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\DOI{10.5802/crchim.261}
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\dateposted{2024-04-19}
\begin{document}

\begin{noXML}
%
%\makeatletter
%\def\TITREspecial{\relax}
%\def\cdr@specialtitle@english{Breaking Barriers in Chemical Biology -- Toulouse 2022}
%%\def\cdr@specialtitle@french{Briser les barri\`eres entre la chimique et la biologie -- Toulouse 2022}
%\makeatother

\CDRsetmeta{articletype}{review}

\title{Functionalized oligonucleotides, synthetic catalysts as enzyme
mimics}

\alttitle{Oligonucl\'{e}otides Fonctionnalis\'{e}s, catalyseurs
synth\'{e}tiques comme mimes enzymatiques}

\author{\firstname{Crystalle} \lastname{Chardet}}
\address{Laboratoire de Synth\`{e}se et Physico-Chimie de Mol\'{e}cules
d'Int\'{e}r\^{e}t Biologique, UMR CNRS 5068, Universit\'{e} Paul
Sabatier, 118 Route de Narbonne, CEDEX 9, 31062 Toulouse, France}

\author{\firstname{Sandra} \lastname{Serres}}
\addressSameAs{1}{Laboratoire de Synth\`{e}se et Physico-Chimie de Mol\'{e}cules
d'Int\'{e}r\^{e}t Biologique, UMR CNRS 5068, Universit\'{e} Paul
Sabatier, 118 Route de Narbonne, CEDEX 9, 31062 Toulouse, France}

\author{\firstname{Corinne} \lastname{Payrastre}\CDRorcid{0000-0002-4267-0390}}
\addressSameAs{1}{Laboratoire de Synth\`{e}se et Physico-Chimie de Mol\'{e}cules
d'Int\'{e}r\^{e}t Biologique, UMR CNRS 5068, Universit\'{e} Paul
Sabatier, 118 Route de Narbonne, CEDEX 9, 31062 Toulouse, France}

\author{\firstname{Jean-Marc} \lastname{Escudier}\CDRorcid{0000-0001-8850-6441}}
\addressSameAs{1}{Laboratoire de Synth\`{e}se et Physico-Chimie de Mol\'{e}cules
d'Int\'{e}r\^{e}t Biologique, UMR CNRS 5068, Universit\'{e} Paul
Sabatier, 118 Route de Narbonne, CEDEX 9, 31062 Toulouse, France}

\author{\firstname{B\'{e}atrice} \lastname{Gerland}\CDRorcid{0000-0003-3063-1893}\IsCorresp}
\email[B. Gerland]{beatrice.gerland@univ-tlse3.fr}
\addressSameAs{1}{Laboratoire de Synth\`{e}se et Physico-Chimie de Mol\'{e}cules
d'Int\'{e}r\^{e}t Biologique, UMR CNRS 5068, Universit\'{e} Paul
Sabatier, 118 Route de Narbonne, CEDEX 9, 31062 Toulouse, France}

\thanks{This work was supported by a grant from the Agence Nationale de
la Recherche, ANR-18-CE07-0023, to BG}

\keywords{\kwd{Functionalized oligonucleotides} 
\kwd{Enzyme mimics}
\kwd{Chemical modifications}}

\altkeywords{\kwd{Oligonucl\'{e}otides fonctionnalis\'{e}s}
\kwd{Mimes enzymatiques}
\kwd{Modifications chimiques}}

\shortrunauthors

\begin{abstract}
This review describes the chemical rationale behind the modification of
deoxyribonucleotides, which are later incorporated through an enzymatic
or automated supported process, resulting in the synthesis of
functionalized oligonucleotides (FuON). These additional handles
(located on the base or the sugar moiety) were carefully chosen to
enhance the rather limited repertoire of DNA to develop FuONs that
display enzyme-like properties such as catalysis and recognition. We
will exemplify the development of FuON either presenting RNA-cleaving
activity or acting as peroxidase or protease mimics and discuss the
impact of the introduced modifications.
\end{abstract}

\begin{altabstract}
Cette revue d\'{e}crit les choix guidant la modification chimique des
d\'{e}soxyribonucl\'{e}otides, incorpor\'{e}s par la suite par voie
enzymatique ou par synth\`{e}se support\'{e}e automatis\'{e}e pour
conduire \`{a} l'obtention d'oligonucl\'{e}otides fonctionnalis\'{e}s
(FuON). Ces modifications additionnelles (localis\'{e}es sur la base ou
sur le sucre) ont \'{e}t\'{e} pertinemment choisies afin d'\'{e}largir
le r\'{e}pertoire relativement limit\'{e} de l'ADN pour d\'{e}velopper
des FuON pr\'{e}sentant des propri\'{e}t\'{e}s g\'{e}n\'{e}ralement
r\'{e}serv\'{e}es aux enzymes telles que la reconnaissance et la
catalyse. Nous illustrerons ce propos en d\'{e}crivant le
d\'{e}veloppement de nombreux FuON, capables de r\'{e}aliser la coupure
de l'ARN ou d'agir~comme des mimes de peroxydases ou de prot\'{e}ases,
et nous commenterons l'influence des modifications introduites.
\end{altabstract}

