Chapter 3.3. DNA G-quadruplex structures: from sensing to logic gates


Qiuting Loh, Yee Siew Choong, Jörn Glökler, and Theam Soon Lim

Art work
Simple and efficient tests for the detection of the main causes of fever, adapted to different geographical areas, 
could have a great impact decreasing the mortality associated to life-threatening infections in poor countries
I come from a country that no longer exists.
Jose Emilio Pacheco 


Deoxyribonucleic acid (DNA) is known to play a central role in storing the genetic information of every living organism, and DNA replication and transcription mechanisms have been studied intensively. Recently, many findings have shown DNA to be a very dynamic molecule with the ability to form a number of non-canonical spatial arrangements such as single-stranded hairpins, homoduplexes, triplexes, and quadruplexes to regulate other functions [1, 2]. The formation of such secondary and tertiary structures is involved in DNA recombination, regulation of gene expression, and possibly proliferation of tumor cells. Therefore, there is growing interest in studying the structures of nucleic acids and their application in medical science.

The guanine (G)-rich sequence G-quadruplex (Gq) DNA structure has been well studied [3]. The typical base pairing in nucleic acids is Watson–Crick pairing, where hydrogen bonds are formed between guanine and cytosine or adenine and thymine. However, nucleic acids have the ability to form another type of pairing known as Hoogsteen base pairing [4]. This pairing mechanism allows the formation of higher-order structures such as Gq structures. Gq structures are made up of planar stacks of G tetrads, and are stabilized by eight Hoogsteen hydrogen bonds [5, 6]. The G-quartets are arranged in a square planar array that is formed by the linkage of four G bases joined by the phosphodiester backbone, and is stabilized by the presence of specific cations. Recently, the properties of the Gq structure have been well described. The Gq structure has attracted a great deal of attention because it plays an important role in many biological functions including telomeres and the principles exploited in biosensors and therapeutics [7].

 G-Quadruplex Structures

Many nuclear magnetic resonance (NMR) and crystallographic studies have reported that Gq structures are highly polymorphic [8]. They can form many different structural arrangements depending on the length of the DNA, orientation of the chains, position of the loops, and nature of the cations. Gq structures can be found in many different multimeric forms including one, two, or four separate chains [9]. They can also be displayed in a variety of topologies in consequence of many different possible combinations of strands, loops, and sequences. Therefore, the Gq structure can be classified in terms of stoichiometry and in relation to its orientation [10]. Figure 1 shows some of the most common Gq structures: parallel (tetramolecular), hairpin (bimolecular), basket, and chair (unimolecular).

Figure 1. Typical G-quadrplex structures

Generally, four separate stretches of G form a parallel four-stranded structure in a quadruplex [11]. Two chains of G fold into dimeric structures by the dimerization of a pair of hairpins that provide a bimolecular Gq structure with two loops. There are two different loop orientations: “edgewise” loops connect adjacent antiparallel chains and “diagonal” loops connect cross-over antiparallel chains. As reported, the NMR spectra show different structures in the presence of different cations (K+ and Na+). In the presence of K+ ions, the Gq structure has “edgewise” loops, but in the presence of Na+ ions, the Gq structure has “diagonal” loops [12, 13].

A unimolecular G-quadruplex structure is made up of a single-stranded, G-rich sequence with four G repeats. It tends to fold and form an intramolecular Gq with three loops in the presence of cations. Owing to the steric hindrance and electrostatic repulsion caused by these loops, the orientation of the three chains is not entirely antiparallel. An example of a unimolecular Gq structure is that which contains the human telomeric sequence dTTAGGGG. The telomeric repeat is able to form another loop, namely a TTA loop, in the intramolecular structure. This allows the four chains to exist in a parallel orientation. Another example is the thrombin-binding aptamer that folds into an intramolecular quadruplex with three lateral loops, often depicted as a chair structure. Another structure that is formed by the Oxytricha telomere sequence has a parallel and antiparallel neighbor that results in three different groove widths: wide, narrow, and medium grooves. Finally, the Tetrahymena telomere repeat forms an unusual quadruplex structure. These variations in structure exist in nature for specific functions [10].

The polymorphism in Gq is the result of a subtle balance between several stabilizing factors. Primarily, Gq structures are determined by monovalent cations such as K+ and Na+ because these structures are dependent on cations for stability. However, other forces such as hydrogen bonding, base-stacking forces, and hydrophobic effects do play an important role in determining Gq structure. Such remarkable polymorphism has driven the use of Gq in many applications, especially in the fields of medicine, biology, and material science. In addition to the various topologies, Gq structures are not perfectly rectangular but generally form so-called “quadruple helices” with varying pitch [14].

