A red light-triggered chemical tool for sequence-specific alkylation of G-quadruplex and I-motif DNA.

The importance of non-canonical DNA structures such as G-quadruplexes (G4) and intercalating-motifs (iMs) in the fine regulation of a variety of cellular processes has been recently demonstrated. As the crucial roles of these structures are being unravelled, it is becoming more and more important to develop tools that allow targeting these structures with the highest possible specificity. While targeting methodologies have been reported for G4s, this is not the case for iMs, as evidenced by the limited number of specific ligands able to bind the latter and the total absence of selective alkylating agents for their covalent targeting. Furthermore, strategies for the sequence-specific covalent targeting of G4s and iMs have not been reported thus far. Herein, we describe a simple methodology to achieve sequence-specific covalent targeting of G4 and iM DNA structures based on the combination of (i) a peptide nucleic acid (PNA) recognizing a specific sequence of interest, (ii) a pro-reactive moiety enabling a controlled alkylation reaction, and (iii) a G4 or iM ligand orienting the alkylating warhead to the reactive residues. This multi-component system allows for the targeting of specific G4 or iM sequences of interest in the presence of competing DNA sequences and under biologically relevant conditions.

Furan-based (photo)oxidation reactions and their application in nucleic acid and protein targeting.

Oligonucleotides (ODNs) find applications as diagnostic and therapeutic tools due to their unique ability to interact, thanks to Watson-Crick base pairing, with a specific DNA or RNA target strand. Although most of the tools available today rely on mere hydrogen bond formation, chemical modifications to enable covalent interstrand-crosslinking (ICL) have been reported, and are gaining a place under the spotlight as they potentially offer a series of advantages over the state of the art, including a higher potency and selectivity. This methodological paper focuses on the use of a pro-reactive furan moiety and its subsequent oxidation for applications in ODN targeting. The design of effective capture and targeting probes to ensure high ICL yields is discussed and the mechanisms underlying the (photo)chemical oxidation of furan are explained. Furthermore, examples of furan-containing DNAs designed for different applications, including DNA-DNA or DNA-RNA ICL and DNA-peptide/protein targeting, are provided. The paper highlights the advantages of using different oxidative chemical triggers, such as N-bromosuccinimide or singlet oxygen, to offer additional selectivity control over the ICL reaction.

Furan-modified PNA probes for covalent targeting and ligation of nucleic acids.

While natural oligonucleotides (ONs) are increasingly used as therapeutic and diagnostic tools, they still face certain challenges such as low resistance to enzymatic degradation, potential immunogenicity, and delivery issues, which can limit their applications. Peptide Nucleic Acids (PNAs) are promising alternatives due to their high affinity for DNA and RNA, the high resistance to enzymatic degradation, and the easy introduction of a wide range of potential modifications. Chemical modifications that enable the covalent targeting of specific DNA and RNA strands offer additional advantages, including enhanced potency. The current study focuses on the utilization of furan-PNAs as pro-reactive probe systems and their applications to DNA and RNA targeting. Specifically, in this methodological paper, we provide practical insights into the design, synthesis, and application of furan-containing PNA probes for achieving efficient PNA-DNA and PNA-RNA interstrand crosslinking (ICL), as well as ON-templated PNA-PNA ligation systems. Furthermore, we discuss the applications of these probes in targeting DNA secondary structures, such as G-quadruplexes and i-motifs, target pull-down assays, and on-surface detection.

Beyond small molecules: targeting G-quadruplex structures with oligonucleotides and their analogues.

G-Quadruplexes (G4s) are widely studied secondary DNA/RNA structures, naturally occurring when G-rich sequences are present. The strategic localization of G4s in genome areas of crucial importance, such as proto-oncogenes and telomeres, entails fundamental implications in terms of gene expression regulation and other important biological processes. Although thousands of small molecules capable to induce G4 stabilization have been reported over the past 20 years, approaches based on the hybridization of a synthetic probe, allowing sequence-specific G4-recognition and targeting are still rather limited. In this review, after introducing important general notions about G4s, we aim to list, explain and critically analyse in more detail the principal approaches available to target G4s by using oligonucleotides and synthetic analogues such as Locked Nucleic Acids (LNAs) and Peptide Nucleic Acids (PNAs), reporting on the most relevant examples described in literature to date.

A Photosensitized Singlet Oxygen (1O2) Toolbox for Bio-Organic Applications: Tailoring 1O2 Generation for DNA and Protein Labelling, Targeting and Biosensing.

Singlet oxygen (1O2) is the excited state of ground, triplet state, molecular oxygen (O2). Photosensitized 1O2 has been extensively studied as one of the reactive oxygen species (ROS), responsible for damage of cellular components (protein, DNA, lipids). On the other hand, its generation has been exploited in organic synthesis, as well as in photodynamic therapy for the treatment of various forms of cancer. The aim of this review is to highlight the versatility of 1O2, discussing the main bioorganic applications reported over the past decades, which rely on its production. After a brief introduction on the photosensitized production of 1O2, we will describe the main aspects involving the biologically relevant damage that can accompany an uncontrolled, aspecific generation of this ROS. We then discuss in more detail a series of biological applications featuring 1O2 generation, including protein and DNA labelling, cross-linking and biosensing. Finally, we will highlight the methodologies available to tailor 1O2 generation, in order to accomplish the proposed bioorganic transformations while avoiding, at the same time, collateral damage related to an untamed production of this reactive species.

