Biophysical evaluation of antiparallel triplexes for biosensing and biomedical applications.

Polypyrimidine sequences can be targeted by antiparallel clamps forming triplex structures either for biosensing or therapeutic purposes. Despite its successful implementation, their biophysical properties remain to be elusive. In this work, PAGE, circular dichroism and multivariate analysis were used to evaluate the properties of PPRHs directed to SARS-CoV-2 genome. Several PPRHs designed to target various polypyrimidine sites within the viral genome were synthesized. These PPRHs displayed varying binding affinities, influenced by factors such as the length of the PPRH and its GC content. The number and position of pyrimidine interruptions relative to the 4 T loop of the PPRH was found a critical factor, affecting the binding affinity with the corresponding target. Moreover, these factors also showed to affect in the intramolecular and intermolecular equilibria of PPRHs alone and when hybridized to their corresponding targets, highlighting the polymorphic nature of these systems. Finally, the functionality of the PPRHs was evaluated in a thermal lateral flow sensing device showing a good correspondence between their biophysical properties and detection limits. These comprehensive studies contribute to the understanding of the critical factors involved in the design of PPRHs for effective targeting of biologically relevant genomes through the formation of triplex structures under neutral conditions.

Rapid and Highly Efficient Separation of i-Motif DNA Species by CE-UV and Multivariate Curve Resolution.

The i-motif is a class of nonstandard DNA structure with potential biological implications. A novel capillary electrophoresis with an ultraviolet absorption spectrophotometric detection (CE-UV) method has been developed for the rapid analysis of the i-motif folding equilibrium as a function of pH and temperature. The electrophoretic analyses are performed in reverse polarity of the separation voltage with 32 cm long fused silica capillaries permanently coated with hydroxypropyl cellulose (HPC), after an appropriate conditioning procedure was used to achieve good repeatability. However, the electrophoretic separation between the folded and unfolded conformers of the studied cytosine-rich i-motif sequences (i.e., TT, Py39WT, and nmy01) is compromised, especially for Py39WT and nmy01, which result in completely overlapped peaks. Therefore, deconvolution with multivariate curve resolution-alternating least-squares (MCR-ALS) has been required for the efficient separation of the folded and unfolded species found at different concentration levels at pH 6.5 and between 12 and 40 °C, taking advantage of the small dissimilarities in the electrophoretic mobilities and UV spectra levels. MCR-ALS has also provided quantitative information that has been used to estimate melting temperatures (Tm), which are similar to those determined by UV and circular dichroism (CD) spectroscopies. The obtained results demonstrate that CE-UV assisted by MCR-ALS may become a very useful tool to get novel insight into the folding of i-motifs and other complex DNA structures.

Aggregation induced nucleic acids recognition by homodimeric asymmetric monomethyne cyanine fluorochromes in mesenchymal stem cells.

In the light of recent retrovirus pandemics, the issue of discovering new and diverse RNA-specific fluorochromes for research and diagnostics became of acute importance. The great majority of nucleic acid-specific probes either do not stain RNA or cannot distinguish between DNA and RNA. The versatility of polymethine dyes makes them suitable as stains for visualization, analysis, and detection of nucleic acids, proteins, and other biomolecules. We synthesized the asymmetric dicationic homodimeric monomethine cyanine dyes 1,1'-(1,3-phenylenebis(methylene))bis(4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)pyridin-1-ium) bromide (Т1) and 1,1'-(1,3-phenylenebis(methylene))bis(4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium) bromide (M1) and tested their binding specificity, spectral characteristics, membrane penetration in living and fixed cells, cellular toxicity, and stability of fluorescent emission. Mesenchymal cells have diverse phenotypes and extensive proliferation and differentiation properties. We found dyes T1 and M1 to show high photochemical stability in living mesenchymal stem cells from apical papilla (SCAP) with a strong fluorescent signal when bound to nucleic acids. We found M1 to perform better than control fluorochrome (Hoechst 33342) for in vivo DNA visualization. T1, on the other hand, stains granular cellular structures resembling ribosomes in living cells and after permeabilization of the nuclear membrane stains the nucleoli and not the chromatin in the nucleus. This makes T1 suitable for the visualization of structures rich in RNA in living and fixed cells.

Exploring the stabilizing effect on the i-motif of neighboring structural motifs and drugs.

