RESUMEN
Synthetic siRNA molecules without chemical modifications are easily degraded in the body, and 2'-O-modifications are frequently introduced to enhance stability. However, such chemical modifications tend to impact the gene knockdown potency of siRNA negatively. To circumvent this problem, we previously developed a prodrug-type siRNA bearing 2'-O-methyldithiomethyl (MDTM) groups, which can be converted into unmodified siRNA under the reductive environment in cells. In this study, we developed a nuclease-resistant prodrug-type 2'-O-MDTM siRNA for deployment in future animal experiments. To rationally design siRNA modified with a minimal number of 2'-O-MDTM nucleotide residues, we identified the sites susceptible to nuclease digestion and tolerant to 2'-O-methyl (2'-OMe) modification in the antisense strand of apolipoprotein B-targeted siRNA. Subsequently, we optimized the positions where the 2'-OMe and 2'-O-MDTM groups should be incorporated. siRNA bearing the 2'-O-MDTM and 2'-OMe groups at their respective optimized positions exhibited efficient knockdown potency in vitro and enhanced stability in serum.
Asunto(s)
Profármacos , ARN Interferente Pequeño/química , Profármacos/farmacología , Profármacos/química , Silenciador del Gen , Apolipoproteínas B/genética , Apolipoproteínas B/metabolismoRESUMEN
Various chemical modifications have been developed to create new antisense oligonucleotides (AONs) for clinical applications. Our previously designed prodrug-type phosphotriester-modified oligonucleotide with cyclic disulfides (cyclic SS PTE ON) can be converted into unmodified ON in an intracellular-mimetic reducing environment. However, the conversion rate of the cyclic SS PTE ON was very low, and the AON with cyclic SS PTE modifications showed much weaker antisense activity than corresponding to the fully phosphorothioate-modified AON. In this study, we synthesized several types of PTE ONs containing linear disulfides (linear SS PTE ONs) and evaluated their conversion rates under reducing conditions. From the results, the structural requirements for the conversion of the synthesized linear SS PTE ONs were elucidated. Linear SS PTE ON with promising promoieties showed a nuclease resistance up to 4.8-fold compared to unmodified ON and a cellular uptake by endocytosis without any transfection reagent. In addition, although the knockdown activity of the linear SS PTE gapmer AON is weaker than that of the fully phosphorothioate-modified gapmer AON, the knockdown activity is slightly stronger than that of the cyclic SS PTE gapmer AON. These results suggest that the conversion rates may be related to the expression of the antisense activity.
Asunto(s)
Oligonucleótidos , Profármacos , Oligonucleótidos/química , Profármacos/farmacología , Profármacos/química , Disulfuros , Oligonucleótidos Antisentido/química , TransfecciónRESUMEN
MAP(Aib)-cRGD, which is a conjugate of an α-aminoisobutyric acid (Aib)-containing amphipathic helical peptide [MAP(Aib)] with a αv ß3 integrin binding ligand, cRGD, at the C-terminus of the helical peptide, has been developed for siRNA delivery into cells. In this work, we synthesized three peptides containing 19 (PI), 18 (PII), and 17 (PIII) amino acid residues in the helical peptide, which lack Aib, Leu-Aib, and Lys-Leu-Aib residues present in the C-terminus of the helical peptide of the parent MAP(Aib)-cRGD, respectively. MAP(Aib)-cRGD showed the siRNA delivery into cells and the RNAi effect both in the presence and in the absence of serum in reaction media. In contrast, PI delivered siRNA into cells, and this was followed by the RNAi effect in only serum-free reaction media. On the other hand, siRNA delivery was abolished by the further reduction of the number of residues (PII and PIII) in the C-terminus. Our data indicate that the Aib-containing helical part requires 20 residues in the conjugation of the helical peptide with cRGD for the construction of carrier for siRNA delivery into cells.
Asunto(s)
Aminoácidos , Péptidos , Aminoácidos/metabolismo , Péptidos/química , Conformación Proteica , Interferencia de ARN , ARN Interferente Pequeño/metabolismoRESUMEN
Replacement therapy with tumor suppressive microRNA (TS-miRNA) might be the next-generation oligonucleotide therapy; however, a novel drug delivery system (DDS) is required. Recently, we developed the cell-penetrating peptide, model amphipathic peptide with α-aminoisobutyric acid (MAP(Aib)), as a carrier for oligonucleotide delivery to cells. In this study, we examined whether a modified MAP(Aib) analogue, MAP(Aib)-cRGD, could be a DDS for TS-miRNA replacement therapy. MIR145-5p, a representative TS-miRNA especially in colorectal cancer, was selected. The MAP(Aib)-cRGD dose was adjusted for MIR145-5p delivery to cells using peripheral blood mononuclear cells and degradation analysis. AlexaFluor488-labeled MIR145-5p incorporation into cells and negative regulation of MIR145-5p-targeting genes demonstrated MAP(Aib)-cRGD's functionality as a miRNA DDS. Treating MIR145-5p with MAP(Aib)-cRGD also revealed various anticancer effects, such as cell viability, invasion inhibition, and apoptosis induction in WiDr cells. Altogether, these findings suggest that MAP(Aib)-cRGD could be a DDS for TS-miRNA replacement therapy, but in vivo investigations are required.
