**3.3. Mechanism and factors influencing the catalytic activity**

faces of the guanine moieties. The additional π-stacking interactions between G-quartets, and

edifice.[10, 26, 27] In a structural point of view, the G-quadruplexes can mainly differ from the total numbers of constitutive G-quartets (from two to several thousands)[28–30] or strands (from one to four),[31–35] from the orientation of the strands (leading to several conformations named antiparallel, parallel, and hybrid), and finally from the length, DNA bases composition, and position of the loop(s) (which can be edgewise, diagonal, or chain-reverse).[36–41] All of these parameters are linked to the global stability of the edifice, like the number of G-quartets

**Figure 1.** Schematic representation of the self-assembly of guanines *via* the Watson–Crick and Hoogsteen faces to form

These noncanonical structures are well known in a biological context, because they are strongly suspected to play important roles in key cellular events, like chromosomal instability, or regulation of gene expression. These aspects are far from the scope of this chapter, and author

In 1996, Y. Li and D. Sen developed and published the fourth known DNAzyme system,[45] able to catalyze the incorporation of metals (*i.e.*, Cu(II) and Zn(II)) into a specific porphyrin, named mesoporphyrin IX, or MPIX. To select the best DNA catalyst for their system, they used the *in vitro* SELEX (for *systematic evolution of ligands by exponential enrichment*) method that highlighted one sequence, termed PS5.ST1, from an initial pool of DNA sequences.[45, 46]

**b.** the presence of alkaline cations was required (with a catalytic activity 300 times higher

**c.** the addition of another porphyrin derivative, the *N*-methyl mesoporphyrin IX (or NMM), well known for its interaction with G-quadruplexes and unable to be metallated due to the steric hindrance of the methyl group,[47–49] inhibited the incorporation of the metals into the MPIX. Altogether, these data suggested for the very first time that G-quadruplexes

incites curious readers to have a look to some reviews cited hereafter.[26, 27, 42–44]

**3.2. The seeds of the G-quartet ability to catalyze peroxidase-like reactions**

Interestingly, three main observations were crucial:

), and

**a.** the sequence of the strand was guanine-rich,

than with Na+

could adopt catalytic properties.

), increase the global stability of the tridimensional

, Na+

(as it is discussed later in this chapter), and are interdependent.

the bonding of cationic ions (*e.g*., K+

450 Advanced Catalytic Materials - Photocatalysis and Other Current Trends

G-quartets, and G-quadruplexes.

with K+

After almost 20 years of research on the G-quadruplex peroxidase-mimicking systems (Figure 2), the precise mechanism of the catalytic cycle is not fully understood. Nevertheless, in 2012, L. Stefan *et al*. proposed a first mechanism in nine main steps focused on the iron–porphyrin complex (*i.e.*, the hemin).[56] It was built on the basis of plenty of work published for the hemoprotein systems (primarily peroxidases and catalases).[57, 58] The mechanism is not scrutinized here, and readers are urged to see the references for additional information. However, the main factors influencing the catalysis are described hereafter and might be used as a roadmap.

**Figure 2.** Schematic representation of the DNAzyme activity promoted by a G-quadruplex.

As explained before, the morphology of the G-quadruplexes used (*i.e*., number of G-quartets, strands, type and length of loops, etc.) influences the catalytic efficiency of the reaction. The activation of the hemin rests on the presence of a hydrophobic binding site, playing the role of the "binding pocket" in enzymes. In this DNAzyme context, accessible G-quartets favor the interaction with hemin and, besides, protect the porphyrin from the oxidative degradation due to the oxygen peroxide. The existence of an axial ligand giving electronic density to the iron atom is another major key point.[11] Like in the native HRP in which an histidine has this function, it is assumed that in the G-quadruplex, one of the guanines of the external G-quartet is devoted to this, by flipping out of the plan (exactly like what was observed in another study with platinum complex binding to a G-quadruplex).[59, 60] Moreover, based on molecular modeling, it was also supposed that this effect could be due, in special cases, to a cytosine from a nearby loop, intercalated between the accessible G-quartet and the hemin, and creating πstacking interactions.[55] This insertion of the loop is clearly not a *sine qua non* condition, because several G-quadruplexes without loops and/or cytosine are effective as biotechnolog‐ ical catalysts (*e.g*., d((G3T*n*)3G3) (*n* = 1–4),[54] d((TG4)4),[56] d((T4G4)4),[23]or d((T4G6T4)4)).[61] Notwithstanding, their activities are less high than G-quadruplexes with loops, for which the catalytic efficiency can be sorted as follows: antiparallel ones > hybrid forms > parallel ones. [62] The presence of a polar environment and several H-bond donors and acceptors on the distal face of the hemin also constitute a positive point.[11] All these aforementioned criteria are directly linked to the inherent design of the DNA sequence,[63] but other experimental parameters can be used to modulate the pseudo-enzymatic activity of the G-quadruplex. Among all the conditions (non-exhaustive list), chemists can easily inflect the pH, the nature of the buffer, the nature and concentration of salts (*e.g*., K+ and Na+ are important for the Gquadruplex structuration), the presence and nature of a surfactant (*e.g.*, Triton X-100, Tween 20, Brij 56), the temperature, and, last but not least, the adjunction of an "additive."[9, 50, 64– 66] Indeed, some additional compounds can be used to amplify the response of the catalysis. In particular, the use of adenosine triphosphate (or ATP), and its derivatives, was studied by the groups of D.-M. Kong and D. Monchaud.[56, 67] Interestingly, the role of this small molecule is tricky, and Monchaud's team tried to decipher its actual role. In fact, ATP was proposed to favor several equilibria in the hemin oxidation/reduction process, as it was described in detail in 2012.[56] The four main effects of ATP are:

