The synthesis of solid supports carrying base labile linkers to generate 3'-phosphate oligonucleotides.

Oligonucleotides carrying 3'-terminal phosphates and conjugates are important tools in molecular biology and diagnostic purposes. We described the preparation of solid supports carrying the base labile linker 4-((2-hydroxyethyl)sulfonyl)benzamide for the solid-phase synthesis of 3'-phosphorylated oligonucleotides. These supports are fully compatible with the phosphoramidite chemistry yielding the desired 3'-phosphate oligonucleotides in excellent yields. The use of mild deprotection conditions allows the generation of partially protected DNA fragments.

Defined covalent attachment of three cancer drugs to DNA origami increases cytotoxicity at nanomolar concentration.

DNA nanostructures have captured great interest as drug delivery vehicles for cancer therapy. Despite rapid progress in the field, some hurdles, such as low cellular uptake, low tissue specificity or ambiguous drug loading, remain unsolved. Herein, well-known antitumor drugs (doxorubicin, auristatin, and floxuridine) were site-specifically incorporated into DNA nanostructures, demonstrating the potential advantages of covalently linking drug molecules via structural staples instead of incorporating the drugs by noncovalent binding interactions. The covalent strategy avoids critical issues such as an unknown number of drug-DNA binding events and premature drug release. Moreover, covalently modified origami offers the possibility of precisely incorporating several synergetic antitumor drugs into the DNA nanostructure at a predefined molar ratio and to control the exact spatial orientation of drugs into DNA origami. Additionally, DNA-based nanoscaffolds have been reported to have a low intracellular uptake. Thus, two cellular uptake enhancing mechanisms were studied: the introduction of folate units covalently linked to DNA origami and the transfection of DNA origami with Lipofectamine. Importantly, both methods increased the internalization of DNA origami into HTB38 and HCC2998 colorectal cancer cells and produced greater cytotoxic activity when the DNA origami incorporated antiproliferative drugs. The results here present a successful and conceptually distinct approach for the development of DNA-based nanostructures as drug delivery vehicles, which can be considered an important step towards the development of highly precise nanomedicines.

Single-Step Functionalization Strategy of Graphene Microtransistor Array with Chemically Modified Aptamers for Biosensing Applications.

Graphene solution-gated field-effect transistors (gSGFETs) offer high potential for chemical and biochemical sensing applications. Among the current trends to improve this technology, the functionalization processes are gaining relevance for its crucial impact on biosensing performance. Previous efforts are focused on simplifying the attachment procedure from standard multi-step to single-step strategies, but they still suffer from overreaction, and impurity issues and are limited to a particular ligand. Herein, a novel strategy for single-step immobilization of chemically modified aptamers with fluorenylmethyl and acridine moieties, based on a straightforward synthetic route to overcome the aforementioned limitations is presented. This approach is benchmarked versus a standard multi-step strategy using thrombin as detection model. In order to assess the reliability of the functionalization strategies 48-gSGFETs arrays are employed to acquire large datasets with multiple replicas. Graphene surface characterization demonstrates robust and higher efficiency in the chemical coupling of the aptamers with the single-step strategy, while the electrical response evaluation validates the sensing capability, allowing to implement different alternatives for data analysis and reduce the sensing variability. In this work, a new tool capable of overcome the functionalization challenges of graphene surfaces is provided, paving the way toward the standardization of gSGFETs for biosensing purposes.

Chemical Modifications in Nucleic Acids for Therapeutic and Diagnostic Applications.

The last decade has witnessed the blooming of nucleic acids for therapeutic and diagnostic applications. In the present article, we describe the most important results from our group in this area covering the international context that surrounded this research. These include the study of modifications at the terminal and internal positions of siRNA duplexes to enhance nuclease resistance, increase loading of the antisense strand to RISC and avoid side effects such as activation of immune response and sense strand misloading. Then, we describe the design of novel lipid, carbohydrate and peptide conjugates to enhance cellular uptake. Finally, we describe the use of nanostructures for drug delivery and for the controlled deposition of matter on surfaces. We invite the readers to submerge into a highly interdisciplinary discipline that combines organic chemistry, biochemical assays, pharmacology issues as well as materials chemistry and structural studies in order to increase the applications of nucleic acids.

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.

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.

Study of conformational transitions of i-motif DNA using time-resolved fluorescence and multivariate analysis methods.

