RESUMEN
Recent success of RNA therapeutics has reinvigorated interest in chemical modifications of RNA. As exemplified by the phosphorothioates, modifications of sugar-phosphate backbone have been remarkably impactful but relatively underexplored in therapeutic RNAs. The present study reports synthesis, thermal stability, and RNA interference activity of RNAs modified with thioamide linkages. Compared to the previously studied amide-modified RNA, thioamide linkages strongly destabilized a short self-complementary RNA model duplex. However, in short interfering RNAs amides and thioamides had a similar effect on duplex stability and target RNA cleavage activity and specificity. Hence, the thioamide may be added to the toolbox of chemical biologist as a useful backbone modification well tolerated by the RNA interference machinery.
Asunto(s)
ARN , Tioamidas , Tioamidas/química , Tioamidas/síntesis química , ARN/química , ARN Interferente Pequeño/química , ARN Interferente Pequeño/metabolismo , ARN Interferente Pequeño/síntesis químicaRESUMEN
Nucleic acids in the form of siRNA, antisense oligonucleotides or mRNA are currently explored as new promising modalities in the pharmaceutical industry. Particularly, the success of mRNA-vaccines against SARS-CoV-2, along with the successful development of the first sugar-modified siRNA therapeutics has inspired the field. The development of nucleic acid therapeutics requires efficient chemistry to link oligonucleotides to chemical structures that can improve stability, boost cellular uptake, or enable specific targeting. For the siRNA therapeutics currently in use, modification of the 3'-end of the oligonucleotides with triple-N-acetylgalactosamine (GalNAc)3 was shown to be of significance. This modification is currently achieved through cumbersome multistep synthesis and subsequent loading onto the solid support material. Herein, we report the development of a bifunctional click-reactive linker that allows the modification of oligonucleotides in a tandem click reaction with multiple sugars, regardless of the position within the oligonucleotide, with remarkable efficiency and in a one-pot reaction.
Asunto(s)
Química Clic , Cobre , Oligonucleótidos , Cobre/química , Oligonucleótidos/química , Oligonucleótidos/síntesis química , Catálisis , Acetilgalactosamina/química , SARS-CoV-2 , ARN Interferente Pequeño/química , ARN Interferente Pequeño/síntesis químicaRESUMEN
Cubane molecules hold great potential for medicinal chemistry applications due to their inherent stability and low toxicity. In this study, we report the synthesis of a cubane derivative phosphoramidite for the incorporation of cubane into small interfering RNAs (siRNAs). Synthetic siRNAs rely on chemical modifications to improve their pharmacokinetic profiles. However, they are still able to mediate sequence-specific gene silencing via the endogenous RNA interference pathway. We designed a library of siRNAs bearing cubane at different positions within the sense and antisense strands. All siRNAs showed excellent gene-silencing activity, with IC50 values ranging from 45.4 to 305â pM. Incorporating the cubane modification in both the sense and antisense strand led to viable duplexes with good biological activity. To the best of our knowledge, this is the first report of siRNAs bearing a cubane derivative within the backbone.
Asunto(s)
Compuestos Organofosforados/química , ARN Interferente Pequeño/síntesis química , ARN Interferente Pequeño/genética , Silenciador del Gen , Células HeLa , Humanos , Estructura Molecular , ARN Interferente Pequeño/químicaRESUMEN
GalNAc oligonucleotide conjugates demonstrate improved potency in vivo due to selective and efficient delivery to hepatocytes in the liver via receptor-mediated endocytosis. GalNAc-siRNA and GalNAc-antisense oligonucleotides are at various stages of clinical trials, while the first two drugs were already approved by FDA. Also, GalNAc conjugates are excellent tools for functional genomics and target validation in vivo. The number of GalNAc residues in a conjugate is crucial for delivery as cooperative interaction of several GalNAc residues with asialoglycoprotein receptor enhances delivery in vitro and in vivo. Here we provide a robust protocol for the synthesis of triple GalNAc CPG solid support and GalNAc phosphoramidite, synthesis and purification of RNA conjugates with multiple GalNAc residues either to 5'-end or 3'-end and siRNA duplex formation.
