**4.3. Applications of synthetic G-quartets**

50 μM, respectively, corresponding to an average increase of efficiency of 2600 %. At this stage, PNADOTASQ is the more active TASQ catalyst for peroxidase-like reactions, and is closer to the activity of G-quadruplex, with a factor of 11 between it and 22AG (*i.e.*, in the experiments, 22 μM PNADOTASQ offers the same catalytic response than 2 μM of the natural G-quadruplex). PNADOTASQ confirms the role of intramolecular G-quartets to activate hemin and to perform DNAzyme-like catalysis, and also that improvement of the efficiency is just at the very beginning of the development. The modification of the arms is a positive point, but the role of the template, directly linked to the ability of synthetic G-quartet formation, is by consequence

RAFT-G4 as a pre-catalyst: To evaluate the role of the template on the DNAzyme-like activity, the TASQ developed by E. Defrancq's team was tested.[197] Termed RAFT-G4, the molecule is composed of a cyclodecapeptide and four guanosine arms in which one triazole per arm is intercalated between the ribose and the peptide scaffold (playing a role in the stabilization of the intramolecular G-quartet).[13] As a first step, UV–Vis titrations of hemin by RAFT-G4 were performed to check the interaction, and compared with DOTASQ-C5. Interestingly, very similar signals shifted from the signal of hemin alone were obtained in the presence of both TASQ for the two peaks at 363 nm and 394 nm, corresponding to the aggregated and disag‐

Afterwards, catalytic experiments were carried out with 1 μM hemin, 2 mM ABTS, and 600 μM H2O2 in Caco.KTD buffer, with concentrations of DOTASQ-C5 and RAFT-G4 from 10 mM to 100 mM. Positively, the results revealed the ability of RAFT to be a catalyst, even if the efficiency was lower than for the DOTASQ-C5. To optimize the catalysis, the use of 10 mM ATP in Caco.KTD buffer at pH 4.8 (protocol based on previous studies with G-quadruplexes) was performed and showed the positive effect of these modifications of the experimental conditions. More precisely, this optimization led to an improvement of DOTASQ and RAFT-

These series of experiments triggered two main conclusions. First, the ability of a TASQ to be a good catalyst is dependent on the template, the nature of the guanine arms, and the stability of the G-quartet, and it seems to be for now difficult to rationalize their impacts. The multi‐ plication of examples of TASQ in the literature will be an invaluable chance to understand the role of each structural part of the molecules. Author wagers that new molecules like Pyro‐ TASQ[16] or NaphtoTASQ[200] will help to decipher a little more the reasons of these

Another nice synthetic G-quartet system used as DNAzyme-like catalyst was developed by H. O. Sintim's team.[141] In this case, the molecule is not a TASQ forming an intramolecular G-quartet, but the cyclic diguanylic acid (also termed c-di-GMP), composed of two guanine residues. The authors proposed that c-di-GMP are able to form a discrete G-quadruplex formed by two intermolecular G-quartets (*i.e*., from four c-di-GMP molecules) at the micromolar level, but with the *sine qua non* presence of the intercalating proflavin molecule. In this case, it can be considered that proflavin assumes the role of a non-covalently linked template, unlike in TASQ. Experiments carried out with 0.5 μM hemin, 30 μM proflavin, 2 mM ABTS, and 2 mM H2O2 in Tris-HCl buffer (50 mM, pH 7.9) revealed that a catalytic activity can be distinguished

gregated forms of the hemin (*i.e.*, catalytically active), respectively.[50]

G4 efficiency by factors 1.8 and 5.1, respectively.[197]

differences.

indirectly linked to the catalytic properties.

462 Advanced Catalytic Materials - Photocatalysis and Other Current Trends

One of the main topics of natural G-quartets is the biological role of G-quadruplex structures, found in many sequences in the human genome (estimated at about 350.000 sequences).[207, 208] This alternative higher-order DNA structure (*i.e*., a noncanonical double-helix one)[209] is strongly suspected to play important roles in key cellular events, like chromosomal insta‐ bility or regulation of gene expression.[26, 27, 42–44] Notably, the extremities of chromosomes, termed telomeres, are composed of a G-overhang strand with several (TTAGGG)*n* repeats from an average of 100–200 bases.[26, 27, 210] Interestingly, this single strand (stabilized by a protein complex named shelterin) is the substrate for a key enzyme, the telomerase.[211–213] Although this field is of particular interest and extremely exciting, it is far from the scope of this chapter, and author recommends some excellent reviews to go further insight. In few words, telomerase is able to synthesize TTAGGG repeats, leading to the elongation of the telomeres. It is however only active in a vast majority of cancer cells (85 % of the tested cell lines) but inactive in somatic healthy cells.[214] Telomerase is thus considered as a cancer marker with a strong therapeutic potential.[152, 153] Consequently, direct or indirect evaluation of the activity of this enzyme from cell lysates could be of the utmost importance for the tumor diagnosis.

Based on the DNAzyme technology, R. Freeman *et al*. developed an assay to detect telomerase activity in an indirect way. If it is active, a primer is elongated to long (TTAGGG)*n* strands able to form G-quadruplexes.[154] However, long single-strand sequences can form multimers, [215] composed of several G-quadruplex structures, with a "stacking"[216–218] or a "beadson-a-string"[219, 220] global structure. In the first case, several G-quadruplexes from the same strand are stacked together, while in the second case, G-quadruplexes are independent, without any interactions between them.