\maketitle

\twocolumngrid

\end{noXML}

\section{Introduction}

In Nature, enzymes are proteins that ensure the efficiency and
selectivity of complex chemical transformations under physiological
aqueous conditions. The key feature for catalysis is an appropriate
macromolecular 3D folding to bring in close proximity catalytic
partners (amino acid side-chains, co-factors\,{\ldots}) while
discriminating between potential substrates and assuring the final
specificity outcome. Nucleic acids also display 3D complex structures
with the unmatched advantages of a programable folding and dynamic
renaturation processes (unlike~proteins). Until the beginning of the
80s and the discovery of RNA acting as an enzyme with self-splicing
properties (coining the term RNAzyme), the role of nucleic acids was
thought to be only devoted to the storage and transfer of genetic
information~\cite{1a,1b}. DNA was not even primarily considered, assuming
that an inert chemical nature would provide an evolutionary advantage
by ensuring the absence of undesired alterations of genetic
instructions. The major hurdles to overcome were four canonical
nucleotides with limited functionalities (mostly involved into Watson
and Crick base pairing) and no hydroxyl group at the
2$^{\prime}$-position of the sugar. It took another decade to
demonstrate that DNAzymes, single-stranded deoxyoligonucleotides (ODN)
with no in vivo counterparts, were also capable of catalytic activities
that could match those of enzymes~\cite{2a,2b}. Both aptamers
(oligonucleotides able to bind a selected target but with no catalytic
\mbox{properties)} and DNAzymes could be selected in vitro by the iterative
and powerful SELEX method~\cite{3a,3b}, relying on the use of unmodified
nucleosides 5$^{\prime}$-triphosphate (dNTP). These nucleotides are
substrates of (mutant) DNA polymerases that can be integrated during
the DNA amplification step of the polymerase chain reaction (PCR). It
is wisely assumed that a library composed of 10$^{15}$
sequences (obtained from a randomized region made of 25 nucleotides) is
sufficient to identify a sequence of interest by exploring a
sufficiently diverse chemical space~\cite{4}. To achieve such catalytic
properties, DNAzymes were able to compensate for the absence of the
2$^{\prime}$-hydroxyl group (in comparison with RNA counterparts) by a
less restrictive sugar puckering, leading to complex
architectures~\cite{5a,5b,5c} or unusual secondary structures such as triplex
structures~\cite{6}. DNA secondary structures adopt local conformations
that could influence the pKa values of functional groups in the same
magnitude as that observed in enzymes~\cite{7}. Taking these remarks
into consideration, it has been experimentally confirmed that DNAzymes
could be as efficient as their RNA counterparts in the Diels--Alder
reaction~\cite{8}. Moreover, DNA displays intrinsic qualities in terms
of fast and effective automated supported synthesis (SPS) combined with
increased chemical stability compared with RNA. Nucleic-based
biomimetic catalysis could definitively benefit from these former
qualities combined with the DNA aptamer property and capacity to be
selected owing to in vitro biomolecular methodologies.\looseness=-1

However, it rapidly emerged that modifications of the native DNAzyme
backbone were required when moving toward biological and/or clinical
applications. Switching to a phosphorothioate linkage combined to a
3$^{\prime}$-inverted nucleotide was proven to be efficient in avoiding
nuclease degradation, relying on the solutions already developed for
the antisens approach~\cite{9}. Most of the work was devoted to
structural modifications of the sugar moiety, alongside the classical
2$^{\prime}$-\textit{O}-methyl modification. For example, DNAzymes
based on XNAs (for Xenobiotic Nucleic Acid)~\cite{10a,10b} and in particular
on FANAs (2$^{\prime}$-Fluoro-Arabino Nucleic Acid) were demonstrated to be less
sensitive to nuclease digestion while maintaining catalytic
RNA-ligating~\cite{11} or -cleaving\break activities~\cite{12a,12b}. FANAzymes
present the additional advantage of being obtained by in vitro
evolution using non-engineered DNA polymerases. It is also of great
interest to mention the ligase-catalyzed oligonucleotide polymerization
(LOOPER) method developed by Hili's laboratory~\cite{13}.
Pentanucleotides anticodons encode modifications through
functionalization of the 5$^{\prime}$-end nucleotide with chemical
scaffolds varying from alkyl chains and phenyl rings to organic
functions while maintaining a 
\mbox{3$^{\prime}$-end} variable dinucleotide sequence for
degeneracy. The combination of SELEX and LOOPER processes allows the
selection of functional aptamers against human alpha-thrombin bearing
diverse chemical groups with high binding affinity and not relying on
the usual G-quadruplex structuration. Although this approach was
concentrated to improve aptamer affinities, it also emphasizes the
added value of increasing the DNA chemical repertoire toward its
functional role in terms of recognition and ultimately biomimetic
catalysis. We decided to narrow the scope of the review to covalently
modified monomers (either dNTP or phosphoramidites) that lead to
functionalized (deoxy)oligonucleotides (FuON) with catalytic
enhancement purposes to mimic biochemical reactions. We will then first
describe FuON with enzyme-like properties that catalyze reactions on
nucleic acids substrates, such as the extensively studied DNA
catalyzed-RNA cleavage. The focus will then be shifted to a broader
reactivity scope as described by the development of FuON, which
displays peroxidase-mimicking activity and DNA-based proteases.

As the chemical strategies to introduce modifications have been
reviewed in the literature by numerous groups~\cite{14a,14b} and
ourselves~\cite{15}, we will only present the functionalized monomers
of interest in charge of the catalytic activities.

\section{Functionalized oligonucleotides as ribonuclease A mimics}

Screening for new DNA catalysts candidates in the general context of
degrading viral RNA in a sequence-specific fashion was inspired by the
ribonuclease A (RNase A) responsible for cleaving the phosphodiester
bond. The target, being a RNA substrate, presents the tremendous
advantage of bypassing the recognition hurdle, as DNA is indeed
complementary to RNA. RNase A catalyzes the cleavage of the
P-5$^{\prime}$-\textit{O}-bond to give two RNA  products bearing a
5$^{\prime}$-OH termini and a 2$^{\prime}$,3$^{\prime}$-cyclic
phosphodiester on the 3$^{\prime}$-side of a pyrimidine nucleotide,
which is further hydrolyzed to a 3$^{\prime}$-monophosphate~\cite{16}.
During the first step of the
2$^{\prime}$-\textit{O}-trans-phosphorylation, the imidazole ring of
Histidine 12 (His12) acts as a general base to increase the
nucleophilic character of the 2$^{\prime}$-hydroxyl, allowing
intramolecular in-line attack on the adjacent phosphorus atom. On the
other hand, the leaving ability of the 5$^{\prime}$-oxygen is increased
because of the protonation provided by the imidazolium side chain of
His19. 

