas developed
developed surrounding
surrounding the the basis
basis of
of the
the interaction
interaction
between fluorophore thioflavin T (ThT) (see Figure 15) and G4. This involved
between fluorophore thioflavin T (ThT) (see Figure 15) and G4. This involved the inducement of the inducement of G4
G4
flanking sites, which results in selective
flanking sites, which results in selective detection detection of the target sequences since ThT has
detection of the target sequences since ThT has a specific a specific
light-up fluorescence probe for G4 structures.
light-up fluorescence probe for G4 structures. ThT ThT gives
gives out
out very
very weak
weak fluorescent
fluorescent signal
signal in
in aqueous
aqueous
solution but a high fluorescent signal upon binding to G4. This structural
solution but a high fluorescent signal upon binding to G4. This structural specificity specificity of ThT
specificity of ThT forfor G4
G4
was taken advantage of to
was taken advantage of to produce produce a sensitive
produce aa sensitive assay.
sensitive assay. The
assay. The formation
The formation
formation of of G4/ThT
of G4/ThT complex
G4/ThT complex due
complex due to theto the
hybridization of mutant DNA with the target DNA induced the G4
hybridization of mutant DNA with the target DNA induced the G4 structure formation instructure formation in the
the probe
probe
causing the ThT fluorophore to light up. This readout was used to elucidate the
causing the ThT fluorophore to light up. This readout was used to elucidate the amount of a mutant amount of a mutant
ion in
ion in aa target
target sequence
sequence with
with high
high sensitivity
sensitivity and
and specificity
specificity [82].
specificity [82].
[82].

Figure 15. Chemical

Figure Chemical structure of
of thioflavin T
T (ThT).
Figure 15.
15. Chemical structure
structure of thioflavin
thioflavin T (ThT).
(ThT).

Thiazole orange
Thiazole orange (TO)
orange (TO) (see
(see Figure
Figure 16a)
16a) is
is one
one of
of the
the most
most commonly
commonly used used fluorescent
fluorescent probes
probes
among the reported studies due
among the reported studies due to its to its high
its high fluorescence
high fluorescence quantum yield. However,
fluorescence quantum yield. However, TO lacks the ability
However, TO lacks the ability
ability
to distinguish G4 from other types of DNA structures since it is a universal nucleic acid
to distinguish G4 from other types of DNA structures since it is a universal nucleic acid fluorescence fluorescence
dye [83].
dye [83]. It
It was
It was reported
was reported that
reported that the
that the introduction
the introduction
introduction of of hydrocarbon
hydrocarbon rings into
hydrocarbon rings into the
the chromophore
chromophore of of thiazole
thiazole
orange alters the planarity of the chromophore and the binding
orange alters the planarity of the chromophore and the binding affinity affinity for G4 as well. With a suitable
affinity for G4 as well. With a suitable
molecular framework
molecular framework to accommodate
frameworktotoaccommodate
accommodate the
the the TO chromophore,
TO chromophore,
TO chromophore, the altered
the altered
the altered TO possesses
TO possesses
TO possesses highly
highly
highly selective
selective fluorescence
selective fluorescence
fluorescence response
response response to G4
to G4 [84].to G4 [84].
[84].

Figure 16.
Figure Chemical structures
16. Chemical structures of
of thiazole
thiazole orange
orange (TO).
(TO).