A study from L. Stefan *et al*. [155] showed that mainly for a 46-base long strand (d[AG3(T2AG3)7] composed of two G-quadruplexes separated only by three nucleobases), two different kinds of G-quartets able to activate hemin can be defined. Indeed, the first accessible G-quartets are the external ones, and are identical to the ones found in a single G-quadruplex. However, the second hemin interaction site is at the interface between two G-quadruplexes. The teams of L. Petraccone and H. Sugiyama demonstrated that this "pocket" (composed of two G-quartets) is more hydrophobic than the classic external G-quartet and favors therefore the interactions with hydrophobic organic molecules, like hemin.[221, 222] Interestingly, this internal binding site clearly has a positive effect on the hemin binding and on its catalytic activity. The efficiency of the DNAzyme response is due to the hydrophobic properties of this internal site that favors hemin fixation and its protection against degradation. Moreover, once stacked to an internal G-quartet, the distal face of the Fe(III)–porphyrin is "through contact with" the second G-quartet that can play a key role during the oxidation/reduction process of the iron during the catalysis (*e.g*., mimicking the action of histidine like in the natural proteins, favoring H2O2 deprotonation).[11] This hydrophobic site, composed of two native G-quartets able to "sandwich" the hemin, is thus more active than only one G-quartet. The increase of activity leads surely to a better detection limit.

With this in mind, it was decided by D. Monchaud's group to create an artificial high-activity hemin binding site to improve the detection of telomeric G-quadruplex sequences, thanks to the use of TASQ. For the detection of 22AG, the addition of TASQ was expected to modify the characteristics of one or two of the external sites, to one or two pseudo-internal sites. Indeed, thanks to a like-likes-like process, TASQ interact with G-quadruplexes *via* a synthetic Gquartet/native G-quartet recognition, identical to a classic G-quartet/G-quartet interaction. Thus, the "binding pocket" created between the external native G-quartet of the G-quadruplex and the intramolecular synthetic G-quartet of a TASQ is an artificial high-activity hemin binding site.[155]

To verify it, a DNAzyme experiment was carried out in Caco.KTD in a 96-well plate with 1 μM hemin, 2 mM ABTS, and 600 μM H2O2, in the presence or absence of 50 μM DOTASQ-C5, with different concentrations of 22AG (from 65 nM to 8 μM). Results highlighted the fact that the catalysis is more efficient in the presence of TASQ, and offer the possibility to decrease the detection limit from 4 μM to 500 nM. This improvement confirms the concept of the pseudointernal high-activity hemin binding site that can be considered as an equivalent of the "binding pocket" of natural enzymes. In term of initial rates, whereas the optimal concentra‐ tion of DOTASQ-C5 can increase it by factor 2.7 at 40 equivalents, PNADOTASQ was able to double it only at one equivalent.[155]

To conclude, TASQ can be used as "boosters" of the catalytic activity of DNAzyme, and permit to improve the detection limit by speeding up the rate of oxidation of the substrate (*e.g*., ABTS or TMB). This effect could be very interesting to detect smaller concentrations of G-quadru‐ plexes, in particular of telomeric G-quadruplexes, to determine with a better signal-to-noise ratio telomerase concentrations from cell lysates.

Another application detailed here is the development of a DNAzyme-mimicking system to detect the bacterial signaling molecule c-di-GMP, published by H. O. Sintim *et al*.[141] The detection of this molecule, able to form biofilms in several clinical relevant bacterial pathogens, is crucial to limit hospital infections. Interestingly, the target molecule is also, by itself, the catalyst of the peroxidation reaction (see section 4.2), because of its ability to self-assemble to form G-quartets. This method was validated with *E. coli* overexpressing a diguanylate cyclase WspRD70E from crude bacterial lysates.

The last but not the least application is the use of TASQ to create fully synthetic process able to mimic nature. Indeed, from a fully natural process with the *horseradish peroxidase*, DNA strands and hemin are still natural products in DNAzyme. However, in TASQ-based catalysis, only hemin is natural, because organic synthetic molecules (*i.e*., TASQ) replace DNA. To pursue the evolution, another extremely soluble Fe(III)–porphyrin, the 5,10,15,20-tetrakis(4 sulfonatophenyl)porphyrin (also termed FeTPPS), was used instead of hemin. This molecule was well known for its excellent water solubility, its stability, its resistance to highly oxidative conditions, and known to perform H2O2-mediated oxidations for more than two decades.[223– 225] After a first titration of the FeTPPS with DOTASQ-C5, the catalytic experiments were carried out in Caco.K (without Triton X-100 and DMSO, because of the excellent solubility of this porphyrin compared to hemin). The same protocol was used in the presence of ABTS, and results showed an initial rate of 0.36 h–1 for DOTASQ-C5, while no activity was detected with 22AG. This point must be due to electrostatic repulsions between the four negative charges of the sulfonate derivative and the negatively charged phosphate groups of the DNA strand. In sum, the use of FeTPPS with a TASQ was the last step to create a fully synthetic process mimicking a well-known natural process of peroxidation.[205]