PNA-Based MicroRNA Detection Methodologies.

MicroRNAs (miRNAs or miRs) are small noncoding RNAs involved in the fine regulation of post-transcriptional processes in the cell. The physiological levels of these short (20-22-mer) oligonucleotides are important for the homeostasis of the organism, and therefore dysregulation can lead to the onset of cancer and other pathologies. Their importance as biomarkers is constantly growing and, in this context, detection methods based on the hybridization to peptide nucleic acids (PNAs) are gaining their place in the spotlight. After a brief overview of their biogenesis, this review will discuss the significance of targeting miR, providing a wide range of PNA-based approaches to detect them at biologically significant concentrations, based on electrochemical, fluorescence and colorimetric assays.

Exploiting G-Quadruplex-DNA Damage as a Tool to Quantify Singlet Oxygen Production.

G-Quadruplexes (G4s) are highly dynamic and polymorphic nucleic acid structures that can adopt a variety of conformations. When exposed to oxidative conditions, more specifically singlet oxygen, the guanosine nucleobases can be oxidized, which in turn can affect the conformation and folding of the G4. Based on this peculiar phenomenon, it is rationalized that G4s can serve as quantification sensors for the production of singlet oxygen. Here, a method for determining the quantum yield of singlet oxygen generation for visible as well as UV-light excited photosensitizers, using a short G4 DNA sequence, readily available from common DNA companies, as a biological and water-soluble probe, is presented.

Synthesis and structure-activity relationship of peptide nucleic acid probes with improved interstrand-crosslinking abilities: application to biotin-mediated RNA-pulldown.

The development of interstrand-crosslinking (ICL) probes for the covalent targeting of DNA and RNA sequences of interest has been extensively reported in the past decade. However, most of the reactions reported so far induce the formation of a stable adduct that cannot be reverted, thus rendering these chemistries less useful in applications where the reversibility of the reaction is needed for further downstream processing of the targeted and isolated sequences, such as enzymatic amplification steps. In this work, we report on the reversibility of the furan-mediated ICL reaction. ICL formation can be conveniently triggered by either chemical (N-bromo succinimide, NBS) or luminous stimuli (visible light irradiation in presence of a photosensitizer) and quantitative reversion can be achieved by heating the crosslinked sample at 95 °C, while maintaining the structure of the DNA/RNA targets intact. As a proof-of-concept and showing the benefits of the ICL reversibility, we apply furan-mediated ICL to the pulldown of a target RNA strand from cell lysate.

Hydrolysis of 5-methylfuran-2-yl to 2,5-dioxopentanyl allows for stable bio-orthogonal proximity-induced ligation.

Ligation methodologies featuring bio-orthogonal units and leading to the formation of a stable adduct are the ideal candidates for being applied in a biological context. However, most of the available strategies rely on highly reactive species that require careful handling, or on the activation of pro-reactive functional groups. We here report on a proximity-induced ligation reaction that relies on a stable 2,5-dione, that can be conveniently generated under acidic conditions from a 2,5-dialkylfuran building block, and hydrazine nucleophiles. This bio-orthogonal ligation, which proceeds under physiological conditions, does not require any stimulus or trigger and leads to the formation of a pyridazinium adduct that demonstrates excellent stability under harsh conditions (24 h at 90 °C). The reaction was applied to the formation of PNA-PNA adducts, DNA- and RNA-templated ligations, and for the formation of peptide-peptide adducts in solution. This convenient methodology was further implemented on plastic and glass surfaces to realize self-addressable covalent constructs.

Teaching photosensitizers a new trick: red light-triggered G-quadruplex alkylation by ligand co-localization.

We propose a bimolecular approach for G-quadruplex alkylation, using a pro-reactive furan-containing ligand, activated by red-light irradiation of a proximate G4-binding photosensitizer. G4- over dsDNA alkylation can be achieved selectively and proves high-yielding at low ligand excess. HPLC and modelling studies allowed identifying potential residues involved in the alkylation.

New (Iso)quinolinyl-pyridine-2,6-dicarboxamide G-Quadruplex Stabilizers. A Structure-Activity Relationship Study.