Recently, the presence of i-motif structures at C-rich sequences in human cells and their regulatory functions have been demonstrated. Despite numerous steady-state studies on i-motif at neutral and slightly acidic pH, the number and nature of conformation of this biological structure are still controversial. In this work, the fluorescence lifetime of labelled molecular beacon i-motif-forming DNA sequences at different pH values is studied. The influence of the nature of bases at the lateral loops and the presence of a Watson-Crick-stabilized hairpin are studied by means of time-correlated single-photon counting technique. This allows characterizing the existence of several conformers for which the fluorophore has lifetimes ranging from picosecond to nanosecond. The information on the existence of different i-motif structures at different pH values has been obtained by the combination of classical global decay fitting of fluorescence traces, which provides lifetimes associated with the events defined by the decay of each sequence and multivariate analysis, such as principal component analysis or multivariate curve resolution based on alternating least squares. Multivariate analysis, which is seldom used for this kind of data, was crucial to explore similarities and differences of behaviour amongst the different DNA sequences and to model the presence and identity of the conformations involved in the pH range of interest. The results point that, for i-motif, the intrachain contact formation and its dissociation show lifetimes ten times faster than for the open form of DNA sequences. They also highlight that the presence of more than one i-motif species for certain DNA sequences according to the length of the sequence and the composition of the bases in the lateral loop.

Evaluation of Floxuridine Oligonucleotide Conjugates Carrying Potential Enhancers of Cellular Uptake.

Conjugation of small molecules such as lipids or receptor ligands to anti-cancer drugs has been used to improve their pharmacological properties. In this work, we studied the biological effects of several small-molecule enhancers into a short oligonucleotide made of five floxuridine units. Specifically, we studied adding cholesterol, palmitic acid, polyethyleneglycol (PEG 1000), folic acid and triantennary N-acetylgalactosamine (GalNAc) as potential enhancers of cellular uptake. As expected, all these molecules increased the internalization efficiency with different degrees depending on the cell line. The conjugates showed antiproliferative activity due to their metabolic activation by nuclease degradation generating floxuridine monophosphate. The cytotoxicity and apoptosis assays showed an increase in the anti-cancer activity of the conjugates related to the floxuridine oligomer, but this effect did not correlate with the internalization results. Palmitic and folic acid conjugates provide the highest antiproliferative activity without having the highest internalization results. On the contrary, cholesterol oligomers that were the best-internalized oligomers had poor antiproliferative activity, even worse than the unmodified floxuridine oligomer. Especially relevant is the effect induced by palmitic and folic acid derivatives generating the most active drugs. These results are of special interest for delivering other therapeutic oligonucleotides.

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.

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.

Detection of a G-Quadruplex as a Regulatory Element in Thymidylate synthase for Gene Silencing Using Polypurine Reverse Hoogsteen Hairpins.

Thymidylate synthase (TYMS) enzyme is an anti-cancer target given its role in DNA biosynthesis. TYMS inhibitors (e.g., 5-Fluorouracil) can lead to drug resistance through an autoregulatory mechanism of TYMS that causes its overexpression. Since G-quadruplexes (G4) can modulate gene expression, we searched for putative G4 forming sequences (G4FS) in the TYMS gene that could be targeted using polypurine reverse Hoogsteen hairpins (PPRH). G4 structures in the TYMS gene were detected using the quadruplex forming G-rich sequences mapper and confirmed through spectroscopic approaches such as circular dichroism and NMR using synthetic oligonucleotides. Interactions between G4FS and TYMS protein or G4FS and a PPRH targeting this sequence (HpTYMS-G4-T) were studied by EMSA and thioflavin T staining. We identified a G4FS in the 5'UTR of the TYMS gene in both DNA and RNA capable of interacting with TYMS protein. The PPRH binds to its corresponding target dsDNA, promoting G4 formation. In cancer cells, HpTYMG-G4-T decreased TYMS mRNA and protein levels, leading to cell death, and showed a synergic effect when combined with 5-fluorouracil. These results reveal the presence of a G4 motif in the TYMS gene, probably involved in the autoregulation of TYMS expression, and the therapeutic potential of a PPRH targeted to the G4FS.

Stabilization of c-KIT G-Quadruplex DNA Structures by the RNA Polymerase I Inhibitors BMH-21 and BA-41.

