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Rudolph L. Juliano - One of the best experts on this subject based on the ideXlab platform.
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The delivery of therapeutic Oligonucleotides.
Nucleic Acids Research, 2016Co-Authors: Rudolph L. JulianoAbstract:: The Oligonucleotide therapeutics field has seen remarkable progress over the last few years with the approval of the first antisense drug and with promising developments in late stage clinical trials using siRNA or splice switching Oligonucleotides. However, effective delivery of Oligonucleotides to their intracellular sites of action remains a major issue. This review will describe the biological basis of Oligonucleotide delivery including the nature of various tissue barriers and the mechanisms of cellular uptake and intracellular trafficking of Oligonucleotides. It will then examine a variety of current approaches for enhancing the delivery of Oligonucleotides. This includes molecular scale targeted ligand-Oligonucleotide conjugates, lipid- and polymer-based nanoparticles, antibody conjugates and small molecules that improve Oligonucleotide delivery. The merits and liabilities of these approaches will be discussed in the context of the underlying basic biology.
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cellular uptake and intracellular trafficking of Oligonucleotides
Advanced Drug Delivery Reviews, 2015Co-Authors: Rudolph L. Juliano, Kyle CarverAbstract:Oligonucleotides manifest much promise as potential therapeutic agents. However, understanding of how Oligonucleotides function within living organisms is still rather limited. A major concern in this regard is the mechanisms of cellular uptake and intracellular trafficking of both ‘free’ Oligonucleotides and Oligonucleotides associated with various polymeric or nanocarrier delivery systems. Here we review basic aspects of the mechanisms of endocytosis and intracellular trafficking and how insights from these processes can be used to understand Oligonucleotide delivery. In particular we discuss opportunities for escape of Oligonucleotides from endomembrane compartments and describe recent studies using small molecules to enhance Oligonucleotide effects.
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cellular uptake and intracellular trafficking of Oligonucleotides implications for Oligonucleotide pharmacology
Nucleic Acid Therapeutics, 2014Co-Authors: Rudolph L. Juliano, Kyle Carver, Xin Ming, Brian LaingAbstract:One of the major constraints on the therapeutic use of Oligonucleotides is inefficient delivery to their sites of action in the cytosol or nucleus. Recently it has become evident that the pathways of cellular uptake and intracellular trafficking of Oligonucleotides can strongly influence their pharmacological actions. Here we provide background information on the basic processes of endocytosis and trafficking and then review recent literature on targeted delivery and subcellular trafficking of Oligonucleotides in that context. A variety of approaches including molecular scale ligand-Oligonucleotide conjugates, ligand-targeted nanocarriers, and the use of small molecules to enhance Oligonucleotide effects are discussed.
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Conjugates of antisense Oligonucleotides with the Tat and Antennapedia cell-penetrating peptides: Effects on cellular uptake, binding to target sequences, and biologic actions
Pharmaceutical Research, 2002Co-Authors: Anna Astriab-fisher, Dimitri Sergueev, Barbara Ramsay Shaw, Michael Fisher, Rudolph L. JulianoAbstract:PURPOSE: The attainment of effective intracellular delivery remains an important issue for pharmacologic applications of antisense Oligonucleotides. Here, we describe the synthesis, binding properties, and biologic properties of peptide-Oligonucleotide conjugates comprised of the Tat and Ant cell-penetrating peptides with 2'-O-methyl phosphorothioate Oligonucleotides. METHODS: The biologic assay used in this study measures the ability of the antisense molecule to correct splicing of an aberrant intron inserted into the Luciferase gene; thus, this assay clearly demonstrates the delivery of functional antisense molecules to the splicing machinery within the nucleus. The binding affinities of the conjugates to their target sequences were measured by surface plasmon resonance (BIAcor) techniques. RESULTS: The peptide-Oligonucleotide conjugates progressively entered cells over a period of hours and were detected in cytoplasmic vesicles and in the nucleus. Peptide-Oligonucleotide conjugates targeted to the aberrant splice site, but not mismatched controls, caused an increase in Luciferase activity in a dose-responsive manner. The kinetics of Luciferase appearance were consistent with the course of the uptake process for the conjugates. The effects of peptide conjugation on the hybridization characteristics of the Oligonucleotides were also examined using surface plasmon resonance. The peptide-Oligonucleotide conjugates displayed binding affinities and selectivities similar to those of unconjugated Oligonucleotides. CONCLUSIONS: Conjugation with cell-penetrating peptides enhances Oligonucleotide delivery to the nucleus without interfering with the base-pairing function of antisense Oligonucleotides.
