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Synthesis and in Vitro Characterization of an ABC …

Synthesis and in vitro Inhibition Properties of siRNA Conjugates Carrying Acridine and Quindoline Moieties

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functional in vitro validation, siRNA encapsulation, ..

The ability to specifically down-regulate gene expression using the RNAi pathway in mammalian cells has tremendous potential in therapy and in basic science. However, delivery systems capable of efficient and biocompatible delivery of siRNA to target cells are not yet satisfactory. Here, we report the synthesis and in vitro characterization of ABC triblock copolymers that self-assemble with siRNA based on electrostatics and with each other by hydrophobic interactions. The ABC triblock copolymer is based on poly(ethylene glycol) (PEG), poly(propylene sulfide) (PPS), and a positively charged peptide (PEG−PPS−peptide). The diblock copolymer PEG45−PPS5,10 was synthesized using anionic polymerization of propylene sulfide upon a PEG macroinitiator, and the peptide domain was coupled to the PPS terminus using a disulfide exchange reaction with an N-terminal cysteine residue on the peptide. The peptides were designed to interact electrostatically with siRNA, selecting the TAT peptide domain of HIV (RKKRRQRRR) and an oligolysine (Lys9). The resulting triblock copolymers were able to self-assemble with siRNA as demonstrated by dynamic light scattering and gel electrophoresis. Complex size was found to be dependent on the amount of polymer used (charge ratio) and the length of the hydrophobic PPS block, achieving sizes ranging from 171 nm to 601 nm. Cell internalization and gene expression down-regulation studies showed that the triblock copolymers are able to transport siRNA inside the cell and mediate gene expression down-regulation, with the amount of internalization and gene transfer affected by charge ratio, PPS length, and the presence of serum. The proposed triblock was able to mediate gene expression down-regulation of GAPDH, achieving up to 90.5% ± 0.02% down-regulation.

Synthesis and in Vitro Characterization of an ABC Triblock Copolymer for siRNA ..

At Altogen Labs, our scientists apply advanced biotechnological techniques to develop new RNAi products and RNAi services. We are committed to offer the highest quality RNAi services for scientific research at the lowest price. Altogen Labs’ life science research services include a wide variety of studies, and , stable , and complete A-to-Z gene silencing services including gene targeting, siRNA synthesis, functional in vitro validation, siRNA encapsulation, in vivo siRNA protection and tissue-targeted delivery (mouse and rat models). We also perform RNAi library-based high-throughput screening, which expedites finding corresponding genes for particular phenotypes, accelerating the process of scientific discovery.

(custom siRNA synthesis using patented ..

21. Walker GF, Fella C, Pelisek J, Fahrmeir J, Boeckle S, Ogris M. . Toward synthetic viruses: endosomal pH-triggered deshielding of targeted polyplexes greatly enhances gene transfer in vitro and in vivo. 2005;11:418-25

Methoxy-terminated DMPE-PEG, DPPE-PEG, DSPE-PEG, DOPE-PEG, and ceramide-PEG were obtained from Avanti Polar Lipids, and methoxy-terminated DMG-PEG, DPG-PEG, and DSG-PEG were from NOF American. The PEG molecular weight is 2 kDa. Ethylenediamine core-poly (amidoamine) (PAMAM) generation 0 dendrimer (G0), D-Luciferin, and bovine serum albumin were purchased from Sigma-Aldrich. Ester-terminated poly(D,L-lactide--glycolide) (PLGA, viscosity of 0.26-0.54 dL/g) was obtained from Durect Corporation. Iodine solution was obtained from Alfa Aesar. Transfection agent lipofectamine2000 (Lipo2000) was purchased from Invitrogen. Steady-Glo luciferase assay system was purchased from Promega. Luciferase siRNA (siLuc) and fluorophore-labeled siRNA (DY547-siRNA, DY647-siRNA, and DY677-siRNA) were acquired from Dharmacon. The siLuc sequence is: 5'-CUU ACG CUG AGU ACU UCG AdTdT-3' (sense) and 5'-UCG AAG UAC UCA GCG UAA GdTdT-3' (antisense). DY547 and DY647 were labeled at the 5'-end of the sense strand of siLuc. DY677 was labeled at the 5'-end of both the sense and antisense strands of siLuc.