Asunto(s)
Ácidos Aminoisobutíricos/química , Péptidos de Penetración Celular/química , MicroARNs/química , Péptidos Cíclicos/química , Línea Celular , Línea Celular Tumoral , Sistemas de Liberación de Medicamentos/métodos , Humanos , Leucocitos Mononucleares/efectos de los fármacos , MicroARNs/farmacología , Oligonucleótidos/químicaRESUMEN
The cRGD-conjugated Aib-containing amphipathic helical peptide, MAP(Aib) derivative (PI), has been reported to be a useful carrier for siRNA delivery into cells. We have conducted a series of structure-activity relationship studies of the influence of the balance between hydrophobicity and basicity on the amphipathicity of PI, and synthesized peptides having a larger number of Lys residues than PI. Increasing the number of basic residues in the amphipathic helix suppressed the ability to deliver siRNA into cells. It was concluded that the balance between hydrophobicity and basicity in the PI helix was important for siRNA delivery into cells. Furthermore, the siRNA delivering ability of PI was specific to cancer cells, such as A549, U-87 MG, and WiDr cells, and was low in normal cells, namely, NIH3T3 cells. Next, we examined the potential of PI as a carrier for the delivery of microRNA-133b (miR-133b), which is known to be an anti-oncomiR. PI enhanced the delivery of miR-133b into WiDr cells, which resulted in the suppression of endogenous protein expression.
Asunto(s)
Lisina/química , Péptidos/química , ARN/química , Secuencia de Aminoácidos , Humanos , Relación Estructura-ActividadRESUMEN
Small interfering RNAs (siRNAs) are an active agent to induce gene silencing and they have been studied for becoming a biological and therapeutic tool. Various 2'-O-modified RNAs have been extensively studied to improve the nuclease resistance. However, the 2'-O-modified siRNA activities were often decreased by modification, since the bulky 2'-O-modifications inhibit to form a RNA-induced silencing complex (RISC). We developed novel prodrug-type 2'-O-methyldithiomethyl (MDTM) siRNA, which is converted into natural siRNA in an intracellular reducing environment. Prodrug-type 2'-O-MDTM siRNAs modified at the 5'-end side including 5'-end nucleotide and the seed region of the antisense strand exhibited much stronger gene silencing effect than non-prodrug-type 2'-O-methyl (2'-O-Me) siRNAs. Furthermore, the resistances for nuclease digestion of siRNAs were actually enhanced by 2'-O-MDTM modifications. Our results indicate that 2'-O-MDTM modifications improve the stability of siRNA in serum and they are able to be introduced at any positions of siRNA.
Asunto(s)
Silenciador del Gen/efectos de los fármacos , Profármacos/farmacología , ARN Interferente Pequeño/efectos de los fármacos , Humanos , Profármacos/química , ARN Interferente Pequeño/genéticaRESUMEN
We previously reported that reducing-environment-responsive prodrug-type small interfering RNA (siRNA) bearing 2'-O-methyldithiomethyl (2'-O-MDTM) uridine exhibits efficient knockdown activity and nuclease resistance. In this report, we describe the preparation of 2'-O-MDTM oligonucleotides modified not only at uridine but also at adenosine, guanosine and cytidine residues by post-synthetic modification. Precursor oligonucleotides bearing 2'-O-(2,4,6-trimethoxybenzylthiomethyl) (2'-O-TMBTM) adenosine, guanosine, and cytidine were reacted with dimethyl(methylthio)sulfonium tetrafluoroborate to form 2'-O-MDTM oligonucleotides in the same manner as the oligonucleotide bearing 2'-O-TMBTM uridine. Furthermore, the oligonucleotides bearing 2'-O-MDTM adenosine, guanosine, and cytidine were efficiently converted into corresponding natural 2'-hydroxy oligonucleotides under the cytosol-mimetic reducing condition.
