RESUMO
A versatile method for the automated synthesis of composites containing DNA-oligonucleotides and boron cluster scaffolds and their assembly into functional nanoparticles is described. The obtained, torus-like nanoparticles carry antisense oligonucleotides that target two different oncogenes simultaneously. The nanoparticles exhibited notable silencing efficiency inâ vitro in a pancreatic carcinoma cell line PANC-1 toward EGFR and c-Myc genes at the mRNA level, and a significant efficiency at the protein level. The proposed approach may be an attractive alternative to methods currently used, including one therapeutic nucleic acid, one genetic target, or the use of cocktails of therapeutic nucleic acids.
Assuntos
Genes myc , Nanopartículas , Boro , DNA , Anticorpos , RNA MensageiroRESUMO
New boron carriers with high boron content and targeted cancer-cell delivery are considered the first choice for boron neutron capture therapy (BNCT) for cancer treatment. Previously, we have shown that composites of antisense oligonucleotide and boron clusters are functional nanoparticles for the downregulation of expression of epidermal growth factor receptor (EGFR) and can be loaded into EGFR-overexpressing cancer cells without a transfection factor. In this study, we hypothesize that free cellular uptake is mediated by binding and activation of the EGFR by boron clusters. Proteomic analysis of proteins pulled-down from various EGFR-overexpressing cancer cells using short oligonucleotide probes, conjugated to 1,2-dicarba-closo-dodecaborane (1,2-DCDDB, [C2B10H12]) and [(3,3'-Iron-1,2,1',2'-dicarbollide)-] (FESAN, [Fe(C2B9H11)2]-), evidenced that boron cage binds to EGFR subdomains. Moreover, inductively coupled plasma mass spectrometry (ICP MS) and fluorescence microscopy analyses confirmed that FESANs-highly decorated B-ASOs were efficiently delivered and internalized by EGFR-overexpressing cells. Antisense reduction of EGFR in A431 and U87-MG cells resulted in decreased boron accumulation compared to control cells, indicating that cellular uptake of B-ASOs is related to EGFR-dependent internalization. The data obtained suggest that EGFR-mediated cellular uptake of B-ASO represents a novel strategy for cellular delivery of therapeutic nucleic acids (and possibly other medicines) conjugated to boron clusters.
Assuntos
Terapia por Captura de Nêutron de Boro , Ácidos Nucleicos , Boro/química , Proteômica , Terapia por Captura de Nêutron de Boro/métodos , Receptores ErbB/genética , Receptores ErbB/metabolismo , Compostos de BoroRESUMO
Boron cluster-conjugated antisense oligonucleotides (B-ASOs) have already been developed as therapeutic agents with "two faces", namely as potential antisense inhibitors of gene expression and as boron carriers for boron neutron capture therapy (BNCT). The previously observed high antisense activity of some B-ASOs targeting the epidermal growth factor receptor (EGFR) could not be rationally assigned to the positioning of the boron cluster unit: 1,2-dicarba-closo-dodecaborane (0), [(3,3'-Iron-1,2,1',2'-dicarbollide) (1-), FESAN], and dodecaborate (2-) in the ASO chain and its structure or charge. For further understanding of this observation, we performed systematic studies on the efficiency of RNase H against a series of B-ASOs models. The results of kinetic analysis showed that pyrimidine-enriched B-ASO oligomers activated RNase H more efficiently than non-modified ASO. The presence of a single FESAN unit at a specific position of the B-ASO increased the kinetics of enzymatic hydrolysis of complementary RNA more than 30-fold compared with unmodified duplex ASO/RNA. Moreover, the rate of RNA hydrolysis enhanced with the increase in the negative charge of the boron cluster in the B-ASO chain. In conclusion, a "smart" strategy using ASOs conjugated with boron clusters is a milestone for the development of more efficient antisense therapeutic nucleic acids as inhibitors of gene expression.