Kyle Carver - One of the best experts on this subject based on the ideXlab platform.
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cellular uptake and intracellular trafficking of Oligonucleotides
Advanced Drug Delivery Reviews, 2015Co-Authors: Rudolph L. Juliano, Kyle CarverAbstract:Oligonucleotides manifest much promise as potential therapeutic agents. However, understanding of how Oligonucleotides function within living organisms is still rather limited. A major concern in this regard is the mechanisms of cellular uptake and intracellular trafficking of both ‘free’ Oligonucleotides and Oligonucleotides associated with various polymeric or nanocarrier delivery systems. Here we review basic aspects of the mechanisms of endocytosis and intracellular trafficking and how insights from these processes can be used to understand Oligonucleotide delivery. In particular we discuss opportunities for escape of Oligonucleotides from endomembrane compartments and describe recent studies using small molecules to enhance Oligonucleotide effects.
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cellular uptake and intracellular trafficking of Oligonucleotides implications for Oligonucleotide pharmacology
Nucleic Acid Therapeutics, 2014Co-Authors: Rudolph L. Juliano, Kyle Carver, Xin Ming, Brian LaingAbstract:One of the major constraints on the therapeutic use of Oligonucleotides is inefficient delivery to their sites of action in the cytosol or nucleus. Recently it has become evident that the pathways of cellular uptake and intracellular trafficking of Oligonucleotides can strongly influence their pharmacological actions. Here we provide background information on the basic processes of endocytosis and trafficking and then review recent literature on targeted delivery and subcellular trafficking of Oligonucleotides in that context. A variety of approaches including molecular scale ligand-Oligonucleotide conjugates, ligand-targeted nanocarriers, and the use of small molecules to enhance Oligonucleotide effects are discussed.
Kenneth J. Rothschild - One of the best experts on this subject based on the ideXlab platform.
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Photocleavable Biotin Phosphoramidite for 5′-End-Labeling, Affinity Purification and Phosphorylation of Synthetic Oligonucleotides
Nucleic Acids Research, 1996Co-Authors: Jerzy Olejnik, Edyta Krzymanska-olejnik, Kenneth J. RothschildAbstract:We report the design, synthesis and evaluation of a non-nucleosidic photocleavable biotin phosphoramidite (PCB-phosphoramidite) which provides a simple method for purification and phosphorylation of Oligonucleotides. This reagent introduces a photocleavable biotin label (PCB) on the 54-terminal phosphate of synthetic Oligonucleotides and is fully compatible with automated solid support synthesis. HPLC analysis shows that the PCB moiety is introduced predominantly on full-length sequences and is retained during cleavage of the synthetic Oligonucleotide from the solid support and during subsequent deprotection with ammonia. The full-length 54-PCB-labeled Oligonucleotide can then be selectively isolated from the crude Oligonucleotide mixture by incubation with immobilized streptavidin. Upon irradiation with 300‐350 nm light the 54-PCB moiety is cleaved with high efficiency in
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photocleavable biotin phosphoramidite for 5 end labeling affinity purification and phosphorylation of synthetic Oligonucleotides
Nucleic Acids Research, 1996Co-Authors: Jerzy Olejnik, Edyta Krzymanskaolejnik, Kenneth J. RothschildAbstract:We report the design, synthesis and evaluation of a non-nucleosidic photocleavable biotin phosphoramidite (PCB-phosphoramidite) which provides a simple method for purification and phosphorylation of Oligonucleotides. This reagent introduces a photocleavable biotin label (PCB) on the 54-terminal phosphate of synthetic Oligonucleotides and is fully compatible with automated solid support synthesis. HPLC analysis shows that the PCB moiety is introduced predominantly on full-length sequences and is retained during cleavage of the synthetic Oligonucleotide from the solid support and during subsequent deprotection with ammonia. The full-length 54-PCB-labeled Oligonucleotide can then be selectively isolated from the crude Oligonucleotide mixture by incubation with immobilized streptavidin. Upon irradiation with 300‐350 nm light the 54-PCB moiety is cleaved with high efficiency in <4 min, resulting in rapid release of affinity-purified 54-phosphorylated Oligonucleotides into solution. 54-PCB-labeled Oligonucleotides should be useful in a variety of applications in molecular biology, including cassette mutagenesis and PCR. As an example, PCB-phosphoramidite has been used for the synthesis, purification and phosphorylation of 50and 60mer Oligonucleotides.