Asunto(s)
Acetilgalactosamina/síntesis química , Ácidos Nucleicos Inmovilizados/síntesis química , Oligodesoxirribonucleótidos/síntesis química , Compuestos Organofosforados/síntesis química , ARN Interferente Pequeño/síntesis química , Acetilgalactosamina/análogos & derivados , Proyectos de Investigación , Flujo de TrabajoRESUMEN
Nucleic acid conjugates are promising drugs for treating gene-related diseases. Conjugating specific units like lipids, cell-penetrating peptides, polymers, antibodies, and aptamers either at the 3'- or 5'-termini of a siRNA duplex molecule has resulted in a plethora of siRNA bioconjugates with improved stabilities in bloodstream and better pharmacokinetic values than unmodified siRNAs. In this sense, lipid-siRNA conjugates have attracted a remarkable interest for their potential value in facilitating cellular uptake. In this chapter, we describe a series of protocols involving the synthesis of siRNA oligonucleotides carrying either neutral or cationic lipids at the 3'- and 5'-termini. The resulting lipid-siRNA conjugates are aimed to be used as exogenous effectors for inhibiting gene expression by RNA interference. A protocol for the formulation of lipid siRNA using sonication in the presence of serum is described yielding interesting transfection properties for cell culture without the use of transfecting agents.
Asunto(s)
Técnicas de Transferencia de Gen , Lípidos/química , Oligonucleótidos/síntesis química , Compuestos Organofosforados/química , Interferencia de ARN , ARN Interferente Pequeño/síntesis química , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Células HeLa , Humanos , Ratones , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proyectos de Investigación , Sonicación , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/metabolismo , Flujo de TrabajoRESUMEN
BACKGROUND: Hepatitis B virus (HBV) infection is difficult to cure. HBV-specific immune tolerance plays a key role in HBV persistence, and enhancing cellular and humoral immunity will improve the control of HBV infection. The purpose of the study was to explore the anti-HBV and immunostimulatory effects of msiRNAs that introduce unpaired uridine bulges in the passenger strand. METHODS: msiRNAs targeting the HBV S and X genes were designed and named msiHBs and msiHBx, respectively. HepG2 cells were cotransfected with siRNA or msiRNA and the HBV replication-competent plasmid pHY106-wta or pHY106-X15. HepG2.215 cells were transfected with siRNA or msiRNA. The levels of HBsAg, HBeAg, and the cytokines TNF-α, IFN-α, IFN-ß, IL-1α, and IL-6 in the culture supernatant was detected by ELISA. The levels of intracellular HBV RNA, nuclear HBV replication intermediates, and HBV DNA in the supernatant were measured by quantitative RT-PCR and PCR. The levels of HBV replication intermediates were detected by Southern blotting. Peripheral blood mononuclear cells were transfected with siRNA or msiRNA, and the levels of secreted cytokines IFN-α and IFN-ß were detected by ELISA. The bioactivity of type I interferons in the supernatants was detected by the virus protection assay. RESULTS: msiHBx treatment led to a significant decrease in HBsAg (to a negative level) and HBV DNA (95.5%) in the supernatant and intrahepatocellular HBV replication intermediates (89.8%) in HepG2 cells with transient HBV replication and in HepG2.2.15 cells. There was no significant difference between msiHBx and siHBx in terms of the reduction in HBV proteins and HBV replication (P > 0.05). Compared with siHBx, msiHBx treatment of HepG2 cells transfected with the HBV replication-competent plasmid led to a significant increase in the levels of the antiviral cytokines TNF-α (3.3-fold), IFN-α (1.4-fold), and IFN-ß (2.5-fold) (P < 0.01), without upregulation of the proinflammatory cytokines IL-1α and IL-6. The virus protection assay results showed msiHBx-mediated type I interferons effectively protected L929 cells against ECMV infection. CONCLUSIONS: msiHBx could effectively inhibit HBV expression and replication and induce an antiviral innate immune response without proinflammatory activation. The dual RNAi and immunostimulatory activity of msiRNAs may play an important role in the control of HBV infection.
