Utilization of G-Quadruplex-Forming Aptamers for theConstruction of Luminescence Sensing PlatformsDik-Lung Ma,*[a] Wanhe Wang,[a] Zhifeng Mao,[a] Tian-Shu Kang,[b] Quan-Bin Han,[c]

Philip Wai Hong Chan,[d, e] and Chung-Hang Leung*[b]

ChemPlusChem 2017, 82, 8 – 17 T 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim8

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Introduction

The fundamental role of DNA in biological systems is to act as

a storage medium for the genetic information of the cell.[1]

DNA can be replicated to produce genetically identical proge-

ny or transcribed into mRNA for the synthesis of proteins.[2]

However, over the past several decades, scientists have exploit-

ed the unique structural and recognition properties of DNA to

achieve a variety of scientific or technological applications thatare wholly unrelated to the biological purpose of DNA.[3–6] For

example, DNA origami structures have been proposed asbuilding blocks of the construction of nanomaterials,[7] whereas

DNA has also been explored for the use of molecular logicgates or computers.[8–11]

Another interesting application of DNA has been the devel-

opment of aptamers, which are nucleic acid sequences capableof recognizing and binding to cognate targets with high affini-

ty. Aptamers were initially discovered by the systematic evolu-tion of ligands by exponential enrichment (SELEX) process in

the 1990s,[12, 13] and since then a vast number of aptamers havebeen identified for a variety of small molecules, metal ions,

proteins, and even entire cells.[14] The discovery of aptamers

has spawned an entire discipline in analytical chemistry thatrevolves around the use of DNA—and, to a lesser extent,

RNA—aptamers for sensing a myriad of targets. Aptamerscompare favorably to protein antibodies for sensing applica-

tions because they are generally more stable under a variety ofenvironmental conditions, reusable, and also cheaper and sim-

pler to synthesize and modify.[14]

At the same time, the G-quadruplex structure is a non-can-onical nucleic acid structure that is formed from guanine-richoligonucleotides. The core G-quadruplex unit is the guaninetetrad, which is formed from four guanine bases linked byHoogsteen hydrogen bonding. Stacking of two or more gua-nine tetrads generates a G-quadruplex motif, which can be fur-ther stabilized by cations in the central ionic channel.[15] In con-

trast to double-helical DNA, which exists predominantly in the

B form, G-quadruplexes can exhibit a diverse array of structuraltopologies depending the number of oligonucleotides that

comprise the structure, the directionality of the nucleic acidstrands, and the length and nature of the intervening loops,

among other factors.[16]

Interestingly, a number of aptamers have been reported to

undergo a conformational change within the G-quadruplex

motif upon binding to their respective targets. In this Minire-view, we highlight recent examples of the use of G-quadru-

plex-forming aptamers in aptamer-based assays in recentyears. Luminescent sensing offers an attractive combination of

sensitivity and simplicity and also provides an optical outputfor the easy visualization of analyte detection.[17] This Minire-

view aims not to be exhaustive but rather intends to highlight

the diversity of luminescent G-quadruplex aptamer-based strat-egies that have been reported over the past three years, with

a view towards sampling different types of mechanisms thathave been employed. For the sake of brevity, we do not con-

sider G-quadruplex-based sensing platforms that do not direct-ly involve the analyte-induced conversion of an aptamer into

a G-quadruplex motif. These include various assays for

Ag+ [18–20] or Hg2 + ,[21] small molecules such as cocaine,[22] andDNA sequences.[11, 23–27] We instead direct the reader to excel-

lent review articles that have been published and discuss G-quadruplex probes or the development of luminescent G-

quadruplex-based sensing platforms more generally.[28, 29]

Principle of luminescent G-quadruplexaptamer-based assays

The conversion of a nucleic acid aptamer from an unstructuredor non-quadruplex conformation into a G-quadruplex motif

upon ligand binding can be transduced into a luminescentoutput in a number of ways. Initial studies in the field of DNA-

based sensing typically employed doubly labeled oligonucleo-

tides that possessed a fluorophore and a quencher moiety attheir termini (“molecular beacons”),[30] such that a relative

movement between the two chromophores would lead to analteration of the fluorescence signal. Alternatively, the use of

two fluorophores allows the fluorescence energy resonancetransfer (FRET) signal to be monitored, which can be an ad-

Aptamers are nucleic acid sequences that can recognize andbind to analytes with high affinity and selectivity. Intriguingly,

a number of aptamers undergo a conformational changewithin the G-quadruplex motif upon ligand binding. This Mini-

review aims to highlight interesting examples of luminescent

G-quadruplex aptamer-based probes that have been devel-oped in recent years. Various mechanisms and sensing modes

are described, and the outlook and future directions of thisfield are also discussed.

