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Quadruplex-Duplex (Q-D) junctions are unique structural motifs garnering increasing interest as drug targets, due to their frequent occurrence in genomic sequences. The viral HIV LTR-III sequence was chosen as a Q-D junction model to study the affinity of the selected compounds BMH-21, namitecan (ST-1968), and doxorubicin (DOXO), all containing a planar polycyclic aromatic moiety, linked to either one short aminoalkyl or an aminoglycosyl group. A multidisciplinary approach that combines NMR spectroscopy, molecular modelling, circular dichroism (CD) and fluorescence spectroscopy was employed. The studied ligands induced moderate but clear stabilization to the Q-D junction by interacting with the interfacial tetrad. DOXO was found to be the best Q-D junction binder. Interestingly, the removal of the aminoglycosyl group significantly changed the pattern of the interactions, indicating that highly polar substituents have a stronger affinity with the exposed regions of the Q-D junction, particularly at the level of the interfacial tetrad.
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Doxorrubicina , G-Cuádruplex , Doxorrubicina/química , Duplicado del Terminal Largo de VIH/genética , Dicroismo Circular , Camptotecina/química , Camptotecina/análogos & derivados , Modelos Moleculares , Humanos , Espectroscopía de Resonancia MagnéticaRESUMEN
One of the most appealing approaches for cancer treatment is targeted therapy, which is based on the use of drugs able to target cancer cells without affecting normal ones. This strategy lets to overcome the major limitation of conventional chemotherapy, namely the lack of specificity of anticancer drugs, which often leads to severe side effects, decreasing the therapy effectiveness. Delivery of cell-killing substances to tumor cells is one-way targeted drug therapy can work. Generally, monoclonal antibodies are combined with chemotherapeutic drugs, allowing cellular uptake through the binding to their targets on the surface of cancer cells. Aptamer-drug conjugates represent a promising alternative solution to antibodies to minimize off-target effects, considering the remarkable selective binding capabilities of aptamers. In this study, to enhance the therapeutic efficacy of the antineoplastic agent 5-fluoro-2'-deoxyuridine (FdU) in various cancer cells, we focused on the development of a novel conjugate using the antiproliferative aptamer T30923 (INT) as a drug vehicle. Three derivatives composed of T30923 conjugated with a different number of FdU units were synthesized, and their structural and biological properties were thoroughly characterized, highlighting their potential for targeted and synergistic anticancer responses.
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Antineoplásicos , Aptámeros de Nucleótidos , Proliferación Celular , Desoxiuridina , Sinergismo Farmacológico , Humanos , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/farmacología , Aptámeros de Nucleótidos/administración & dosificación , Antineoplásicos/farmacología , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Línea Celular Tumoral , Desoxiuridina/análogos & derivados , Desoxiuridina/administración & dosificación , Desoxiuridina/farmacología , Desoxiuridina/química , Proliferación Celular/efectos de los fármacos , Sistemas de Liberación de Medicamentos/métodosRESUMEN
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.
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Oligonucleótidos , Técnicas de Síntesis en Fase Sólida , Oligonucleótidos/química , Oligonucleótidos/síntesis química , Fosfatos/química , Benzamidas/química , Benzamidas/síntesis química , Compuestos Organofosforados/química , Compuestos Organofosforados/síntesis química , Fosforilación , Estructura MolecularRESUMEN
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.
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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.
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Antineoplásicos , Nanoestructuras , Neoplasias , Antineoplásicos/farmacología , ADN/química , Sistemas de Liberación de Medicamentos , Doxorrubicina/farmacología , Doxorrubicina/química , Nanoestructuras/química , Conformación de Ácido Nucleico , NanotecnologíaRESUMEN
Nucleosides and their analogues constitute an important family of molecules with potential antiviral and antiproliferative activity. The enantiomers of natural nucleosides, l-nucleoside derivatives, which have comparable biological activity but more favorable toxicological properties and greater metabolic stability than d-nucleosides, have emerged as a new class of therapeutic agents. Furthermore, l-nucleosides can be used as a building block to prepare l-oligonucleotides, which have identical physical properties in terms of solubility, hybridization kinetics, and duplex thermal stability as d-oligonucleotides but completely orthogonal in nature. Consequently, they are resistant to nuclease degradation, nontoxic, and immunologically passive, which are desirable properties for biomedical applications. Herein, we describe the synthesis of several 2'-O-methyl/2'-O-MOE-l-nucleoside pyrimidine derivatives and their incorporation into G-rich oligonucleotides. Finally, we evaluated the stability and resistance against nucleases of these new G-quadruplexes, demonstrating the potential of the l-nucleosides described in this work in providing enhanced nuclease resistance with a minimal impact in the nucleic acid structural properties.