Asunto(s)
Virus de la Hepatitis B/genética , Virus de la Hepatitis B/inmunología , Hepatitis B/inmunología , Inmunidad Innata , ARN Interferente Pequeño/química , ARN Interferente Pequeño/inmunología , Uridina/metabolismo , Genes Virales , Células Hep G2 , Humanos , Inmunización , Leucocitos Mononucleares/metabolismo , ARN Interferente Pequeño/síntesis química , ARN Interferente Pequeño/genética , Transfección , Uridina/genética , Replicación ViralRESUMEN
Cancer recurrence presents a huge challenge in cancer patient management. Immune escape is a key mechanism of cancer progression and metastatic dissemination. CD25 is expressed in regulatory T (Treg) cells including tumor-infiltrating Treg cells (TI-Tregs). These cells specially activate and reinforce immune escape mechanism of cancers. The suppression of CD25/IL-2 interaction would be useful against Treg cells activation and ultimately immune escape of cancer. Here, software, web servers and databases were used, at which in silico designed small interfering RNAs (siRNAs), de novo designed peptides and virtual screened small molecules against CD25 were introduced for the prospect of eliminating cancer immune escape and obtaining successful treatment. We obtained siRNAs with low off-target effects. Further, small molecules based on the binding homology search in ligand and receptor similarity were introduced. Finally, the critical amino acids on CD25 were targeted by a de novo designed peptide with disulfide bond. Hence we introduced computational-based antagonists to lay a foundation for further in vitro and in vivo studies.
Asunto(s)
Subunidad alfa del Receptor de Interleucina-2/antagonistas & inhibidores , Interleucina-2/antagonistas & inhibidores , Péptidos/farmacología , ARN Interferente Pequeño/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Programas Informáticos , Bases de Datos Factuales , Humanos , Interleucina-2/química , Interleucina-2/inmunología , Subunidad alfa del Receptor de Interleucina-2/química , Subunidad alfa del Receptor de Interleucina-2/inmunología , Péptidos/síntesis química , Péptidos/química , ARN Interferente Pequeño/síntesis química , ARN Interferente Pequeño/química , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunologíaRESUMEN
RNA interference (RNAi) is the standard method of suppressing gene expression because of its target specificity, potency, and ability to silence the expression of virtually any gene. Using 21-mer small interfering RNA (siRNA) is the general approach for inducing RNAi, as siRNA can be easily prepared using a DNA/RNA synthesizer. Synthetic siRNA can be chemically modified to increase the potency of RNAi activity and abrogate innate immune stimulation. However, designing chemically modified siRNA requires substantial experimentation. A practical method for understanding the interaction of siRNA and RNAi-related proteins and how modifications affect RNA-protein interactions is therefore needed. Plasmid DNA (pDNA) expressing short hairpin RNA (shRNA) can also be used to induce RNAi. pDNA produces numerous shRNAs that induce RNAi with potent and longterm RNAi activity, even if only one pDNA molecule is delivered to the nucleus. However, this approach has some drawbacks with regard to its therapeutic application, such as a low pDNA transfection efficiency due to its huge molecular size and innate immune responses induced by extra genes, such as CpG motifs. To overcome these issues with RNAi inducers (siRNA and pDNA), our group developed some chemical approaches using chemically modified oligonucleotides. This article focuses on our two original approaches. The first involves the groove modification of siRNA duplexes to understand siRNA-protein interactions using 7-bromo-7-deazaadenosine and 3-bromo-3-deazaadenosine as chemical probes, while the second involves the generation of RNAi medicine using chemically modified DNA, known as an intelligent shRNA expression device (iRed).
