**3.4. Applications of G-quadruplex-based DNAzymes**

The *horseradish peroxidase*, mimicked by G-quadruplex-based DNAzyme systems, is a very well-known protein in a biochemical context in which, grafted to a biomolecule (mainly an antibody), it is used to oxidize a colorimetric or fluorogenic substrate in ELISA (for *enzymelinked immunosorbent assay*). This high-throughput assay is, for example, used for the clinical detection of anti-HIV antibodies from biological samples.[69, 70] Moreover, the *horseradish peroxidase* is also used for the purification of waste waters,[71] to develop biosensors, or as a reagent for organic synthesis. This popular enzyme, extracted from the root of the horseradish, is able to oxidize many substrates (*e.g*., aromatics, phenols, indoles, amines) in the presence of hydrogen peroxide. Its properties were discovered by L. A. Planche in 1810, who noticed that a tincture of guaiac resin became blue when a piece of fresh horseradish root was placed in it. The chemical reaction occurred here is now expected to be due to the peroxidase-catalyzed oxidation of 2,5-di-(4-hydroxy-3-methoxyphenyl)-3,4-diphenylfuran to the corresponding bismethylenequinone (also known as "guaiacum blue").[72]

Due to all the advantages described before concerning the use of DNA instead of protein to catalyze reactions, chemists have used for more than 15 years DNAzyme to develop and create plenty of new applications. Indeed, the far better modularity of the G-quadruplex-based peroxidase-mimicking systems constitutes a new powerful and invaluable tool for scientists, mainly for *in vitro* detection of a large range of molecules.

stacking 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

quadruplex 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

and Na+

are important for the G-

of the buffer, the nature and concentration of salts (*e.g*., K+

452 Advanced Catalytic Materials - Photocatalysis and Other Current Trends

described in detail in 2012.[56] The four main effects of ATP are:

**a.** to facilitate the fixation of H2O2 on the iron atom of hemin,

**c.** to protect the reaction partners against oxidation, and

**3.4. Applications of G-quadruplex-based DNAzymes**

methylenequinone (also known as "guaiacum blue").[72]

**b.** to fuel the two one-electron transfer steps of the catalytic cycle,

**d.** to modulate the pH of the reaction mixture, like what is observed in cells.[68]

The *horseradish peroxidase*, mimicked by G-quadruplex-based DNAzyme systems, is a very well-known protein in a biochemical context in which, grafted to a biomolecule (mainly an antibody), it is used to oxidize a colorimetric or fluorogenic substrate in ELISA (for *enzymelinked immunosorbent assay*). This high-throughput assay is, for example, used for the clinical detection of anti-HIV antibodies from biological samples.[69, 70] Moreover, the *horseradish peroxidase* is also used for the purification of waste waters,[71] to develop biosensors, or as a reagent for organic synthesis. This popular enzyme, extracted from the root of the horseradish, is able to oxidize many substrates (*e.g*., aromatics, phenols, indoles, amines) in the presence of hydrogen peroxide. Its properties were discovered by L. A. Planche in 1810, who noticed that a tincture of guaiac resin became blue when a piece of fresh horseradish root was placed in it. The chemical reaction occurred here is now expected to be due to the peroxidase-catalyzed oxidation of 2,5-di-(4-hydroxy-3-methoxyphenyl)-3,4-diphenylfuran to the corresponding bis-

Due to all the advantages described before concerning the use of DNA instead of protein to catalyze reactions, chemists have used for more than 15 years DNAzyme to develop and create plenty of new applications. Indeed, the far better modularity of the G-quadruplex-based Trying to be as exhaustive as possible, a list of the principal applications found in the literature is detailed hereinafter.


method has a sensitivity up to 2 pg.mL–1, 50 times better than the results obtained with ELISA (with a limit of 100 pg.mL–1). Likewise, L. Stefan *et al*. reported the very first steps of a general strategy to make G-quadruplex DNA catalysts easily immobilizable, for the development of high-throughput ELISA-type assay.[126] In this study, a 96-well plate coated with streptavidin was functionalized using a cyclododecapeptide termed RAFT (for *regioselectivity addressable functionalized template*),[127] equipped on one side with a Gquadruplex (intra- or intermolecular), and with a biotin on the other side. The first step was the optimization of experimental conditions to access the best detection limit. In this case, it was found that for a catalysis limited to 2 hours (a fixed time decided by the authors to have a catalytic response in an acceptable amount of time), reactions have to be carried out in a cacodylic buffer (10 mM, with 10 mM KCl and 90 mM NaCl) at pH 4.4, with 1 μM hemin, 400 μM TMB, and finally 2 mM H2O2 to trigger the process. Using this protocol compatible with biochemical applications, the authors used only 2 pmol of the catalysis per well to detect streptavidin from a commercially available pre-coated microplate (300 pmol/well). This default of RAFT-quadruplex was chosen both to avoid unspecific associations and to limit the consumption of the catalyst. To propose scientists different ways to work with these kinds of DNAzyme systems, two protocols were developed. The more user-friendly one is a three-step procedure: first, a 200 μL solution of RAFTquadruplex + hemin solubilized in the *ad hoc* buffer described before was poured in the wells; after an incubation time of half an hour, a washing step (using a cacodylic buffer at pH 7.2) was performed to remove all unbound materials; and finally, a solution, including the luminescent probe TMB, was put inside all the wells. Reactions started when hydrogen peroxide solutions were put inside. The variation of absorbance at 370 nm was monitored using a 96-plate UV–Vis reader with one measure every 2 minutes. This work highlighted the fact that surface-immobilized DNAzymes are interesting alternatives to develop practically convenient, simple, and rapid biophysical assays. Nevertheless, this work constitutes another brick in the wall of the development of effective DNAzyme-based assays.[126]