[a] Dr. D.-L. Ma, W. Wang, Z. MaoDepartment of ChemistryHong Kong Baptist University224 Waterloo Road, Kowloon Tong, Hong Kong 852 (P. R. China)E-mail : edmondma@hkbu.edu.hk

[b] T.-S. Kang, Dr. C.-H. LeungState Key Laboratory of Quality Research in Chinese MedicineInstitute of Chinese Medical SciencesUniversity of Macau, Macao 999078 (P. R. China)E-mail : duncanleung@umac.mo

[c] Dr. Q.-B. HanSchool of Chinese MedicineHong Kong Baptist University, Kowloon, Hong Kong 852 (P. R. China)

[d] Dr. P. W. H. ChanSchool of ChemistryMonash University, Clayton, VIC 3800 (Australia)

[e] Dr. P. W. H. ChanDepartment of ChemistryUniversity of Warwick, Coventry CV4 7AL (United Kingdom)

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vantage owing to the possibility of a ratiometric mode of de-tection.[31] The labeled DNA strategy can also be adapted to G-

quadruplex aptamers by the incorporation of fluorophore moi-eties or fluorophore–quencher pairs at judicious locations of

the aptamer, such that the folding of the oligonucleotide intoa G-quadruplex motif can bring two chromophores closer to-

gether or further apart. More complex schemes that involvemultiple labeled DNA oligonucleotides can also be envisioned,

as shall be seen below.

Alternatively, a label-free strategy can be employed in whichthe formation of the nascent G-quadruplex is recognized by an

external dye.[32] To achieve a luminescent mode of detection,the dye must be non-emissive when in aqueous solution or in

the presence of non-quadruplex forms of DNA but “light up”when bound to G-quadruplex DNA. In recent years, a number

of luminescent G-quadruplex-selective probes have been de-

veloped to serve this purpose.[33–37] The advantage of the label-free strategy is that unmodified oligonucleotides can be used;

these are generally much cheaper and easier to prepare thanmodified oligonucleotides. However, drawbacks of the label-

free strategy are the lack of true multiplex capability as well asthe possibility that the dye might become sequestered by in-

terfering species in the sample environment. Thus, it is impor-

tant for the dyes to be highly selective toward G-quadruplexDNA. A DNAzyme-based approach that uses the oxidizing abili-

ty of the hemin–G-quadruplex assembly is another way totransfer the DNA switching event into the luminescence signal.

Once formed, the hemin–G-quadruplex assembly could oxidizea non-emissive precursor compound into an emissive fluoro-

phore.[38, 39]

Traditional techniques for ion, small molecule, and protein

detection include atomic absorption/emission spectroscopy(AAS/AES),[40] inductively-coupled plasma mass spectrometry(ICP-MS),[41] ion-selective electrodes,[42] high-performance liquid

chromatography (HPLC), and gas chromatography (GC) in co-operation with a mass spectrometer. However, these methodsgenerally require expensive instrumentation and labor-inten-sive sample preparation protocols that are not suitable for on-site testing. Meanwhile, compared with assays that use otherDNA structures (commonly dsDNA), G-quadruplex-based

assays have the potential to exhibit greater specificity and sen-sitivity. This is because organic dyes capable of binding todsDNA and producing a strong fluorescence response also

usually recognize G-quadruplex DNA, whereas G-quadruplex-selective ligands are usually less likely to intercalate into DNA

owing to their relatively larger sizes. Additionally, as the majorDNA conformation in living organisms, dsDNA is more likely to

be encountered as an interfering species in the sample matrix,

and therefore assays based on duplex formation might bemore susceptible to false positives. In contrast, the utilization

of G-quadruplex for the construction of a sensing platformcould potentially reduce the problem of non-specific DNA

binding.

Luminescent G-quadruplex aptamer-basedprobes for metal ions

G-quadruplex structures are stabilized by the presence of cat-ionic ions in the central channel of the stacked G-quartets.

Thus, G-quadruplex sequences can also be considered as ap-tamers for metal ions. Interestingly, some G-quadruplex se-

quences only respond to specific metal ions, whereas others

might show distinct quadruplex topologies with differentmetal ions. For example, the well-known human telomeric se-

quence adopts a mixed-type/propeller confirmation in thepresence of K+ ions but prefers an anti-parallel basket configu-ration with Na+ ions.[41] The sensitivity of G-quadruplex confor-mation to metal ions has naturally enabled the development

of both labeled and label-free G-quadruplex-based probes formetal ions in recent years.