Method for the in vitro synthesis of short double …

Different from the stimuli-triggered de-PEGylation strategies, our alternative approach avoids the design of TME stimuli-responsive chemistry, which may introduce additional complexities in the synthesis and scale-up of therapeutic NPs. Notably, the self-assembly of lipid-PEGs on the hybrid NP surface is also very robust, thus offering a convenient approach for high-throughput screening of lipid-PEG molecules. The optimization of other factors, such as the PEG length and the combination of multiple different lipid-PEGs, may further lead to better efficacy of the hybrid RNAi NPs. By enabling effective silencing of specific genes in tumor tissues following systemic administration, this NP platform could serve as a robust toolkit for fundamental cancer research and rapid validation of potential therapeutic targets in cancer pathogenesis, in particular those considered as 'undruggable'. In addition, the hybrid siRNA NPs can also simultaneously encapsulate small molecular drugs (e.g., taxanes and cisplatin prodrugs) within the PLGA polymer core[-], and the co-delivery of siRNA and drug combinations is expected to have a synergistic anti-tumor effect[, ]. In summary, we successfully demonstrated that surface lipid-PEG dissociation controls the and performance of the lipid-polymer hybrid siRNA NPs, and we expect this robust NP platform to be highly useful in cancer research and treatment.

PURExpress® is a reconstituted protein synthesis system based on the PUREsystem™ (Shimizu et al., 2001) where all necessary components needed for in vitro transcription and translation are purified from E. coli.

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  • Gene Synthesis service-USD0.19/bp-Peptide Synthesis

    (157b) Synthesis and in Vitro Characterization of a Triblock Copolymer Carrier for Sirna

  • Adipose Tissue Macrophage-Derived ..

    01/09/2003 · Simple and rapid synthesis of siRNA derived from in vitro transcribed shRNA

  • In vitro production and testing of sgRNA (single guide …

    Functional Genomics & RNAi

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In Vitro Transcription and Screening Kits for sgRNA

AB - We report the conjugation of the natural lipid squalene (SQ) with a small interfering RNA (siRNA), against the junction oncogene RET/PTC1, usually found in papillary thyroid carcinoma (PTC). The acyclic isoprenoid chain of squalene has been covalently coupled with siRNA RET/PTC1 at the 3′-terminus of the sense strand via maleimide-sulfhydryl chemistry. Remarkably, the linkage of siRNA RET/PTC1 to squalene led to an amphiphilic molecule that self-organized in H2O as siRNA-SQ RET/PTC1 nanoparticles (NPs). The siRNA-SQ RET/PTC1 NPs, stable in H2O, were used for biological studies. In vitro, they did not show any cytotoxicity. Interestingly, in vivo, on a mice xenografted RET/PTC1 experimental model, RET/PTC1-SQ NPs were found to inhibit tumor growth and RET/PTC1 oncogene and oncoprotein expression after 2.5 mg/kg cumulative dose intravenous injections. In conclusion, these results showed that the "squalenoylation" offers a new noncationic plate-form for the siRNA delivery.

Pharmaceutical & Medicinal Chemistry

Conjugation of small interfering RNA (siRNA) to an asialoglycoprotein receptor ligand derived from -acetylgalactosamine (GalNAc) facilitates targeted delivery of the siRNA to hepatocytes and . The ligands derived from GalNAc are compatible with solid-phase oligonucleotide synthesis and deprotection conditions, with synthesis yields comparable to those of standard oligonucleotides. Subcutaneous (SC) administration of siRNA–GalNAc conjugates resulted in robust RNAi-mediated gene silencing in liver. Refinement of the siRNA chemistry achieved a 5-fold improvement in efficacy over the parent design with a median effective dose (ED50) of 1 mg/kg following a single dose. This enabled the SC administration of siRNA–GalNAc conjugates at therapeutically relevant doses and, importantly, at dose volumes of ≤1 mL. Chronic weekly dosing resulted in sustained dose-dependent gene silencing for over 9 months with no adverse effects in rodents. The optimally chemically modified siRNA–GalNAc conjugates are hepatotropic and long-acting and have the potential to treat a wide range of diseases involving liver-expressed genes.

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N2 - We report the conjugation of the natural lipid squalene (SQ) with a small interfering RNA (siRNA), against the junction oncogene RET/PTC1, usually found in papillary thyroid carcinoma (PTC). The acyclic isoprenoid chain of squalene has been covalently coupled with siRNA RET/PTC1 at the 3′-terminus of the sense strand via maleimide-sulfhydryl chemistry. Remarkably, the linkage of siRNA RET/PTC1 to squalene led to an amphiphilic molecule that self-organized in H2O as siRNA-SQ RET/PTC1 nanoparticles (NPs). The siRNA-SQ RET/PTC1 NPs, stable in H2O, were used for biological studies. In vitro, they did not show any cytotoxicity. Interestingly, in vivo, on a mice xenografted RET/PTC1 experimental model, RET/PTC1-SQ NPs were found to inhibit tumor growth and RET/PTC1 oncogene and oncoprotein expression after 2.5 mg/kg cumulative dose intravenous injections. In conclusion, these results showed that the "squalenoylation" offers a new noncationic plate-form for the siRNA delivery.

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