Asunto(s)
Desarrollo de Medicamentos/métodos , Interferencia de ARN , ARN Interferente Pequeño/síntesis química , ADN , Inmunidad Innata , Oligonucleótidos/química , Dominios y Motivos de Interacción de Proteínas , ARN Interferente Pequeño/química , Tratamiento con ARN de Interferencia , Tubercidina/químicaRESUMEN
Various chemical modifications have been identified that enhance potency of small interfering RNAs (siRNAs) and that reduce off-target effects, immune stimulation, and toxicities of metabolites of these therapeutic agents. We previously described 5'-C-methyl pyrimidine nucleotides also modified at the 2' position of the sugar. Here, we describe the synthesis of 2'-position unmodified 5'-(R)- and 5'-(S)-C-methyl guanosine and evaluation of these nucleotides in the context of siRNA. The (R) isomer provided protection from 5' exonuclease and the (S) isomer provided protection from 3' exonuclease in the context of a terminally modified oligonucleotide. siRNA potency was maintained when these modifications were incorporated at the tested positions of sense and antisense strands. Moreover, the corresponding 5' triphosphates were not substrates for mitochondrial DNA polymerase. Models generated based on crystal structures of 5' and 3' exonuclease oligonucleotide complexes with 5'-(R)- and 5'-(S)-C-methyl substituents attached to the 5'- and 3'-terminal nucleotides, respectively, provided insight into the origins of the observed protections. Structural properties of 5'-(R)-C-methyl guanosine incorporated into an RNA octamer were analysed by X-ray crystallography, and the structure explains the loss in duplex thermal stability for the (R) isomer compared with the (S) isomer. Finally, the effect of 5'-C-methylation on endoribonuclease activity has been explained.
Asunto(s)
Guanosina/análogos & derivados , ARN Interferente Pequeño , Isomerismo , Modelos Moleculares , Conformación de Ácido Nucleico , ARN Interferente Pequeño/síntesis química , ARN Interferente Pequeño/químicaRESUMEN
Surface-modified cellulose nanocrystals (CNCs) were developed for efficient delivery of polymeric siRNA in cancer cells. Cationic CNCs were synthesized using the sequential process of hydrothermal desulfation and chemical modification following which, polymeric siRNA obtained using from a two-step process of rolling circle transcription and Mg2+ chelation was complexed with the modified CNCs by electrostatic interaction. The complexation efficiency was optimized for high drug loading and release in the cytoplasmic environment. The resultant nanocomplex showed significantly enhanced enzymatic stability, gene knockdown efficacy, and apoptosis-induced in vitro therapeutic effect. Our results suggest CNCs as a promising carbohydrate-based delivery platform which could be utilized for RNAi-mediated cancer therapeutics.
Asunto(s)
Antineoplásicos/farmacología , Cationes/química , Celulosa/química , Preparaciones de Acción Retardada/farmacología , Nanopartículas/química , Polímeros/síntesis química , ARN Interferente Pequeño/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Preparaciones de Acción Retardada/uso terapéutico , Liberación de Fármacos , Técnicas de Silenciamiento del Gen/métodos , Humanos , Neoplasias/tratamiento farmacológico , Polímeros/química , ARN Interferente Pequeño/síntesis química , Electricidad Estática , Propiedades de SuperficieRESUMEN
Chemical modifications of small interfering (si)RNAs are used to enhance their stability and potency, and to reduce possible off-target effects, including immunogenicity. We have earlier introduced highly effective antiviral siRNA swarms against herpes simplex virus (HSV), targeting 653 bp of the essential UL29 viral gene. Here, we report a method for enzymatic production and antiviral use of 2'-fluoro-modified siRNA swarms. Utilizing the RNA-dependent RNA polymerase from bacteriophage phi6, we produced 2'-F-siRNA swarms containing either all or a fraction of modified adenosine, cytidine or uridine residues in the antisense strand of the UL29 target. The siRNA containing modified pyrimidines demonstrated high resistance to RNase A and the antiviral potency of all the UL29-specific 2'-F-siRNA swarms was 100-fold in comparison with the unmodified counterpart, without additional cytotoxicity. Modest stimulation of innate immunity signaling, including induced expression of both type I and type III interferons, as well as interferon-stimulated gene 54, by 2'-F-cytidine and 2'-F-uridine modified siRNA swarms occurred at early time points after transfection while the 2'-F-adenosine-containing siRNA was similar to the unmodified antiviral siRNA swarm in this respect. The antiviral efficacy of the 2'-F-siRNA swarms and the elicited cellular innate responses did not correlate suggesting that innate immunity pathways do not significantly contribute to the observed enhanced antiviral activity of the modified siRNAs. The results support further applications of enzymatically produced siRNA molecules with incorporated adenosine nucleotides, carrying fluoro-modification on ribose C2' position, for further antiviral studies in vitro and in vivo.