3.2.2. Non-Covalent Fluorescence

3.2.2. Non-Covalent
Non-Covalent Aptamer-Based
Aptamer-Based G-Quadruplex
Fluorescence Aptamer-Based G-Quadruplex Sensing Sensing System
System
3.2.2. Fluorescence G-Quadruplex Sensing System
Aptamers
Aptamers are are folded
are folded nucleic
folded nucleic acids
nucleic acids with
with aaa single-stranded
acids with single-stranded structure
single-stranded structure (RNA
structure (RNA
(RNA or or ssDNA),
or ssDNA), generally
ssDNA), generally
generally
Aptamers
designed
designed from
from 25
25 to
to 60
60 bases
bases in
in length.
length. The
The sequence
sequence variation
variation allows
allows the
the display
display of
of a
a large
large number
number
designed from 25 to 60 bases in length. The sequence variation allows the display of a large number
of structural
of structural arrangements.
structural arrangements.
arrangements. This This variability
This variability allows
variability allows for
allows for sufficient
for sufficient structural
sufficient structural diversity
structural diversity
diversity toto generate
generate the
to generate the
of the
specific
specific recognition
recognition cavities
cavities against
against a
a wide
wide range
range of
of targets
targets which
which includes
includes small
small molecules,
molecules, proteins,
proteins,
specific recognition cavities against a wide range of targets which includes small molecules, proteins,
ions, whole
ions, whole cells
whole cells and
cells and even
and even entire
entire bacteria
even entire and
bacteria and viruses
and viruses [85,86].
viruses [85,86]. Aptamers
[85,86]. Aptamers
Aptamers can can
can bebe isolated
be isolated from
from aaa large
isolated from large
large
ions, bacteria
random-sequence
random-sequence librarylibrary
library ofof either
of either RNA
either RNA
RNA or or DNA
or DNA
DNA by by an
by an evolution
an evolution process
evolution process called
process called systemic
called systemic evolution
systemic evolution
evolution
random-sequence
of
of ligands
ligands by
by exponential
exponential enrichment
enrichment (SELEX).
(SELEX). Through
Through SELEX,
SELEX, a
a vast
vast number
number of
of aptamers
aptamers have
have
of ligands by exponential enrichment (SELEX). Through SELEX, a vast number of aptamers have
been identified
been identified and
identified and
and itit was
it was reported
was reported that
reported that a number
that aa number
number of of aptamers
of aptamers underwent
aptamers underwent a conformational
underwent aa conformational
conformational changechange
change
been
within
within the
the G4
G4 motif
motif upon
upon ligand
ligand binding
binding [87].
[87]. DNA
DNA SELEX
SELEX has
has mainly
mainly yielded
yielded more
more G4
G4 aptamers
aptamers
within the G4 motif upon ligand binding [87]. DNA SELEX has mainly yielded more G4 aptamers
with
with high
high affinities. G4 have
affinities. G4
G4 have a higher
higher electrostatic
electrostatic potential
potential per
per unit
unit length
length compared
compared to to a duplex.
duplex.
with high affinities. have aa higher electrostatic potential per unit length compared to aa duplex.
When compared
When compared to to aa double
double helix,
helix, G4
G4 have
have twice
twice the
the negative
negative charge
charge density
density perper unit
unit length.
length. Hence,
Hence,
Molecules 2019, 24, x FOR PEER REVIEW 17 of 29
Molecules 2019, 24, 1079 17 of 30
G4 represent a good arrangement for differential interaction with small molecules or cationic proteins
[88].
When compared to a double helix, G4 have twice the negative charge density per unit length. Hence, G4
It is also assumed that in the absence of other possible interactions in RNA due to its free 2’-OH
represent a good arrangement for differential interaction with small molecules or cationic proteins [88].
groups, the high stability of G4s are often required for DNA aptamers to adopt more complex non-
It is also assumed that in the absence of other possible interactions in RNA due to its free
canonical structures for ligand binding. Upon ligand binding, a nucleic acid aptamer converts from
20 -OH groups, the high stability of G4s are often required for DNA aptamers to adopt more complex
a non-quad or unstructured conformation into a G4 motif. This can then be effectively monitored
non-canonical structures for ligand binding. Upon ligand binding, a nucleic acid aptamer converts
through transduction into various outputs such as fluorescence and luminescence in a number of
from a non-quad or unstructured conformation into a G4 motif. This can then be effectively monitored
ways [87]. Aptamers are interesting alternative binders in sensory applications due to their simplicity,
through transduction into various outputs such as fluorescence and luminescence in a number of
reusability and stability under a variety of environmental conditions. They are also cheaper to be
ways [87]. Aptamers are interesting alternative binders in sensory applications due to their simplicity,
synthesized and modified [89]. Aptamers also offer other advantages such as a lower molecular
reusability and stability under a variety of environmental conditions. They are also cheaper to be
weight, reproducible synthesis, controllable labeling, flexible design, low immunogenicity and fast
synthesized and modified [89]. Aptamers also offer other advantages such as a lower molecular
tissue penetration [90].
weight, reproducible synthesis, controllable labeling, flexible design, low immunogenicity and fast
Since the conjugation of aptamers may lead to other unwanted issues and considering the
tissue penetration [90].
unique feature of aptamers to adopt G4 structures upon binding to their cognate ligand, attempts
Since the conjugation of aptamers may lead to other unwanted issues and considering the unique
have been made to develop a detection strategy devoid of aptamer modifications or without the use
feature of aptamers to adopt G4 structures upon binding to their cognate ligand, attempts have been
of enzymes. Various studies have been conducted utilizing non-covalent fluorescence on aptamer-
made to develop a detection strategy devoid of aptamer modifications or without the use of enzymes.
based detection system. In one of these studies, a switch-on method for the detection of adenosine
Various studies have been conducted utilizing non-covalent fluorescence on aptamer-based detection
triphosphate (ATP) using aptamer based on a duplex-to-quadruplex conversion strategy was
system. In one of these studies, a switch-on method for the detection of adenosine triphosphate (ATP)
reported. Abnormal ATP levels from an overproduction of ATP by creatinine kinase has been
using aptamer based on a duplex-to-quadruplex conversion strategy was reported. Abnormal ATP
associated with diseases like angiocardiopathy, making accurate detection of ATP a critical
levels from an overproduction of ATP by creatinine kinase has been associated with diseases like
assessment for angiocardiopathy [91]. The ATP detection method applied a label-free fluorescence
angiocardiopathy, making accurate detection of ATP a critical assessment for angiocardiopathy [91].
switch-on assay, utilizing crystal violet (CV) (see Figure 17a) to monitor the conversion of the ATP
The ATP detection method applied a label-free fluorescence switch-on assay, utilizing crystal violet
aptamer in the presence of ATP (see Figure 17b). It was also suggested that such a G4-based design
(CV) (see Figure 17a) to monitor the conversion of the ATP aptamer in the presence of ATP (see
could reduce the non-specific binding of the probe, while maintaining the simplicity and cost
Figure 17b). It was also suggested that such a G4-based design could reduce the non-specific binding
efficiency of label-free detection. In this system, the ATP aptamer and its complementary sequence
of the probe, while maintaining the simplicity and cost efficiency of label-free detection. In this system,
were initially hybridized. In the absence of ATP, the binding of CV to the duplex DNA was weak
the ATP aptamer and its complementary sequence were initially hybridized. In the absence of ATP,
resulting in a low emission of fluorescence signal. The duplex conformation was claimed to be in
the binding of CV to the duplex DNA was weak resulting in a low emission of fluorescence signal.
equilibrium with a small population of dissociated single strands allowing the ATP aptamer to form
The duplex conformation was claimed to be in equilibrium with a small population of dissociated
a G4 structure. Through population-shift mechanism, the presence of ATP will trigger the
single strands allowing the ATP aptamer to form a G4 structure. Through population-shift mechanism,
dissociation of the duplex and the formation of the aptamer-target complex as ATP only stabilizes
the presence of ATP will trigger the dissociation of the duplex and the formation of the aptamer-target
the G4 structure of the aptamer. A strong interaction between the CV and the G4 was formed,
complex as ATP only stabilizes the G4 structure of the aptamer. A strong interaction between the CV
producing a significant fluorescence response to ATP [92].
and the G4 was formed, producing a significant fluorescence response to ATP [92].

