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Molecules (Basel, Switzerland) Apr 2015This manuscript reviews the possibilities offered by 2,5-dimethylfuran-protected maleimides. Suitably derivatized building blocks incorporating the exo Diels-Alder... (Review)
Review
This manuscript reviews the possibilities offered by 2,5-dimethylfuran-protected maleimides. Suitably derivatized building blocks incorporating the exo Diels-Alder cycloadduct can be introduced at any position of oligonucleotides, peptide nucleic acids, peptides and peptoids, making use of standard solid-phase procedures. Maleimide deprotection takes place upon heating, which can be followed by either Michael-type or Diels-Alder click conjugation reactions. However, the one-pot procedure in which maleimide deprotection and conjugation are simultaneously carried out provides the target conjugate more quickly and, more importantly, in better yield. This procedure is compatible with conjugates involving oligonucleotides, peptides and peptide nucleic acids. A variety of cyclic peptides and oligonucleotides can be obtained from peptide and oligonucleotide precursors incorporating protected maleimides and thiols.
Topics: Click Chemistry; Cyclization; Maleimides; Oligonucleotides; Peptide Nucleic Acids; Peptides; Peptides, Cyclic
PubMed: 25867825
DOI: 10.3390/molecules20046389 -
Current Protocols in Nucleic Acid... Jun 2020This protocol describes a method based on iodine and a base as mild coupling reagents to synthetize deoxyribonucleic guanidines (DNGs)-oligodeoxynucleotide analogues...
This protocol describes a method based on iodine and a base as mild coupling reagents to synthetize deoxyribonucleic guanidines (DNGs)-oligodeoxynucleotide analogues with a guanidine backbone. DNGs display unique properties, such as high cellular uptake with low toxicity and increased stability against nuclease degradation, but have been impeded in their development by the requirement for toxic and iterative manual synthesis protocols. The novel synthesis method reported here eliminates the need for the toxic mercuric chloride and pungent thiophenol that were critical to previous DNG synthesis methods and translates their synthesis to a MerMade 12 automated oligonucleotide synthesizer. This method can be used to synthesize DNG strands up to 20 bases in length, along with 5'-DNG-DNA-3' chimeras, at 1- to 5-μmol scales in a fully automated manner. We also present detailed and accessible instructions to adapt the MerMade 12 oligonucleotide synthesizer to enable the parallel synthesis of DNG and DNA/RNA oligonucleotides. Because DNG linkages alter the overall charge of the oligonucleotides, we also describe purification strategies to generate oligonucleotides with varying lengths and numbers of DNGs, based on extraction or preparative-scale gel electrophoresis, along with methods to characterize the final products. Overall, this article provides an overview of the synthesis, purification, and handling of DNGs and mixed-charge DNG-DNA oligonucleotides. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of a MerMade synthesizer for guanidine couplings Basic Protocol 2: Synthesis of DNG strands on a MerMade synthesizer Basic Protocol 3: Purification of DNG strands using preparative acetic acid urea (AU) PAGE Basic Protocol 4: Characterization of DNG strands using MALDI-TOF MS Basic Protocol 5: Characterization of DNG strands using AU PAGE Support Protocol 1: Synthesis of initiator-functionalized CPG Support Protocol 2: Synthesis of thiourea monomer.
Topics: Automation; Guanidine; Nucleic Acid Conformation; Oligonucleotides
PubMed: 32530578
DOI: 10.1002/cpnc.110 -
Angewandte Chemie (International Ed. in... Apr 2021Nucleosidic and oligonucleotidic diarylethenes (DAEs) are an emerging class of photochromes with high application potential. However, their further development is...
Nucleosidic and oligonucleotidic diarylethenes (DAEs) are an emerging class of photochromes with high application potential. However, their further development is hampered by the poor understanding of how the chemical structure modulates the photochromic properties. Here we synthesized 26 systematically varied deoxyuridine- and deoxycytidine-derived DAEs and analyzed reaction quantum yields, composition of the photostationary states, thermal and photochemical stability, and reversibility. This analysis identified two high-performance photoswitches with near-quantitative, fully reversible back-and-forth switching and no detectable thermal or photochemical deterioration. When incorporated into an oligonucleotide with the sequence of a promotor, the nucleotides maintained their photochromism and allowed the modulation of the transcription activity of T7 RNA polymerase with an up to 2.4-fold turn-off factor, demonstrating the potential for optochemical control of biological processes.