G-quadruplex (G4)-interactive small molecules have a wide range of potential applications, not only as drugs, but also as sensors of quadruplex structures. The purpose of this work is the synthesis of analogues of the bis-methylquinolinium-pyridine-2,6-dicarboxamide G4 ligand 360A, to identify relevant structure-activity relationships to apply to the design of other G4-interactive small molecules bearing bis-quinoline or bis-isoquinoline moieties. Thermal denaturation experiments revealed that non-methylated derivatives with a relative 1,4 position between the amide linker and the nitrogen of the quinoline ring are moderate G4 stabilizers, with a preference for the hybrid h-Telo G4, a 21-nt sequence present in human telomeres. Insertion of a positive charge upon methylation of quinoline/isoquinoline nitrogen increases compounds' ability to selectively stabilize G4s compared to duplex DNA, with a preference for parallel structures. Among these, compounds having a relative 1,3-position between the charged methylquinolinium/isoquinolinium nitrogen and the amide linker are the best G4 stabilizers. More interestingly, these ligands showed different capacities to selectively block DNA polymerization in a PCR-stop assay and to induce G4 conformation switches of hybrid h-Telo G4. Molecular dynamic simulations with the parallel G4 formed by a 21-nt sequence present in k-RAS gene promoter, showed that the relative spatial orientation of the two methylated quinoline/isoquinoline rings determines the ligands mode and strength of binding to G4s.

Visible-light triggered templated ligation on surface using furan-modified PNAs.

Oligonucleotide-templated reactions are frequently exploited for target detection in biosensors and for the construction of DNA-based materials and probes in nanotechnology. However, the translation of the specifically used template chemistry from solution to surfaces, with the final aim of achieving highly selective high-throughput systems, has been difficult to reach and therefore, poorly explored. Here, we show the first example of a visible light-triggered templated ligation on a surface, employing furan-modified peptide nucleic acids (PNAs). Tailored photo-oxidation of the pro-reactive furan moiety is ensured by the simultaneous introduction of a weak photosensitizer as well as a nucleophilic moiety in the reacting PNA strand. This allows one to ensure a localized production of singlet oxygen for furan activation, which is not affected by probe dilution or reducing conditions. Simple white light irradiation in combination with target-induced proximity between reactive functionalities upon recognition of a short 22mer DNA or RNA sequence that functions as a template, allows sensitive detection of nucleic acid targets in a 96 well plate format.

Exploiting Double Exchange Diels-Alder Cycloadditions for Immobilization of Peptide Nucleic Acids on Gold Nanoparticles.

The generation of PNA-decorated gold nanoparticles (AuNPs) has revealed to be more difficult as compared to the generation of DNA-functionalized ones. The less polar nature of this artificial nucleic acid system and the associated tendency of the neutral poly-amidic backbone to aspecifically adsorb onto the gold surface rather than forming a covalent bond through gold-thiol interaction, combined with the low solubility of PNAs itself, form the main limiting factors in the functionalization of AuNP. Here, we provide a convenient methodology that allows to easily conjugate PNAs to AuNP. Positively charged PNAs containing a masked furan moiety were immobilized via a double exchange Diels-Alder cycloaddition onto masked maleimide-functionalized AuNPs in a one-pot fashion. Conjugated PNA strands retain their ability to selectively hybridize with target DNA strands. Moreover, the duplexes resulting from hybridization can be detached through a retro-Diels-Alder reaction, thus allowing straightforward catch-and-release of specific nucleic acid targets.

Combining 1,3-Ditriazolylbenzene and Quinoline to Discover a New G-Quadruplex-Interactive Small Molecule Active against Cancer Stem-Like Cells.

Quadruplex nucleic acids are promising targets for cancer therapy. In this study we used a fragment-based approach to create new flexible G-quadruplex (G4) DNA-interactive small molecules with good calculated oral drug-like properties, based on quinoline and triazole heterocycles. G4 melting temperature and polymerase chain reaction (PCR)-stop assays showed that two of these compounds are selective G4 ligands, as they were able to induce and stabilize G4s in a dose- and DNA sequence-dependent manner. Molecular docking studies have suggested plausible quadruplex binding to both the G-quartet and groove, with the quinoline module playing the major role. Compounds were screened for cytotoxicity against four cancer cell lines, where 4,4'-(4,4'-(1,3-phenylene)bis(1H-1,2,3-triazole-4,1-diyl))bis(1-methylquinolin-1-ium) (1 d) showed the greater activity. Importantly, dose-response curves show that 1 d is cytotoxic in the human colon cancer HT-29 cell line enriched in cancer stem-like cells, a subpopulation of cells implicated in chemoresistance. Overall, this study identified a new small molecule as a promising lead for the development of drugs targeting G4 in cancer stem cells.

Design of Modular G-quadruplex Ligands.

Guanine-rich nucleic acid sequences able to form four-stranded structures (G-quadruplexes, G4) play key cellular regulatory roles and are considered as promising drug targets for anticancer therapy. On the basis of the organization of their structural elements, G4 ligands can be divided into three major families: one, fused heteroaromatic polycyclic systems; two, macrocycles; three, modular aromatic compounds. The design of modular G4 ligands emerged as the answer to achieve not only more drug-like compounds but also more selective ligands by targeting the diversity of the G4 loops and grooves. The rationale behind the design of a very comprehensive set of ligands, with particular focus on the structural features required for binding to G4, is discussed and combined with the corresponding biochemical/biological data to highlight key structure-G4 interaction relationships. Analysis of the data suggests that the shape of the ligand is the major factor behind the G4 stabilizing effect of the ligands. The information here critically reviewed will certainly contribute to the development of new and better G4 ligands with application either as therapeutics or probes.