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\DOI{10.5802/crchim.256}
\datereceived{2023-02-07}
\daterevised{2023-08-24}
\dateaccepted{2023-08-24}
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\dateposted{2023-10-04}
\begin{document}

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%\makeatletter
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%\def\cdr@specialtitle@english{Chemical Biology}
%\def\cdr@specialtitle@french{Ch\'emobiologie}
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\title{Chemical Biology of G-quadruplex and i-motif DNA: use of
topologically constrained DNA}

\alttitle{Utilisation de syst\`emes ADN contraints pour l'\'etude des
G-quadruplexes et des i-motifs}

\author{\firstname{J\'{e}r\^{o}me} \lastname{Dejeu}\CDRorcid{0000-0002-2434-2959}}
\address{Universit\'{e} Grenoble-Alpes, CNRS, UMR 5250, D\'{e}partement
de Chimie Mol\'{e}culaire (DCM), 38000 Grenoble, France}
\address{SUPMICROTECH, Universit\'{e} de Franche-Comt\'{e}, CNRS,
Institut FEMTO-ST, F-25000 Besan\c{c}on, France}
\email[J. Dejeu]{jerome.dejeu@femto-st.fr}

\author{\firstname{Eric} \lastname{Defrancq}\CDRorcid{0000-0002-3911-6241}\IsCorresp}
\addressSameAs{1}{Universit\'{e} Grenoble-Alpes, CNRS, UMR 5250, D\'{e}partement
de Chimie Mol\'{e}culaire (DCM), 38000 Grenoble, France}
\email[E. Defrancq]{eric.defrancq@univ-grenoble-alpes.fr}

\keywords{\kwd{G-quadruplex}
\kwd{i-motif}
\kwd{DNA}
\kwd{SPR}
\kwd{BLI}
\kwd{Oligonucleotide conjugate}
\kwd{Click chemistry}}

\altkeywords{\kwd{G-quadruplex}
\kwd{i-motif}
\kwd{ADN}
\kwd{SPR}
\kwd{BLI}
\kwd{Oligonucl\'eotides contraints}
\kwd{Chimie click}}

\thanks{The authors wish to acknowledge the institutions for providing
financial support: Universit\'{e} Grenoble-Alpes, CNRS, Labex ARCANE
(ANR-11-LABX-0003-01) and CBH-EUR-GS (ANR-17-EURE-0003), Agence
Nationale de la Recherche (Quarpdiems grant ANR-12-BSV8-0008-04,
G4-TopiPro grant ANR-16-CE11-0006-01, iCARE grant ANR-21-CE44-0005-02)
and R\'{e}gion Auvergne-Rh\^{o}ne-Alpes.}

\begin{abstract}
Tetrameric DNA structures such as G-quadruplex (\textbf{G4}) and
i-motif (\textbf{i-DNA}) have attracted increasing interest in the last
decades. They are indeed involved in many biological processes
including translation regulation, pre-mRNA processing, mRNA targeting,
telomere maintenance, etc. We have developed chemical tools named TASQ
(Template-Assembled Synthetic Quadruplex) to address the following
scientific goals: (i) identify unambiguous (i.e., affine and specific)
\textbf{G4}- and \textbf{i-DNA}-interacting ligands, (ii) identify
proteins interacting with those structures and determine their cellular
relevance and (iii) select specific antibodies for \textbf{G4} and
\textbf{i-DNA}. This review reports on our works over the past decade.
\end{abstract}

\begin{altabstract}
Les structures d'ADN t\'etram\'eriques telles que le G-quadruplex (G4) et
le i-motif (i-DNA) ont suscit\'e un int\'er\^et croissant au cours des
derni\`eres d\'ecennies. Elles sont en effet impliqu\'ees dans de nombreux
processus biologiques dont la r\'egulation de la traduction, le
traitement des pr\'e-ARNm, le ciblage des ARNm, la maintenance des
t\'elom\`eres, etc. Nous avons d\'evelopp\'e des outils chimiques nomm\'es TASQ
(\textit{Template-Assembled Synthetic Quadruplex}) pour r\'epondre aux
objectifs scientifiques suivants : (i) identifier des ligands
interagissant sans ambigu\"it\'e (c'est-\`a-dire affins et sp\'ecifiques) avec
l'ADN G-quadruplex ou l'ADN i-motif, (ii) identifier les prot\'eines
interagissant avec ces structures et d\'eterminer leur pertinence
cellulaire et (iii) s\'electionner des anticorps sp\'ecifiques pour le G4
et l'i-ADN. Cette revue rend compte de nos travaux au cours de la
derni\`ere d\'ecennie.
\end{altabstract}

\maketitle

%{\vspace*{4pt}}

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\section{Introduction}\label{sec1}

The double-helical structure of DNA in which two antiparallel strands
are held together through canonical A/T and G/C base pairing was
established over half a century ago. However, the last decades brought
accumulating evidence of the existence and biological relevance of
four-stranded nucleic acid structures namely G-quadruplex (\textbf{G4})
and i-motif (\textbf{i-DNA}). \textbf{G4} structures could be formed
from guanine rich \mbox{sequences} and consist in stacked tetrads of Hoogsteen
hydrogen-bonded guanine nucleobases (i.e. G-tetrad or quartet, 
Figure~\ref{fig1}A), connected by {various}
{loop-forming} sequences, and
stabilized through the coordination of physiologically abundant cations
(Na$^{+}$, K$^{+}$)~\cite{1,2}.