Topics: Bacteriophage T7; DNA-Directed RNA Polymerases; Drug Development; Enzyme Inhibitors; Ethylenes; Oligonucleotides; Photochemical Processes; Pyrimidine Nucleosides; Viral Proteins
PubMed: 33476096
DOI: 10.1002/anie.202014878 -
ALTEX 2022Oligonucleotide therapeutics (ONTs) encompass classes of medicines that selectively target and potentially ameliorate previously untreatable and often rare diseases.... (Review)
Review
Oligonucleotide therapeutics (ONTs) encompass classes of medicines that selectively target and potentially ameliorate previously untreatable and often rare diseases. Several unique classes of ONTs provide versatility, enabling direct modulation of gene expression by virtue of Watson-Crick base pairing or modulation of cell signaling through structural mimicry or interference with protein-receptor interactions. Due to a lack of suitable in vitro models capable of recapitulating or predicting in vivo effects of ONTs, their discovery and optimization has relied heavily on animal studies for predicting efficacy and safety in humans. Since ONTs often lack cross-species activity, animal models with genetic humanization and/or species-specific surrogate ONTs are often required. Human microphysiological systems (MPS) offer an opportunity to reduce the use of animals and may enable evaluation of drug mechanisms, optimization of cell and tissue targeting ligands or delivery vehicles, and characterization of pharmacokinetics (PK), pharmacodynamics (PD), and safety of candidate ONTs. The lack of published examples for MPS applications with ONT demonstrates the need for a focused effort to characterize and build confidence in their utility. The goals of this review are to summarize the current landscape of ONTs and highlight potential opportunities and challenges for application of MPS during ONT discovery and development. In addition, this review aims to raise awareness with ONT drug developers and regulatory authorities on the potential impact of MPS with respect to characterizing pharmacology, ADME, and toxicity and to educate MPS platform developers on unique design attributes needed to fully appreciate MPS advantages in ONT development.
Topics: Animals; Oligonucleotides; Pharmaceutical Preparations
PubMed: 34766620
DOI: 10.14573/altex.2108241 -
Chemical Society Reviews Dec 2011Hybrid lipid oligonucleotide conjugates are finding more and more biotechnological applications. This short critical review highlights their synthesis, supramolecular... (Review)
Review
Hybrid lipid oligonucleotide conjugates are finding more and more biotechnological applications. This short critical review highlights their synthesis, supramolecular organization as well as their applications in the field of biotechnology (111 references).
Topics: Animals; Biomedical Research; Cell Membrane; Chemistry Techniques, Synthetic; Drug Design; Lipid Metabolism; Oligonucleotides
PubMed: 21611637
DOI: 10.1039/c1cs15038c -
BMC Molecular Biology Apr 2013The double-stranded conformation of cellular DNA is a central aspect of DNA stabilisation and protection. The helix preserves the genetic code against chemical and... (Review)
Review
The double-stranded conformation of cellular DNA is a central aspect of DNA stabilisation and protection. The helix preserves the genetic code against chemical and enzymatic degradation, metabolic activation, and formation of secondary structures. However, there are various instances where single-stranded DNA is exposed, such as during replication or transcription, in the synthesis of chromosome ends, and following DNA damage. In these instances, single-stranded DNA binding proteins are essential for the sequestration and processing of single-stranded DNA. In order to bind single-stranded DNA, these proteins utilise a characteristic and evolutionary conserved single-stranded DNA-binding domain, the oligonucleotide/oligosaccharide-binding (OB)-fold. In the current review we discuss a subset of these proteins involved in the direct maintenance of genomic stability, an important cellular process in the conservation of cellular viability and prevention of malignant transformation. We discuss the central roles of single-stranded DNA binding proteins from the OB-fold domain family in DNA replication, the restart of stalled replication forks, DNA damage repair, cell cycle-checkpoint activation, and telomere maintenance.
Topics: Cell Cycle; DNA Repair; DNA Replication; DNA-Binding Proteins; Genomic Instability; Humans; Oligonucleotides
PubMed: 23548139
DOI: 10.1186/1471-2199-14-9 -
Scientific Reports Mar 2021Antibody-Oligonucleotide Conjugates (AOCs) represent an emerging class of functionalized antibodies that have already been used in a wide variety of applications. While...