G-quadruplex aptamer-based probes for K++

Potassium is one of the alkali metals, and it plays key roles inbiological systems, such as reducing the risk of high blood

pressure and stroke, and balancing pH. An unbalance of K+

ions is associated with arrhythmia.[44, 45] Fluorescence polariza-tion (FP) is a widely employed technique for the analysis of

small molecules in a variety of application fields. Hu and co-workers have developed a simple and rapid FP method for K+

detection based on a target-induced DNA conformationalswitch.[46] Their sensing strategy utilized two complementary

Dik-Lung Ma completed his PhD in

2004 at the University of Hong Kong

under the supervision of Prof. C.-M.

Che. Between 2005 and 2009, he

worked at the University of Hong

Kong, the Hong Kong Polytechnic Uni-

versity, and the Scripps Research Insti-

tute with Prof. C.-M. Che, Prof. K.-Y.

Wong, and Prof. R. Abagyan. His re-

search mainly focuses on luminescent

sensing of biomolecules and metal

ions, computer-aided drug discovery,

and inorganic medicine. He is currently an Associate Professor at

Hong Kong Baptist University.

Chung-Hang Leung completed his

PhD in 2002 at the City University of

Hong Kong. After completing a five-

year postdoctoral fellowship at the De-

partment of Pharmacology, Yale Uni-

versity, he was appointed Research As-

sistant Professor at the University of

Hong Kong and then at Hong Kong

Baptist University. He is currently an

Associate Professor at the University of

Macau. His primary research interests

are in anticancer and anti-inflammato-

ry drug discovery and molecular biol-

ogy.

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DNA probes: probe A: 5’- CTA2C3GTAGAT13CTACG3T2AG3T2-

AG3T2AG3T-3’ and probe B: 5’-TACG3T2AG-FAM-3’. The hairpinstructure of probe A contained four regions: a K+ aptamer se-

quence (G3T2AG3T2AG3T2AG3), a probe B-complementary DNA

sequence (BC-DNA), an extend-DNA (E-DNA) sequence, anda 12 bp T–T region (Figure 1a). The E-DNA sequence was de-

signed to be complementary to the BC-DNA sequence to stabi-lize the hairpin form of probe A so as to limit the hybridization

of BC-DNA with probe B in the absence of K+ . Probe B is com-plementary to BC-DNA and was also labeled with fluorescein-amidite (FAM). The addition of K+ ions transforms probe A

from a hairpin conformation into a G-quadruplex motif, there-by causing the oligonucleotide to adopt an open configura-tion. The exposed BC-DNA region then hybridizes withprobe B, thereby resulting in an increase in the FP signal of the

FAM dye. The system exhibited a detection limit for K+ ions of4.5 nm, and was also selective toward K+ over a range of other

metal ions. Thus, this system acts as a K+ sensor in aqueoussolution. However, it is unclear whether such assays couldfunction effectively in biological samples in which the molecu-

lar rotation of the tracer molecule could be easily perturbednon-specifically.

Another aptamer-based fluorescent method for K+ detectionwas developed by Verdian-Doghaei and co-workers.[47] The

30 bp insulin-binding aptamer (IBA) sequence converts from

a random coil into an intramolecular parallel G-quadruplexstructure in the presence of K+ ions (Figure 1d). The triple-

helix molecular switch consists of a central IBA sequenceflanked by two arm segments and a dual-labeled oligonucleo-

tide that is complementary to the arm segment sequences.Upon addition of K+ ions, the IBA-containing sequence folds

into a G-quadruplex confirmation, thereby disrupting the tri-

plex helix. The dual-labeled oligonucleotide is then releasedand folds into a hairpin conformation. This brings the fluoro-

phore (FAM) and quencher (BHQ1, black hole quencher 1) into

close proximity, thereby resulting in a quenching of the fluo-rescence signal. Hence, this system functions as a switch-off

probe for K+ ions.A label-free DNAzyme-based fluorescent method for K+ de-

tection was reported by Li and co-workers in 2012 (Fig-ure 1b).[48] In the absence of K+ , the G-rich nucleic acid existsin a random coil state, and therefore the active DNAzyme is

not formed. However, the addition of K+ transforms the G-richnucleic acid into a G-quadruplex structure that binds to heminto form an active DNAzyme. This allows the oxidation of 3-(4-hydroxyphenyl)-2-oxopropanoic acid (HPPA) by H2O2 to takeplace, and the fluorescence intensity of the oxidative productincreases along with the increase in the concentration of K+ .

Recently, Chen and co-workers reported a label-free strategyfor K+ detection that utilizes PicoGreen as a fluorescent duplexprobe.[49] PicoGreen is a small molecule organic dye that inter-

acts with duplex DNA to produce an enhanced luminescencesignal (Figure 1c). The addition of K+ ion causes the aptamer

to convert into a G-quadruplex motif, which reduces theamount of duplex DNA that can be formed upon subsequent

addition of a complementary strand. This leads to an associat-

ed decrease in the fluorescence of PicoGreen, which bindsonly to duplex, but not G-quadruplex, DNA. Hence, this probe

acts as a switch-off fluorescence sensor for K+ ions. Further-more, the authors demonstrated the ability of the probe to

detect K+ in tap water and human urine.

Figure 1. (a) Schematic illustration of the strategy for K+ determination using FP enhancement. (b) Schematic illustration of the strategy for K+ determinationusing fluorophore and quencher. (c) Schematic illustration of the label-free fluorescent aptasensor for detection of K+ . (d) Schematic illustration of the label-free fluorescent strategy for detecting K+ or OTA based on aptamers and PicoGreen dye.

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G-quadruplex aptamer-based probes for Pb2++

The Pb2 + ion is an acutely toxic metal ion that causes adversehealth effects in humans, including delayed physical and

mental developmental in infants and children, kidney disease,and high blood pressure in adults.[50, 51] In addition to K+ ions,

Pb2+ ions can also effectively stabilize G-quadruplex structures,thereby generating more compact G-quadruplex motifs thanthose stabilized by K+ .[52, 53] Wang and co-workers recently de-

veloped an FP-based G-quadruplex-based assay for Pb2 + ionsusing the thrombin-binding aptamer (TBA) G-quadruplex se-quence (5’-G2T2G2TGTG2T2G2-3’) (Figure 2a).[54] TBA is internallylabeled with a fluorophore (TMR) at the seventh thymine nu-

cleotide of TBA (T7-TMR-TA15) to allow the FP of the system tobe measured. In the absence of Pb2 + , the TBA sequence exists

in a random-coil structure. Intramolecular interactions of the

TMR label with adjacent guanine bases by photoinduced elec-tron transfer (PET) results in the probe having a large FP value

in the absence of Pb2 + . However, the addition of the target in-duces a conformational change from a random-coil structure

into a highly ordered G-quadruplex. This weakens the interac-tion between TMR and adjacent nucleotides, thereby allowing

the label to rotate faster and reducing the FP signal of the

probe.Piccirilli and co-workers used an interesting RNA aptamer

termed “spinach” for the construction of a label-free Pb2 +

assay (Figure 2b).[55] Spinach contains a G-quadruplex motif

that can be induced into a G-quadruplex conformation by theaddition of Pb2 + ions. In the absence of the target, the apta-

mer exists in a hairpin conformation that is incapable of bind-

ing to 3,5-difluoro-4-hydroxybenzylidene imidazolinone(DFHBI), and thus the fluorescence of the dye is low. However,

the addition of Pb2 + transforms the spinach aptamer intoa two-layered G-quadruplex system, which binds to and acti-

vates the fluorescence of DFHBI. Thus, this probe functions asa switch-on sensor for Pb2 + ions. Moreover, the ability of the

probe to detect spiked Pb2 + ions in tap water was demonstrat-

ed. One drawback of the probe is that it relies on RNA aptam-ers, which are generally less stable and more susceptible todegradation by exogenous RNAses than DNA aptamers.

Our group previously developed a label-free assay for Pb2 +

ions based on the specific interaction between a G-quadru-

plex-selective iridium(III) complex and the PS2.M G-quadruplex(5’-GTG3TAG3CG3T2G2-3’; Figure 2c).[56] The PS2.M oligonucleo-

tide is a guanine-rich sequence that can be induced into an in-tramolecular G-quadruplex structure in the presence of Pb2 +

ions. In the absence of Pb2 + ions, PS2.M exists in a random-coilconfiguration, and the weak interaction of the iridium(III) com-plex with the G-quadruplex motif results in a weak lumines-

cence signal. In the presence of Pb2 + ions, however, the PS2.Moligonucleotide is induced into a G-quadruplex structure thatis recognized by the iridium(III) complex, thereby resulting ina switch-on luminescence response. This assay also functionedeffectively in spiked river water samples.

