Author: Justin Mahoney Wolfe
Publisher:
ISBN:
Category :
Languages : en
Pages : 419
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Book Description
The intracellular delivery of functional macromolecules remains an outstanding challenge in biomedicine. While small molecules can diffuse through the plasma membrane, many large therapeutic molecules are not internalized to an appreciable extent. One strategy to improve cell uptake involves linking the molecule of interest to a cell-penetrating peptide (CPP). CPPs are widely employed to enhance macromolecule delivery, with hundreds of different peptides and modifications reported to improve cellular uptake. In this thesis, CPPs were systematically investigated and chemically altered to facilitate the delivery of antisense oligonucleotides. To accurately compare the existing CPPs, 64 CPP sequences were synthesized, conjugated to oligonucleotides, and assayed for delivery. These CPPs showed a range of effectiveness, with some CPPs hindering the delivery of oligonucleotide cargo and others leading to a 10-fold increase in oligonucleotide activity. To help identify which CPPs might be valuable for oligonucleotide delivery specifically, a computational model was developed to predict, de novo, whether or not a CPP will be effective. When experimentally validated, this model successfully predicted which sequences would improve oligonucleotide delivery greater than 3-fold. Multiple strategies were employed to improve CPP effectiveness. First, arginine-rich CPPs were chemically modified with perfluoroarenes. Cyclic and bicyclic CPPs were synthesized by linking multiple cysteine residues together with a perfluoroarene. After oligonucleotide conjugation, these peptides led to a 14-fold increase in delivery. Second, two different CPPs were combined into one long chimeric sequence. The CPP chimeras were highly active, leading to a 20-fold increase in oligonucleotide delivery. Third, the idea of combining multiple CPPs led to the development of a method for the rapid synthesis combinatorial peptide conjugates. Using the judicious choice of bioconjugation chemistry, highly-active modular constructs were synthesized that contain three peptides linked to one oligonucleotide. In addition to CPPs for oligonucleotide delivery, one section of this thesis employed perfluoroaryl macrocyclic peptides to address the challenge of peptide delivery across the blood-brain barrier. An additional section developed a new peptide conjugation strategy that uses palladium-peptide oxidative addition complexes as solid, storable, and water-soluble reagents for bioconjugation.
Author: Jens Kurreck
Publisher: Royal Society of Chemistry
ISBN: 0854041168
Category : Medical
Languages : en
Pages : 362
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Book Description
This book provides a compelling overall update on current status of RNA interference
Author: Colin MacLaine Fadzen
Publisher:
ISBN:
Category :
Languages : en
Pages : 325
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Book Description
Many nucleic acids, peptides, and small molecules struggle to become clinically viable therapeutics as a result of poor delivery. Biological barriers such as the plasma membrane and the blood-brain barrier (BBB) contribute to this challenge as they can limit the passage of macromolecules. Cell-penetrating peptides (CPPs) that interact with membranes can improve the uptake of macromolecules across biological barriers. Here we explore methods for the peptide-mediated delivery of antisense oligonucleotides (ASOs) and chemotherapeutics. First, we address the issue that the optimal peptide sequence for the delivery of a macromolecular cargo is often context-dependent and specific to that cargo. With one class of ASO, we develop a paradigm that combines systematic screening of known CPPs in a functional assay for ASO delivery with machine learning methods. Using our computational model, we identify five novel sequences that increase ASO activity at least three-fold. Next, we demonstrate that combining CPPs of different classes generates chimeric peptides with synergistic effects on ASO delivery. These chimeras improve ASO activity twenty-fold, which is greater than any literature-reported sequence. Then, we examine peptide cyclization with perfluoroaryl-cysteine SNAr chemistry to improve the stability and delivery of peptide-ASO conjugates. We extend our SNAr chemistry to the synthesis of arginine-rich bicyclic peptides, which are more stable to proteolysis than single cycles. Both perfluoroaryl cyclic and bicyclic arginine-rich peptides improve ASO activity fourteen-fold. Consequently, we demonstrate that peptide cyclization with perfluoroaryl-cysteine SNAr chemistry enhances the ability of peptides to cross the BBB. We prepare macrocyclic analogues of both a CPP and a therapeutic peptide. We show that a subset of the macrocycles cross the BBB in both a cellular spheroid model of the BBB, as well as after intravenous injection in mice. Finally, we conjugate a platinum (IV) prodrug of the chemotherapeutic cisplatin to a brain-penetrating perfluoroaryl macrocycle and show that the amount of platinum in the mouse brain is fifteen-fold greater than cisplatin after five hours. In summary, we explore strategies to improve the peptide-mediated delivery of ASOs and small molecule chemotherapeutics across biological barriers. In the future, we envision extending these approaches to other macromolecular cargos of therapeutic interest.