Asunto(s)
Antivirales/farmacología , Supervivencia Celular , Herpesvirus Humano 1/efectos de los fármacos , Inmunidad Innata , ARN Interferente Pequeño/farmacología , ARN Polimerasa Dependiente del ARN/metabolismo , Adenosina/metabolismo , Bacteriófago phi 6/enzimología , Línea Celular , Línea Celular Tumoral , Citidina/metabolismo , Proteínas de Unión al ADN/antagonistas & inhibidores , Herpesvirus Humano 1/inmunología , Humanos , ARN Interferente Pequeño/síntesis química , Transfección , Uridina/metabolismo , Proteínas Virales/antagonistas & inhibidoresRESUMEN
Small interfering RNA (siRNA) has been recognized as a powerful gene-silencing tool. For therapeutic application, chemical modification is often required to improve the properties of siRNA, including its nuclease resistance, activity, off-target effects, and tissue distribution. Careful siRNA guide strand selection in the RNA-induced silencing complex (RISC) is important to increase the RNA interference (RNAi) activity as well as to reduce off-target effects. The passenger strand-mediated off-target activity was previously reduced and on-target activity was enhanced by substitution with acyclic artificial nucleic acid, namely serinol nucleic acid (SNA). In the present study, the reduction of off-target activity caused by the passenger strand was investigated by modifying siRNAs with SNA. The interactions of SNA-substituted mononucleotides, dinucleotides, and (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO)-labeled double-stranded RNA (dsRNA) with the MID domain of the Argonaute 2 (AGO2) protein, which plays a pivotal role in strand selection by accommodation of the 5'-terminus of siRNA, were comprehensively analyzed. The obtained nuclear magnetic resonance (NMR) data revealed that AGO2-MID selectively bound to the guide strand of siRNA due to the inhibitory effect of the SNA backbone located at the 5' end of the passenger strand.