Luminescent G-quadruplex aptamer-basedprobes for small molecules

A number of aptamer sequences undergo a conformationalchange within the G-quadruplex motif upon binding to specif-

ic small molecules. This has stimulated the development of G-quadruplex aptamer-based luminescent probes for small mole-

cules in recent years.

G-quadruplex aptamer-based probes for ATP

ATP is the most important energy carrier in all living cells. It

plays crucial roles in the regulation of cellular metabolism andhas been widely used as an index in biomass determinations

in clinical microbiological assays, food quality control, and en-

vironmental analyses.[57, 58] Ge and co-workers have developedan ATP assay based on the target-triggered strand displace-

ment reaction (SDR) cycle (Figure 3a).[59] In the absence of ATP,the “catalyst” strand is hybridized with the ATP aptamer and

the SDR cycle is not initiated. Therefore, the fluorescence ofthe system is low on account of the quenching of the FAM flu-

orophore on the substrate strand by the DABCYL moiety of

the hybridized C-2 strand. However, the addition of ATP formsan aptamer–ATP G-quadruplex conformation that releases thecatalyst strand to initiate the SDR cycle. The catalyst strand

Figure 2. (a) Schematic illustration of the use of a TBA G-quadruplex for the FP-based detection of Pb2 + , (b) the Spinach sensor for the detection of Pb2 + , and(c) the G-quadruplex-based luminescent switch-on detection strategy for Pb2 + ions using a G-quadruplex-selective iridium(III) complex.

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first displays the C-1 strand to form an intermediate duplex.This is followed by a subsequent exchange of a “fuel” strand,

thereby displacing both C-2 and the catalyst strand to forma fuel–substrate duplex. The removal of the DABCYL-linked C-2

allows the FAM-linked substrate strand to become emissive.

The released catalyst strand can initiate another round of DNASDR, thereby resulting in the amplification of the signal.

In 2012, our group reported a label-free assay for ATP thatutilized crystal violet (CV) as a G-quadruplex probe (Fig-

ure 3b).[60] In the absence of ATP, the ATP aptamer remains hy-bridized to its complementary sequence and the fluorescenceof CV is low on account of its weak interaction with duplex

DNA. However, the addition of ATP induces the dissociation ofthe duplex and subsequent formation of ATP G-quadruplexaptamer structure. The strong binding of CV to the nascent G-quadruplex allows the system to function as a switch-on probe

for ATP. The system exhibited a detection limit for ATP of 5 mmand was selective toward ATP over other nucleotides, small

molecules, or protein. Later we improved upon this design by

utilizing an iridium(III) complex instead of CV. As the iridium(III)complex was more selective toward G-quadruplex DNA than

CV, the background signal of the system in the absence of ATPcould be reduced. Under optimized conditions, the assay ex-

hibited a detection limit of 2.5 mm. This demonstrates the gen-eral principle that the sensitivity of label-free G-quadruplex

aptamer-based assays can be increased by the use of more se-

lective G-quadruplex probes.

G-quadruplex aptamer-based probes for ochratoxin A

Ochratoxin A (OTA) is a mycotoxin that is found in a numberof foodstuffs, including grains, coffee, wines, and beer. As

a consequence, the sensitive and rapid detection of OTA is im-portant in food safety, environmental monitoring, and quality

control.[61, 62] Zhao and co-workers have described an assay forOTA detection that utilizes a singly labeled OTA aptamer (Fig-

ure 3c).[63] Interestingly, the fluorescence response of the apta-

mer to OTA binding varied depending on the position of theattached fluorophore. Presumably, the conformational switch

of the OTA aptamer from a random coil into a G-quadruplexconformation alters the environment of the fluorophore, there-

by resulting in a change in the fluorescence behavior of thedye. Both switch-on and switch-off systems were developed.

The most sensitive aptamer exhibited a detection limit of

about 5 nm, and the ability of the probe to detect spiked OTAin red wine was also demonstrated. A later study by the same

group measured the FP signal of the aptamer in response toOTA binding.[64] Various fluorophore positions were tested, and

the optimum position was found to be when the aptamer waslabeled with TMR at the tenth thymine nucleotide. The system

showed a detection limit for OTA of 3 nm and could detectspiked OTA in both diluted red wine and urine samples.

