**5. Conclusion**

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

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

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

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

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

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,

activity leads surely to a better detection limit.

464 Advanced Catalytic Materials - Photocatalysis and Other Current Trends

binding site.[155]

double it only at one equivalent.[155]

ratio telomerase concentrations from cell lysates.

WspRD70E from crude bacterial lysates.

As demonstrated all along this chapter, native and synthetic G-quartets are powerful catalysts for peroxidase-like process.

On the one hand, the DNAzyme field is fed by the numerous examples of G-quadruplexes used as a native catalytic platform, that found dozens of applications, from the detection of products in biological samples, to the evaluation of heavy metal concentrations, proteins activity, or to develop logic gates for new DNA-based nanotechnologies.[7, 23, 24] G-quadru‐ plex DNA are extremely versatile structures that can be folded from plenty of sequences, in several media, and their functionalization to add probes, functional groups, or to graft them on a solid support are important and invaluable advantages. The design of the G-quadruplex structures, that is, of the catalyst, is far easier than for enzymes, because small modifications of a protein commonly lead to a modification of the active site and then, consequently, to a partial or total loss of activity. Indeed, all the G-quadruplexes are virtually able to catalyze peroxidase-mimicking reactions, because the key part of these noncanonical structures is one of the external G-quartet which is, by definition, the basic unit of G-quadruplexes.[10, 180] As a result, these easy-to-use DNAzyme systems are ready to be applied in chemistry and biology laboratories, mainly for their adaptability and stability, but also in the medical field in which DNAzyme can be considered as a cheaper alternative to the natural *horseradish peroxidase*, mainly to tag relevant biomolecules, like antibodies in ELISA protocols.

On the other hand, the only use of the minimal catalytically active part of the G-quadruplexes, the G-quartet, was presented in this chapter, mainly thanks to the use of TASQ. These templateassembled synthetic G-quartets, able to form an intramolecular G-quartet, proved that a small synthetic molecule can selectively interact with hemin to catalyze peroxidase-mimicking reactions. Interestingly, all the TASQ highlighted here (DOTASQ,[14, 205] PNADOTASQ, [15, 206] or RAFT-G4)[13, 197]) offer different activities, and the best edifice is definitely PNADOTASQ, which is closer to the efficiency of G-quadruplex-based DNAzymes, even if a decrease by a factor of 10, approximately, is observed. However, the use of synthetic molecules instead of natural structures (*i.e*., enzymes or DNA) is an undeniable advantage, in particular because these TASQ are easily synthesized in four straightforward steps from commercially available products, with good yields. Their modifications and improvements are only limited by imagination, and the new molecules designed by D. Monchaud's team clearly demonstrate this point.[16, 200] Interestingly, a fully synthetic system based only on nonnatural compo‐ nents was described in this chapter for its ability to reproduce the natural enzymatic process. In other words, TASQ permit to mimic a natural catalysis originally made by the *horseradish peroxidase* with only synthetic molecules made by chemists.[55] It can be postulated that the mechanism behind this reaction is probably very close to the natural one, even if more data are needed to confirm this proposition.

To conclude, this chapter showed the role of nature-mimicking catalytic systems, using noncanonical DNA G-quadruplex structures as native G-quartets, or synthetic G-quartets with TASQ. Even if the efficiency of these systems is for now not as high as the natural *horseradish peroxidase*, it is weighted against plenty of advantages in terms of applications, experimental conditions, versatility, and chemical modifications. Step by step, the scientific community puts new bricks in the wall and paves the way to more efficient nature-mimicking catalytic systems, closer and closer to what nature is able to do.