Cytosine-rich DNA sequences may fold into a structure known as i-motif, with potential in vivo modulation of gene expression. The stability of the i-motif is residual at neutral pH values. To increase it, the addition of neighboring moieties, such as Watson-Crick stabilized loops, tetrads, or non-canonical base pairs have been proposed. Taking a recently described i-motif structure as a model, the relative effect of these structural moieties, as well as several DNA ligands, on the stabilization of the i-motif has been studied. To this end, not only the original sequence but different mutants were considered. Spectroscopic techniques, PAGE, and multivariate data analysis methods have been used to model the folding/unfolding equilibria induced by changes of pH, temperature, and the presence of ligands. The results have shown that the duplex is the moiety that is responsible of the stabilization of the i-motif structure at neutral pH. The T:T base pair, on the contrary, shows little stabilization of the i-motif. From several selected DNA-binding ligands, the G-quadruplex ligand BA41 is shown to interact with the duplex moiety, whereas non-specific interaction and little stabilization has been observed within the i-motif.

Detection of pyrimidine-rich DNA sequences based on the formation of parallel and antiparallel triplex DNA and fluorescent silver nanoclusters.

In this work, the use of DNA-stabilized fluorescent silver nanoclusters for the detection of target pyrimidine-rich DNA sequences by formation of parallel and antiparallel triplex structures is studied by molecular fluorescence spectroscopy. In the case of parallel triplexes, the probe DNA fragments are Watson-Crick stabilized hairpins, and whereas in the case of antiparallel triplexes, the probe fragments are reverse-Hoogsteen clamps. In all cases, the formation of the triplex structures has been assessed by means of polyacrylamide gel electrophoresis, circular dichroism, and molecular fluorescence spectroscopies, as well as multivariate data analysis methods. The results have shown that it is possible the detection of pyrimidine-rich sequences with an acceptable selectivity by using the approach based on the formation of antiparallel triplex structures.

Detection of SARS-CoV-2 Virus by Triplex Enhanced Nucleic Acid Detection Assay (TENADA).

SARS-CoV-2, a positive-strand RNA virus has caused devastating effects. The standard method for COVID diagnosis is based on polymerase chain reaction (PCR). The method needs expensive reagents and equipment and well-trained personnel and takes a few hours to be completed. The search for faster solutions has led to the development of immunological assays based on antibodies that recognize the viral proteins that are faster and do not require any special equipment. Here, we explore an innovative analytical approach based on the sandwich oligonucleotide hybridization which can be adapted to several biosensing devices including thermal lateral flow and electrochemical devices, as well as fluorescent microarrays. Polypurine reverse-Hoogsteen hairpins (PPRHs) oligonucleotides that form high-affinity triplexes with the polypyrimidine target sequences are used for the efficient capture of the viral genome. Then, a second labeled oligonucleotide is used to detect the formation of a trimolecular complex in a similar way to antigen tests. The reached limit of detection is around 0.01 nM (a few femtomoles) without the use of any amplification steps. The triplex enhanced nucleic acid detection assay (TENADA) can be readily adapted for the detection of any pathogen requiring only the knowledge of the pathogen genome sequence.

Exploring the Interaction of G-quadruplex Binders with a (3 + 1) Hybrid G-quadruplex Forming Sequence within the PARP1 Gene Promoter Region.

The enzyme PARP1 is an attractive target for cancer therapy, as it is involved in DNA repair processes. Several PARP1 inhibitors have been approved for clinical treatments. However, the rapid outbreak of resistance is seriously threatening the efficacy of these compounds, and alternative strategies are required to selectively regulate PARP1 activity. A noncanonical G-quadruplex-forming sequence within the PARP1 promoter was recently identified. In this study, we explore the interaction of known G-quadruplex binders with the G-quadruplex structure found in the PARP gene promoter region. The results obtained by NMR, CD, and fluorescence titration, also confirmed by molecular modeling studies, demonstrate a variety of different binding modes with small stabilization of the G-quadruplex sequence located at the PARP1 promoter. Surprisingly, only pyridostatin produces a strong stabilization of the G-quadruplex-forming sequence. This evidence makes the identification of a proper (3+1) stabilizing ligand a challenging goal for further investigation.