(a)
Figure 17. Cont.
Molecules
Molecules 2019,
2019, 24,
24, x1079
FOR PEER REVIEW 1818ofof 29
30
Molecules 2019, 24, x FOR PEER REVIEW 18 of 29

(b)
(b)
Figure 17. (a) Chemical structure of crystal violet. (b) Schematic representation of the aptamer-based
Figure17.
Figure (a)Chemical
17.(a) Chemicalstructure
structureofofcrystal
crystalviolet.
violet.(b)
(b)Schematic
Schematicrepresentation
representationofofthe
theaptamer-based
aptamer-based
fluorescent turn-on strategy for ATP detection using G-quadruplex probe crystal violet (CV). ATP
fluorescentturn-on
fluorescent turn-onstrategy
strategyfor
forATP
ATPdetection
detectionusing
usingG-quadruplex
G-quadruplexprobe
probecrystal
crystalviolet
violet(CV).
(CV).ATP
ATP
promotes duplex dissociation and incudes formation of the aptamer-target G-quadruplex structure,
promotesduplex
promotes duplexdissociation
dissociationand
andincudes
incudesformation
formationofofthe
theaptamer-target
aptamer-targetG-quadruplex
G-quadruplexstructure,
structure,
which is detected by CV.
whichisisdetected
which detectedbybyCV.
CV.

A fluorescent dye, N-methylmesoporphyrin IX (NMM) (see Figure 18a) waswas used to detect the
AAfluorescent
fluorescentdye, dye, N-methylmesoporphyrin
N-methylmesoporphyrin IX IX (NMM)
(NMM) (see(see Figure
Figure 18a)18a)was usedused to detect
to detect the
presence of
the presence thrombin [93].
of thrombin The NMM
[93].NMM was
The was
NMM designed to
was designed bind to the formed G4 structure upon toehold
presence of thrombin [93]. The designed to bind to bind
to the to the G4
formed formed G4 structure
structure upon toeholdupon
strand
toeholddisplacement. The
strand displacement. DNA formation will assemble to multiple G4 structures, emitting amplified
strand displacement. The DNA The DNA formation
formation will assemble willtoassemble
multipleto G4multiple
structures,G4 emitting
structures, emitting
amplified
fluorescent
amplified signals for signals
fluorescent improved for sensitive
improved detection.
sensitive The systemThe
detection. allowed
system forallowed
G4-forming
for sequences
G4-forming
fluorescent signals for improved sensitive detection. The system allowed for G4-forming sequences
to be initially locked in the hairpin structures, meanwhile the initiation strand (IS) and the thrombin-
tosequences
be initiallytolocked
be initially
in thelocked
hairpininstructures,
the hairpin structures,
meanwhile themeanwhile the initiation
initiation strand (IS) and strand (IS) and
the thrombin-
binding aptamer
the thrombin-binding sequence
aptamerwere partially hybridized. No free IS could be generated when the
binding aptamer sequence weresequence
partiallywere partiallyNo
hybridized. hybridized.
free IS couldNo free be IS could bewhen
generated generated
the
thrombin
when thewas was absent
thrombin and the G4-forming sequences remained engaged in the hairpin structures,
thrombin absent was
and absent and the G4-forming
the G4-forming sequencesengaged
sequences remained remained engaged
in the hairpinin structures,
the hairpin
avoiding
structures,the association of NMM. Low fluorescent emission could be expected in this case. The
avoiding theavoiding
associationthe of
association
NMM. Low of NMM. Low emission
fluorescent fluorescent emission
could could beinexpected
be expected this case.inThethis
presence
case. The ofpresence
thrombin of promotes
thrombin the release the
promotes of IS as a result
release of IS of athe
as formation
result of the of the G4 of
formation aptamer-
the G4
presence of thrombin promotes the release of IS as a result of the formation of the G4 aptamer-
thrombin complex. complex.
aptamer-thrombin
thrombin complex.

(a)
(a)

(b)
(b)
Figure 18.
Figure (a)Chemical
18. (a) Chemicalstructure
structureof N-methylmesoporphyrin IX
ofN-methylmesoporphyrin IX (NMM).
(NMM). (b)
(b)Structure
Structure of
ofthe
thecomplex
complex
Figure 18. (a) Chemical structure of N-methylmesoporphyrin IX (NMM). (b) Structure of the complex
of G-quadruplex
of G-quadruplex and N-methylmesoporphyrin IX
and N-methylmesoporphyrin IX (NMM)
(NMM) (PDB: 4FXM).
of G-quadruplex and N-methylmesoporphyrin IX (NMM) (PDB: 4FXM).
 Molecules 2019, 24, x FOR PEER REVIEW 19 of 29
Molecules 2019, 24, 1079 19 of 30
The liberated IS will then hybridize with the first hairpin strand and unfolds the hairpin
structure, forming the toehold for strand displacement by the second hairpin strand. The second and
The liberated
unfolded hairpin IS will then
strands willhybridize
hybridizewith
and the
the first
IS ishairpin strand and
then displaced viaunfolds
toeholdthe hairpin
strand structure,
displacement
forming the toehold for strand displacement by the second hairpin strand. The second
mechanism. The displaced IS will then hybridize again with the initial hairpin sequence to initiateand unfolded
hairpin strands
the toehold will hybridize
strand and the
displacement. IS will
This is then displaced
finally resultvia
in toehold
a massivestrand displacement
generation mechanism.
of dsDNA strands
The
withdisplaced
both endsIS will
havingthen hybridize
active again with
G4-forming the initial
sequences. Thehairpin sequence
G4-forming to initiate
sequences andthe
NMMtoehold
will
strand displacement. This will finally result in a massive generation of dsDNA strands
associate to produce a sensitive monitoring system for thrombin by giving out remarkably intensified with both
ends havingemission
fluorescent active G4-forming
[93]. Figuresequences.
18b showsThe G4-forming
the structural sequences
complex of a and NMM
G4 and NMM. will associate to
produce a sensitive monitoring system for thrombin by giving out remarkably intensified fluorescent
3.3. Luminescence-Based
emission [93]. Figure 18bG-quadruplex Sensing System
shows the structural complex of a G4 and NMM.