Our group has developed a label-free OTA assay using the

luminescent G-quadruplex-selective iridium(III) probe.[49] (Fig-ure 3b) The oligonucleotide sequence ON1, which contains the

OTA aptamer (5’-GATCG3TGTG3TG2CGTA3G3AGCATCG2ACA-3’), isinitially hybridized with a partially complementary DNA strand

ON2 (5’-C2ACAC3GATC-3’). The addition of OTA causes dissocia-

tion of the ON1–ON2 duplex owing to the formation of theOTA aptamer G-quadruplex. The OTA G-quadruplex aptamer is

then bound by the G-quadruplex-selective iridium(III) complexwith an increased emission response, thereby allowing this

system to act as a switch-on label-free luminescent detectionplatform for OTA. Compared to the random-coil-to-quadruplex

Figure 3. (a) Reaction mechanism of SDR-based catalytic cycle and schematic illustration of the FRET-based detection of ATP. (b) Schematic illustration of theaptamer-based fluorescent turn-on strategy for ATP, OTA, and HNE detection using a G-quadruplex probe CV or an iridium(III) complex. (c) Schematic illustra-tion of the principle for fluorescent sensing of OTA with a single fluorophore-labeled aptamer.

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approach described above, this duplex-to-quadruplex strategyis advantageous because the initial duplex substrate might be

more resistant to the presence of interfering ions, such as K+

and Na+ , which might induce the formation of a G-quadruplex

motif even in the absence of OTA.Another label-free OTA sensing strategy was developed by

Li and co-workers (Figure 1d).[65] In the absence of OTA, theaptamer remains in a single-stranded conformation and is freeto hybridize with its complementary sequence. This allows the

intercalation of the PicoGreen into the duplex motif, therebyresulting in a high fluorescence signal. In the presence of OTA,

the aptamer switches from a random-coil structure to a G-quadruplex motif. As a result, less of a duplex structure isformed and the fluorescence of PicoGreen is reduced. Hence,this system acts as a switch-off probe for OTA. Additionally, the

ability of the assay to detect OTA in 1 % beer was shown.

Luminescent G-quadruplex aptamer-basedprobes for protein

Intriguingly, aptamers have also been reported for a number of

proteins. This has paved the way for the development of vari-ous luminescent G-quadruplex aptamer-based protein detec-

tion methods.

G-quadruplex aptamer-based probes for thrombin

Thrombin, a multifunctional serine protease that produces in-

soluble fibrin through proteolytic cleavage of a soluble fibrino-gen, plays an essential role in coagulation and cardiovascular

disease therapy.[66] TBA has been widely used as a sensing ele-ment for the construction of thrombin aptasensors. TBA folds

into a compact G-quadruplex structure in the presence of K+

or thrombin. This G-quadruplex can bind hemin to generate

a DNAzyme with peroxidase-like activity. Li and co-workers

have utilized this property to develop a luminescent assay forthrombin.[67] In the presence of thrombin, the hemin–G-quad-

ruplex aptamer–hemin complex catalyzes the oxidation of thia-mine, a non-fluorescent substrate, into the fluorescent thio-chrome, by means of H2O2. Under optimized conditions, theassay exhibited a detection limit for thrombin of 1 pm, which

was superior to that of a corresponding colorimetric assay thatutilized the ABTS-H2O2 system (20 nm).[68]

Recently, a toehold strand-displacement-based thrombin de-tection assay was developed by Xiang and co-workers (Fig-ure 4a).[69] The G-quadruplex sequences are initially locked in

the hairpin structures of HP1 and HP2, whereas the initiationstrand (IS) is partially hybridized with the thrombin aptamer se-

quence. In the absence of thrombin, the G-quadruplex-formingsequences remain in the hairpin state, and the fluorescence of

the G-quadruplex-specific dye porphyrin N-methyl mesopor-

phyrin IX (NMM) is low. However, the addition of thrombintriggers the release of IS owing to the formation of the G-

quadruplex aptamer–thrombin complex. The released IS thenbinds to and unfolds HP1, thus leading to the formation of

a toehold for strand displacement by HP2. Subsequently, HP2

hybridizes with the unfolded HP1 and displaces the IS through

the toehold strand-displacement mechanism. IS then entersanother catalytic cycle to unfold more hairpin structures. The

unmasked G-quadruplex-forming sequences are then able tofold into G-quadruplex motifs, thereby allowing the fluores-

cence of NMM to be significantly increased. This assay wasalso able to detect spiked thrombin in human serum.