miR-141 (a biological marker of the human prostate cancer) by naked eyes. Furthermore, scientists created DNAzyme sensors to detect small nucleotides like adenosine triphos‐ phate (or ATP),[137–140] cyclic diguanilate c-di-GMP,[141] and also 8-OHdG (for 8 hydroxy-2′-deoxyguanosine) from the urine, that is associated with various cancers, diabetes, and neurological diseases.[142]

method has a sensitivity up to 2 pg.mL–1, 50 times better than the results obtained with ELISA (with a limit of 100 pg.mL–1). Likewise, L. Stefan *et al*. reported the very first steps of a general strategy to make G-quadruplex DNA catalysts easily immobilizable, for the development of high-throughput ELISA-type assay.[126] In this study, a 96-well plate coated with streptavidin was functionalized using a cyclododecapeptide termed RAFT (for *regioselectivity addressable functionalized template*),[127] equipped on one side with a Gquadruplex (intra- or intermolecular), and with a biotin on the other side. The first step was the optimization of experimental conditions to access the best detection limit. In this case, it was found that for a catalysis limited to 2 hours (a fixed time decided by the authors to have a catalytic response in an acceptable amount of time), reactions have to be carried out in a cacodylic buffer (10 mM, with 10 mM KCl and 90 mM NaCl) at pH 4.4, with 1 μM hemin, 400 μM TMB, and finally 2 mM H2O2 to trigger the process. Using this protocol compatible with biochemical applications, the authors used only 2 pmol of the catalysis per well to detect streptavidin from a commercially available pre-coated microplate (300 pmol/well). This default of RAFT-quadruplex was chosen both to avoid unspecific associations and to limit the consumption of the catalyst. To propose scientists different ways to work with these kinds of DNAzyme systems, two protocols were developed. The more user-friendly one is a three-step procedure: first, a 200 μL solution of RAFTquadruplex + hemin solubilized in the *ad hoc* buffer described before was poured in the wells; after an incubation time of half an hour, a washing step (using a cacodylic buffer at pH 7.2) was performed to remove all unbound materials; and finally, a solution, including the luminescent probe TMB, was put inside all the wells. Reactions started when hydrogen peroxide solutions were put inside. The variation of absorbance at 370 nm was monitored using a 96-plate UV–Vis reader with one measure every 2 minutes. This work highlighted the fact that surface-immobilized DNAzymes are interesting alternatives to develop practically convenient, simple, and rapid biophysical assays. Nevertheless, this work constitutes another brick in the wall of the development of effective DNAzyme-based

454 Advanced Catalytic Materials - Photocatalysis and Other Current Trends

**e.** Detection of nucleic acids is also the target of several G-quadruplex DNAzyme process. Indeed, they are applied for the detection of single-strand DNA,[128] or other DNA analytes,[62] including the smart "DNA machine" developed by I. Willner's team in which its sensitivity is equal to 10–14 M of the target sequence (a 29-mer corresponding to a domain of single-strand DNA hepatitis B viral gene).[129] The detection of genetically modified organisms can also be done, as proved by B. Qiu *et al*. with both cauliflower mosaic virus 35S promoter, and lectin gene.[130] This approach was improved in 2014 to reach a detection limit of 5 nM.[131] Interestingly, a nonclassic fluorescence probe, the 2′,7′ dichlorodihydrofluorescein diacetate (or H2DCFDA), was used instead of the usual ABTS,

**f.** Moreover, DNAzyme is a powerful biotechnological tool to detect micro RNA (also termed mRNA),[132–134] particularly when it is coupled to a rolling circle amplification which allows a sensitivity of 0.3 fM,[135] or as "DNAzyme Ferris wheel" like nanostruc‐ tures, as proposed by W. Zhou *et al*. in 2015,[136] with a detection limit of 5x10–13 M for

assays.[126]

TMB, or luminol.


To summarize, all these cases, which represent the range from the more applied to the more conceptual scientific applications of the same DNAzyme catalysis, illustrate how using DNA instead of enzyme to catalyze a reaction puts out a new avenue in terms of polyvalence. It is believed that this list will increase more and more in the next years. But the precise under‐ standing of the mechanism constitutes also an exciting challenge. On the one hand, this progress should offer scientists the possibility to fine-tune the experimental conditions (*e.g*., sequence of the G-quadruplex DNA strand(s), length of the loop(s), addition of a boosting agent, etc.). On the other hand, a better comprehension should help to enlighten chemists about the mechanistic aspect of the oxidation states of the hemin, in both biological and DNAzyme systems.