3.3. Luminescence-Based
3.3.1. G-Quadruplex Sensing
Conventional Luminescence-Based System
G-Quadruplex Sensing System

3.3.1. The characteristics

Conventional of luminescentG-Quadruplex
Luminescence-Based heavy metal Sensing
complexes have gained large interest and
System
attention especially towards the fabrication of light-emitting materials and numerous sensing
The characteristics of luminescent heavy metal complexes have gained large interest and attention
applications due to its versatility, low cost, simplicity and high sensitivity. Their luminescence can be
especially towards the fabrication of light-emitting materials and numerous sensing applications due to
detected in profoundly fluorescent media as a result of their long emission life-time, with the
its versatility, low cost, simplicity and high sensitivity. Their luminescence can be detected in profoundly
utilization of time-resolved luminescence spectroscopy. Besides photophysical properties and
fluorescent media as a result of their long emission life-time, with the utilization of time-resolved
interaction with biomolecules, they require no complicated synthetic procedures and are readily
luminescence spectroscopy. Besides photophysical properties and interaction with biomolecules, they
available for fine-tuning. In addition, self-quenching can be prevented as they possess remarkable
require no complicated synthetic procedures and are readily available for fine-tuning. In addition,
stokes shifts [94]. As a consequence, the potential of several luminescent heavy metal complexes such
self-quenching can be prevented as they possess remarkable stokes shifts [94]. As a consequence,
as ruthenium (II), iridium (III), and platinum (II), as molecular light switches for nucleic acids
the potential of several luminescent heavy metal complexes such as ruthenium (II), iridium (III), and
including G4 DNA have been widely studied.
platinum (II), as molecular light switches for nucleic acids including G4 DNA have been widely studied.
Silver ions develop cytotoxicity in humans [95] and are associated to a number of medical
Silver ions develop cytotoxicity in humans [95] and are associated to a number of medical
complications including organ failure [96]. This highlights the importance of silver ion level
complications including organ failure [96]. This highlights the importance of silver ion level
determination in the body. To carry out silver (I) ion detection in aqueous solution, a luminescence-
determination in the body. To carry out silver (I) ion detection in aqueous solution, a luminescence-
based G4 sensing system was developed utilizing chloro(2-phenyl-1,10-phenanthroline)-platinum(II)
based G4 sensing system was developed utilizing chloro(2-phenyl-1,10-phenanthroline)-platinum(II)
(complex 1). As Ag+ ions are able to destabilize the G4 structure [28], G4 structures can undergo
(complex 1). As Ag+ ions are able to destabilize the G4 structure [28], G4 structures can undergo
conformational change from G4 to a duplex structure. The formed duplex structure allows the
conformational change from G4 to a duplex structure. The formed duplex structure allows the
intercalation of complex 1, producing a high emission intensity (see Figure 19). However, low
intercalation of complex 1, producing a high emission+ intensity (see Figure 19). However, low emission
emission intensity is observed in the absence of Ag owing to the weak interaction of complex 1 with
intensity is observed in the absence of Ag+ owing to the weak interaction of complex 1 with G4.
G4. Upon addition of various metal ions other than silver, the emission intensity only increases
Upon addition of various metal ions other than silver, the emission intensity only increases slightly
slightly suggesting the selectivity of the system +
for Ag+ ions [97].
suggesting the selectivity of the system for Ag ions [97].

Figure 19. A schematic

Figure19. schematic illustration
illustrationofofluminescence-based
luminescence-based G-quadruplex
G-quadruplex assay
assay forfor aqueous
aqueous silver
silver ions
ions detection.
detection. (a)The(a)The initial
initial structure
structure of G-quadruplex.
of G-quadruplex. (b) The
(b) The poorpoor interaction
interaction of G-quadruplex
of G-quadruplex and
and complex 1. (c) The silver ions induce the G4-to-duplex
complex 1. (c) The silver ions induce the G4-to-duplex conformational conformational change, allowing the
allowing the
intercalation
intercalationofofcomplex
complex1 1andandresulting
resultingininthe
theemission
emissionofofintense
intensephosphorescence.
phosphorescence.
Molecules 2019, 24, x FOR PEER REVIEW 20 of 29
Molecules 2019, 24, 1079 20 of 30