Properties of Parallel Tetramolecular G-Quadruplex Carrying N-Acetylgalactosamine as Potential Enhancer for Oligonucleotide Delivery to Hepatocytes.

The development of oligonucleotide conjugates for in vivo targeting is one of the most exciting areas for oligonucleotide therapeutics. A major breakthrough in this field was the development of multifunctional GalNAc-oligonucleotides with high affinity to asialoglycoprotein receptors (ASGPR) that directed therapeutic oligonucleotides to hepatocytes. In the present study, we explore the use of G-rich sequences functionalized with one unit of GalNAc at the 3'-end for the formation of tetrameric GalNAc nanostructures upon formation of a parallel G-quadruplex. These compounds are expected to facilitate the synthetic protocols by providing the multifunctionality needed for the binding to ASGPR. To this end, several G-rich oligonucleotides carrying a TGGGGGGT sequence at the 3'-end functionalized with one molecule of N-acetylgalactosamine (GalNAc) were synthesized together with appropriate control sequences. The formation of a self-assembled parallel G-quadruplex was confirmed through various biophysical techniques such as circular dichroism, nuclear magnetic resonance, polyacrylamide electrophoresis and denaturation curves. Binding experiments to ASGPR show that the size and the relative position of the therapeutic cargo are critical for the binding of these nanostructures. The biological properties of the resulting parallel G-quadruplex were evaluated demonstrating the absence of the toxicity in cell lines. The internalization preferences of GalNAc-quadruplexes to hepatic cells were also demonstrated as well as the enhancement of the luciferase inhibition using the luciferase assay in HepG2 cell lines versus HeLa cells. All together, we demonstrate that tetramerization of G-rich oligonucleotide is a novel and simple route to obtain the beneficial effects of multivalent N-acetylgalactosamine functionalization.

Study of the interaction of the palmatine alkaloid with hybrid G-quadruplex/duplex and i-motif/duplex DNA structures.

There is an increasing interest in the study of guanine or cytosine-rich sequences that may fold into G-quadruplex (G4) or i-motif (iM) structures showing a short hairpin (or stem-loop) stabilized by Watson-Crick base pairs. These hybrid spatial arrangements may be target of ligands that have been shown to interact strongly with B-DNA. In this work, the interaction of the palmatine alkaloid with several sequences forming different G4s, iMs, and hybrid structures has been studied by means of spectroscopic and separation techniques, as well as multivariate data analysis methods. At the experimental conditions used in this work, the results have shown that this ligand strongly stabilizes parallel G4 structures, whereas a weaker interaction was observed with the antiparallel G4 adopted by the thrombin-binding aptamer or iMs. The presence of hairpins within the loops scarcely affects the affinity of this ligand for the hybrid G4/duplex or iM/duplex structures. Fluorescence measurements have provided evidence of a certain interaction with iMs at pH 5.1, despite the absence of thermal stabilization effects.

Correction: Clua et al. Properties of Parallel Tetramolecular G-Quadruplex Carrying N-Acetylgalactosamine as Potential Enhancer for Oligonucleotide Delivery to Hepatocytes. Molecules 2022, 27, 3944.

In the original article [...].

Alkaloid Escholidine and Its Interaction with DNA Structures.

Berberine, the most known quaternary protoberberine alkaloid (QPA), has been reported to inhibit the SIK3 protein connected with breast cancer. Berberine also appears to reduce the bcl-2 and XIAP expression-proteins responsible for the inhibition of apoptosis. As some problems in the therapy with berberine arose, we studied the DNA binding properties of escholidine, another QPA alkaloid. CD, fluorescence, and NMR examined models of i-motif and G-quadruplex sequences present in the n-myc gene and the c-kit gene. We provide evidence that escholidine does not induce stabilization of the i-motif sequences, while the interaction with G-quadruplex structures appears to be more significant.

Investigation of the Complexes Formed between PARP1 Inhibitors and PARP1 G-Quadruplex at the Gene Promoter Region.