Assuntos
Boro , Oligonucleotídeos Antissenso , Oligonucleotídeos Antissenso/farmacologia , Boro/metabolismo , Cinética , RNA Complementar , Ribonuclease H/genética , Ribonuclease H/metabolismo , Inativação Gênica , Oligonucleotídeos , Receptores ErbB/metabolismo , Pirimidinas , Ferro/metabolismoRESUMO
Epidermal growth factor receptor (EGFR) is one of the most promising molecular targets for anticancer therapy. We used boron clusters as a platform for generation of new materials. For this, functional DNA constructs conjugated with boron clusters (B-ASOs) were developed. These B-ASOs, built from 1,2-dicarba-closo-dodecaborane linked with two anti-EGFR antisense oligonucleotides (ASOs), form with their complementary congeners torus-like nanostructures, as previously shown by atomic force microscope (AFM) and transmission electron cryo-microscopy (cryo-TEM) imaging. In the present work, deepened studies were carried out on B-ASO's properties. In solution, B-ASOs formed four dominant complexes as confirmed by non-denaturing polyacrylamide gel electrophoresis (PAGE). These complexes exhibited increased stability in cell lysate comparing to the non-modified ASO. Fluorescently labeled B-ASOs localized mostly in the cytoplasm and decreased EGFR expression by activating RNase H. Moreover, the B-ASO complexes altered the cancer cell phenotype, decreased cell migration rate, and arrested the cells in the S phase of cell cycle. The 1,2-dicarba-closo-dodecaborane-containing nanostructures did not activate NLRP3 inflammasome in human macrophages. In addition, as shown by inductively coupled plasma mass spectrometry (ICP MS), these nanostructures effectively penetrated the human squamous carcinoma cells (A431), showing their potential applicability as anticancer agents.
Assuntos
Antineoplásicos/farmacologia , Boranos/farmacologia , Regulação Neoplásica da Expressão Gênica , Nanopartículas/química , Oligonucleotídeos Antissenso/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Boranos/síntese química , Boranos/metabolismo , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Movimento Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Células HeLa , Humanos , Cinética , Células MCF-7 , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Fase S/efeitos dos fármacos , Fase S/genética , Transdução de SinaisRESUMO
Antisense oligonucleotides conjugated with boron clusters (B-ASOs) have been described as potential gene expression inhibitors and carriers of boron for boron neutron capture therapy (BNCT), providing a dual-action therapeutic platform. In this study, we tested the nucleolytic stability of DNA oligonucleotides labeled with metallacarborane [(3,3'-iron-1,2,1',2'-dicarbollide)(-1)]ate [Fe(C2B9H11)2] (FESAN) against snake venom phosphodiesterase (svPDE, 3'â5'-exonuclease). Contrary to the previously observed protective effect of carborane (C2B10H12) modifications, the B-ASOs containing a metallacarborane moiety at the 5'-end of the oligonucleotide chain were hydrolyzed faster than their parent nonmodified oligomers. Interestingly, an enhancement in the hydrolysis rate was also observed in the presence of free metallacarborane, and this reaction was dependent on the concentration of the metallacarborane. Microscale thermophoresis (MST) analysis confirmed the high affinity (Kd nM range) of the binding of the metallacarborane to the proteins of crude snake venom and the moderate affinity (Kd µM range) between the metallacarborane and the short single-stranded DNA. We hypothesize that the metallacarborane complex covalently bound to B-ASO holds DNA molecules close to the protein surface, facilitating enzymatic cleavage. The addition of metallacarborane alone to the ASO/svPDE reaction mixture provides the interface to attract freely floating DNA molecules. In both cases, the local DNA concentration around the enzymes increases, giving rise to faster hydrolysis. It was experimentally shown that an allosteric effect, possibly attributable to the observed boost in the 3´â5´-exonucleolytic activity of snake venom phosphodiesterase, is much less plausible.