3.3.2. Luminescence Aptamer-Based G-Quadruplex Sensing System

3.3.2.
HumanLuminescence
neutrophil Aptamer-Based
elastase (HNE) G-Quadruplex
is a type ofSensing serine System
protease that degrades a variety of
functional Human and neutrophil
structural elastase
proteins,(HNE)whichisincludes
a type of collagen, proteoglycan,
serine protease that degrades fibronectin
a varietyand oflaminin.
functional
However,
and structural proteins, which includes collagen, proteoglycan, fibronectin and laminin.condition
the destruction of normal tissues may occur if the HNE is overproduced. This However,
has been associated with the development of various autoimmune
the destruction of normal tissues may occur if the HNE is overproduced. This condition has been disorders. For this reason, an
accurate detection platform for the diagnosis of HNE-related diseases
associated with the development of various autoimmune disorders. For this reason, an accurate is crucial. A selective and
sensitive
detection switch-on
platform assay
for to
thedetect sub-nanomolar
diagnosis of HNE-related HNE in homogeneous
diseases is crucial.solution was reported.
A selective This
and sensitive
assay was conducted
switch-on usingsub-nanomolar
assay to detect a label-free aptamer-based
HNE in homogeneous strategy utilizing
solution awas G4-selective
reported. Thisluminescent
assay was
iridium (III) complex [Ir(ppy)2(dpp)] + (where ppy = 2-phenylpyridine and dpp = 2,9-diphenyl-1,10-
conducted using a label-free aptamer-based strategy utilizing a G4-selective luminescent iridium (III)
phenanthroline) as a signal-transducing
complex [Ir(ppy)2(dpp)] + (where ppy =element [98]. In this
2-phenylpyridine andsystem,
dpp = either in an aqueous solution or
2,9-diphenyl-1,10-phenanthroline)
in asthea presence of dsDNA element
signal-transducing containing the In
[98]. HNEthisaptamer,
system, Complex
either in 1, an[Ir(ppy)2(dpp)]
aqueous solution
+, is weakly
or in the
emissive.
presenceThe dissociation
of dsDNA of the the
containing DNA HNEduplex was Complex
aptamer, induced with the addition +of, isHNE.
1, [Ir(ppy)2(dpp)] weakly Theemissive.
HNE
aptamer is then released
The dissociation of theandDNA will subsequently
duplex was induced fold into
witha the
G4 structure.
addition of An enhanced
HNE. The HNEluminescence
aptamer is
response that could be readily viewed under UV-illumination
then released and will subsequently fold into a G4 structure. An enhanced luminescence was produced due to the response
strong
interaction of Complex 1 with the HNE-aptamer. Therefore, a linear
that could be readily viewed under UV-illumination was produced due to the strong interaction of relationship was observed
between
Complex the1HNEwithconcentration
the HNE-aptamer. and the luminescence
Therefore, intensity
a linear of Complex
relationship 1. The range
was observed betweenand limit
the HNEof
detection of this system was of the same standard as the commercial ELISAs.
concentration and the luminescence intensity of Complex 1. The range and limit of detection of this Although the MB-based
approach
system was was of reported
the same to standard
be more sensitive than this assay
as the commercial ELISAs. [99], this assay
Although the is MB-based
consideredapproach
useful due was
to reported
the lower cost, simplicity and eliminates the involvement of the
to be more sensitive than this assay [99], this assay is considered useful due to the lower cost, covalent labeling of
oligonucleotides [98].
simplicity and eliminates the involvement of the covalent labeling of oligonucleotides [98].
Chemiluminescence
Chemiluminescence is is luminescence
luminescence where
whereenergyenergyis issupplied
suppliedbybychemical chemicalreactions.
reactions.In Ina a
reported
reported study,
study, a thrombin
a thrombin aptamer
aptamer waswasused
used toto develop
develop a cost
a cost effective
effective sensitive
sensitive aptasensor
aptasensor with
with
guanine
guanine chemiluminescence detection [100]. This system is capable of quantifying thrombininin
chemiluminescence detection [100]. This system is capable of quantifying thrombin
human
human serum
serum without
without thetheneed
need ofof
tedious
tedious procedures
procedures andandexpensive
expensive nanoparticles.
nanoparticles. InInthethepresence
presence
of of
tetra-n-propylammonium hydroxide (TPA), guanine of G4 TBA-conjugated
tetra-n-propylammonium hydroxide (TPA), guanine of G4 TBA-conjugated carboxyfluorescein carboxyfluorescein (6-
FAM) that is bound with thrombin would not react with 3,4,5-trimethoxylphenylglyoxal
(6-FAM) that is bound with thrombin would not react with 3,4,5-trimethoxylphenylglyoxal (TMPG) (TMPG) (see
Figure 20). Guanines
(see Figure of freeof
20). Guanines TBAfreeand
TBATBA-conjugated
and TBA-conjugated 6-FAM6-FAM will rapidly react react
will rapidly with withTMPG by
TMPG
immobilization on the surface of graphene oxide to emit light. As a
by immobilization on the surface of graphene oxide to emit light. As a result of the formation result of the formation of G4 TBA-
conjugated 6-FAM bound with
of G4 TBA-conjugated 6-FAM thrombin
boundinwith a sample, the brightness
thrombin in a sample, of guanine chemiluminescence
the brightness of guanine
was quenched. The reaction
chemiluminescence between The
was quenched. guanines of TBA
reaction and guanines
between TMPG inof theTBApresence of TPA
and TMPG in formed an
the presence
intermediate of high energy that was capable of emitting dim light
of TPA formed an intermediate of high energy that was capable of emitting dim light by itself. by itself. Consequently, based on
theConsequently,
principle of chemiluminescence
based on the principle energyoftransfer (CRET), energy
chemiluminescence was transferred
energy to 6-FAM
transfer (CRET), to emit
energy was
a bright light. Using the technology reported in this study, various types
transferred to 6-FAM to emit a bright light. Using the technology reported in this study, various types of G4 DNA aptasensors
capable
of G4 DNA of specifically
aptasensors sensing
capable a target molecule could
of specifically sensing beadeveloped
target molecule [100]. could
Thesebe target molecules
developed [100].
mayTheseinclude
target ATP, HIV, ochratoxin,
molecules may include potassium
ATP, HIV,ions, and thrombin.
ochratoxin, potassium ions, and thrombin.