DNA repair inhibitors are one of the latest additions to cancer chemotherapy. In general, chemotherapy produces DNA damage but tumoral cells may become resistant if enzymes involved in DNA repair are overexpressed and are able to reverse DNA damage. One of the most successful drugs based on modulating DNA repair are the poly(ADP-ribose) polymerase 1 (PARP1) inhibitors. Several PARP1 inhibitors have been recently developed and approved for clinical treatments. We envisaged that PARP inhibition could be potentiated by simultaneously modulating the expression of PARP 1 and the enzyme activity, by a two-pronged strategy. A noncanonical G-quadruplex-forming sequence within the PARP1 promoter has been recently identified. In this study, we explored the potential binding of clinically approved PARP1 inhibitors to the G-quadruplex structure found at the gene promoter region. The results obtained by NMR, CD, and fluorescence titration confirmed by molecular modeling demonstrated that two out the four PARP1 inhibitors studied are capable of forming defined complexes with the PARP1 G-quadruplex. These results open the possibility of exploring the development of better G-quadruplex binders that, in turn, may also inhibit the enzyme.

Exploring the Interaction of Curaxin CBL0137 with G-Quadruplex DNA Oligomers.

Curaxins and especially the second-generation derivative curaxin CBL0137 have important antitumor activities in multiple cancers such as glioblastoma, melanoma and others. Although most of the authors suggest that their mechanism of action comes from the activation of p53 and inactivation of NF-kB by targeting FACT, there is evidence supporting the involvement of DNA binding in their antitumor activity. In this work, the DNA binding properties of curaxin CBL0137 with model quadruplex DNA oligomers were studied by 1H NMR, CD, fluorescence and molecular modeling. We provided molecular details of the interaction of curaxin with two G-quadruplex structures, the single repeat of human telomere d(TTAGGGT)4 and the c-myc promoter Pu22 sequence. We also performed 1H and 31P NMR experiments were also performed in order to investigate the interaction with duplex DNA models. Our data support the hypothesis that the interaction of curaxin with G-quadruplex may provide a novel insight into the DNA-binding properties of CBL0137, and it will be helpful for the design of novel selective DNA-targeting curaxin analogues.

Structural Effects of Incorporation of 2'-Deoxy-2'2'-Difluorodeoxycytidine (Gemcitabine) in A- and B-Form Duplexes.

We report the structural effect of 2'-deoxy-2',2'-difluorocytidine (dFdC) insertions in the DNA strand of a DNA : RNA hybrid duplex and in a self-complementary DNA : DNA duplex. In both cases, the modification slightly destabilizes the duplex and provokes minor local distortions that are more pronounced in the case of the DNA : RNA hybrid. Analysis of the solution structures determined by NMR methods show that dFdC is an adaptable derivative that adopts North type sugar conformation when inserted in pure DNA, or a South sugar conformation in the context of DNA : RNA hybrids. In this latter context, South sugar pucker favors the formation of a 2'F⋅⋅H8 attractive interaction with a neighboring purine, which compensates the destabilizing effect of base pair distortions. These interactions share some features with pseudohydrogen bonds described previously in other nucleic acids structures with fluorine modified sugars.

G-quadruplex binding properties of a potent PARP-1 inhibitor derived from 7-azaindole-1-carboxamide.

Poly ADP-ribose polymerases (PARP) are key proteins involved in DNA repair, maintenance as well as regulation of programmed cell death. For this reason they are important therapeutic targets for cancer treatment. Recent studies have revealed a close interplay between PARP1 recruitment and G-quadruplex stabilization, showing that PARP enzymes are activated upon treatment with a G4 ligand. In this work the DNA binding properties of a PARP-1 inhibitor derived from 7-azaindole-1-carboxamide, (2-[6-(4-pyrrolidin-1-ylmethyl-phenyl)-pyrrolo[2,3-b]pyridin-1-yl]-acetamide, compound 1) with model duplex and quadruplex DNA oligomers were studied by NMR, CD, fluorescence and molecular modelling. We provide evidence that compound 1 is a strong G-quadruplex binder. In addition we provide molecular details of the interaction of compound 1 with two model G-quadruplex structures: the single repeat of human telomeres, d(TTAGGGT)4, and the c-MYC promoter Pu22 sequence. The formation of defined and strong complexes with G-quadruplex models suggests a dual G4 stabilization/PARP inhibition mechanism of action for compound 1 and provides the molecular bases of its therapeutic potential.

Studies on the interactions of Ag(i) with DNA and their implication on the DNA-templated synthesis of silver nanoclusters and on the interaction with complementary DNA and RNA sequences.