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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.
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This Special issue is dedicated to the memory of Enrique Pedroso, Professor Emeritus of Organic Chemistry at University of Barcelona, who passed away at the age of 72 in September 2020 [...].
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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.
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ADN , Electroforesis Capilar , Espectrofotometría , Espectrofotometría Ultravioleta/métodos , Temperatura , Electroforesis Capilar/métodosRESUMEN
This study aims to explore the stability of lipo-polymeric niosomes/niosome-based pCMS-EGFP complexes under different storage temperatures (25 °C, 4 °C, and -20 °C). To date, the question of nucleic acid-complex stability is one of the most vital issues in gene delivery applications. The need for stable vaccines during the COVID-19 pandemic has merely highlighted it. In the case of niosomes as gene carriers, the scientific literature still lacks comprehensive stability studies. In this study, the physicochemical features of niosomes/nioplexes in terms of size, surface charge, and polydispersity index (PDI), along with transfection efficiency, and cytotoxicity in NT2 cells were evaluated for 8 weeks. Compared to day 0, the physicochemical features of the niosomes stored at 25 °C and -20 °C changed dramatically in terms of size, zeta potential, and PDI, while remaining in reasonable values when stored at 4 °C. However, niosomes and nioplexes stored at 4 °C and -20 °C showed nearly stable transfection efficiency values, yet an obvious decrease at 25 °C. This article provides a proof of concept into the stability of polymeric cationic niosomes and their nioplexes as promising gene delivery vehicles. Moreover, it highlights the practical possibility of storing nioplexes at 4 °C for up to 2 months, as an alternative to niosomes, for gene delivery purposes.
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COVID-19 , Liposomas , Humanos , Liposomas/química , Pandemias , Plásmidos , ADN , PolímerosRESUMEN
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.
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ADN , G-Cuádruplex , Ligandos , ADN/química , Emparejamiento Base , Concentración de Iones de Hidrógeno , Conformación de Ácido NucleicoRESUMEN
MicroRNAs (miRNAs) are endogenous, short RNA oligonucleotides that regulate the expression of hundreds of proteins to control cells' function in physiological and pathological conditions. miRNA therapeutics are highly specific, reducing the toxicity associated with off-target effects, and require low doses to achieve therapeutic effects. Despite their potential, applying miRNA-based therapies is limited by difficulties in delivery due to their poor stability, fast clearance, poor efficiency, and off-target effects. To overcome these challenges, polymeric vehicles have attracted a lot of attention due to their ease of production with low costs, large payload, safety profiles, and minimal induction of the immune response. Poly(N-ethyl pyrrolidine methacrylamide) (EPA) copolymers have shown optimal DNA transfection efficiencies in fibroblasts. The present study aims to evaluate the potential of EPA polymers as miRNA carriers for neural cell lines and primary neuron cultures when they are copolymerized with different compounds. To achieve this aim, we synthesized and characterized different copolymers and evaluated their miRNA condensation ability, size, charge, cytotoxicity, cell binding and internalization ability, and endosomal escape capacity. Finally, we evaluated their miRNA transfection capability and efficacy in Neuro-2a cells and rat primary hippocampal neurons. The results indicate that EPA and its copolymers, incorporating ß-cyclodextrins with or without polyethylene glycol acrylate derivatives, can be promising vehicles for miRNA administration to neural cells when all experiments on Neuro-2a cells and primary hippocampal neurons are considered together.
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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.
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ADN , Plata , Secuencia de Bases , ADN/genética , ADN/química , Pirimidinas , Colorantes , Conformación de Ácido Nucleico , Dicroismo CircularRESUMEN
Antisense and small interfering RNA (siRNA) oligonucleotides have been recognized as powerful therapeutic compounds for targeting mRNAs and inducing their degradation. However, a major obstacle is that unmodified oligonucleotides are not readily taken up into tissues and are susceptible to degradation by nucleases. For these reasons, the design and preparation of modified DNA/RNA derivatives with better stability and an ability to be produced at large scale with enhanced uptake properties is of vital importance to improve current limitations. In the present study, we review the conjugation of oligonucleotides with lipids and peptides in order to produce oligonucleotide conjugates for therapeutics aiming to develop novel compounds with favorable pharmacokinetics.