Figure
Figure 20.20. Schematic
Schematic illustration
illustration of of G-quadruplex-TBA
G-quadruplex-TBA aptasensor
aptasensor with
with guanine
guanine chemiluminescence
chemiluminescence
detection principle.
detection principle.
Molecules 2019, 24, 1079 21 of 30

3.4. G-Quadruplex-Based Higher Order Sensor and Actuator Approaches

As G4s can form high affinity interactions with otherwise separate oligonucleotide strands,
other high molecular weight approaches in sensing and actuation have been explored. For instance,
the potassium-dependent contraction of a long human telomeric repeat sequence was observed
by surface plasmon resonance (SPR) that could measure the change of mass within its evanescent
field [101]. Technically, fluorescence energy transfer (FRET) can be used to directly measure the
structural change but with the disadvantage of using labels, or by direct probing using atomic force
microscopy. In contrast, using this principle of SPR, an on-line and direct measurement of structural
change can be performed without the need of labels. The changes in refractive index resulting from
the binding events or in the structure of biomolecules such as RNA are able to be measured too [101].
Thus, it may be possible to detect interactions that influence the formation of G4s by mere mechanical
means. At the same time, it is conceivable that such a higher order conformational change can be
employed as an actuator responding to a specific trigger as well.
Alternatively, the principle shown in Figure 5 has been expanded to generate high molecular
weight complexes based on hybridization chain reaction (HCR) [102] that has more recently been
devised into a label free method [103]. Unlike the strategy of splitting-DNAzyme, locking the intact G4
sequence into hairpin probes of HCR was proposed as an attractive nucleic acid detection system which
is protein-free, isothermal and capable of self-amplifying [102]. In this approach, the G4 was closed
one-third in the stem of one of the GQ-HCR hairpin probes and two-thirds in the loop. The GQ-HCR
probes stayed as inactive meta-stable hairpin structures in the absence of the target molecule resulting
in an inert G4. As HCR could be initiated by cross-opening of G4 probes, when the GQ-HCR probes
comes across the target molecule the closed G4 could be freed.
Even higher order structures have been selectively assembled or switched based on G4 formation.
DNA Origami technology shows some prospect in terms of new applications for G4s. A FRET-based
sensor system using a triangular scaffold was shown to be highly responsive to potassium ion
concentrations [104]. This hints at the potential application of G4s for conditional release or capture of
molecules when entering a cell that normally has higher potassium concentrations than the outside
medium. Yet complete macromolecular folding blocks can rearrange in response to analytes to gain an
enzymatic function. A potassium-dependent switch of such blocks aptly termed “dominoes” has been
demonstrated most recently by the research group of Willner [105]. Given that such rearrangements
can be achieved depending on the presence of specific analytes, it may be possible to detect the
formation of a regular higher order structure in liquid crystals by the naked eye that can be applied
in completely novel bottom-up sensor concepts. This highlights the tremendous freedom one can be
accorded when utilizing G4 systems for sensing and actuation applications.
The ability for G4 structures to be adapted for different sensing platforms allows great flexibility
in sensor designs. Table 2 summarizes the different sensing platform that can be designed with the
utilization of G4 structures. The many different G4 derived sensing platforms highlights the usefulness
to apply G4 as an alternative sensing platform for diagnostic applications.
Molecules 2019, 24, 1079 22 of 30

Table 2. Characteristics of different G4-based detection platforms.

Facilitating Limit of
Sensing System Target Molecules Advantages Disadvantages References
Ligands Detection
ssDNA (SNP) • Simple, fast analysis and low cost 0.95 nM [56]
• Easy to prepare and purify as compared with natural
protein enzymes
• Requires no expensive and sophisticated instrumentation
• No tedious covalent labelling
G4/Hemin-
Hemin • Instant and visible colour change -
basedDNAzyme Sodium ions 0.6 µM [58]
• High sensitivity and selectivity
• More desirable because in some cases, organic dyes undergo
photobleaching and possess poor aqueous solubility
• In contrast to nanomaterials-based peroxidase mimics,
G4/hemin DNAzymes are commonly water-soluble

• Exhibits higher catalytic activity than G4/hemin-based

• When the concentration of H2 S is
DNAzyme induced by other commonly-used cations
Hemin, Ag+ and higher than a certain level, Ag+ would
Hydrogen sulfide • High selectivity as it is not affected by the presence of not be able to transform into AgS
Tb3+ (as substitute other anions 13 nM [29]
(H2 S) causing the absorbance of ABTS-H2 O2
for K+ )
• Can be applied on serum sample without affecting solution to remain the same.
the precision

• Probe with multiple GGG repeats

could form an intramolecular G4
structure and produce a significant
background signal (Problem was
ssDNA solved by adding a sequence to its
DNAzyme-based • Split probes exhibit excellent selectivity against nucleic acid
(rifampin-resistant 50 -end that is partially complementary - [106]
colorimetric split G4
M.tb) to the 30 -end )
• Probe could form a stable secondary
structure and result in a low
signal readout
Hemin
3.1 × 10−10
nrDNA ITS • Does not involve fluorophore/quencher complex labeling as - [70]
mol·L−1
conventional molecular beacons do
DNAzyme-based • Does not involve chemical modification
colorimetric • DNAzyme MB can act as a primer for polymerization
G4/Hemin p53 DNA • Provide sequence specific recognition in a - 25 fM [71]
Molecular Beacon straightforward fashion
• DNAzyme MB is able to exhibit SDA effect, act as target
DNA recognition element and generate signal
Molecules 2019, 24, 1079 23 of 30

Table 2. Cont.