G-quadruplex aptamer-based probes for human neutrophilelastase

Human neutrophil elastase (HNE) is a serine protease that de-grades a variety of structural and functional proteins such ascollagen, fibronectin, laminin, and proteoglycan. However, theoverproduction of HNE can lead to the destruction of normal

tissues and thus has been implicated in the pathogenesis ofvarious autoimmunological disorders such as rheumatoid ar-thritis, idiopathic pulmonary fibrosis, emphysema, and adultrespiratory distress syndrome.[70, 71] Our group has developed

a G-quadruplex aptamer-based assay for HNE that utilizes aniridium(III) complex as a G-quadruplex probe (Figure 3b).[72] In

this system, the HNE aptamer is initially hybridized with a par-

tially complementary strand, and the weak binding of the com-plex to the DNA duplex results in a weak luminescence signal.

However, the presence of HNE induces the dissociation of theduplex structure to generate the HNE-G-quadruplex aptamer

complex. The aptamer G-quadruplex is then recognized by theiridium(III) with enhanced luminescence response.

Figure 4. (a) Schematic illustration of the toehold strand displacement-driven assay for thrombin. (b) Schematic illustration of allosteric molecularbeacon probe for sensitive and selective detection of AGR2 based on fluo-rescent flow cytometry analysis.

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G-quadruplex aptamer-based probes for anterior gradienthomologue 2

The anterior gradient homologue 2 (AGR2) is a cancer bio-

marker that has been found to be overexpressed in a widerange of human cancers, including carcinomas of the esopha-

gus, pancreas, breast, prostate, and lung.[73–75] Yang and co-workers developed an aptamer-based assay for AGR2 (Fig-ure 4b).[76] In this assay, a DNA oligonucleotide is designed thatcontains the streptavidin (SA) aptamer sequence, the AGR2aptamer sequence, an SA aptamer complementary sequence,and a fluorophore. A stable hairpin structure is formed by in-tramolecular hybridization between the SA aptamer sequenceand the complementary sequence, which prevents the oligo-nucleotide from binding to the SA beads. However, the addi-

tion of AGR2 changes the conformation of the DNA owing to

the formation of the AGR2-G-quadruplex aptamer complex,thereby revealing the SA aptamer sequence that binds to the

beads. The beads will therefore fluorescence strongly owing tothe FAM label on the bound probes. The target molecules

could be detected and quantified by measuring the fluores-cence intensity of SA beads by means of flow cytometry.

Outlook and Summary

In this Minireview, we have highlighted a number of different

strategies that have been employed for the development of G-quadruplex aptamer-based probes in the past three years

(Table 1). We observe that a number of different luminescentmodes of detection have been employed, including FP, as well

as both switch-on and switch-off luminescent intensity for-

mats. In general, we consider a switch-on mode of detectionto be superior to a switch-off mode of detection because it is

less susceptible to false positives that arise from non-specificquenching mechanisms in the environment.

In this context of the label-free strategy, we note that bothorganic (e.g. , NMM, CV)[60] and inorganic (e.g. , iridium(III) com-

plexes)[49, 56, 72] G-quadruplex probes have been utilized for the

development of G-quadruplex aptamer-based assays. The para-mount goal of any G-quadruplex probe is to achieve high se-

lectivity for G-quadruplex DNA over other DNA conformations,and thus we envision that further gains in sensitivity and selec-tivity of these assays can be achieved as superior G-quadruplexdyes are developed.

Additionally, we find that the G-quadruplex aptamer-basedassays reported in recent years vary over a wide spectrum ofcomplexity. At one end are the simple random-coil-to-G-quad-

ruplex-based aptamer assays, such as the OTA detection assayreported by Zhao and co-workers (Figure 3c),[63] whereas at the

other end are the complex catalytic schemes that involve mul-tiple rounds of DNA conformational changes, such as the ATP

assay developed by Ge and co-workers (Figure 3a).[59] A catalyt-ic mechanism allows for amplification of the analyte signal,

which can greatly improve the sensitivity of the system. How-

ever, one drawback is that increasing the number of movingparts in the mechanism will necessarily increase the unit cost

per assay, as well as increase the time and resources neededfor the optimization of the probe.

Looking toward the future, we envision that the focus of G-quadruplex aptamer-based assays should be shifted towards

the practical application of such assays for the detection of an-

alytes in biological or environmental milieux. To date, researchon nucleic acid-based sensors has usually been in vitro and in

cellulo applications are rare. This is primarily due to major mo-lecular barriers faced in vivo, such as 1) efficient delivery and

targeting to the site of interest, 2) stability of externally intro-duced DNA species, and 3) their potential toxicity in the host

organism. In this context, Takenaka and co-workers have re-

ported using a peptide–thrombin binding aptamer conjugateto visualize K+ concentration changes in living cells.[77] Al-

though not G-quadruplex-related, a DNA nanomachime devel-oped by Koushika, Krishnan, and co-workers has been used for

the spatiotemporal recognition of pH changes in living organ-isms.[78] Moreover, the sensitivity, selectivity, and robustness of

these assays are all critically important. Here, luminescent

metal complexes have a salient advantage over organic dyes

Table 1. A summary table of all the current assays that used luminescent G-quadruplex aptamers.