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BACKGROUND: Nucleoside and nucleobase antimetabolites are an important class of chemotherapeutic agents for the treatment of cancer as well as other diseases. INTRODUCTION: In order to avoid undesirable side effects, several prodrug strategies have been developed. In the present review, we describe a relatively unknown strategy that consists of using oligonucleotides modified with nucleoside antimetabolites as prodrugs. METHODS: The active nucleotides are generated by enzymatic degradation once incorporated into cells. This strategy has attracted large interest and is widely utilized at present due to the continuous developments made in therapeutic oligonucleotides and the recent advances in nanomaterials and nanomedicine. RESULTS: A large research effort was made mainly in the improvement of the antiproliferative properties of nucleoside homopolymers, but recently, chemically modified aptamers, antisense oligonucleotides and/or siRNA carrying antiproliferative nucleotides have demonstrated a great potential due to the synergetic effect of both therapeutic entities. In addition, DNA nanostructures with interesting properties have been built to combine antimetabolites and enhancers of cellular uptake in the same scaffold. Finally, protein nanoparticles functionalized with receptor-binders and antiproliferative oligomers represent a new avenue for a more effective treatment in cancer therapy. CONCLUSION: It is expected that oligonucleotides carrying nucleoside antimetabolites will be considered as potential drugs in the near future for biomedical applications.
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Nucleósidos , Profármacos , Humanos , Nucleósidos/química , Antimetabolitos , Profármacos/química , Oligonucleótidos , NucleótidosRESUMEN
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.
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COVID-19 , Ácidos Nucleicos , Humanos , SARS-CoV-2/genética , COVID-19/diagnóstico , Oligonucleótidos/química , Reacción en Cadena de la Polimerasa , ARN Viral/genética , ARN Viral/análisis , Técnicas de Amplificación de Ácido Nucleico/métodosRESUMEN
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.
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G-Cuádruplex , Dicroismo Circular , Reparación del ADN , Ligandos , Regiones Promotoras GenéticasRESUMEN
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.
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Acetilgalactosamina , G-Cuádruplex , Acetilgalactosamina/química , Receptor de Asialoglicoproteína/metabolismo , Células HeLa , Hepatocitos , Humanos , Oligonucleótidos/metabolismoRESUMEN
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.
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Nanoestructuras , Ácidos Nucleicos , Sistemas de Liberación de Medicamentos , ARN Interferente Pequeño/químicaRESUMEN
Current therapy in acute myeloid leukemia (AML) is based on chemotherapeutic drugs administered at high doses, lacking targeting selectivity and displaying poor therapeutic index because of severe adverse effects. Here, we develop a novel nanoconjugate that combines a self-assembled, multivalent protein nanoparticle, targeting the CXCR4 receptor, with an Oligo-Ara-C prodrug, a pentameric form of Ara-C, to highly increase the delivered payload to target cells. This 13.4 nm T22-GFP-H6-Ara-C nanoconjugate selectively eliminates CXCR4+ AML cells, which are protected by its anchoring to the bone marrow (BM) niche, being involved in AML progression and chemotherapy resistance. This nanoconjugate shows CXCR4-dependent internalization and antineoplastic activity in CXCR4+ AML cells in vitro. Moreover, repeated T22-GFP-H6-Ara-C administration selectively eliminates CXCR4+ leukemic cells in BM, spleen and liver. The leukemic dissemination blockage induced by T22-GFP-H6-Ara-C is significantly more potent than buffer or Oligo-Ara-C-treated mice, showing no associated on-target or off-target toxicity and, therefore, reaching a highly therapeutic window. In conclusion, T22-GFP-H6-Ara-C exploits its 11 ligands-multivalency to enhance target selectivity, while the Oligo-Ara-C prodrug multimeric form increases 5-fold its payload. This feature combination offers an alternative nanomedicine with higher activity and greater tolerability than current intensive or non-intensive chemotherapy for AML patients.