Asunto(s)
Productos Biológicos/química , Nucleósidos/química , Oligonucleótidos/síntesis química , Profármacos/síntesis química , Estructura Molecular , Oligonucleótidos/química , Profármacos/químicaRESUMEN
We synthesized prodrug-type phosphotriester (PTE) oligonucleotides containing the six-membered cyclic disulfide moiety by using phosphoramidite chemistry. Prodrug-type oligonucleotides named "Reducing-Environment-Dependent Uncatalyzed Chemical Transforming (REDUCT) PTE oligonucleotides" were converted into natural oligonucleotides under cytosol-mimetic reductive condition. Furthermore, the REDUCT PTE oligonucleotides were robust to nuclease digestion and exhibited good cell membrane permeability.
Asunto(s)
Endonucleasas/metabolismo , Oligonucleótidos/química , Profármacos/síntesis química , Células A549 , Secuencia de Bases , Permeabilidad de la Membrana Celular/efectos de los fármacos , Disulfuros/química , Resistencia a Medicamentos , Colorantes Fluorescentes/química , Silenciador del Gen/efectos de los fármacos , Genes Reporteros , Humanos , Microscopía Confocal , Oligonucleótidos/síntesis química , Oligonucleótidos/farmacología , Compuestos Organofosforados/química , Profármacos/química , Profármacos/farmacologíaRESUMEN
The conjugation of Aib-containing amphipathic helical peptide with cyclo(-Arg-Gly-Asp-d-Phe-Cys-) (cRGDfC) at the C-terminus of the helix peptide (PI) has been reported to be useful for constructing a carrier for targeted siRNA delivery into cells. In order to explore structure-activity relationships for the development of potential carriers for siRNA delivery, we synthesized conjugates of Aib-containing amphipathic helical peptide with cRGDfC at the N-terminus (PII) and both the N- and C-termini (PIII) of the helical peptide. Furthermore, to examine the influence of PI helical chain length on siRNA delivery, truncated peptides containing 16 (PIV), 12 (PV), and 8 (PVI) amino acid residues at the N-terminus of the helical chain were synthesized. PII and PIII, as well as PI, could deliver anti-luciferase siRNA into cells to induce the knockdown of luciferase stably expressed in cells. In contrast, all of the truncated peptides were unlikely to transport siRNA into cells.
Asunto(s)
Ácidos Aminoisobutíricos/química , Portadores de Fármacos/química , Oligopéptidos/química , Péptidos Cíclicos/química , ARN Interferente Pequeño/metabolismo , Células A549 , Secuencia de Aminoácidos , Dicroismo Circular , Colorantes Fluorescentes/química , Fluorobencenos/química , Humanos , Microscopía Fluorescente , Interferencia de ARN , ARN Interferente Pequeño/química , Relación Estructura-Actividad , Transfección/métodosRESUMEN
RNAs bearing various 2'-modifications have been synthesized in an effort to improve nuclease resistance. However, the gene silencing activity of small interfering RNAs (siRNAs) has been decreased or sometimes completely suppressed by the chemical modifications. We previously developed a post-synthetic approach for the synthesis of 2'-O-methyldithiomethyl-modified RNA, which can be converted into unmodified RNA under reducing conditions, and named it Reducing-Environment-Dependent Uncatalyzed Chemical Transforming RNA (REDUCT RNA). Here, the gene silencing activity of REDUCT siRNA bearing 2'-O-methyldithiomethyl groups was evaluated. REDUCT siRNA showed more effective gene silencing than unmodified siRNA regardless of the modification site. This result suggests that REDUCT siRNA is converted into unmodified siRNA inside cells as a prodrug-type siRNA.
Asunto(s)
Silenciador del Gen , Profármacos/química , ARN Interferente Pequeño/química , Secuencia de Bases , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión , Genes Reporteros , Humanos , Profármacos/metabolismo , ARN Interferente Pequeño/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
To achieve the targeted delivery of siRNA, five conjugates of Aib-containing amphipathic helical peptides with mono-, di-, and trivalent cRGDfC [cyclo(-Arg-Gly-Asp-d-Phe-Cys-)], which is known to bind to αVß3 integrin, at several positions of the amphipathic helical peptide were designed and synthesized. Among the five conjugates, the monovalent cRGDfC conjugating at position 20 of the amino acid sequence of the helical peptide through the formation of a disulfide bond (PI) and the divalent cRGDfC conjugating at positions 2 and 14 of the amino acid sequence of the helical peptide through the formation of disulfide bonds (PIII) significantly enhanced the delivery of fluorescence-labeled siRNA into A549 cells as the peptide/siRNA complex formed by electrostatic interaction. The cellular uptake of the PI/siRNA complex was mediated by both endocytic and non-endocytic pathways, whereas that of the PIII/siRNA complex was enabled by endocytosis. Furthermore, the cellular uptake of the PI/siRNA complex might involve specific interactions of the RGD group with the αVß3 integrin receptor. Next, the RNAi effect of the peptide/siRNA complex on luciferase expression in A549-Luc cells was examined. Luciferase expression was significantly decreased in the presence of the complex at the concentration of 1.0µM PI/10nM siRNA. In contrast, the PIII/siRNA complex did not show the RNAi effect under the same conditions. However, extending the incubation time led to the suppression of the luciferase expression in the presence of the PIII/siRNA complex. Considering that the cellular uptake of the PIII/siRNA complex is mediated by the endocytic pathway, the release of siRNA from the endosome into the cytosol might require a long time. We present herein a useful and unique tool for the delivery of siRNA.