The stabilization of G-quadruplex DNA structures by small molecules with affinity to oncogene promoters has emerged as a promising anticancer strategy, due to a potential role in gene expression regulation. We explored the ability of BMH-21 (1) and its analogue BA-41 (2) to bind the G-quadruplex structure present in the c-KIT promoter by biophysical methods and molecular modeling. We provide evidence that both compounds interact with the c-KIT 21-mer sequence. The stable monomeric intramolecular parallel G-quadruplex obtained by the mutation of positions 12 and 21 allowed the precise determination of the binding mode by NMR and molecular dynamics studies. Both compounds form a complex characterized by one ligand molecule positioned over the tetrad at the 3'-end, stabilized by an extensive network of π-π interactions. The binding constants (Kb) obtained with fluorescence are similar for both complexes (around 106 M-1). Compound BA-41 (2) showed significant antiproliferative activity against a human lymphoma cell line, SU-DHL4, known to express substantial levels of c-KIT. However, the partial inhibition of c-KIT expression by Western blot analysis suggested that the interaction of compound 2 with the c-KIT promoter is not the primary event and that multiple effects provide a contribution as determinants of biological activity.

siRNA Modified with 2'-Deoxy-2'-C-methylpyrimidine Nucleosides.

(2'S)-2'-Deoxy-2'-C-methyluridine and (2'R)-2'-deoxy-2'-C-methyluridine were incorporated in the 3'-overhang region of the sense and antisense strands and in positions 2 and 5 of the seed region of siRNA duplexes directed against Renilla luciferase, whereas (2'S)-2'-deoxy-2'-C-methylcytidine was incorporated in the 6-position of the seed region of the same constructions. A dual luciferase reporter assay in transfected HeLa cells was used as a model system to measure the IC50 values of 24 different modified duplexes. The best results were obtained by the substitution of one thymidine unit in the antisense 3'-overhang region by (2'S)- or (2'R)-2'-deoxy-2'-C-methyluridine, reducing IC50 to half of the value observed for the natural control. The selectivity of the modified siRNA was measured, it being found that modifications in positions 5 and 6 of the seed region had a positive effect on the ON/OFF activity.

Multiple Multicomponent Reactions: Unexplored Substrates, Selective Processes, and Versatile Chemotypes in Biomedicine.

Multiple multicomponent reactions rapidly assemble complex structures. Despite being very productive, the lack of selectivity and the reduced number of viable transformations restrict their general application in synthesis. Hereby, we describe a rationale for a selective version of these processes based in the preferential generation of intermediates which are less reactive than the initial substrates. In this way, applying the Groebke-Blackburn-Bienaymé reaction on a range of α-polyamino-polyazines, we prepared a family compact heterocyclic scaffolds with relevant applications in medicinal and biological chemistry (live cell imaging probes, selective binders for DNA quadruplexes, and antiviral agents against human adenoviruses). The approach has general character and yields complex molecular targets in a selective, tunable and direct manner.

Study of light-induced formation of photodimers in the i-motif nucleic acid structure by rapid-scan FTIR difference spectroscopy and hybrid hard- and soft-modelling.

The i-motif is a DNA structure formed by cytosine-rich sequences, very relevant from a biochemical point of view and potentially useful in nanotechnology as pH-sensitive nanodevices or nanomotors. To provide a different view on the structural changes and dynamics of direct excitation processes involving i-motif structures, the use of rapid-scan FTIR spectroscopy is proposed. Hybrid hard- and soft-modelling based on the Multivariate Curve Resolution by Alternating Least Squares (MCR-ALS) algorithm has been used for the resolution of rapid-scan FTIR spectra and the interpretation of the photochemically induced time-dependent conformational changes of i-motif structures. The hybrid hard- and soft-modelling version of MCR-ALS (HS-MCR), which allows the introduction of kinetic models to describe process behavior, provides also rate constants associated with the transitions modeled. The results show that UV irradiation does not produce degradation of the studied sequences but induces the formation of photodimers. The presence of these affect much more the stability of i-motif structures formed by short sequences than that of those formed by longer sequences containing additional structural stabilizing elements, such as hairpins.

Naturally occurring quaternary benzo[c]phenanthridine alkaloids selectively stabilize G-quadruplexes.