 Functions of G-Quadruplex structures

G-quadruplex DNAzymes, originally known as catalytic enzymes or DNA enzymes, have catalytic activity [15]. DNAzymes can catalyze many reactions such as DNA modification, ligation [16, 17], cleavage of DNA or RNA [18, 19], and metalation of porphyrin rings [5, 20]. Such distinctive characteristics have driven the use of DNAzymes in many applications, especially in the fields of medicine, biology, and material science. Unlike the classical protein enzymes that are usually active in a narrow temperature range, DNAzymes are stable over a broad temperature range, even at high temperatures. DNAzymes can be easily prepared by chemical synthesis or by polymerase chain reaction (PCR) in contrast to protein enzymes, which require tedious preparation and purification processes.

Figure 2. DNAzyme activity of G-quadruplex structures

One of the most important functions of DNAzyme quadruplex-forming oligonucleotides is the promotion of peroxidase-like activity [21]. More specifically, the DNAzyme exhibits peroxidase-like activities when hemin is bound to the quadruplex [22, 23]. This complex, for instance, may catalyze the peroxide-mediated oxidation of the 2,2ʹ-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS). In the presence of hydrogen peroxide (H2O2), oxidation of the colorless ABTS results in a colored product (Figure 2). This characteristic has promoted the use of DNAzyme with peroxidase-like activity in many applications in various fields. The colorimetric change has facilitated the development of assays that detect metal ions, aptamer–substrate complexes, and even proteins [24-27].

Zhou et al. (2010) have described the detection of silver ions (Ag+) using a Gq structure. Ag+ stabilizes cytosine–cytosine (C-C) mismatches and forms C-Ag+-C base pairs. Therefore, in the absence of Ag+, the G-rich sequence forms an intramolecular duplex; however, the addition of Ag+ to the mixture enables the G-rich sequence to fold into a quadruplex structure. This structure subsequently binds to hemin and regulates DNAzyme activity [28].

The detection of mercury ions (Hg2+) has also been carried out using G-rich sequences, whereby the ion binds specifically to the thymine–thymine (T-T) mismatch in a DNA duplex. In the absence of Hg2+, the properly folded G-quadruplex binds to hemin and regulates a colorimetric reaction. The addition of Hg2+ disrupts the proper folding of the G-quadruplex structure, which prevents the generation of a colorimetric signal [29].

The photophysical properties of the structures are highly sensitive to the bound metal ions owing to the coordination of ions in the center of the quadruplex that change the location of the nucleobase electrons of the structure. The G-quadruplex can exhibit two- to ten-fold higher quantum fluorescence yields, whereby it allows the structure to serve as an energy donor to energy acceptors in close proximity. Thus, using this approach, the Gq can serve as a donor in fluorescence resonance energy transfer (FRET) systems [30-32].

Moreover, the Gq structure can serve as an internal fluorescent probe, whereby its nucleobases are modified or attached to fluorescent dyes. Intercalating agents can also bind with the Gq structure through the end-stacking interaction, resulting in noncovalent fluorescence probes [31]. The G-tetrads have a large surface area that is exposed to solvents, and presents receptors to aromatic cations that stack to the surface. Classical intercalating agents such as ethidium bromide bind to double-stranded DNA and can enhance the fluorescence intensity by 30-fold. Unlike classical probes, Gq noncovalent fluorescent probes tend to exhibit quenched fluorescence that is affected by the G residues. Therefore, the characteristics of Gq structures have allowed the development of several molecule-based logic gate systems that perform a set of Boolean operations, “AND”, “OR”, and “NOT”, in response to chemical or physical inputs [33-36]. Such variations in structure, fluorescence, and color provide researchers with a wide variety of tools for the development of diagnostics.

 Application of G-Quadruplex for Diagnostics

Highly sensitive and selective detection of biomarkers is the main objective in diagnostics. Typically, the most sensitive immunoassay systems use naturally occurring enzymes such as horseradish peroxidase (HRP) and alkaline phosphatase, which are commercially available as reporter systems. Although natural enzymes are used extensively, many limitations are associated with them. A common problem is the loss of catalytic activity during storage caused by conformational changes of the enzymes. Another issue concerning such enzymes is the cost involved in their production and purification. The enzymes are not able to function by themselves in assays because they must be conjugated to either antibodies or antigens before use. Enzyme-linked immunosorbent assays (ELISAs) are strongly dependent on the successful conjugation of the enzymes to the capture or donor molecules.

The chemical processes involved in protein conjugation may also result in a loss of protein function, and, more importantly, a low yield of successful conjugates. Recently, the development of enzyme mimics using G-quadruplex structures has become the focus of attention because they can achieve catalysis with high accuracy, stability, and reusability.