Silver nanoclusters (AgNCs) prepared by the reduction of silver ions in the presence of DNA oligonucleotides have attracted great interest as potential diagnostic tools for their tunable and high fluorescent properties. In this work, three DNA sequences that consist of a 12-nucleotide long probe sequence at the 5'-end linked to the complementary sequence to three miRNAs are studied. First, the interaction of these sequences with Ag(i) was characterized by means of circular dichroism spectroscopy. By applying multivariate methods to the analysis of spectroscopic data, two complexes with different Ag(i) : DNA ratios were resolved. Secondly, the impact of several experimental variables, such as temperature, borohydride concentration and reaction time, on the formation of AgNCs templated by these three sequences was studied. Finally, the fluorescence properties of the duplexes formed by DNA probes with complementary DNA or miRNA sequences were studied. The results presented here highlight the role of the secondary structure adopted by the DNA probe on the fluorescence properties of DNA-stabilized AgNCs which, in turn, affect the development of methods for miRNA detection.

Tuning G-Quadruplex Nanostructures with Lipids. Towards Designing Hybrid Scaffolds for Oligonucleotide Delivery.

Two G-quadruplex forming oligonucleotides [d(TG4T)4 and d(TG6T)4] were selected as two tetramolecular quadruplex nanostructures because of their demonstrated ability to be modified with hydrophobic molecules. This allowed us to synthesize two series of G-quadruplex conjugates that differed in the number of G-tetrads, as well as in the terminal position of the lipid modification. Both solution and solid-phase syntheses were carried out to yield the corresponding lipid oligonucleotide conjugates modified at their 3'- and 5'-termini, respectively. Biophysical studies confirmed that the presence of saturated alkyl chains with different lengths did not affect the G-quadruplex integrity, but increased the stability. Next, the G-quadruplex domain was added to an 18-mer antisense oligonucleotide. Gene silencing studies confirmed the ability of such G-rich oligonucleotides to facilitate the inhibition of target Renilla luciferase without showing signs of toxicity in tumor cell lines.

Influence of pH and a porphyrin ligand on the stability of a G-quadruplex structure within a duplex segment near the promoter region of the SMARCA4 gene.

In a previous work, the formation of G-quadruplex structures in a 44-nucleotide long sequence found near the promoter region of the SMARCA4 gene was reported. The central 25 nucleotides were able to fold into an antiparallel G-quadruplex structure, the stability of which was pH-dependent. In the present work, the effect of the presence of lateral nucleotides and the complementary cytosine-rich strand on the stability of this G-quadruplex has been characterized. Moreover, the role of the model ligand TMPyP4 has been studied. Spectroscopic and separation techniques, as well as multivariate data analysis methods, have been used with these purposes. The results have shown that stability of the G-quadruplex as a function of pH or temperature is greatly reduced in the presence of the lateral nucleotides. The influence of the complementary strand does not prevent the formation of the G-quadruplex. Moreover, attempts to modulate the equilibria by an external ligand led us to determine the influence of the TMPyP4 porphyrin on these complex equilibria. This study could eventually help to understand the regulation of SMARCA4 expression.

A pH-dependent bolt involving cytosine bases located in the lateral loops of antiparallel G-quadruplex structures within the SMARCA4 gene promotor.

Some lung and ovarian tumors are connected to the loss of expression of SMARCA4 gene. In its promoter region, a 44-nucleotides long guanine sequence prone to form G-quadruplex structures has been studied by means of spectroscopic techniques (circular dichroism, molecular absorption and nuclear magnetic resonance), size exclusion chromatography and multivariate analysis. The results have shown that the central 21-nucleotides long sequence comprising four guanine tracts of disparate length is able to fold into a pH-dependent ensemble of G-quadruplex structures. Based on acid-base titrations and melting experiments of wild and mutated sequences, the formation of a C·C+ base pair between cytosine bases present at the two lateral loops is shown to promote a reduction in conformational heterogeneity, as well as an increase in thermal stability. The formation of this base pair is characterized by a pKa value of 7.1 ± 0.2 at 20 °C and 150 mM KCl. This value, higher than those usually found in i-motif structures, is related to the additional stability provided by guanine tetrads in the G-quadruplex. To our knowledge, this is the first thermodynamic description of this base pair in loops of antiparallel G-quadruplex structures.