\begin{figure*}
\includegraphics{fig01}
\caption{\label{fig1}(A) G-tetrad (or G-quartet), (B) schematic
representations of some G-quadruplex topologies with three G-tetrads
from reference~\cite{2}.}
\vspace*{-2pt}
\end{figure*}

Bioinformatics studies suggested that the human genome contains around
370,000 sequences having the potential to form stable G-quadruplex
structures  (PQS)~\cite{3}. This was reevaluated using a novel
algorithm and high-resolution sequencing-based method (termed
``G4-seq''), which identified more than 700,000 PQS within the
genome~\cite{4,5}. \mbox{Interestingly,} these putative \textbf{G4s} are not
distributed randomly in the genome. Indeed a statistically significant
enrichment of PQS was found in several relevant domains of the genome.
DNA \textbf{G4}-forming sequences can be found in the telomeric region
where their \mbox{stabilization} has been shown to inhibit activity of
telomerase, which is over-expressed in 80\% of cancer cells, thus
evidencing their potential as anticancer drug targets~\cite{6,7}.
However
\textbf{G4} formation is not limited to the telomeric region: they are
also over-represented in the promoter regions of a number of genes,
including proto-oncogenes c-Myc, c-Kit, bcl-2 and KRAS~\cite{8}.
Furthermore,
the majority of the 250,000 human replication origins are close to
\textbf{G4} motifs suggesting that the formation of stable \textbf{G4}
structures participates in the initiation of replication~\cite{9}.
It has been
reported that certain pathologies or chronic diseases caused by cell
dysfunction might involve the presence of \textbf{G4}. \textbf{G4}
formation has been linked to genetic disorders (diabetes, fragile X
disorder, Bloom syndrome), age-related degenerative illness (ALS, FTD)
and cancer (telomere, MYC, Kit, BCL-2). \textbf{G4} formation has also
been evidenced in the genomes of viruses suggesting functional
significance~\cite{10}. Besides \textbf{G4-DNA},
G-rich RNA sequences are also prone to fold into stable \textbf{G4}
architectures (\textbf{G4-RNA})~\cite{11}. \textbf{G4-RNA}-forming
sequences can be found in the $5'$- and $3'$-untranslated regions of
many genes, and also in the open reading frame of some mRNAs~\cite{11}.
To date, the formation of \textbf{G4-RNA} has been involved in several
biological processes linked to RNA metabolism such as translation
regulation, pre-mRNA processing, and mRNA targeting. Owing to the
single-stranded nature of transcribed RNA, in vivo formation of
\textbf{G4-RNA} is expected to occur more easily than \textbf{G4-DNA}.
Strong arguments have been provided that argue in favor of the
formation of DNA and RNA \textbf{G4} structures within cells, by using
\textbf{G4}-specific antibodies~\cite{12}, in vivo  NMR~\cite{13}, and
binding-activated fluorescent \textbf{G4}-targeting \unskip\break
ligands~\cite{14}.

An essential feature of \textbf{G4} is their intrinsic polymorphic
nature: numerous in vitro studies have revealed their
susceptibility to adopt different topologies, which are in equilibrium.
Indeed, depending on the length and composition of the sequence, as
well as the environmental conditions (including the nature and
concentration of metal cations, and local molecular crowding), a
G-quadruplex-forming sequence can adopt different topologies in which
the strands are in parallel or antiparallel conformations, with the
co-existence of different types of loops (lateral, diagonal or
propeller) with variable lengths \unskip\break(Figure~\ref{fig1}B)~\cite{1}.

Cytosine, the complementary nucleobase of guanine, is also prone to
assemble to form four-stranded structures named i-motif DNA
(\textbf{i-DNA}) in which cytosines are intercalated via a stack of
hemi-protonated (CH$^{+}$:C) base pairs  (Figure~\ref{fig2})~\cite{15}.

\begin{figure}
{\vspace*{2pt}}
\includegraphics{fig02}
{\vspace*{2pt}}
\caption{\label{fig2}(A) (CH$^{+}$:C) base pairing, (B)~schematic
representation of \textbf{i-DNA}.}
\end{figure}

A major characteristic of \textbf{i-DNA} is the strong pH-dependency of
its stability and formation. Indeed, \textbf{i-DNA} structures are
typically observed in vitro at acidic pH, a particularity that has cast
doubt on their existence in cell. However, two independent studies have
recently demonstrated the stability of exogenous \textbf{i-DNA}
structures in human cells through in cellulo NMR 
spectroscopy~\cite{16} as well as the presence of endogenous
\textbf{i-DNA} in the nuclei of human cells through immunofluorescence
using an antibody \mbox{(\textit{i-Mab})} raised against the
i-motif~\cite{17}.

The biological relevance of \textbf{i-DNA} has been less investigated
mainly due to the scientific community's skepticism about the existence
of i-motifs in vivo. A recent review from Brown and Kendrick provides
some insight into the biological function of \textbf{i-DNA} 
structure~\cite{18}. In the context of bcl-2 oncogene, a
transcriptional activation was reported, caused by the formation of
stable i-motif through the interaction with IMC-48 compound, a
cholestane derivative (selected from a screening of a NCI diversity
set library)~\cite{19}. In contrast with bcl-2 oncogene, a
transcriptional repression was reported with c-MYC  i-motif~\cite{20}.
Also, proteins such as hnRNP LL have been identified to interact with
i-motif structures acting as an activating transcription 
factor~\cite{21}.

The intrinsic polymorphism associated with the formation of \textbf{G4}
and \textbf{i-DNA} as well as the pH-dependency of \textbf{i-DNA}
stability represent severe bottlenecks for the studies of those
tetrameric DNA structures. Indeed, the polymorphism could lead to
intricate structural mixtures in solution that can complicate the
rationalization of the relationships between \textbf{G4} or i-motif
structures and recognition by proteins and ligands. Likewise, low-pH
conditions used to induce the formation of \textbf{i-DNA} could lead to
the protonation of many ligands (e.g., proteins), strongly increasing
their non-specific nucleic acid binding. The design of chemical tools
able to reduce the structural heterogeneity of \textbf{G4} and
\textbf{i-DNA} as well as able to improve i-motif stability in
physiological conditions is thus of high interest.

In this context, we developed some years ago an innovative concept that
consists to constrain the accessible topologies of a
G-quadruplex-forming sequence to a single one~\cite{22}.  This strategy
named TASQ for Template-Assembled Synthetic \mbox{Quadruplex,} is based on the
use of a rigid cyclic peptide scaffold with two independently
functionalizable faces, resulting from the orientation of the lysine
side-chains. One face is dedicated to the anchoring of different
oligonucleotide sequences to obtain the desired \textbf{G4} topology
and a biotin residue is incorporated on the other side for attachment
to streptavidin immobilized surfaces for various applications
(Figure~\ref{fig3}). This template concept allowed the formation of
very stable G-quadruplex motifs in a unique conformation, in aqueous
medium. It was next extended to the formation of constrained\break i-motif
DNA.

\begin{figure}
\includegraphics{fig03}
\caption{\label{fig3}Schematic representation of the TASQ concept. The
different oligonucleotide sequences forming the target structure are
attached to the cyclopeptide scaffold through different ligation
techniques (see below) on the top of the cyclopeptide and a biotin
residue is incorporated in the lower face of the cyclopeptide.}
\end{figure}

In this article, we describe our contribution for the design of
efficient chemical tools based on constrained nucleic acids for the
study of \textbf{G4} and i-motif DNA. Different applications and
perspectives of those chemical tools are then described.

\section{Results and discussions}

\subsection{Synthesis of the various constrained G-quadruplex and i-DNA
systems}

The design of constrained DNA in the chemical biology research domain
has already been investigated. As an example, Escudier \etal\ have
developed modified oligonucleotides in which the phosphodiester
internucleotidic linkage is replaced with a
dioxo-1,3,2-oxaza-phosphorinane moiety resulting in conformationally
constrained nucleotides  (CNA)~\cite{23}. In the case of G-quadruplex,
the use of various templates to pre-organize G-quartet assemblies has
also been described by different  groups~\cite{24,25,26,27}.

Our approach consisted in the use of cyclic peptide scaffolds based on
the TASP concept (Template-Assembled Synthetic Proteins) for the design
of folded proteins developed by Mutter in 1985~\cite{28}. The
chemoselectively addressable cyclic peptide template is the key
intermediate as it exhibits two independent and chemically addressable
domains, which allows the sequential and regioselective assembly of the
different oligonucleotides forming the tetrameric nucleic acid target
on one face, the other face serving for the attachment on surfaces. By
using sequential ligation techniques we were able to prepare different
G-quadruplex systems\break (Figure~\ref{fig4}).

\begin{figure*}
\includegraphics{fig04}
\caption{\label{fig4}Structure of the different constrained tetrameric
nucleic acid structures \textbf{1}--\textbf{5} and unconstrained control
\textbf{6}.}
{\vspace*{-1pt}}
\end{figure*}

Conjugate \textbf{1} was the first constrained \textbf{G4} 
prepared~\cite{22}. It may mimic intermolecular-like G-quadruplexes.
Four oligonucleotides derived from human telomeric sequence
d(${5'}$-TTAGGGT-${3{'}}$) were attached onto the peptide scaffold by using
oxime bond formation from $3'$-aldehyde-containing oligonucleotides and
the peptide scaffold bearing four aminooxy residues. By using CD
melting studies, we demonstrated that the peptide template allows the
formation of a very stable \textbf{G4} motif in a unique parallel
conformation, in aqueous medium. Using the same oxime ligation (OL)
method, we later synthetized the corresponding \textbf{RNA G4}
\textbf{2} from telomeric sequence (TERRA) and found again that the
template allows stabilization of the desired parallel
topology~\cite{29}.

The synthesis of antiparallel topology required the use of two
successive ligation reactions for the attachment of oligonucleotides at
both $3'$ and $5'$ extremities. This was achieved through \mbox{sequential} oxime
(OL) and Cu(I)-catalyzed azide--alkyne cycloaddition (CuAAc) reactions.
The antiparallel topologies of G-quadruplex DNA from telomeric sequence
\textbf{3} was obtained by the reaction of $3'$-aldehyde, $5'$-alkyne
bis-functionalized oligonucleotides with the suitable cyclopeptide and
was shown to exhibit high stability and reduced polymorphism~\cite{30}.

Next, we expanded the TASQ approach to stabilize biologically relevant
viral \textbf{G4} structures such as the one found in sequence
(${5{'}}$-TGGCCTGGGCGGGACTGGG-${3{'}}$) derived from the LTR
region of HIV-1~\cite{31}. Unlike with telomeric \textbf{G4}-forming
conjugates \textbf{1-3}, the site-specific attachment onto the
cyclopeptide scaffold of the two G-rich DNA oligonucleotides with
sequences  ${5{'}}$-TGGCCTGGGC-${3{'}}$ and
${5{'}}$-GGACTGGG-${3{'}}$, respectively, mimicking the
sequence from LTR region of HIV-1, implied the use of an additional
orthogonal chemical ligation step along with OL and CuAAC. This was
achieved using a SN2-thiol coupling reaction (TC). Conjugate \textbf{4}
was thus prepared through successive conjugations, first with a
functionalized oligonucleotide bearing an aldehyde at its $3'$-end and an
alkyne at its $5'$-end, then with another one bearing a thiol function at
its $5'$-end and an alkyne at its $3'$-end~\cite{32}.

Lastly, the construction of conjugate \textbf{5}, a mimic of i-motif
DNA formed from the telomeric sequence, was carried out. The synthesis
of the i-motif structural mimic \textbf{5} was achieved via the
stepwise assembly of peptide--DNA conjugates through four successive
ligations with one OL, one TC and two CuAAC reactions~\cite{33}. The
resulting conjugate \textbf{5} was found by CD to fold, at room
temperature, into an i-motif structure which is stable at acidic and
neutral pH. It may therefore be used to study, at physiologically
relevant pH, the interaction of the i-motif with putative i-motif
targeting ligands (i.e., small molecules or \mbox{proteins).}

\subsection{Applications of the constrained DNA chemical tools}

The different constrained G-quadruplex and i-motif DNA \textbf{1-5}
were used to study previously described ligands or new molecules able
to interact with the \textbf{G4} or \textbf{i-DNA} targets. The main
objectives of those studies were to investigate the interactions of the
ligands with different DNA targets and to assess their \mbox{selectivity}
versus other DNA structures as well as versus different G-quadruplex
topologies.

With these chemical tools in our hands, another goal was to identify,
by classical capture methods, and characterize proteins which bind to a
predetermined single \textbf{G4} topology, and study their interactions
with the diverse structural motifs (i.e., loops, grooves, quartets) of
the quadruplexes by \mbox{comparing} their binding properties to different
defined constructs. This application is now extended to\break \textbf{i-DNA}.

We also envision to use the constrained G-quadruplex to produce and
characterize antibodies for a given \textbf{G4} topology by using the
above-described chemical tools. We believe that preventing the
equilibrium between the different conformations that are associated
with G-quadruplex-forming sequences will facilitate the production of
specific antibodies. Again this will be extended to \textbf{i-DNA}.

\subsection{Use of constrained systems to study the interactions with
ligands}

Most of the knowledge of the impact of \textbf{G4-DNA} secondary
structures on cell metabolism resulted from the use of selective
chemical probes that bind or modulate the formation of such
structures~\cite{34}. A major challenge in G{-}quadruplex ligand
synthesis is the development of compounds that are able to distinguish
G{-}quadruplexes from duplex DNA and also discriminate between various
\textbf{G4} topologies. A detailed picture of quadruplex structure is
emerging from crystallographic and NMR studies, and together with
computer modeling, it is possible to develop a rational approach toward
the design and optimization of quadruplex-stabilizing
compounds~\cite{35}. The desirable features of these stabilizing
molecules are (i) a ${\uppi}${-}delocalized system that is able to
stack on the face of a guanine quartet; (ii) a partial positive charge
that lies in the center of the quartet, increasing stabilization by
substituting for the cationic charge of the potassium or sodium that
would normally occupy that site; and (iii) positively charged
substituents that will interact with the grooves and loops of the
quadruplex and the negatively charged backbone phosphates.

In contrast to \textbf{G4} ligands, relatively few molecules were
reported to interact with \textbf{i-DNA}, and a controversy concerning
their binding mode, affinity, and selectivity persists in the
literature~\cite{36,37}. The main challenges in this regard are the
strong pH-dependency, flexibility and polymorphism of \textbf{i-DNA},
introducing potential bias into screening methods. Indeed, low-pH
conditions used to induce the formation of i-DNA lead to the
protonation of many ligands, strongly increasing their non-specific
nucleic acid binding.

To investigate the interactions of our constrained tetrameric nucleic
acids with potential ligands, two optical techniques were used: surface
plasmon resonance (SPR) and bio-layer interferometry (BLI). Those two
label-free techniques are widely used to study the interactions of
ligands (including proteins, nucleic acids, sugars, and small
molecules) with \mbox{analytes.} The ligand is immobilized on the surface
while the analyte is injected close to the surface via a micro-fluidic
system for SPR or deposited in microplate for BLI. The sensorgram
fittings provide the association and dissociation kinetic constants,
and the responses obtained at the steady state (\textit{Req}) afford
the equilibrium dissociation constant ($K_D$). Those two techniques
display a number of advantages, including the non-use of special
radioactive or fluorescent labeling of the molecules, the time
efficiency, the use of very low quantity of materials associated with a
high sensitivity, the access to a variety of commercial surface
sensors and the possibility to assemble homemade sensors bearing
specific chemical functionalities. Possible drawbacks of BLI/SPR
techniques are related to the relatively high cost of such equipment as
well as the requirement of a good expertise (i.e., to not
over/mis-interpret the results).

\subsubsection{Study of the interaction with well-known G-quadruplex
ligands}

A large number of G-quadruplex ligands have been reported in the
literature and most of them interact with G-quadruplex DNA by
${\uppi}$-stacking interactions with the external G-quartet of the
quadruplex~\cite{38,39}. We have used some
of them with the aim of verifying if the constrained G-quadruplex
systems could act as efficient mimics before the investigation of
unknown ligands. Moreover, we envisioned that our different
G-quadruplex systems could afford some information about the mode of
interaction. The following reported ligands TMPyP4~\cite{40},
MMQ1~\cite{41}, distamycin~\cite{42}, FRHR~\cite{43}, and
DODC~\cite{44} were first studied
(Figure~\ref{fig5}). As anticipated, ligands displaying a
${\uppi}$-stacking binding mode such as TMPyP4 showed a higher binding
affinity for intermolecular-like G-quadruplex \textbf{1} due to the
absence of loops which could prevent the interactions, whereas ligands
with other binding modes (groove and/or loop binding) such as
distamycin showed no significant difference in their binding affinities
for the constrained quadruplex \textbf{1} and unconstrained control
\textbf{6}~\cite{45}.
In addition, the method has also provided information about the
selectivity of ligands for G-quadruplex DNA over the duplex DNA through
comparative studies with DNA hairpin duplex. Further studies with other
well-known G-quadruplex ligands such as Phen-DC3~\cite{46}, 
PDS~\cite{47}, BRACO-19~\cite{48} and NMM~\cite{49} (Figure~\ref{fig5})
were carried out next. The use of constrained or not constrained
\textbf{G4} systems also allowed to obtain some information about the
selectivity for the ligands with a single \textbf{G4} topology. Most of
the described ligands do not show any \textbf{G4} topology preference,
except NMM. We have demonstrated the high selectivity of NMM for the
parallel \textbf{G4} structure with a dissociation constant at least
ten times lower than those of other \textbf{G4} topologies as well as
the ability of this ligand to shift the \textbf{G4} conformation from
both the hybrid and antiparallel topologies toward the parallel
structure~\cite{50}.

\begin{figure*}
\includegraphics{fig05}
\caption{\label{fig5}Structure of the different reported
ligands.}
{\vspace*{-2pt}}
\end{figure*}

The studies with well-known G-quadruplex ligands thus validated that
the constrained systems are useful tools for investigating the
interactions with G-quadruplexes. With those chemical tools in hands,
we next investigated the interactions with various ligands designed by
our collaborators.



\subsubsection{Study of the interaction of different families of
G-quadruplex ligands based on metal complexes}

In the field of targeting G-quadruplex nucleic acids structures with
small molecules, hundreds of ligands have been reported~\cite{38,39}.
Most of them interact with G-quadruplex DNA by ${\uppi}$-stacking
interactions with the external G-quartet of the quadruplex. The design
of metal complexes targeting G-quadruplex DNA has also attracted
intense interest~\cite{51}. In comparison to organic compounds, metal
complexes show many advantages, such as a net positive charge (i.e.,
able to increase the interactions with DNA), tunable geometry, and,
most interestingly, some of them display potentially useful
photochemical properties. In that context, we were interested in the
design and study of different classes of G-quadruplex binders based on
metal complexes including metal porphyrin derivatives, salophens, and
ruthenium and iridium photoreactive\break complexes.

\paragraph{Porphyrin-based ligands.} 
One of the first reported G-quadruplex ligands was the non-metalated
porphyrin, meso-5,10,15,20-tetrakis(4-N-methylpyridiniumyl)-porphyrin
(TMPyP4, Figure~\ref{fig5}). The main disadvantage of TMPyP4 is its weak
selectivity for \textbf{G4} versus all other DNA structure. In the aim
of improving selectivity, Pratviel \etal\ have designed new porphyrin
derivatives through the insertion of a metal ion (Ni$^{2+}$, Co$^{3+}$,
or Mn$^{3+}$) into the porphyrin core (TMPyP4 series \mbox{\textbf{8--10})} or
by \mbox{modification} of the meso substituents R of the porphyrin with a
phenyl-N-methylpyridinium group (TMPyP4-PP series \textbf{11--14}) and
a guanidinium group (TMPyP4-PG \textbf{15}--\textbf{17}) as depicted in\break
Figure~\ref{fig6}~\cite{52,53,54}.

\begin{figure*}
\includegraphics{fig06}
\caption{\label{fig6}Structure of metalated TMPyP4 derivatives. The
un-metalated ligands TMPyP4 \textbf{7}, TMPyPP4-PP \textbf{11} and
TMPyP4-PG \textbf{15} correspond to those structures without M.}
\end{figure*}

By using the constrained \textbf{G4} system \textbf{1}, we have
demonstrated that the insertion of a metal ion into TMPyP4 (Ni, Co or
Mn) or modification of the porphyrin meso substituents could
drastically \mbox{modify} affinity and selectivity for the \textbf{G4}. For
example TMPyP4 derivatives \textbf{9} and \textbf{10} with cobalt and
manganese metal respectively, showed a high selectivity for
G-quadruplexes \textbf{1} and \textbf{6} versus duplex DNA as no
interaction occurs with duplex DNA, whereas for TMPyP4 derivative
\textbf{8} with nickel metal as well as for parent TMPyP4 \textbf{7} an
interaction for duplex DNA quite equivalent to that for G-quadruplex
\textbf{6} was observed (Figure~\ref{fig7}). That was because
water/hydroxo as axial ligands on the cobalt and manganese derivatives
could preclude the intercalation of the porphyrin moiety between the
base pairs of duplex. For TMPyP4-PP \textbf{11}--\textbf{14} and
TMPyP4-PG \mbox{\textbf{15}--\textbf{17}} \mbox{series,} the presence
of bulky substituents should also prevent the intercalation between the
base pairs of duplex DNA leading to a weak interaction of those
compounds with duplex DNA in comparison with quadruplexes \textbf{1}
and \textbf{6} (Figure~\ref{fig7}). 

\begin{figure}
\includegraphics{fig07}
\caption{\label{fig7} $K_D$ values obtained with the different
porphyrin derivatives \textbf{7}--\textbf{17} in interaction with
G-quadruplexes \textbf{1} (black), unconstrained control \textbf{6}
(red) and duplex DNA (orange). \textbf{7}: TMPyP4, \textbf{8}:
Ni-TMPyP4, \textbf{9}: Co-TMPyP4, \textbf{10}: Mn-TMPyP4, \textbf{11}:
TMPyP4-PP, \textbf{12}: Ni-TMPyP4-PP, \textbf{13}: Co-TMPyP4-PP,
\textbf{14}: Mn-TMPyP4-PP, \textbf{15}: TMPyP4-PG, \textbf{16}:
Ni-TMPyP4-PG, \textbf{17}: Mn-TMPyP4-PG. No histogram for duplex DNA
means that the $K_D$ value could not be determined due to too weak
interaction. The reported values are the means of representative
independent experiments, and the errors provided are standard
deviations from the mean. Each experiment was repeated at least three
 times.}
\end{figure}

Guanidinium phenyl porphyrin derivatives \textbf{15--17} exhibit
moderate cytotoxicity toward cells in culture. Strikingly, the nickel
porphyrin derivative \textbf{15} was able to displace hPOT1 sheltering
protein from telomeres in human cells~\cite{53}.


\paragraph{Ni-Salphen derivatives} 
Pioneering works by Neidle \etal~revealed that salphen derivatives bind
strongly to the human telomeric G-quadruplexes and inhibit the
telomerase activity with EC$_{50}$ of roughly 
0.1~${\upmu}$M~\cite{55}. In order to study the impact on binding
affinity of the length of the side-chains and their positions on the
salphen scaffold, Thomas \etal\ have prepared new family of
G-quadruplex binders based on the nickel(II) salphen platform
(Figure~\ref{fig8}). The side-chains are alkyl-imidazolium arms
connected at para, ortho or meta positions of the phenol
moieties~\cite{56,57}. The affinity for G-quadruplex DNA \textbf{1} and
\textbf{6} as well as the selectivity versus duplex DNA were evaluated
by using SPR.

\begin{figure*}
{\vspace*{2pt}}
\includegraphics{fig08}
{\vspace*{2pt}}
\caption{\label{fig8}Structure of the different Ni-Salphen 
derivatives.}
{\vspace*{2pt}}
\end{figure*}

The different salphen derivatives \textbf{18--29} showed $K_D$ values in
the 0.1--2~${\upmu}$M range for both G-quadruplex \textbf{1} and
\textbf{6}, which are in the range of those reported for related
compounds interacting with the HTelo sequence~\cite{58} and most of
them do not bind tightly to duplex DNA (Figure~\ref{fig9}).

\begin{figure}
\includegraphics{fig09}
{\vspace*{-2pt}}
\caption{\label{fig9}$K_D$ values obtained with the Ni-salphen
derivatives \textbf{18}--\textbf{29} in interaction with G-quadruplexes
\textbf{1} (black), \textbf{6} (red) and duplex DNA (orange). No
histogram for duplex DNA means that the $K_D$ value could not be
determined due to too weak interaction. The reported values are the
means of representative independent experiments, and the errors
provided are standard deviations from the mean. Each experiment was
repeated at least three  times.}
{\vspace*{-2pt}}
\end{figure}


SPR studies with our systems \textbf{1} and \textbf{6} allowed to
obtain some useful information. We observed that the shorter the side
arms, the higher the affinity for \textbf{G4}. Furthermore the
introduction of a third anchor on the diaminobenzene bridge also
improved the affinity for \textbf{G4}. The difference
of affinity for \textbf{1} versus \textbf{6} also suggested that the
compounds interact both by ${\uppi}$-stacking over the tetrad and
electrostatic interactions in the grooves. From the SPR studies, we
have concluded that salphen \textbf{21}, \textbf{25} and \textbf{29}
were the optimal G-quadruplex binders: they were also the best salphen
derivatives able to inhibit telomerase activity and in particular with
an IC$_{50}$ value measured from TRAP-G4 assays of 70 nM for
\textbf{29}.

\paragraph{Ruthenium and iridium complexes}
Ruthenium(II) and iridium metal complexes have been investigated as
\textbf{G4}-ligands and some of them have shown good affinity and
selectivity toward \textbf{G4s}~\cite{51,59,61,62}. In this context,
Elias \etal\ have developed several ruthenium and iridium complexes
which are able to target G-quadruplex DNA (Figure~\ref{fig10}). The
affinity for G-quadruplexes and the selectivity versus DNA duplexes
were investigated using BLI and SPR \mbox{(Figure~\ref{fig11}).}

\begin{figure*}
\includegraphics{fig10}
{\vspace*{2pt}}
\caption{\label{fig10}Structure of ruthenium and iridium complexes from
Elias collaboration.}
\end{figure*}

\begin{figure}
\includegraphics{fig11}
{\vspace*{-2pt}}
\caption{\label{fig11}$K_D$ values obtained with different ruthenium
and iridium derivatives \textbf{30}--\textbf{39} in interaction with
G-quadruplexes \textbf{1} (black), \textbf{3} (blue) and \textbf{6}
(red) and duplex DNA (orange). No histogram for duplex DNA means that
the $K_D$ value could not be determined due to too weak interaction.
The reported values are the means of representative independent
experiments, and the errors provided are standard deviations from the
mean. Each experiment was repeated at least three times.}
{\vspace*{-2pt}}
\end{figure}

A first series based on dph (dph $=$
dipyrazino\ubreak[2,3-a:2',$3'$-h]phenazine) and bpph (bpph =
benzo[a]{\ubreak}pyrazino[2,3-h]phenazine) ancillary ligands (complexes
\textbf{30}--\textbf{33}) were studied using G-quadruplex systems
\textbf{1} and \textbf{6}~\cite{63,64}. It was shown that the removal
of two non-chelating nitrogen atoms from the dph ligand in complex
\textbf{30} to form the bpph ligand in complex \textbf{31} led to a
huge impact on the interactions with \textbf{G4} and duplex DNA.
Indeed, a decrease of the affinity toward \textbf{G4} structure
\textbf{6} was observed ($K_D = 9~{\upmu}$M and  32~${\upmu}$M for
complexes \textbf{30} and \textbf{31}, respectively), while a weak
affinity for duplex DNA could be measured with complex \textbf{31}
($K_D = 102~{\upmu}$M) thus leading to a partial loss of selectivity.
The dph analogues therefore appeared to be slightly more selective
toward \textbf{G4} versus duplex DNA compared to their respective bpph
analogues.\looseness=-1


Another series was based on the CPIP
(2-(4-chlorophenyl)-1H-imidazo[4,5-f][1,10]phenanthrol{\unskip-\break}ine) and CPIPTAP
ligands (complexes \textbf{34--37}). Investigation by BLI indicated that
these complexes displayed a good affinity for G-quadruplex DNA ($K_D$
around 1 and $3~\upmu$M for \textbf{36} and \textbf{35,}
respectively, for \textbf{G4} system \textbf{3}) and selectivity over
duplex DNA. It was also noticed that their affinities were higher for
G-quadruplex structures \textbf{3} and \textbf{6} which contain TTA
loops, than for parallel-stranded quadruplex \textbf{1}. This is
consistent with interactions of the complexes with G-quadruplexes
through mixed ${\uppi}$-stacking with the guanine tetrad and further
interactions with loops and grooves. Due to their photophysical
properties, these complexes are able to react with DNA through type II
photoreaction (i.e., formation of singlet oxygen) or through
photo-induced charge transfer (PET). Interestingly, complexes
\textbf{34} and \textbf{36} elicited a dramatic photo-cytotoxic effect,
as 100\% mortality was obtained upon irradiation of U2OS osteosarcoma
cells in their presence, whereas very low mortality was observed in the
dark at the same drug concentration~\cite{65}.


The interaction of iridium complexes \textbf{38} and \textbf{39} with
G-quadruplex DNA was also investigated~\cite{66}. $K_D$ values in the
micromolar range was obtained for the G-quadruplex structures that fall
within the range of those reported for similar ruthenium(II) complexes.
However, a weak selectivity for the\unskip\break G-quadruplex structure versus
duplex DNA was observed. This could be explained by the net positive
charge of the Ir$^{\mathrm{III}}$ complexes in comparison with the 
Ru$^{\mathrm{II}}$
complexes that could favor non-specific ionic interactions with DNA.

\subsubsection{Study of the interaction with i-motif DNA}
As described below, the stability of \textbf{i-DNA} strongly depends on
pH. Conditions typically used for screening of ligand candidates with
native \textbf{i-DNA} sequences employ low pH which, at the same time,
leads to protonation of the ligands and favors their non-specific
interactions with nucleic acids. In that context, we have recently
designed constrained \textbf{i-DNA} \textbf{5} which shows an increased
stability and invariability of the i-motif structure in a broad pH
range, allowing to use this scaffold as an \textbf{i-DNA} substrate at
physiologically relevant conditions~\cite{33}. This constrained i-DNA
\textbf{5} was thus used to investigate the interaction between
previously reported i-motif DNA ligands (Figures~\ref{fig5} 
and~\ref{fig12}) and folded
or unfolded \textbf{i-DNA} in acidic (pH 5.5) and near-neutral (pH 6.5)
conditions by using~BLI.\looseness=-1

\begin{figure*}
\includegraphics{fig12}
\caption{\label{fig12}Structure of compounds used for the studies of i-motif interactions.
\textbf{[Ru(Phen)}$_2$\textbf{dppz}]$^{2+}$~\cite{69}, 
\textbf{BisA}~\cite{70}, \textbf{Berberine}~\cite{71}, 
\textbf{IMC-48}~\cite{19}, \textbf{RHPS4}~\cite{72}
and \textbf{mitoxantrone}~\cite{73}.}
\end{figure*}

Very interestingly, we have observed that, despite several ligands such
as macrocyclic bis-acridine (BisA) and pyridostatin (PDS) showing good
affinities for the telomeric i-motif forming sequence, none of the
ligands displayed selective interactions with the \textbf{i-DNA}
structure nor were able to promote its \mbox{formation}  
(Figure~\ref{fig13})~\cite{67}. More recently we have reported that
\textbf{IMC-48}, although described as \textbf{i-DNA} ligand, is a very
weak ligand of \textbf{i-DNA} as no quantifiable interaction or
significant stabilization of i-motif structures could be observed,
stimulating a quest for an alternative mechanism of its biological
activity~\cite{68}. All together, these results further emphasize the
need for efforts to identify specific i-motif ligands. In this context,
the use of constrained \textbf{i-DNA} \textbf{5}, which ensures an
i-motif folding, represents an interesting alternative to identify
unambiguous (i.e., affine and specific) i-DNA-interacting ligands.

\begin{figure}
\vspace*{-2pt}
\includegraphics{fig13}
\vspace*{-4pt}
\caption{\label{fig13}$K_D$ values obtained with different ligands in
interaction with I-motif \textbf{6} (blue), h-Telo sequence (orange) at
pH 5.5 and 6.5 fill and sparse pattern. No histogram for duplex
berberine, [Ru(Phen)$_{2}$dppz]$^{2+}$ and RHPS4 means that the $K_D$
value could not be determined due to too weak interaction. The reported
values are the means of representative independent experiments, and the
errors provided are standard deviations from the mean. Each experiment
was repeated at least three times.}
\vspace*{-2pt}
\end{figure}

\subsection{Use of constrained G4 for protein fishing}

Given the increasing roles of \textbf{G4} structures in cellular
metabolism, extensive researches have been conducted in the last years
to identify new \textbf{G4}-dependent mechanisms. Notably classical
pull-down approaches identified hundreds of proteins associated to
G-rich oligonucleotides forming  \textbf{G4}  structures~\cite{74}.
However, in solution, G-rich single-stranded molecules are in
equilibrium between unfolded and folded states, and thus numerous
identified \textbf{G4} binding proteins are also able to recognize
unfolded G-rich sequences~\cite{75}. In this context, we have used the
constrained G-quadruplex \textbf{3} which folds into the single
antiparallel topology.

Moreover, such locked \textbf{G4} displays a thermal stability
significantly higher than unconstrained \textbf{G4} which strongly
reduces the possibility to form unfolded single-stranded sequences. We
identified through affinity purifications coupled to mass spectrometry
(MS)-based quantitative proteomics a set of human proteins associated
to locked \textbf{G4} structures. Notably, this approach allowed us to
identify NELF proteins as a new \textbf{G4}-interacting complex,
leading us to investigate the impact of RNA-Pol II pausing mechanism in
the response to \textbf{G4} stabilization by \textbf{G4}
ligands~\cite{76}.

\section{Conclusion}

The different constrained G-quadruplex systems have proved efficient
tools for the identification of \textbf{G4} structure-specific
synthetic ligands. They could also give some interesting information
about the mode of interaction and the selectivity versus duplex and
also versus \textbf{G4} topologies. The latter could be crucial to
design more specific \textbf{G4} ligands associated with less
off-target side effects. The constrained \textbf{i-DNA} represents also
an interesting tool. Indeed, our recent study has demonstrated that all
the molecules described so far as i-motif ligands, are not able to
discriminate between folded and unfolded i-motif structures. The
constrained \textbf{i-DNA} will thus be used to identify unambiguous
(i.e., affine and specific) \textbf{i-DNA}-interacting ligands.

The pull-down strategy using constrained G-quadruplex DNA was proved
efficient to identify proteins selective for a single \textbf{G4}
topology. Future directions of our approach will concern the
construction of constrained \textbf{G4} structures mimicking parallel
\textbf{G4} topologies in order to refine the impact of loops on
protein binding. We will also use constrained i-DNA \textbf{5} to
identify proteins interacting with the non-canonical secondary
structure i-motif.

Lastly, given the fact that constrained G-quadruplexes reduce the
unfolding to single-stranded sequences, we envisioned to use system
\textbf{3} for the selection of topologically specific \textbf{G4}
antibody. In the same way, due to the lower sensitivity of constrained
\textbf{i-DNA} to pH conditions, we will also use this system for the
selection of specific i-motif antibodies.

\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.

\section*{Acknowledgements}
The NanoBio-ICMG platforms
(UAR 2607) are acknowledged for their support. We thank also our
collaborators for providing the different samples of products: M. P.
Teulade-Fichou and A. Grand from Institut Curie Orsay, B.~Elias from
Universit\'{e} catholique de Louvain, G.~Pratviel from CNRS Toulouse,
F. Thomas and O.~Jarjayes from Universit\'{e} Grenoble-Alpes. The
authors are also grateful to all the students who contributed to those
works.

\CDRGrant[ANR]{ANR-11-LABX-0003-01}
\CDRGrant[ANR]{ANR-17-EURE-0003}
\CDRGrant[ANR]{ANR-12-BSV8-0008-04}
\CDRGrant[ANR]{ANR-16-CE11-0006-01}
\CDRGrant[ANR]{ANR-21-CE44-0005-02}

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