Author: Victoria Steven
Publisher:
ISBN:
Category :
Languages : en
Pages : 0
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Book Description
Modifed oligonucleotides are routinely employed as bioanalytical probes for use in diagnostics; however, a major limiting factor in oligonucleotide-based diagnostics is poor cellular uptake. This can be overcome by covalent attachment of a cell penetrating peptide. Diels-Alder cycloaddition is an attractive methodology for oligonucleotide peptide conjugation; the reaction is fast and chemoselective and the reaction rate is greatly enhanced in aqueous media. An oligonucleotide sequence has been derivatised with a series of dienes at the 5'- terminus, through chemical synthesis of a series of unique dienyl modified phosphoramidites. Investigation into the effect of diene type on the efficiency of conjugation to a maleimido derivatised Tat peptide derivative, via the Diels-Alder cycloaddition, has been performed. The optimal diene for biomolecule conjugation was found to be cyclohexadiene in the conjugation of oligonucleotides to a cell penetrating peptide via Diels-Alder cycloaddition for the first time. An oligonucleotide sequence has also been derivatised with cyclohexadiene internally; Diels-Alder cycloadditions of these oligonucleotides to a maleimido derivatised Tat peptide derivative were also successful. However, oligonucleotide conjugation to Tat peptide via Diels-Alder cycloaddition of fluorescently labelled oligonucleotides for visualisation in cell studies has not been as successful as for unlabelled oligonucleotides. Generation of a biocatalytic DNA sequence for the acceleration of Diels-Alder cycloadditions has been attempted. Through a SELEX-type process, a DNA sequence has been isolated for experimental determination of its potential as a biological catalyst for Diels-Alder cycloadditions. Oligonucleotide conjugation to Tat peptide via gold nanoparticles has been achieved. Chemical synthesis of a dithiolated, long-chain PEGylated ligand allowed covalent attachment of Tat peptide to gold nanoparticles. Bifunctionalisation of gold nanoparticles with oligonucleotides and this ligand, followed by covalent attachment of Tat peptide generated the desired conjugate. The conjugates have been shown to hybridise successfully with the complementary oligonucleotide sequence, thereby displaying potential for their use as bioanalytical probes. Methods for quantification of both oligonucleotides and Tat peptide conjugated to gold nanoparticles, by enzyme hydrolysis, have been developed.
Author: Alagarsamy Pandikumar
Publisher: Elsevier
ISBN: 0128230347
Category : Technology & Engineering
Languages : en
Pages : 570
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Book Description
Nanoscale Graphitic Carbon Nitride focuses on multi-functional applications including energy conversion, storage and healthcare. Polymeric graphitic carbon nitride materials have attracted much attention in recent years because of their similarity to graphene. They are composed of carbon, nitrogen and some minor hydrogen content. In contrast to graphene, g-Graphitic carbon nitride is a medium band-gap semiconductor and in that role an effective photocatalyst and chemical catalyst for a broad variety of reactions and applications. This book covers the fundamentals and applications of graphitic carbon nitride (g-C3N4) in different sectors. It also covers the application of graphitic carbon nitride-based composites with metal, metal oxides, metal sulphide and carbon-based materials. This is an important resource for researchers in the fields of materials science, engineering, energy storage and chemical engineering who want to understand how nanoscale graphitic carbon nitride is being used for a range of industrial applications and processes. Outlines the major properties of nanoscale graphitic carbon nitride, along with their major application areas Assesses the challenges of manufacturing graphitic carbon nitride on a mass scale Explains major synthesis methods for nanoscale graphitic carbon nitride
Author: Jordi Agramunt Pi
Publisher:
ISBN:
Category :
Languages : en
Pages : 0
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"2,2-Disubstituted cyclopent-4-ene-1,3-diones (CPDs), which have been as non-hydrolysable maleimide analogs, have been found to possess an unexpected reactivity with N-terminal cysteines. Whilst maleimides react in an irreversible manner with all types of thiol nucleophiles, the Michael-type addition between CPDs and N-terminal cysteines, furnish a stable product with a mass 20 Da lower than the expected Michael-type adduct. This newly found reactivity can be applied for different purposes. In first instance, CPD-derivatized peptides can be used to synthesize conjugates by reaction with cystine-derivatized biomolecules such as peptides, peptide nucleic acids (PNAs) or oligonucleotides. In addition, the combination of the CPD-Cys reaction with other "click" reactions has been explored. In the first instance, the CPD-Cys reaction was paired the thia-Michael addition of thiols to maleimides using peptides containing two cysteines (one at the N-terminus). By first performing a CPD-Cys reaction at the N-terminus and then the addition of a maleimide, double conjugation could be easily achieved. In another case, combinations with oxime ligation and copper(I) catalyzed azide-alkyne (CuAAC) cycloadditions have been accomplished with a different degree of success. In the latter case, the CPD-Cys reaction has to be strictly performed before the CuAAC. In a further examination why this was the case, it was found out that CPDs react with azides to furnish two stable