Facilitating Limit of
Sensing System Target Molecules Advantages Disadvantages References
Ligands Detection
DNA (base
number of DNA) • Protoporyphrin • Sensitive to single-nucleotide addition - [79]
IX (PPIX) • Binding affinity between PPIX and G4 is largely dependent
Conventional Copper (II) ion on the integrity of G4 structure • The fluorescence intensity changes 3.0 nM [80]
non-covalent only little when the fluorescence has
• Does not involve probe fluorophore labeling
fluorescence G4 reached a plateau at a
DNA (EGFR exon and modification
• Thioflavin T certain concentration.
19 deletion • Is not interfered by other metal ions 2.3 nM [82]
(ThT)
mutant) • Exhibit high specificity and sensitivity, enabling efficient
detection of target mutant DNA
• Reduce the non-specific binding of the probe, while
maintaining the simplicity and cost efficiency of
label-free detection.
• The interfering NaCl or glucose deso not induce significant
fluorescent changes in the system
ATP Crystal violet (CV) - 5 µM [92]
• Potential to be applied for the detection of ATP in
real samples.
Non-covalent • Eliminates the requirement for fluorescent labeling of DNA
fluorescence aptamer while the robustness is still maintained due to the
aptamer-based G4 selective CV–G4 interaction.
• Enzyme-free
• Non-labeled, exhibits better binding affinity since aptamer
N-Methyl modification may alter their binding affinity
Thrombin mesoporphyrin (IX) • Able to assay thrombin in real samples - 5 pM [93]
(NMM) • High selectivity which is associated with the highly specific
binding between the target thrombin and the
corresponding aptamers.
Conventional Chloro(2-phenyl-
• Simple and cost-effective
luminescence-based Silver ion 1,10-phenanthroline)- - 20 nM [97]
• Sensitive and highly selective
G4 platinum(II)
• Its selectivity is comparable to oligonucleotide-based systems
• Possesses excellent solubility in aqueous solution
• The range and limit of detection of this are of the same
Iridium(III) standard as the commercial ELISAs. • MB-based approach was reported to be
Enzyme complex • Eliminates the involvement of the covalent labeling more sensitive than this assay - [98]
Luminescence
[Ir(ppy)2 (dpp)]+ of oligonucleotides
aptamer-based G4
• Excellent sensitivity compared to molecular beacon-based
methods
Molecules 2019, 24, 1079 24 of 30

Table 2. Cont.

Facilitating Limit of
Sensing System Target Molecules Advantages Disadvantages References
Ligands Detection
• Simple, rapid and cost-effective
• Highly sensitive guanine chemiluminescence detection
3,4,5-trimethoxy-
without expensive and intractable nanoparticles including
Thrombin phenylglyoxal - 12.3 nM [100]
magnetic Fe3 O4 GO nanoparticles
(TMPG)
• Involves no complicated procedures
• Capable of quantifying thrombin in human serum
Molecules 2019, 24, 1079 25 of 30

4. Conclusions
The versatility of the G4 motif has created a prominent impact towards the field of sensing.
The number of potential applications of G4 has vastly expanded with the increasing number of studies
on G4. Compared with antibodies or organic chemosensors, DNA oligonucleotides possess various
remarkable advantages which includes high thermostability and solubility, ease of production and
modification as well as rich structural polymorphism in the presence of particular targets, and can be
responsive. In particular, the systems that have been described in this review focused on ‘label-free’
strategies. These label-free approaches have recently gained popularity due to its simplicity and does
not require time-consuming labelling and immobilization steps.
The discovery of G4 DNA has spawned an entire discipline and new perception of DNA.
The various applications of G4 DNA for the development of different sensing systems highlights the
robustness of G4 to function as a reporter system in future sensor platforms. It is conceivable that G4
reporter systems will play a more active role in sensor developments. Recent combinations of G4s with
DNA Origami may lead to the discovery of entirely novel sensor concepts. In conclusion, G4 based
reporter systems could serve as an attractive alternative to existing reporter systems available to date.

Author Contributions: All authors listed have made a substantial, direct and intellectual contribution to the work,
and approved it for publication.
Funding: T.S.L. acknowledges the Malaysian Ministry of Education for financial support through the Higher
Institution Centre of Excellence (HICoE) Grant [Grant No. 311/CIPPM/4401005].
Acknowledgments: J.I., S.K.C. and Y.Y.L. thank Universiti Sains Malaysia for financial support.
Conflicts of Interest: The authors declare that the research was conducted in the absence of any commercial or
financial relationships that could be construed as a potential conflict of interest.

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