Tatiana S. Oretskaya - One of the best experts on this subject based on the ideXlab platform.
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New phosphoramidite derivatives for the preparation of Oligonucleotides containing a hydrazide group in any specified position of the Oligonucleotide chain
Russian Chemical Bulletin, 2005Co-Authors: S. I. Antsypovich, Tatiana S. Oretskaya, G. Von KiedrowskiAbstract:A new versatile method for the preparation of Oligonucleotides containing hydrazide groups in any position of the Oligonucleotide chain by standard phosphoramidite automated Oligonucleotide synthesis is proposed. The method is based on the use of a series of new modified components for Oligonucleotide synthesis. An original protecting group for the hydrazide group is proposed. The presence of the hydrazide group in the obtained Oligonucleotides and its high reactivity were demonstrated by the reaction with 4-methoxybenzaldehyde in solution.
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Synthesis and applications of Oligonucleotide-carbohydrate conjugates.
Chemistry & Biodiversity, 2004Co-Authors: Timofei S. Zatsepin, Tatiana S. OretskayaAbstract:: Nowadays, Oligonucleotide-carbohydrate conjugates are used in antisense biotechnology and in the study of glycosylated DNA functioning in vitro. The application of mono- and disaccharide phosphoramidites, solid-phase supports with immobilized carbohydrates, glycosylated nucleoside phosphoramidites, and postsynthetic conjugation of reactive sugar derivatives with Oligonucleotides for preparation of Oligonucleotide-carbohydrate conjugates have been systematically studied. The advantages and disadvantages of these approaches are considered. Possible strategies for synthesis of glycoclusters with different topologies conjugated to DNA are discussed. Applications of Oligonucleotide-carbohydrate conjugates are highlighted. Studies of interactions of glycosylated Oligonucleotides with proteins and effective cell-specific delivery of Oligonucleotide-carbohydrate conjugates are discussed.
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Oligonucleotides with 2 o carboxymethyl group synthesis and 2 conjugation via amide bond formation on solid phase
Organic and Biomolecular Chemistry, 2004Co-Authors: Anna V Kachalova, Eugeny M Zubin, Dmitry A Stetsenko, Michael J Gait, Tatiana S. OretskayaAbstract:An efficient method for synthesis of Oligonucleotide 2′-conjugates via amide bond formation on solid phase is described. Protected Oligonucleotides containing a 2′-O-carboxymethyl group were obtained by use of a novel uridine 3′-phosphoramidite, where the carboxylic acid moiety was introduced as its allyl ester. This protecting group is stable to the conditions used in solid-phase Oligonucleotide assembly, but easily removed by Pd(0) and morpholine treatment. 2′-O-Carboxymethylated Oligonucleotides were then efficiently conjugated on a solid support under normal peptide coupling conditions to various amines or to the N-termini of small peptides to give products of high purity in good yield. The method is well suited in principle for the preparation of peptide–Oligonucleotide conjugates containing an amide linkage between the 2′-position of an Oligonucleotide and the N-terminus of a peptide.
Dieter H. Gauss - One of the best experts on this subject based on the ideXlab platform.
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Chemically Modified Oligonucleotides as Probes and Inhibitors
Angewandte Chemie International Edition in English, 1991Co-Authors: Uwe Englisch, Dieter H. GaussAbstract:Oligonucleotides bind specifically to single-stranded nucleic acids to form a double helix if there is a complementary antiparallel nucleotide sequence. In addition, certain Oligonucleotides bind specifically to a variety of proteins. Therefore, biological processes involving these nucleic acids or proteins can be modulated (normally inhibited) by addition of the respective Oligonucleotides. The effects of these Oligonucleotides and the fields of potential application can be broadened by the introduction of chemically modified nucleotides. For instance, replacing Oligonucleotide phosphate groups by methylphosphonates results in the loss of one negative charge per nucleotide and the Oligonucleotide becomes more lipophilic. An Oligonucleotide carrying a reactive group can modify its binding partner. An Oligonucleotide covalently linked to a dye can be localized in a biological specimen. Oligonucleotides attached to an enzyme can be detected in very small amounts since the enzyme can catalyze the formation of large amounts of the substance assayed (e.g., a fluorescent product). An important biochemical application is the detection and localization of a messenger RNA or its gene. Medical applications include the detection of bacterial or viral sequences. There is also great interest in inhibiting the translation of messenger RNA and the transcription and replication of DNA. So-called antisense Oligonucleotides are currently being used for the inhibition of protein biosynthesis and of reverse transcription of retroviruses.