Assuntos
Compostos de Boro/química , DNA Antissenso/análogos & derivados , Diester Fosfórico Hidrolases/metabolismo , Venenos de Serpentes/enzimologia , Hidrólise , Ligação Proteica , Especificidade por SubstratoRESUMO
Nucleic acids are key biomolecules in all life forms. These biomolecules can encode and transfer information via Watson-Crick base-pairing interactions and can form double-stranded structures between complementary sequences with high precision. These properties make nucleic acids extremely successful in applications in materials science as nanoconstruction materials. Herein, we describe a method for the automated synthesis of "oligopeds", which are building blocks based on the boron cluster structure equipped with short DNA adapters; these building blocks assemble into functional nanoparticles. The obtained, well defined, torus-like structures are the first DNA nanoconstructs based on a boron cluster scaffold. The results indicate the potential of boron clusters in DNA nanoconstruction and open the way for the design of entirely new types of buildings blocks based on polyhedral heteroborane geometry and its unique properties. The use of antisense oligonucleotides as DNA adapters illustrates one of the possible applications of the obtained nanoconstructs as vectors for therapeutic nucleic acids.
Assuntos
Boranos/química , Nanopartículas/química , Ácidos Nucleicos/química , Sequência de Bases , Boranos/síntese química , Linhagem Celular Tumoral , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Inativação Gênica , Humanos , Microscopia de Força Atômica , Oligonucleotídeos Antissenso/análise , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , TermodinâmicaRESUMO
A general and convenient approach for the incorporation of different types of boron clusters into specific locations of the DNA-oligonucleotide chain based on the automated phosphoramidite method of oligonucleotide synthesis and post-synthetic "click chemistry" modification has been developed. Pronounced effects of boron-cluster modification on the physico- and biochemical properties of the antisense oligonucleotides were observed. The silencing activity of antisense oligonucleotides bearing a single boron cluster modification in the middle of the oligonucleotide chain was substantially higher than that of unmodified oligonucleotides. This finding may be of importance for the design of therapeutic nucleic acids with improved properties. The proposed synthetic methodology broadens the availability of nucleic acid-boron cluster conjugates and opens up new avenues for their potential practical use.
Assuntos
Boro/química , Receptores ErbB/antagonistas & inibidores , Oligonucleotídeos Antissenso/química , Sequência de Bases , Dicroísmo Circular , Química Click , Receptores ErbB/genética , Receptores ErbB/metabolismo , Inativação Gênica , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Espectroscopia de Ressonância Magnética , Microscopia de Fluorescência , Oligonucleotídeos Antissenso/síntese química , Oligonucleotídeos Antissenso/metabolismo , Compostos Organofosforados/química , Temperatura de TransiçãoRESUMO
Boron cluster-modified therapeutic nucleic acids with improved properties are of interest in gene therapy and in cancer boron neutron capture therapy (BNCT). High metallacarborane-loaded antisense oligonucleotides (ASOs) targeting epidermal growth factor receptor (EGFR) were synthesized through post-synthetic Cu (I)-assisted "click" conjugation of alkyne-modified DNA-oligonucleotides with a boron cluster alkyl azide component. The obtained oligomers exhibited increased lipophilicity compared to their non-modified precursors, while their binding affinity to complementary DNA and RNA strands was slightly decreased. Multiple metallacarborane residues present in the oligonucleotide chain, each containing 18 B-H groups, enabled the use of IR spectroscopy as a convenient analytical method for these oligomers based on the diagnostic B-H signal at 2400-2650 cm-1. The silencing activity of boron cluster-modified ASOs used at higher concentrations was similar to that of unmodified oligonucleotides. The screened ASOs, when used in low concentrations (up to 50 µM), exhibited pro-oxidative properties by inducing ROS production and an increase in mitochondrial activities in HeLa cells. In contrast, when used at higher concentrations, the ASOs exhibited anti-oxidative properties by lowering ROS species levels. In the HeLa cells (tested in the MTT assay) treated (without lipofectamine) or transfected with the screened compounds, the mitochondrial activity remained equal to the control level or only slightly changed (±30%). These findings may be useful in the design of dual-action boron cluster-modified therapeutic nucleic acids with combined antisense and anti-oxidant properties.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Boro/química , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/farmacologia , Antineoplásicos/síntese química , Terapia por Captura de Nêutron de Boro , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Receptores ErbB/genética , Células HeLa , Humanos , Estrutura Molecular , Oligonucleotídeos Antissenso/síntese química , Espécies Reativas de Oxigênio/química , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Modified nucleosides present in the wobble position of the tRNA anticodons regulate protein translation through tuning the reading of mRNA codons. Among 40 of such nucleosides, there are modified uridines containing either a sulfur atom at the C2 position and/or a substituent at the C5 position of the nucleobase ring. It is already evidenced that tRNAs with 2-thiouridines at the wobble position preferentially read NNA codons, while the reading mode of the NNG codons by R5U/R5S2U-containing anticodons is still elusive. For a series of 18 modified uridines and 2-thiouridines, we determined the pKa values and demonstrated that both modifying elements alter the electron density of the uracil ring and modulate the acidity of their N3H proton. In aqueous solutions at physiological pH the 2-thiouridines containing aminoalkyl C5-substituents are ionized in ca. 50%. The results, confirmed also by theoretical calculations, indicate that the preferential binding of the modified units bearing non-ionizable 5-substituents to guanosine in the NNG codons may obey the alternative C-G-like (Watson-Crick) mode, while binding of those bearing aminoalkyl C5-substituents (protonated under physiological conditions) and especially those with a sulfur atom at the C2 position, adopt a zwitterionic form and interact with guanosine via a 'new wobble' pattern.