Asunto(s)
Proteínas Argonautas , Interferencia de ARN , ARN Interferente Pequeño , Complejo Silenciador Inducido por ARN , Proteínas Argonautas/biosíntesis , Proteínas Argonautas/genética , Línea Celular , Humanos , Dominios Proteicos , ARN Bicatenario/genética , ARN Bicatenario/metabolismo , ARN Interferente Pequeño/síntesis química , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Complejo Silenciador Inducido por ARN/genética , Complejo Silenciador Inducido por ARN/metabolismoRESUMEN
RNA interference induced by small interfering RNA (siRNA) is a promising strategy for the treatment of various intractable diseases including cancer. Lipid nanoparticles (LNP) composed of ionizable lipids and siRNA are known as a leading siRNA delivery system. However, LNPs composed of conventional ionizable lipids will be aggregated in the physiological environment because of loss of ionization. Therefore, the inclusion of hydrophilic polymer-conjugated lipids such as polyethylene glycol (PEG)-conjugated lipid is required to improve the LNP stability. Herein, we synthesized a novel charge-reversible lipid derivative, dioleoylglycerophosphate-diethylenediamine conjugate (DOP-DEDA). The surface of LNP composed of DOP-DEDA (DOP-DEDA LNP) was constantly ionized and positively charged at pH 6.0, almost neutral at pH 7.4, and negatively charged at pH 8.0. Importantly, DOP-DEDA LNP were stable in the physiological milieu without PEG-conjugated lipid. DOP-DEDA LNP disrupted the red blood cells only under the low-pH condition in a hemolysis assay, suggesting that the interaction between DOP-DEDA LNP and biological membranes is pH-dependent. DOP-DEDA LNP encapsulating siRNA against polo-like kinase 1 (siPLK1) highly suppressed the expression of PLK1 mRNA and its protein. The cellular uptake of DOP-DEDA LNP was increased in an apolipoprotein E3 (apoE3) dose-dependent manner. In addition, DOP-DEDA LNP was taken up into cancer cells via both clathrin- and caveola-mediated endocytosis pathways. These findings indicate that LNP composed of this charge-reversible lipid should be a highly stable and potent siRNA delivery vector.
Asunto(s)
Técnicas de Transferencia de Gen , Lípidos/síntesis química , Nanopartículas/química , ARN Interferente Pequeño/síntesis química , Línea Celular , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Lípidos/administración & dosificación , Nanopartículas/administración & dosificación , ARN Interferente Pequeño/administración & dosificaciónRESUMEN
We report on the synthesis of siRNAs containing both 2'-5'- and 3'-5'-internucleotide linkages and their effects on siRNA structure, function, and interaction with RNAi proteins. Screening of these siRNAs against their corresponding mRNA targets showed that 2'-5' linkages were well tolerated in the sense strand, but only at a few positions in the antisense strand. Extensive modification of the antisense strand minimally affected 5'-phosphorylation of the siRNA by kinases, however, it negatively affected siRNA loading into human AGO2. Modelling and molecular dynamics simulations were fully consistent with these findings. Furthermore, our studies indicated that the presence of a single 5'p-rN1-(2'-5')-N2 unit in the antisense strand does not alter the 'clover leaf' bend and sugar puckers that are critical for anchoring the 5'-phosphate to Ago 2 MID domain. Importantly, 2'-5'-linkages had the added benefit of abrogating immune-stimulatory activity of siRNAs. Together, these results demonstrate that 2'-5'/3'-5'-modified siRNAs, when properly designed, can offer an efficient new class of siRNAs with diminished immune-stimulatory responses.
Asunto(s)
Interferencia de ARN , ARN Interferente Pequeño/química , Proteínas Argonautas/metabolismo , Conformación de Carbohidratos , Células HeLa , Humanos , Luciferasas de Luciérnaga/genética , Simulación de Dinámica Molecular , Conformación de Ácido Nucleico , ARN Interferente Pequeño/síntesis química , ARN Interferente Pequeño/inmunología , Proteína p53 Supresora de Tumor/genéticaRESUMEN
We designed and synthesized caged siRNAs with photolabile linker and single cRGD peptide modifications for the photoregulation of gene expression. Photolabile linker and cRGD were inserted at 5' terminus of siRNAs to obtain cRGD-modified caged siRNAs. All these caged siRNAs could be activated through light activation to release the native siRNAs and further achieve the photoregulation of gene silencing of two exogenous reporter genes (firefly luciferase and green fluorescent protein, GFP) and one endogenous gene (the mitosis motor protein, Eg5). The intracellular distribution and cellular uptake pathways of these caged siRNAs were also investigated. Tumor-bearing mice were further used to demonstrate the photoregulation of gene silencing with cRGD-modified caged siRNAs in vivo. Overall, the data support the use of this new generation of caged siRNAs in cancer therapy.