compounds one is the product of a 1,3-dipolar cycloaddition and the other results from nitrogen loss. Secondly, the bioconjugation of a splice-switching enhancing molecule known as "Retro-1" to an oligonucleotide with splice-switching activity described as 623 has been explored. In this part of the work, the complete synthesis of described Retro-1 has been accomplished in addition to that of other several analogues containing either a 1,3-diene, a thiol or a phosphoramidite group appending from two positions, nitrogen 4 or carbon 3. For this purpose, two synthetic strategies were followed one making use of valuable intermediates obtained from the Retro-1 synthesis and the other utilizing a properly protected lysine analogue to construct the Retro scaffold and introduce a reactive group at carbon 3. Additionally to all these reactants two oligonucleotides were synthesized: one with the 623 sequence and another with the same nucleobases but different order coined "scrambled". Both oligonucleotides were appended at the 5' position with a protected maleimide phosphoramidite (to obtain the corresponding maleimido-oligonucleotide and allow a Diels-Alder or thia-Michael addition to be performed) or the Retro-phosphoramidite. Finally, in this work the possibility of using 7-oxanorbornenes as dienophiles in the inverse electron-demanding Diels-Alder cycloaddition (IEDDA) with 3,6-disbustituted 1,2,4,5-tetrazines has been explored. The oxanorbornene moiety has been appended at the N-terminus and internal positions in peptides, and at the 5' and 3' positions of oligonucleotides utilizing solid-phase protocols. For the tetrazine counterpart optimization processes have been followed for the synthesis of N-terminal tetrazine peptides and for derivatization with biologically relevant molecules in reasonably good success. Once oxanorbornene- and tetrazine-containing derivatives were obtained it was observed that the IEDDA reaction took place satisfactorily, allowing for the synthesis of peptide and oligonucleotide conjugates with small molecules. In second term, the possibility of double conjugation involving at least one IEDDA reaction has been explored. The IEDDA has been combined with the Diels-Alder cycloaddition between a maleimide and a 1,3-diene, the thia-Michael between a thiol and a maleimide, the CPD-Cys reaction (utilizing CPDs and N-terminal cysteines as previously commented) and the aromatic nucleophilic substitution between a thiol and a chlorotetrazines. In every combination tested the double conjugate target compound was obtained, usually performing both reactions simultaneously, but sometimes in a "one-pot" fashion, with the sequential addition of reactants due to side reactions. In one case, it was necessary to isolate the intermediate monoconjugate because of difficulties related to undesired interferences between reactants." -- TDX.
Author: Nicolay Ferrari
Publisher: John Wiley & Sons
ISBN: 1118537335
Category : Medical
Languages : en
Pages : 576
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Book Description
A comprehensive review of contemporary antisense oligonucleotides drugs and therapeutic principles, methods, applications, and research Oligonucleotide-based drugs, in particular antisense oligonucleotides, are part of a growing number of pharmaceutical and biotech programs progressing to treat a wide range of indications including cancer, cardiovascular, neurodegenerative, neuromuscular, and respiratory diseases, as well as other severe and rare diseases. Reviewing fundamentals and offering guidelines for drug discovery and development, this book is a practical guide covering all key aspects of this increasingly popular area of pharmacology and biotech and pharma research, from the basic science behind antisense oligonucleotides chemistry, toxicology, manufacturing, to safety assessments, the design of therapeutic protocols, to clinical experience. Antisense oligonucleotides are single strands of DNA or RNA that are complementary to a chosen sequence. While the idea of antisense oligonucleotides to target single genes dates back to the 1970's, most advances have taken place in recent years. The increasing number of antisense oligonucleotide programs in clinical development is a testament to the progress and understanding of pharmacologic, pharmacokinetic, and toxicologic properties as well as improvement in the delivery of oligonucleotides. This valuable book reviews the fundamentals of oligonucleotides, with a focus on antisense oligonucleotide drugs, and reports on the latest research underway worldwide. • Helps readers understand antisense molecules and their targets, biochemistry, and toxicity mechanisms, roles in disease, and applications for safety and therapeutics • Examines the principles, practices, and tools for scientists in both pre-clinical and clinical settings and how to apply them to antisense oligonucleotides • Provides guidelines for scientists in drug design and discovery to help improve efficiency, assessment, and the success of drug candidates • Includes interdisciplinary perspectives, from academia, industry, regulatory and from the fields of pharmacology, toxicology, biology, and medicinal chemistry Oligonucleotide-Based Drugs and Therapeutics belongs on the reference shelves of chemists, pharmaceutical scientists, chemical biologists, toxicologists and other scientists working in the pharmaceutical and biotechnology industries. It will also be a valuable resource for regulatory specialists and safety assessment professionals and an important reference for academic researchers and post-graduates interested in therapeutics, antisense therapy, and oligonucleotides.