Zhou et al. (2009) have reported the use of DNAzyme-functionalized gold nanoparticles (GNPs) as nanoprobes in immunoassays to amplify the detection of protein cancer biomarkers. As shown in Figure 3, a “sandwich-type” immunoassay with two types of probes was employed. Magnetic microparticles (MMPs) functionalized with a protein cancer biomarker are effective probes. The researchers applied α-fetoprotein (AFP) monoclonal antibodies for specific detection. GNPs functionalized with double-stranded DNA and AFP polyclonal antibodies are also effective probes. Both GNPs and MMPs were mixed together in excess to capture the AFP effectively and form MMP–AFP–GNP sandwich complexes. These complexes were collected magnetically and thoroughly washed. The double-stranded DNA on the GNPs was then de-hybridized to form active DNAzymes in the presence of hemin. These DNAzymes were then reacted with ABTS as a substrate. The use of DNAzymes as catalytic labels in immunoassays is very promising, because they have significant advantages over conventional reporters: they are inexpensive, stable, and easy to conjugate with nanoparticles [37]

Figure 3.  Schematic diagram of the immunoassay based on DNAzyme-functionalized gold nanoparticles (GNPs) as nanoprobes

Recently, an immunoassay based on a pre-formed reporter system has been reported. Omar et al. (2013) demonstrated that a Gq-containing daunomycin aptamer could exhibit hemin-dependent peroxidase activity independent of daunomycin. In this work, a probe was successfully developed using streptavidin GNPs conjugated with enhanced green fluorescent protein (EGFP) as the biotinylated antigen, together with a biotinylated daunomycin aptamer (DQ), as depicted in Figure 4A. The generation of the reporter system only requires a simple and quick streptavidin–biotin interaction in a one-pot synthesis. Thus, the pre-assay generation of reporter probes allows rapid one-step incubation. This eliminates the multiple tedious steps of incubation required in conventional immunoassay systems (Figure 4B). In this antibody–antigen assay, antibodies against EGFP were coated on the microtiter plates and incubated with the probe. The wells were then developed with ABTS solution. This probe-based system showed significant results in a direct immunoassay comparison with a competitive assay. However, competitive assays are expected to be more suitable for the detection of small haptens such as hormones or drug molecules [38].


Figure 4  Schematic diagram of the immunoassay that based on the preformed-reporter system

 A system with Gq-based DNAzymes for the amplified catalytic detection of thrombin has been developed by Shen et al. (2010) (Figure 5). This system utilizes a guanine-rich thrombin-binding aptamer (TBA) that folds into a Gq structure owing to the thrombin-induced conformational change. It is capable of binding to hemin and acquiring peroxidase-mimicking DNAzyme activity. Therefore, in the presence of thrombin, the peroxidase activity can be sufficiently elevated, resulting in an amplified electrochemical readout [39].

Figure 5. Catalytic detection of thrombin based on G-quadruplex DNAzyme.

DNAzyme-catalyzed reactions can act as reporters in logic gate systems. Many reported logic gate applications exploit the dynamic feature of DNA nanostructures (Figure 6). An example is the switch-off detection platform for thrombin using the TBA. Ethidium bromide can be used as a duplex probe; it intercalates into the duplex structure formed by the hybridization of TBA and a complementary DNA sequence and emits fluorescence. However, in the presence of thrombin, the duplex form of the TBA dissociates, forming thrombin-aptamer Gq. Consequently, the fluorescence signal of the ethidium bromide is greatly reduced owing to its weak interaction with the quadruplex [40] 

Figure 6  Logic Gates Detection Platforms

However, many switch-on detectors in aptamer-based logic gates for the detection of proteins have also been reported. Leung et al. (2013) have recently developed a switch-on detection platform for human neutrophil elastase (HNE). This platform also implements the duplex-to-quadruplex conversion strategy, with an iridium (III) complex being used as the quadruplex probe. Initially, the HNE aptamer is hybridized with the complementary DNA strand. The iridium (III) complex binds only weakly to the duplex, emitting a weak luminescence signal. Therefore, the addition of HNE protein causes the duplex structure to disassociate and form the HNE–aptamer quadruplex. Subsequently, the iridium (III) complex strongly interacts with the newly formed quadruplex and enhances the luminescence signal [41].


Gq systems have emerged as potential alternative reporter systems for various bioanalytical applications. In comparison with the current reporter systems, Gq-based systems provide advantages in terms of cost, thermostability, and ease of synthesis. This makes them very useful for the development of a variety of sensing probes for diagnosis, and they are applicable to both DNA and protein detection. The dynamic Gq structure is also a superior label-free system and it can yield characteristic emission responses upon formation. Moreover, it can be incorporated into an instrument-free reporter system with colorimetric detection to be evaluated by the naked eye. However, depending on the design and sensitivity of the assay, some Gq-based colorimetric systems require additional instrumentation.

Taken together, these advantages make Gq-based assays very attractive for the development of a variety of sensing probes for diagnosis. Such assays are versatile and flexible, and can be adapted to many different types of diagnostic platforms. This makes Gq an appealing alternative to conventional commercially available enzyme-based diagnostic platforms.


The authors would like to acknowledge the Malaysian Ministry of Higher Education Fundamental Research Grant Scheme (Grant No. 203/CIPPM/6711473).


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