Asunto(s)
Péptidos Cíclicos/química , Interferencia de ARN , ARN Interferente Pequeño/química , Línea Celular Tumoral , Técnicas de Química Sintética , Proteínas Fluorescentes Verdes/genética , Humanos , Péptidos Cíclicos/síntesis química , Procesos Fotoquímicos , ARN Interferente Pequeño/síntesis química , ARN Interferente Pequeño/genéticaRESUMEN
Nucleic acids templated on gold (Au) surfaces have led to a wide range of functional materials ranging from microarrays, sensors and probes in addition to drug delivery and treatment. In this application, we describe a simple and novel method for templating amino-functionalized RNA onto Au surfaces and their self-assembly into small, discrete nanoparticles. In our method, sample hybridization with a complementary RNA strand with and without a fatty acid (palmitamide) appendage produced functionalized double-stranded RNA on the Au surface. The resulting Au-functionalized RNA particles were found to be stable under reducing conditions according to UV-Vis spectroscopy. Sample characterization by DLS and TEM confirmed self-assembly into primarily small (â¼10-40 nm) spherical shaped nanoparticles expected to be amenable to cell biology. However, fluorescence emission (λexc: 350 nm, λem: 650 nm) revealed radiative properties which limited cell uptake detection. Introduction of FITC within the Au-functionalized RNA particles produced a bifunctional probe, in which FITC fluorescence emission (λexc: 494 nm, λem: 522 nm) facilitated cell uptake detection, in a time-dependent manner. The dual encapsulation-release profiles of the FITC-labeled Au-functionalized RNA particles were validated by time-dependent UV-Vis spectroscopy and spectrofluorimetry. These experiments respectively indicated an increase in FITC absorption (λabs: 494 nm) and fluorescence emission (λem: 522 nm) with increased sample incubation times, under physiological conditions. The release of Au-functionalized siRNA particles in prostate cancer (PC-3) cells resulted in concomitant knockdown of GRP75, which led to detectable levels of cell death in the absence of a transfection vector. Thus, the formulation of stable, small and discrete Au-functionalized RNA nanoparticles may prove to be valuable bifunctional probes in the theranostic study of cancer cells.
Asunto(s)
Antineoplásicos/farmacología , Oro/farmacología , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Proteínas Mitocondriales/antagonistas & inhibidores , Nanopartículas/química , Neoplasias de la Próstata/tratamiento farmacológico , ARN Interferente Pequeño/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Oro/química , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Masculino , Proteínas Mitocondriales/metabolismo , Estructura Molecular , Células PC-3 , Tamaño de la Partícula , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , ARN Interferente Pequeño/síntesis química , ARN Interferente Pequeño/química , Propiedades de Superficie , Nanomedicina TeranósticaRESUMEN
Herein, polymerization-induced electrostatic self-assembly (PIESA) is conducted to mediate the self-assembly behavior of short interfering RNA (siRNA) for the first time. In PIESA, siRNA not only formed a simple electrostatic polyplex with positively charged polycations, but also facilitated directed self-assembly due to the molecular rigidity of siRNA, leading to appealing nanotubes.
Asunto(s)
ARN Interferente Pequeño/síntesis química , Estructura Molecular , Tamaño de la Partícula , Polimerizacion , ARN Interferente Pequeño/química , Electricidad Estática , Propiedades de SuperficieRESUMEN
Influenza is a worldwide public health problem. Annually, this infection affects up to 15% of the world population; and about half a million people die from this disease every year. Moreover, influenza A and B viruses tend to garner most of the attention, as these types are a major cause of the epidemics and pandemics. Although the influenza virus primarily affects the respiratory tract, it may also affect the cardiovascular and central nervous systems. Several antiviral drugs, that target various stages of viral reproduction, have been considered effective for the treatment and prevention of influenza, but some virus strains become resistant to these medications. Thus, new strategies and techniques should be developed to overcome the antiviral drug resistance. Recent studies suggest that new drugs based on RNA interference (RNAi) appear to be a promising therapeutic approach that regulates the activity of viral or cellular genes. As it is known, the RNAi is a eukaryotic gene regulatory mechanism that can be triggered by a foreign double-stranded RNA (dsRNA) and results in the cleavage of the target messenger RNA (mRNA). This review discusses the prospects, advantages, and disadvantages of using RNAi in carrying out a specific treatment for influenza infection. However, some viruses confer resistance to small interfering RNAs (siRNA) targeting viral genes. This problem can significantly reduce the effectiveness of RNAi. Therefore, applying siRNAs targeting host cell factors required for influenza virus reproduction can be a way to overcome the antiviral drug resistance.