Assay Detecting target Aptamer used Output signal Ref.

Figure 1a potassium ions 5’-G3T2AG3T2AG3T2AG3-3’ fluorescencepolarization

[46]

Figure 1c potassium ions 5’-GTG3TAG3CG3T2G2AC2ACAC2A2C2-3’ fluorescence signal [48]Figure 1d potassium ions 5’-G2T2G2TGTG2T2G2-3’ fluorescence signal [49]Figure 2a lead ions thrombin-binding aptamer (TBA) G-quadruplex sequence (5’-G2T2G2TGTG2T2G2-3’) fluorescence

polarization[54]

Figure 2b lead ions 5’-G4AGA2G2ACG3UC2AGUGCGA3CACGCACUGU2GAGUAGAGUGUGAGCUC3-3’ fluorescence signal [55]Figure 1b potassium ion 5’-CTCTCTG2TG2TG8T2G2TAG3TGTCT2CTCTCTC-3’ fluorescence signal [47]Figure 2c lead ions PS2.M G-quadruplex (5’-GTG3TAG3CG3T2G2-3’). luminescence signal [56]Figure 3a ATP 5’-AC2TG5AGTAT2TGCG2AG2A2G2T-3’ fluorescence signal [59]Figure 3b ATP 5’-A2C2TG5AGTAT2GCG2AG2A2G2T-3’ luminescence signal [60]Figure 3b ochratoxin A 5’-GATCG3TGTG3TG2CGTA3G3AGCATCG2ACA-3’ fluorescence signal [65]Figure 3c ochratoxin A 5’-GATCG3TGTG3TG2CGTA3G3AGCATCG2ACA-3’ luminescence signal [63]Figure 1d ochratoxin A 5’-GATCG3TGTG3TG2CGTA3G3AGCATCG2ACA-3’ fluorescence signal [66]Figure 4a thrombin 5’-TATAGTC2GTG2TAG3CAG2T2G4TGACT-3’ fluorescence signal [70]Figure 3b human neutrophil elastase 5’-TAGCGATACTGCGTG3T2G4CG2TAG3C2AGCAGTCTCGT-3’ luminescence signal [73]Figure 4b anterior gradient homologue 2 5’-C3TG3AGT2GTG9TG3AG3T2-3’ fluorescence signal [77]

ChemPlusChem 2017, 82, 8 – 17 www.chempluschem.org T 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim15

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in that their phosphorescent lifetimes are generally muchlonger than typical fluorescence lifetimes, which could allow

their luminescence to be distinguished from exogenous fluoro-phores in the biological or environmental samples by the use

of time-resolved fluorescence spectroscopy. Another priorityfor the development of bioassays could be the development

of dyes that emit in the red or near-infrared region, as this liesin the “optical window” in which the absorbance of photons

by biological tissues is at a minimum. Given the promising ex-

amples highlighted in this Minireview, we envision that thisfield will continue to expand fruitfully in the years to come.

Acknowledgements

Our research group is supported by Hong Kong Baptist University(FRG2/14-15/004 and FRG2/15-16/002), the Health and MedicalResearch Fund (HMRF/14130522), the Research Grants Council(HKBU/201811, HKBU/204612, and HKBU/201913), the French

Agence Nationale de la Recherche/Research Grants Council JointResearch Scheme (A-HKBU201/12; Oligoswitch ANR-12-IS07-

0001), the National Natural Science Foundation of China

(21575121), Guangdong Province Natural Science Foundation(2015A030313816), the Hong Kong Baptist University Century

Club Sponsorship Scheme 2015, the Interdisciplinary ResearchMatching Scheme (RC-IRMS/14-15/06), the Science and Technolo-

gy Development Fund, Macao SAR (098/2014/A2), and the Uni-versity of Macau (MYRG091(Y3-L2)-ICMS12-LCH, MYRG2015-

00137-ICMSQRCM, MRG023/LCH/2013/ICMS and MRG044/LCH/

2015/ICMS).

Keywords: aptamers · G-quadruplexes · luminescence ·oligonucleotides · sensors

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Manuscript received: January 23, 2016

Revised: March 7, 2016Accepted Article published: March 9, 2016Final Article published: March 21, 2016

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