Assuntos
Biossíntese de Proteínas/genética , RNA Mensageiro/genética , RNA de Transferência/genética , Uridina/genética , Sistemas de Transporte de Aminoácidos Neutros/química , Sistemas de Transporte de Aminoácidos Neutros/genética , Códon/genética , Código Genético , Guanosina/genética , Conformação de Ácido Nucleico , RNA Mensageiro/química , RNA de Transferência/química , Tiouridina/análogos & derivados , Tiouridina/química , Uridina/químicaRESUMO
In recent years, increasing attention has been focused on the posttranscriptional modifications present in transfer RNAs (tRNAs), which have been suggested to constitute another level of regulation of gene expression. The most representative among them are the 5-substituted 2-thiouridines (R5S2U), which are located in the wobble position of the anticodon and play a fundamental role in the tuning of the translation process. On the other hand, sulfur-containing biomolecules are the primary site for the attack of reactive oxygen species (ROS). We have previously demonstrated that under in vitro conditions that mimic oxidative stress in the cell, the S2U alone or bound to an RNA chain undergoes desulfuration to yield uridine and 4-pyrimidinone nucleoside (H2U) products. The reaction is pH- and concentration-dependent. In this study, for the first time, we demonstrate that the substituent at the C5 position of the 2-thiouracil ring of R5S2Us influences the desulfuration pathway, and thus the products ratio. As the substituent R changes, the amount of R5H2U increases in the order H->CH3O->CH3OC(O)CH2->HOC(O)CH2NHCH2-≈ CH3NHCH2-, and this effect is more pronounced at lower pH. The conformational analysis of the resulting R5H2U products indicates that independent of the nature of the R5 substituent, the R5H2U nucleosides predominantly adopt a C2'-endo sugar ring conformation, as opposed to the preferred C3'-endo conformation of the parent R5S2Us.
Assuntos
Pirimidinonas/metabolismo , RNA de Transferência/química , Tiouridina/análogos & derivados , Humanos , Modelos Moleculares , Estresse Oxidativo , Tiouridina/metabolismoRESUMO
2'-Deoxyzebularine and its α-anomer have been efficiently synthesized with relatively high stereoselectivity by a modified procedure of the silyl method of the N-glycosidic bond formation. An SnCl4-catalyzed condensation of silylated pyrimidin-2-one with 1-α-chloro-3,5-di-O-p-toluoyl-2-deoxy-d-ribofuranose under kinetic control condition (-33°C, 1,2-dichloroethane) led to the mixture of ß- and α-anomeric nucleosides in 3:1 ratio. Analogous condensation at +35°C (thermodynamic control conditions) provided mainly p-toluoyl protected α-2'-deoxyzebularine (α:ß=4:1), easily separated by crystallization from the anomeric mixture. The structures of both 2'-deoxyzebularine anomers were confirmed by X-ray analysis of the crystals and conformational studies in solution performed using an NMR method.