Considering the key role of two histidine side chains in the general
acid--base catalytic mechanism, the first modified DNAzyme selected to
cleave RNA was named ``16.2-11'' and decorated with imidazole
groups~\cite{17}. On a suitably protected
5-(3-aminopropenyl)-2$^{\prime}$-deoxyuridine, the 4-imidazoleacrylic acid moiety
was linked through an amide coupling to the primary amine before
converting the resulting nucleoside either to its corresponding
5$^{\prime}$-triphosphate \textbf{1} (for the SELEX process) or phosphoramidite
\textbf{2}, for the solid-phase synthesis of the most active DNAzymes
(Figure~\ref{fig1}).

\begin{figure*}
\includegraphics{fig01}
\caption{\label{fig1}C5-Functionalized nucleotides bearing an imidazole
moiety (CE\ ${=}$\ cyanoethyl).}
\end{figure*}

Introduction of modifications at the C5 position on pyrimidines (or at
the N7 or C8 positions on the purine base) does not impair recognition
by DNA polymerases during the enzymatic polymerization of dNTP inherent
to the SELEX process. Moreover, Watson and Crick base pairing is also
maintained during duplex formation, which is a key feature for
efficient RNA recognition. The overall structure of such functionalized
DNAzymes was described by a catalytic core containing four imidazole
residues located close to the cleavage site and two binding arms
complementary to the RNA substrate. Despite displaying high efficiency
($k_{\mathrm{obs}}<1.5$~min$^{-1}$),
sequence specificity, and turnover (the DNA binding arms being short
enough to denature the duplex structure once the RNA strand was
cleaved), the presence of divalent metal ions (in particular
Zn$^{2+}$) was required. This metal ion dependency could
be explained by Zn$^{2+}$ ions either playing a structural
role in folding the DNAzyme in an active conformation or being part of
the mechanism, activating the 2$^{\prime}$-hydroxyl or a molecule of
water (or both). Although encouraging, this result indicated that
imidazole modifications were not sufficient to bypass the need for
metal cations catalysis.

Lysine 31 (Lys31) was also proposed to be involved in transition state
stabilization, and the lack of a cationic function needed for the
general acid--base mechanism might be the main reason for
M$^{2+}$-dependent catalysis. This rationale developed by
Perrin's laboratory was confirmed by the selection of the first
independent DNAzyme 9$_{25}$-11 bearing both imidazole and
ammonium functionalities with $k_{\mathrm{obs}}<0.2
$~min$^{-1}$~\cite{18a,18b}. This approach relied on two dNTP
\textbf{3} and \textbf{4} to introduce the different modifications on a
purine or pyrimidine base, respectively (Figure~\ref{fig2}).

\begin{figure*}
\includegraphics{fig02}
\caption{\label{fig2}Functionalized nucleotides used for the selection
of RNase A mimics.}
\end{figure*}

First, the substitution of the bromine atom by histamine onto
8-bromo-2-deoxyriboadenosine-5$^{\prime}$-monophosphate 
and subsequent conversion led to the corresponding
(8-(2-(4-imidazolyl)ethylamino)-2$^{\prime}$-deoxyriboadenosine-5$
^{\prime}$-triphosphate
(dA$^{\mathrm{Im}}$TP) \textbf{3}. Along with commercial
5-(3-aminoallyl)-2$^{\prime}$-deoxyribouridine-5$^{\prime}$-triphosphate 
(dU$^{\mathrm{aa}}$TP) \textbf{4}, these compounds were used for in
vitro selection. 9$_{25}$-11 was only able to cleave a
single ribonucleotide bond embedded into a DNA substrate
(Figure~\ref{fig3}, left). To evaluate its \textit{trans}-catalytic
properties, M$^{2+}$-independent 9$_{25}$-11
presenting a minimal yet active structure (represented in italic,
Figure~\ref{fig3}, left) was synthesized de novo by SPS from the
corresponding phosphoramidites of \textbf{3} and \textbf{4}. Kinetic
studies indicated a $k_{\mathrm{cat}}$ of ${\sim}1.5 \times
10^{-2}$~min$^{-1}$, a 3-fold decrease
compared with the previously observed \textit{cis} cleavage. However, a
high substrate specificity against the target d(GCGTGCC)rCd(GTCTGTT)
was observed as a drastic reduced cleavage for degenerate sequences was
measured.

\begin{figure*}
\includegraphics{fig03}
\caption{\label{fig3}Predicted secondary structures of (left) DNAzyme
9$_{25}$-11~\cite{18a} (in italic the mimimal catalytic
core~\cite{18b}) with a single ribophosphodiester substrate; (middle)
DNAzyme clone 32 with an all-RNA target~\cite{19}; (right) DNAzyme
7-38-22t with an all-RNA target~\cite{20}. (Blue letters:
ribonucleotides; black letters: deoxyribonucleotides; star colored
letters: modified nucleotides; black arrows indicate the cleavage
site.)}
\end{figure*}

In contrast, subsequent work by Williams \etal\ led to the cleavage of
substrates made only of RNA~\cite{19} (Figure~\ref{fig3}, middle). They
relied on previously described nucleotide \textbf{1} to introduce
imidazole moieties and designed 7-aminopropynyl modified 7-deaza-dATP
\textbf{5} (Figure~\ref{fig2}). They observed that the introduction of
modifications at this position on the adenine base was less
destabilizing within a duplex, facilitating the PCR step, combined with
a better tolerance of DNA polymerases toward a more rigid linker such
as the propenyl group~\cite{21}. After a palladium-catalyzed coupling
reaction between 7-deaza-7-iodo desoxyadenosine and
\textit{N}-propynyltrifluoracetamide followed by a deprotection step,
the resulting nucleoside was converted into its corresponding dATP
\textbf{5}. This DNAzyme clone 32 did present a
M$^{2+}$-independent \textit{cis} \mbox{catalytic} \mbox{activity} on a
12nt target RNA sequence with a weak $k_{\mathrm{obs}}$ of 0.07
min$^{-1}$, still a 10$^{5}$ rate improvement
\mbox{compared} to the uncatalyzed RNA cleavage. The cleavage was
site-specific, with the scissile bond located opposite the loop region
presenting both imidazole and amino residues. The authors of those
studies concluded on the unknown precise role of those appended
functionalities, as they could either participate directly in the
catalytic mechanism or be involved in the folding DNAzyme process
leading to an active structuration, thus advocating\break for further
investigations. Building on those first discoveries, other
functionalized RNA-cleaving DNAzymes were developed as thoroughly
reviewed by Hollenstein~\cite{22} and others~\cite{23a,23b}. The long-term
research work from Perrin's laboratory was rewarded by the design of an
efficient DNAzyme able to mimic RNase A catalysis in the absence of
divalent metal cations. During selection, the authors combined the
already developed 8-histaminyl-deoxyadenosine
(dA$^{\mathrm{im}}$TP) \textbf{3}, the 5-aminoallyl-deoxycytidine
(dC$^{\mathrm{aa}}$TP) \textbf{6}, an analog
to~dU$^{\mathrm{aa}}$TP, with~5-guanidinoallyl-deoxyuridine
(dU$^{\mathrm{ga}}$TP) \textbf{7}, presenting a cationic group
known to increase thermostability by electrostatic complementarity with
the phosphodiester negative charge (Figure~\ref{fig2}).

DNAzyme 7-38-22 was found to cleave a 19nt long all-RNA substrate in
the absence of M$^{2+}$ ions and with multiple turnovers,
displaying a rate constant of 4.9 min$^{-1}$ for
self-cleavage~\cite{20}. Designed to easily allow the evaluation of
\textit{trans}-catalysis, the DNazyme was able to cleave
($k_{\mathrm{cat}}=0.27$~min$^{-1}$ with multiple
turn-overs) with a 20-fold selectivity the UG bond of a 19nt all-RNA
substrate embedded into a four bases bulge that was located (as usually
observed) in front of a fully modified catalytic core
(Figure~\ref{fig3}, right). They then demonstrated that the presence of
an imidazole ring, which is thought to be required as a key player for
acid--base catalysis, could be bypassed by solely introducing both
cationic amine and guanidine functions to select self-cleaving DNAzymes
with higher activities than some imidazole-decorated
DNAzymes~\cite{24}.

Site-selective modification of the catalytic core of 10-23 DNAzyme was
explored through SPS by introducing functionalized
2$^{\prime}$-deoxy(deaza)adenosine phosphoramidites with imidazole,
guanidinium, or amino group either at the C-6, C-7 or C-8 onto the
purine base. The ``chemical evolution'' of the A9 or A12 position
confirmed the necessity of maintaining the Watson and Crick base
pairing (as expected), whereas other modifications did not improve
cleaving activity~\cite{25}. Other phosphoramidites bearing different
linkers with a terminal amino group at the 2$^{\prime}$-position were
synthesized to evaluate the impact of the modifications of the known
conserved positions of the 8-17 DNAzyme. The catalytic effects of a
Ca$^{2+}$-mediated reaction were found to be
limited~\cite{26}.

Apart from the progress that could arise from the optimization of SELEX
conditions, there is also room for chemical input in designing new
functionalized nucleotides with different linkers or even
functionalities, considering that no clear rationale has emerged to
pinpoint the beneficial positions to modify a given DNAzyme.

As indicated, RNA cleavage mediated by DNA usually requires the
presence of bivalent cations. Consequently, DNAzymes have been selected
for metal detection and biosensing purposes; however, when unmodified,
metal selectivity remains low~\cite{27}. The introduction of additional
coordination sites via an organic ligand onto the ODN could improve
such selectivity. This approach was recently described using a
glycil-histidine functionalized tertiary amine introduced onto a
RNA-cleaving DNAzyme, which enhanced selectivity toward the desired
divalent metallic ions~\cite{28}. Oxidation also requires a metallic
center in charge of the catalytic function. A recent example from
Martell's group demonstrated the use of a DNA double helix as a
scaffold to spatially pre-organize two co-catalysts (4-carboxy-TEMPO
and 4,4$^{\prime}$-dicarboxy-2,2$^{\prime}$-bipyridine)
involved in synergistic Cu-TEMPO
alcohol oxidation~\cite{29}. Conjugated through an amide link at the
5$^{\prime}$-end or 3$^{\prime}$-end, respectively, of a matched DNA
duplex, both are positioned at an optimal distance, as confirmed by the
70-fold activity enhancement in catalyst turnover number compared with
the unscaffolded oxidation reaction; however, no specificity for the
various tested alcohol substrates was observed. We decided to focus on
the field of peroxidase-like mimics because studies have exemplified
the intrinsic DNA properties in terms of structuration, recognition,
and catalysis, which are combined in functionalized oligonucleotides
purposefully designed to recognize their substrates and perform the
subsequent catalytic reaction. 

\section{Functionalized oligonucleotides as\newline horseradish
peroxidase and phosphatase\newline mimics} 


Hemin/G-quadruplex structures were well-known to act as horseradish
peroxidase (HRP)-mimicking catalysts~\cite{30}. These unmodified short
single-stranded oligonucleotides present sub-micromolar affinities
toward the hemin metallofactor they were selected against. The DNA
backbone is organized in a highly ordered G-quadruplex structure,
acting as an apoprotein mimic by interacting in a supramolecular
fashion through ${\uppi}$--${\uppi}$ stacking interactions with the
hemin in charge of the enhanced (NADH) peroxidase activities~\cite{31}.
Most are used for biosensing applications as the oxidation of
substrates, either ABTS
(2,2$^{\prime}$-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) by
H$_{2}${O}$_{2}$ to
ABTS$^{\bullet +}$ or luminol induces a color
change or generates chemoluminescence, respectively~\cite{32}. 

Willner's team developed an elegant concept based initially originated
from combining a DNAzyme (in charge of the catalytic activity) with an
aptamer with a short TATA sequence between the two nucleic parts. In
this case, the aptamer is recognized by hydrogen bonding its substrate
to oxidize, acting as a binding pocket. They anticipated that close
\mbox{proximity} between partners would be beneficial, inspired by
RNA-cleaving DNAzymes in which the catalyst and RNA target substrate
are kept in close proximity through Watson and Crick base pairing.
Those ``nucleoaptazymes'' (as called) mimic the main features in the
active site of native enzymes, namely catalysis and recognition. To the
hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme was
tethered one aptamer selected against L-DOPA to perform
H$_{2}$O$_{2}$-mediated oxidation, leading to the
chromophoric aminochrome with a 20-fold enhanced activity for the
modified DNAzyme~\cite{33}. One of the key features is the facilitated
release of the product after oxidation due to its decreased affinity
toward the aptamer pocket, allowing a possible multiple turnover, as
observed in native enzymes. A modest enantioselectivity was also
observed for the oxidation of L-DOPA against its D-enantiomer, which
was correlated with differences in affinities. However, the development
of efficient unmodified nucleoaptazymes, as defined by Willner's team,
faced two issues~\cite{34}. First, even close proximity between
partners is not sufficient to ensure an optimum (catalytic)
conformation. Second, despite a relatively furnished catalog of
DNAzymes and aptamer sequences, finding relevant DNAzyme--aptamer
couples that can perform a designed reaction is not trivial. 

To overcome this former limitation, the same team proposed a new family
of functionalized nucleoaptazymes by combining the dopamine binding
aptamer (DBA) sequence while substituting the DNAzyme part by a
metallic ligand such as Fe$^{3+}$ or
Cu$^{2+}$-terpyridine derivatives to perform the oxidation
of L-dopamine, mimicking the catechol oxidase activity 
(Scheme~\ref{sch1})~\cite{35}. These oligonucleotides were
functionalized by introducing
a single metal complex ligand to chelate cupric or ferric ions in the
right oxidation state because the native enzyme presents an oxidized
Cu$^{\mathrm{II}}$--Cu$^{\mathrm{II}}$ met state center with
histidine side chains as proximal ligands in both metal binding
sites~\cite{36}. Those FuON were synthetized through a convertible
approach by reacting the 5$^{\prime}$- or 3$^{\prime}$-amino DBA
sequence with the corresponding 4$^{\prime}$-carboxylic acid of
2,2$^{\prime}$:6$^{\prime}$,2$^{\prime\prime}$-terpyridine, resulting in
the formation of a stable amide bond between both\break partners.

\begin{scheme}
\vspace*{5pt}
\includegraphics{sc01}
\vspace*{6pt}
\caption{\label{sch1}Schematic representation of a nucleoaptazyme
constituted by a Dopamine Binding Aptamer linked to a
Cu$^{2+}$-terpyridine moiety at its 5$^{\prime}$-end by a 4T sequence.}
\vspace*{-3pt}
\end{scheme}

They demonstrated the beneficial presence of a covalent flexible linker
made of four thymidines (T4) between the metallic part and the aptamer
binding site because oxidation does not occur efficiently when both
sites are separated. 

The best nucleoaptazymes present the T4 linker at the 5$^{\prime}$-end
with a $k_{\mathrm{cat}}$ of $40\times 10^{-4}$~s$^{-1}$ and
$267\times 10^{-4}$~s$^{-1}$ for the Cu$^{2+}$- and
Fe$^{3+}$-terpyridine complexes, respectively. This result highlights
the impact on catalysis of the nature of the metal ion within the
complex. As expected, the spatial organization adopted by the tethered
partners also played a key role in the catalytic enhancement, as
confirmed by the molecular dynamic simulations. An application of this
concept was described for the bioconjugation of tyrosine derivatives
using \textit{N}-methyl luminol. The impact of this aptamer-assisted
catalysis could be demonstrated by the 12-fold enhancement observed
when the aptamer sequence binds to the tyrosine substrate~\cite{37}.

The presence of metal ions is also crucial in the phosphatase mimics
field because the native mechanism relies on the presence of a
Zn$^{2+}$ dinuclear site. The Zn$^{2+}$ ions
coordinate and activate the hydroxyl side chain of the serine that
attacks the phosphorus atom of the monophosphoester bond while
maintaining the right geometry for substrate binding through a hydrogen
bonding network~\cite{38a,38b}. Several teams have investigated the design
of these challenging DNAzymes as phosphatase mimics. Unfunctionalized
DNAzymes were previously demonstrated by Silverman's team to be either
in charge of the phosphorylation of the tyrosine side chain of a
peptide substrate or able to catalyze the reverse \mbox{process,} i.e.,~the
dephosphorylation step with multiple turnovers and a
$k_{\mathrm{obs}}$ of 0.19 min$^{-1}$~\cite{39a,39b}.
Building on the nucleoaptazyme concept, Willner's team also designed
phosphatase mimics with a bis-Zn$^{2+}$-pyridyl-salen-type
complex tethered to an ATP aptamer~\cite{40}. Relying again on amine
bond formation, an ATP aptamer (pdb 1AW4) bearing an amino function at
the 5$^{\prime}$- or 3$^{\prime}$-end was covalently linked to a
bis-Zn$^{2+}$-pyridyl-salen-type complex. In this case, an
optimal distance composed of a dithymidine linker at the
3$^{\prime}$-end between the two partners is sufficient to ensure both
flexibility and cooperative proximity. As the promising measured
$k_{\mathrm{cat}}$ (7 min$^{-1}$) shows a
10$^{3}$ times lower efficiency than the native enzyme, it
appeared that the future FuON would still have room for improvement.

\section{Functionalized oligonucleotides as serine protease mimics} 

As illustrated with the last described biomimetic approach and for
asymmetric chemical reactions, most of the literature on DNA-based
catalysis relies on oligonucleotides using metallic
co-factors~\cite{41}. Alongside some recent developments for
Ribonuclease A mimics, other examples based only on organic co-factors
in a protein-like fashion were developed for the DNA-based proteases
field. We now describe the synthetic efforts in this area led by the
functionalization of oligonucleotides with amino acid-like functions to
hydrolyze the amide bond.

${\upalpha}$-Chymotrypsin is one of the most representative members of
the serine proteases family that uses three amino acids constitutive of
its catalytic triad, serine 195 (Ser195), histidine 57 (His57), and
aspartate 102 (Asp102), in a well-known cooperative mechanism 
(Scheme~\ref{sch2})~\cite{42}. 

\begin{scheme*}
\includegraphics{sc02}
\vspace*{5pt}
\caption{\label{sch2}Accepted mechanism for chymotrypsin-like serine
proteases.}
\vspace*{3pt}
\end{scheme*}

Acting as a general base catalyst, the proximal imidazole ring from
His57 enhances the nucleophilic character of the hydroxyl of Ser195,
which is consequently sufficiently reactive to attack the carbonyl
center of the peptide bond. The resulting anionic tetrahedral
intermediate is stabilized in the so-called oxyanion hole by hydrogen
bonds originating from the NH of the peptide backbone. It then
collapses into a covalent acyl-enzyme complex, liberating\break the
\textit{N}-part of the peptide. The resulting (ester)\break acyl-enzyme is
now more easily hydrolyzed than the previous amide bond. Thanks to the
general acid catalysis of His57, under its protonated form stabilized
by Asp 102, the active serine is finally regenerated following the
liberation of the \textit{C}-part of the peptide. Although the protease
mimics field has been active for decades, no synthetic scaffolds have
been able to match the enzyme efficiency~\cite{43}. Enzymatic catalysis
and chemical hydrolysis are strictly two different processes. The first
process introduces a two-step sequence to lower energy barriers through
covalent acyl enzyme intermediates. The former is impaired by chemical
factors such as amide bond stabilization by resonance, the combined
poor nucleophily of water, and the poor ability of the amine to
function as a good leaving group. Diverging from small organic
catalysts or micellar systems, DNA was indeed expected to be a suitable
scaffold because the nucleic part of the ribosome performs the reverse
reaction, i.e., peptide bond formation~\cite{44}. Moreover, with the
difference of peptide-based mimics that folding could neither be
accurately predicted nor undergo any renaturation process, DNA has the
unmatched ability to (re)fold into any designed secondary structures
that could mimic the active site of enzymes. Finally, as stated
earlier, phosphoramidite-based ON synthesis allows the precise
introduction of amino acid residues alongside the nucleic backbone to
reproduce the putative spatial organization of the catalytic triad. 

Most of the efforts were concentrated on the SELEX approach, exploring
de facto an expanded chemical space to screen for a functional nucleic
catalyst. The RNAzyme field, which relies only on unmodified NTP,
remains underexplored~\cite{45}. Dr. Hollenstein's work was aimed at
the functionalization of dNTP, such as modified
7-deaza-2$^{\prime}$-deoxyadenosine bearing at the 7-position a
propargylamido-histamine moiety (dA$^{\mathrm{Hs}}$TP) \textbf{8}
to act as a histidine surrogate (Figure~\ref{fig4}). Two
5-modified-2$^{\prime}$-deoxyribopyrimidine-5$^{\prime}$-triphosphates,
either bearing a valeric acid moiety in the cytosine series
(dC$^{\mathrm{Val}}$TP) \textbf{9} or a pentynol arm in the uracil
series (dU$^{\mathrm{POH}}$TP) \textbf{10}, completed the set of
functionalized dNTP, being mimics of aspartate and serine side chains,
respectively~\cite{46}.

\begin{figure*}
\includegraphics{fig04}
\caption{\label{fig4}Functionalized dNTP with imidazole, carboxylate,
or hydroxyl functions.}
\end{figure*}

They were obtained using a Sonogashira cross-coupling step starting
from their commercial 5-iodo-deoxynucleoside counterparts, followed by
the \mbox{necessary} protection/deprotection steps to introduce the
5$^{\prime}$-triphosphate group required for the SELEX process.
Although the modified dNTPs were well tolerated by DNA polymerases, as
demonstrated during primer extension experiments, they were less
accepted during the PCR amplification step. Unfortunately, no SELEX
attempt in the search for a DNAzyme with amidolytic properties was
described.

The key player in the field was Silverman's group~\cite{47}, which has
devoted important efforts to developing DNA-based protease mimics and
overcoming some pitfalls associated with the SELEX process. First, they
were urged to design a sophisticated selection and ``product
capture'' process to avoid the selection of DNA-cleaving DNAzyme, as
previously observed~\cite{48}. By embedding the amide bond into a
nucleic substrate facing the 40-nucleotide random loop region, they
reported in 2013 the selection of unmodified DNAzymes performing esters
hydrolysis ($k_{\mathrm{obs}}=0.54$~h$^{-1}$) leading
to a moderate 240-fold rate enhancement compared with basal hydrolysis
in the required presence of divalent cations such as
Mg$^{2+}$~\cite{49}. Eventually, although a
$k_{\mathrm{obs}}$ of 3.1~h$^{-1}$ was measured for
aromatic amide hydrolysis, no DNAzymes could be selected for cleaving
the amide bond of aliphatic substrates. They then postulated that a
better nucleophile than water might be required, such as an amino
group. They confirmed their hypothesis in a subsequent publication
starting from commercial 5-hydroxymethyl-, 5-amino-allyl- or
(\textit{E})-5-(2-carboxyvinyl)-2$^{\prime}
$-deoxyuridine-5$^{\prime}$-triphosphates \textbf{11},
\textbf{12}, and \textbf{13} (Scheme~\ref{sch3}). 

\begin{scheme*}
\vspace*{2pt}
\includegraphics{sc03}
\vspace*{6pt}
\caption{\label{sch3}Capture process (left) and C5-functionalized
deoxyuridine 5$^{\prime}$-triphosphates (right).}
\vspace*{-4pt}
\end{scheme*}

After rounds of SELEX optimization using one C5-substituted dUTP at the
time, they were able to isolate the most potent DNAzymes decorated with
hydroxyl groups with hydrolysis activity 
($k_{\mathrm{obs}}=0.1$--0.2~h$^{-1}$) on an aliphatic amide embedded between
two DNA fragments~\cite{50}. The two sequences leading to the most
potent DNAzymes were re-synthetized with no modifications for control
\mbox{purposes.} \mbox{Surprisingly,} they still presented substantial catalytic
activity, although such unmodified sequences did not arise during the
previous selections. This twist of events highlights the fact that
SELEX is undoubtedly a powerful method, but it remains a tedious task
prone to selection bias. 

The complementary bottom-up approach is the SPS of functionalized ON
based on the phosphoramidite strategy. Even if the chemical space
explored cannot be as extensive as the one offered by SELEX, DNA-based
structures mimicking the active site of the serine protease could be
designed through careful and deliberate choice of the positions to be
modified. 

Attempts were made to design esterase mimics by solely using an
imidazole pendant as the nucleophilic catalyst.
5-Ethylnyl-imidazole-deoxyuridine-3$^{\prime}$-phosphoramidites were
incorporated during SPS and organized within duplexes, leading to
structures bearing up to three imidazole-modified positions. However,
none of the duplexes displayed catalytic activity above that of
imidazole, which was used as a buffer control. Relying also solely on
imidazole modification, Gorin's team used an approach similar to the
``nucleoaptazyme'' strategy developed by Willner's
laboratory~\cite{51}. They designed DNA-conjugated small-molecule
catalysts (DCats) by functionalizing with an imidazole group two cholic
acid aptamers folded in a three-way junction (3WJ) secondary structure
(Scheme~\ref{sch4}). At different positions alongside each arm of the 
junction, commercially available 2$^{\prime}$-deoxycytidine or thymidine
3$^{\prime}$-phosphoramidites bearing a C6-amino-linker at the C5
position were introduced and then conjugated with disuccinimidyl
glutarate to histamine. 

\begin{scheme*}
\vspace*{-2pt}
\includegraphics{sc04}
\vspace*{8pt}
\caption{\label{sch4}Schematic representation of the ``DCat'' approach
based on a cholic acid aptamer linked to an imidazole moiety.}
\vspace*{-4pt}
\end{scheme*}

Up to ten DCats were obtained by modulating the position of the
modification alongside the 3WJ to evaluate their ability to cleave
after binding cholic acid-derived umbelliferone esters and the possible
impact on the recognition process. This substrate was chosen because
the cholic acid part is supposed to be recognized at the structure
core, while the fluorogenic release of the umbelliferone allows the
monitoring of the reaction evolution. The best DCat displayed a
100-fold enhancement esterase rate compared with the free equimolar
imidazole but only when it was correctly folded, as corroborated by a
turn on-off experiment with a toehold displacement.


{However, only Madder's team developed 2$^{\prime}$-functionalized
deoxyuridine phosphoramidites} \textbf{14}, \textbf{15}{, and}
\textbf{16} {bearing amino acid side chain-like residues such as
suitably protected carboxylic acid, imidazole, and alcohol functions,
respectively, which were tethered to the oligonucleotide via an amide
bond (Figure~\ref{fig5})~\cite{52}. After solid-phase synthesis, up to
three modifications could be introduced by strand, and the rigid but
programable duplex structure was used to organize the residues along
the major groove of the helix to obtain up to six different sequences,
which were then combined into their corresponding duplexes~\cite{53}.
Analysis of the thermal denaturation studies indicated that each
introduction has a negative impact of ${-}$5~\textdegree C on the thermal
value, although one mismatched duplex displayed a} ${\Delta}${Tm of
${-}$5.3~\textdegree C (compared to an expected} ${\Delta}${Tm of
${{\approx}{-}}$15~\textdegree C), compensating and largely overcoming the
destabilization effect. This result could be explained by the fact that
the imidazole and carboxylate moieties, facing each other, were able to
interact and stabilize the structures through electrostatic
interactions. Despite this chemical effort, no proteolytic activity has
been reported.} 

\begin{figure*}
\includegraphics{fig05}
\caption{\label{fig5}2$^{\prime}$-functionalized nucleotides bearing
amino acid side chain-like residues and further introduced in
oligonucleotides organized within a duplex structure.}
\vspace*{9pt}
\end{figure*}

As the structuration properties of DNA have not been exploited to their
full potential, we decided to build a 3 way junction (3WJ) from three
oligonucleotides partially complementary to each other, bearing at its
core the three functional groups involved in the catalytic
triad~\cite{54}.

First, we established our proof of concept by relying on a versatile
convertible approach by introducing organic functionalities not at the
usual 2$^{\prime}$- or 5-position but at the underestimated
5$^{\prime}$-position. As the 2$^{\prime}$- or C5-modifications direct
the chemical handle toward the major groove within a B-DNA duplex, we
demonstrated that, depending on the stereochemistry of the newly
generated 5$^{\prime}$-\textit{C}-stereogenic center, the modification
could point either toward the solvent or the minor groove when the
absolute configuration of the 5$^{\prime}$-carbon is \textit{R}-assigned or
\textit{S}-assigned, respectively~\cite{55}. Second, we decided to
organize our potential DNA-based proteases into a flexible secondary
structure such as a 3WJ. This assembly, bearing three unpaired
thymidines at its core, was expected to bring the three added
modifications in close proximity with sufficient flexibility to avoid
steric hindrance. After the efficient incorporation of the convertible
5$^{\prime}$-\textit{C}-(\textit{S})-propargyl thymidine\break phosphoramidite
\textbf{17} into three different oligonucleotides, each strand
underwent a post-synthetic copper(I)-catalyzed alkyne-azide
cycloaddition (CuAAC) ``click'' reaction with the three azide
derivatives bearing either a carboxylic, alcohol, or imidazole
functions (Scheme~\ref{sch5}). The FuON were then assembled in an equimolar
mixture, forming a DNA 3WJ with a triply modified core.

\begin{scheme*}
\includegraphics{sc05}
\vspace*{4pt}
\caption{\label{sch5}5$^{\prime}$-convertible nucleotide for the
synthesis and functionalization of a 3WJ structure with amino acid side
chain mimics at its core.}
\vspace*{-4pt}
\end{scheme*}

The 3WJ assembly was confirmed by the presence of only one slow
migrated band during non-denaturing polyacrylamide gel electrophoresis
analyses and was also corroborated by the characteristic B-DNA
signature obtained by circular dichro\"{i}sm spectroscopy. Thermal
denaturation data were obtained to evaluate the contribution of each
amino acid side chain-like residue to the overall stability of the
secondary structure. As expected in the context of a polyanionic DNA
backbone and different pKa values for each residue, the presence of a
carboxylate moiety was modestly destabilizing (${-}$1~\textdegree C), neutral
for the alcohol moiety, and slightly stabilizing for the (cationic)
imidazole residue (${+}$1~\textdegree C). Taken together, unmodified, triply
propargyl- or $^{\mathrm{Ser}, \mathrm{His}, \mathrm{Asp}}$3WJs displayed the same Tm
value of 43~\textdegree C, indicating that the size of the three added
cycloaddition products did not destabilize the resulting secondary
structure. This decorated three-way junction ultimately failed to
provide any amide bond hydrolysis. This could be explained by the lack
of cooperative interactions between the three appended functionalities,
as an alleged over-stabilization (that could originate from a close
proximity of the functional groups) was not observed. Moreover, the
stabilization of the\break anionic tetrahedral intermediate in the proximity
of the negatively charged DNA backbone must be considered. We are
investigating new synthetic options to ensure cooperative interactions
between the three functional groups to design a functional DNA-based
protease that can cleave the amide bond.

\section{Conclusion}

Despite the exquisite evolutionary optimization of DNA to perform
reactions thought to be only devoted to enzymes, organic chemists came
into play to increase the DNA catalytic repertoire. As unmodified
DNAzymes were first developed to cleave the phosphodiester bond of RNA,
mimicking the catalytic activity of Ribonucleoase A, chemists rapidly
took inspiration from the active site of such enzyme to functionalize
oligonucleotides with organic groups reminiscent of the side chain of
the amino acids involved in catalysis. Although a reduced catalytic
efficiency was observed on non-nucleic acid substrates, FuON were also
modified with metal complex ligands to confer either peroxidase or
phosphatase properties or organic functions to mimic the amidolytic
cleavage of the peptide bond, as exemplified in this review. Moreover,
the recognition properties of DNA were also exploited to develop DNA
catalysts capable of recognizing their target and performing the
desired reaction in the so-called nucleoaptazyme\break approach.

Despite these promising results, more challenges lie in front of
(bio)chemists in terms of matching the efficiency and selectivity of
enzymes, with DNA catalysts being less potent than their enzymatic
counterparts. Within the nucleoaptazyme approach, improving the binding
properties would consequently increase the effective concentration of
the substrate, leading to more efficient catalysis. Moreover, guidance
could also be provided by in silico design~\cite{56} and
algorithms~\cite{57} to rationally design functional FuON bearing, at
the right positions, catalytic modifications or even to reach new
stable secondary structures. Finally, advances must be made to improve
each step of the process by developing new SELEX protocols and
engineering new enzymes to overcome the limitations of functionalized
nucleotides that are sometimes poorly recognized by polymerases.
Evolution could also be directed toward reactions not yet known to be
catalyzed by DNA, for~example, by \mbox{modulating} the modification status of
RNA, as already explored with RNA-catalyzed RNA bio-orthogonal labeling
reactions~\cite{58}. However, the catalog of enzymatic reactions
remains underexploited, as exemplified by the challenging site-specific
cleavage of DNA by DNAzymes~\cite{48,59}. Organic chemistry is
associated with this ongoing optimization process with recent
developments toward the expansion of the genetic alphabet~\cite{60},
original orthogonal bioconjugation~\cite{61}, or revisiting the solid
phase synthesis process using P(V) chemistry to give ODN with optically
pure phosphorothioate linkages~\cite{62}. Finally, starting from a
single oligonucleotide displaying catalytic properties, recent advances
in their supramolecular assembly in 3D organizations such as DNA
tetrahedral~\cite{63} or origami nanostructures~\cite{64} or their
capacity to mimic the enzymatic cascade process~\cite{65} will
undoubtedly widen the scope and success of their applications in the
foreseeable future.\looseness=-1

\section*{Declaration of interests} 
The authors do not work for, advise, own shares in, or receive funds
from any organization that could benefit from this article, and have
declared no affiliations other than their research organizations.

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