Author: Peter E. Nielsen
Publisher: Humana Press
ISBN: 9780896039766
Category : Science
Languages : en
Pages : 274
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Book Description
Peptide nucleic acids (PNAs) have now existed for slightly more than ten years, with the interest in and applications of this pseudopeptide DNA mimic steadily increasing during the entire period. PNAs have rapidly attracted the attention of scientists from a diversity of fields ranging from (bio)organic and biophysical chemistry to prebiotic evolution, and from molecular biology to genetic diagnostics and drug development. Many of the applications take advantage of the unique properties of PNA—an uncharged pseudopeptide—that distinguish this DNA mimic from more traditional DNA analogs. Rather than trying to create a comprehensive collection of all published methods and protocols involving PNA—many of which have not yet been validated— I have decided to concentrate on select protocols that are either very well established by several groups around the world, such as PCR-clamping and in situ hybridization, or on new methods that may have broader future impact. Basic methods for PNA oligomer synthesis and analyses have also been included. I am very grateful to those friends and colleagues who have enthusiastically contributed their work, discussions, and writing, and thereby made this book possible. Peter E. Nielsen v Contents Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix IINTRODUCTION 1 PNA Technology Peter E. Nielsen. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 II CHEMISTRY 2 Solid Phase Synthesis of PNA Oligomers Frederik Beck. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 3 Synthesis of PNA-Peptide Conjugates Satish Kumar Awasthi and Peter E. Nielsen. . . . . . . . . . . . . . . . . . 43 4 Parallel Synthesis of PNA-Peptide Conjugate Libraries Satish Kumar Awasthi and Peter E. Nielsen. . . . . . . . . . . . . . . . . .
Author: Piet Herdewijn
Publisher: Humana Press
ISBN: 9781617374418
Category : Medical
Languages : en
Pages : 0
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Book Description
A collection of powerful new techniques for oligonucleotide synthesis and for the use of modified oligonucleotides in biotechnology. Among the protocol highlights are a novel two-step process that yields a high purity, less costly, DNA, the synthesis of phosphorothioates using new sulfur transfer agents, the synthesis of LNA, peptide conjugation methods to improve cellular delivery and cell-specific targeting, and triple helix formation. The applications include using molecular beacons to monitor the PCR amplification process, nuclease footprinting to study the sequence-selective binding of small molecules of DNA, nucleic acid libraries, and the use of small interference RNA (siRNA) as an inhibitor of gene expression.
Author: Akira Murakami
Publisher: Springer
ISBN: 364230463X
Category : Technology & Engineering
Languages : en
Pages : 189
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Book Description
New Antisense Strategies: Chemical Synthesis of RNA Oligomers, by Junichi Yano und Gerald E. Smyth Development and Modification of Decoy Oligodeoxynucleotides for Clinical Application, by Mariana Kiomy Osako, Hironori Nakagami und Ryuichi Morishita Modulation of Endosomal Toll-Like Receptor-Mediated Immune Responses by Synthetic Oligonucleotides, by Ekambar R. Kandimalla und Sudhir Agrawal Delivery of Nucleic Acid Drugs, by Yan Lee und Kazunori Kataoka Aptamer: Biology to Applications, by Yoshikazu Nakamura Development and Clinical Applications of Nucleic Acid Therapeutics, by Veenu Aishwarya, Anna Kalota und Alan M. Gewirtz