Asunto(s)
Antivirales/farmacocinética , Virus de la Influenza A/efectos de los fármacos , Gripe Humana/tratamiento farmacológico , ARN Interferente Pequeño/farmacología , Antivirales/síntesis química , Interacciones Huésped-Patógeno , Humanos , Virus de la Influenza A/genética , Virus de la Influenza A/patogenicidad , Gripe Humana/genética , Gripe Humana/virología , Interferencia de ARN , ARN Bicatenario/síntesis química , ARN Bicatenario/farmacología , ARN Interferente Pequeño/síntesis química , Drogas Sintéticas/química , Drogas Sintéticas/farmacología , Replicación Viral/efectos de los fármacosRESUMEN
One of the major hurdles in RNAi research has been the development of safe and effective delivery systems for siRNAs. Although various chemical modifications have been proposed to improve their pharmacokinetic behaviour, their delivery to target cells and tissues presents many challenges. In this work, we implemented a receptor-targeting strategy to selectively deliver siRNAs to cancer cells using folic acid as a ligand. Folic acid is capable of binding to cell-surface folate receptors with high affinity. These receptors have become important molecular targets for cancer research as they are overexpressed in numerous cancers despite being expressed at low levels in normal tissues. Employing a post-column copper-catalyzed alkyne-azide cycloaddition (CuAAC), we report the synthesis of siRNAs bearing folic acid modifications at different positions within the sense strand. In the absence of a transfection carrier, these siRNAs were selectively taken up by cancer cells expressing folate receptors. We show that centrally modified folic acid-siRNAs display enhanced gene-silencing activity against an exogenous gene target (â¼80% knockdown after 0.75 µM treatment) and low cytotoxicity. In addition, these siRNAs achieved potent dose-dependent knockdown of endogenous Bcl-2, an important anti-apoptotic gene.
Asunto(s)
Ácido Fólico/química , Silenciador del Gen , Marcación de Gen/métodos , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Interferente Pequeño/genética , Carbonatos/química , Supervivencia Celular , Receptores de Folato Anclados a GPI/genética , Receptores de Folato Anclados a GPI/metabolismo , Ácido Fólico/metabolismo , Genes Reporteros , Células HT29 , Células HeLa , Humanos , Luciferasas/antagonistas & inhibidores , Luciferasas/genética , Luciferasas/metabolismo , Pargilina/análogos & derivados , Pargilina/química , Potasio/química , Unión Proteica , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Interferente Pequeño/síntesis química , TransfecciónRESUMEN
This study investigated the synthesis and properties of 4'-C-guanidinomethyl-2'-O-methyluridine and RNAs containing the analog. Thermal and thermodynamic stabilities of double-stranded RNAs (dsRNAs) containing the nucleoside analog were examined. It was found that although the analog decreased the thermal and thermodynamic stabilities of dsRNA, it had base-discrimination ability. The 4'-C-guanidinomethyl modification increased stability of RNAs in a buffer containing serum. Furthermore, small interference RNAs incorporating one analog at the passenger strand still preserved their RNA interference activities. It was suggested that the 4'-guanidinomethyl modification significantly improved cell membrane permeability of RNA. Thus, 4'-C-guanidinomethyl-2'-O-methyl analogs may be useful in improving the properties of therapeutic siRNA molecules.