In this work, the interaction of six natural benzo[c]phenanthridine alkaloids (macarpine, sanguilutine, sanguirubine, chelerythrine, sanguinarine and chelirubine) with parallel and antiparallel G-quadruplex DNA structures was studied. HT22 corresponding to the end of human telomeres and the modified promoter oncogene c-kit21 and Pu22 sequences have been used. Spectroscopically-monitored melting experiments and fluorescence titrations, competitive dialysis and nuclear magnetic resonance spectroscopy were used for this purpose. The results showed that these alkaloids stabilized G-quadruplex structures in terms of increments of Tm values (from 15 to 25 °C) with high selectivity over duplexes and unfolded DNA. The mode of binding was mainly by stacking on the terminal G-tetrads with stoichiometries of 1 : 2 (DNA : ligand). The presence of non-specific electrostatic interactions was also observed. Overall, the results pointed to a strong stabilization of G-quadruplex structures by these alkaloids.

Stabilization of Telomeric I-Motif Structures by (2'S)-2'-Deoxy-2'-C-Methylcytidine Residues.

G-quadruplexes and i-motifs are tetraplex structures present in telomeres and the promoter regions of oncogenes. The possibility of producing nanodevices with pH-sensitive functions has triggered interest in modified oligonucleotides with improved structural properties. We synthesized C-rich oligonucleotides carrying conformationally restricted (2'S)-2'-deoxy-2'-C-methyl-cytidine units. The effect of this modified nucleoside on the stability of intramolecular i-motifs from the vertebrate telomere was investigated by UV, CD, and NMR spectroscopy. The replacement of selected positions of the C-core with C-modified residues induced the formation of stable intercalated tetraplexes at near-neutral pH. This study demonstrates the possibility of enhancing the stability of the i-motif by chemical modification.

The effect of l-thymidine, acyclic thymine and 8-bromoguanine on the stability of model G-quadruplex structures.

BACKGROUND: Guanine-rich oligonucleotides are capable of forming tetrahelical structures known as G-quadruplexes with interesting biological properties. We have investigated the effects of site-specific substitution in the loops and in the tetrads model G-quadruplexes using thymine glycol nucleic acid (GNA) units, l-thymidine and 8-Br-2'-deoxyguanosine. METHODS: Modified oligonucleotides were chemically synthesized and spectroscopic techniques were used to determine the relative stability of the modified G-quadruplex. The double 8-BrdG-modified quadruplexes were further characterized by Nuclear Magnetic Resonance. Binding to thrombin of selected quadruplex was analyzed by gel electrophoresis retention assay. RESULTS: The most interesting results were found with a 8-bromoG substitution that had the larger stabilization of the quadruplex. NMR studies indicate a tight relationship between the loops and the tetrads to accommodate 8-bromoG modifications within the TBA. CONCLUSIONS: The substitutions of loop positions with GNA T affect the TBA stability except for single modification in T7 position. Single l-thymidine substitutions produced destabilization of TBA. Larger changes on quadruplex stability are observed with the use of 8-bromoG finding a single substitution with the highest thermal stabilization found in thrombin binding aptamers modified at the guanine residues and having good affinity for thrombin. Double 8-BrdG modification in anti positions of different tetrads produce a conformational flip from syn to anti conformation of 8-Br-dG to favor loop-tetrad interaction and preserve the overall TBA stability. GENERAL SIGNIFICANCE: Modified guanine-rich oligonucleotides are valuable tools for the search for G-quadruplex structures with higher thermal stability and may provide compounds with interesting protein-nucleic acid binding properties. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.

Controlling the Reversible Assembly of Liposomes through a Multistimuli Responsive Anchored DNA.

We present a novel approach to reversibly control the assembly of liposomes through an anchored multistimuli responsive DNA oligonucleotide decorated with an azobenzene moiety (AZO-ON1). We show that liposomes assembly can be simultaneously controlled by three external stimuli: light, Mg(2+), and temperature. (i) Light alters the interaction of AZO-ON1 with liposomes, which influences DNA coating and consequently liposomes assembly. (ii) Mg(2+) induces the assembly, hence variation in its concentration enables for reversibility. (iii) Double-stranded AZO-ON1 is more efficient than single-stranded AZO-ON1 in triggering the assembly of liposomes and temperature has been used for controllable assembly through DNA thermal denaturation. Our multiresponsive AZO-ON1 represents a unique example in which multiple stimuli can be simultaneously applied to regulate the reversible assembly of liposomes.