Asunto(s)
Péptidos Cíclicos/química , ARN Interferente Pequeño/administración & dosificación , Células A549 , Secuencia de Aminoácidos , Diseño de Fármacos , Humanos , Péptidos Cíclicos/metabolismo , ARN Interferente Pequeño/metabolismo , Fracciones Subcelulares/metabolismoRESUMEN
A spermine-conjugated ethyl phosphotriester oligonucleotide was obtained by solid-phase synthesis based on phosphoramidite chemistry. The ethyl phosphotriester linkage was robust to exonuclease digestion and stable in fetal bovine serum. Cell membrane permeability of the spermine-conjugated ethyl phosphotriester oligonucleotide was studied by fluorescence experiments. The effective cell penetrating potency of the spermine-conjugated ethyl phosphotriester oligonucleotide was determined by confocal laser scanning microscopy and measurement of intracellular fluorescence intensity.
Asunto(s)
Permeabilidad de la Membrana Celular , Oligonucleótidos/química , Compuestos Organofosforados/química , Espermina/química , Animales , Bovinos , Línea Celular Tumoral , Humanos , Oligonucleótidos/síntesis química , Oligonucleótidos/metabolismo , Oligonucleótidos/farmacocinética , Compuestos Organofosforados/síntesis química , Compuestos Organofosforados/metabolismo , Compuestos Organofosforados/farmacocinética , Albúmina Sérica Bovina/metabolismo , Técnicas de Síntesis en Fase Sólida , Espermina/síntesis química , Espermina/metabolismo , Espermina/farmacocinéticaRESUMEN
α-Aminoisobutyric acid (Aib)-containing peptide analogs derived from TV-XIIa, a cell-penetrating peptide (CPP), were synthesized to explore structure-activity relationships. The replacement of Aib at position 1, 5, or 9 in the TV-XIIa amino acid sequence with alanine (Ala) suppressed the cellular uptake,whereas the simultaneous substitution of the two proline (Pro) residues at positions 6 and 10 with Aib(P-IV) considerably increased the cellular uptake. In order to explore the potential use of the Aib-containing peptide analogs for the cellular delivery of oligonucleotides (ODNs), we synthesized a covalent conjugate (P-IV-AON) of a 15-mer antisense ODN, which is complementary to luciferase gene, with P-IV, and the antisense effect of the P-IV-AON conjugate on luciferase expression in A549 cells was examined. Luciferase expression was decreased in the presence of the conjugate upon treatment with the reaction buffer at the concentrations of 5 and 10 µM.
Asunto(s)
Ácidos Aminoisobutíricos/química , Péptidos de Penetración Celular/metabolismo , Oligonucleótidos/metabolismo , Péptidos/metabolismo , Secuencia de Aminoácidos , Línea Celular Tumoral , Péptidos de Penetración Celular/síntesis química , Péptidos de Penetración Celular/química , Genes Reporteros , Humanos , Luciferasas/antagonistas & inhibidores , Luciferasas/genética , Luciferasas/metabolismo , Oligonucleótidos/química , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/metabolismo , Péptidos/síntesis química , Péptidos/química , Relación Estructura-ActividadRESUMEN
This unit describes the synthesis of 2'-O-methyldithiomethyluridine-containing oligonucleotides, which can be deprotected to yield the parental oligoribonucleotides under high concentrations of glutathione similar in cytoplasm. The 2'-O-methyldithiomethyl group is sensitive to reductive conditions, so that it is incompatible to 3'-O-phosphoramidite modification in nucleosides. Thus, a novel post-synthetic approach to obtain 2'-O-methyldithiomethyluridine-containing oligonucleotides was developed, in which 2'-O-(2,4,6-trimethoxybenzylthiomethyl)uridine-modified oligonucleotides are readily converted by treatment with dimethyl(methylthio)sulfonium tetrafluoroborate to the 2'-O-methyldithiomethyluridine-modified oligonucleotides. The 2'-O-methyldithiomethyluridine-modified oligonucleotides are readily and cleanly converted to the parental oligonucleotides under high thiol conditions, such as 10 mM glutathione and dithiothreitol.