Antibody-Oligonucleotide Conjugates (AOCs) represent an emerging class of functionalized antibodies that have already been used in a wide variety of applications. While the impact of dye and drug conjugation on antibodies' ability to bind their target has been extensively studied, little is known about the effect caused by the conjugation of hydrophilic and charged payloads such as oligonucleotides on the functions of an antibody. Previous observations of non-specific interactions of nucleic acids with untargeted cells prompted us to further investigate their impact on AOC binding abilities and cell selectivity. We synthesized a series of single- and double-stranded AOCs, as well as a human serum albumin-oligonucleotide conjugate, and studied their interactions with both targeted and non-targeted living cells using a time-resolved analysis of ligand binding assay. Our results indicate that conjugation of single strand oligonucleotides to proteins induce consistent non-specific interactions with cell surfaces while double strand oligonucleotides have little or no effect, depending on the preparation method.
Topics: Antibodies; Cell Line, Tumor; Cell Survival; Humans; Kinetics; Oligonucleotides; Trastuzumab
PubMed: 33723336
DOI: 10.1038/s41598-021-85352-w -
Drug Design, Development and Therapy 2019Hereditary transthyretin amyloidosis is a fatal autosomal dominant disorder characterized by deposition of transthyretin amyloid into the peripheral nervous system,... (Review)
Review
Hereditary transthyretin amyloidosis is a fatal autosomal dominant disorder characterized by deposition of transthyretin amyloid into the peripheral nervous system, heart, kidney, and gastrointestinal tract. Previous treatments using liver transplantation and small molecule stabilizers were not effective in stopping disease progression. Inotersen, a 2'-O-methyoxyethyl-modified antisense oligonucleotide, which acts by reducing the production of transthyretin, was recently demonstrated to improve disease course and quality of life in early hereditary transthyretin amyloidosis polyneuropathy in a 15-month Phase III study.
Topics: Amyloid Neuropathies, Familial; Animals; Humans; Oligodeoxyribonucleotides, Antisense; Oligonucleotides
PubMed: 31118583
DOI: 10.2147/DDDT.S162913 -
Methods in Molecular Biology (Clifton,... 2022The eye is the organ in charge of vision and, given its properties, has become an excellent organ to test genetic therapies, including antisense oligonucleotide (AON)...
The eye is the organ in charge of vision and, given its properties, has become an excellent organ to test genetic therapies, including antisense oligonucleotide (AON) technology. In fact, the first AON receiving FDA and EMA approval was meant to treat an eye condition. Currently, dozens of clinical trials are being conducted for a variety of subtypes of inherited retinal disease. Although most of them are based on gene augmentation therapies, a phase 3 and two phase 1/2 clinical trials using AONs are ongoing. Since the retina is a layered structure of nondividing cells, obtaining human retinal tissue and expanding it in the lab is not possible, unless induced pluripotent stem cell technology is used. Mouse models have helped to elucidate the function of many genes, and the retinal structure is quite similar to that of humans. Thus, drug delivery to the mouse eye can provide valuable information for further optimization of therapies. In this chapter, the protocol for intravitreal injections of AONs is described in detail.
Topics: Animals; Genetic Therapy; Mice; Oligonucleotides; Oligonucleotides, Antisense; Retina; Retinal Diseases
PubMed: 35213028
DOI: 10.1007/978-1-0716-2010-6_22 -
ACS Synthetic Biology Dec 2013High-frequency oligonucleotide-directed recombination engineering (recombineering) has enabled rapid modification of several prokaryotic genomes to date. Here, we...
High-frequency oligonucleotide-directed recombination engineering (recombineering) has enabled rapid modification of several prokaryotic genomes to date. Here, we present a method for oligonucleotide-mediated recombineering in the model eukaryote and industrial production host Saccharomyces cerevisiae , which we call yeast oligo-mediated genome engineering (YOGE). Through a combination of overexpression and knockouts of relevant genes and optimization of transformation and oligonucleotide designs, we achieve high gene-modification frequencies at levels that only require screening of dozens of cells. We demonstrate the robustness of our approach in three divergent yeast strains, including those involved in industrial production of biobased chemicals. Furthermore, YOGE can be iteratively executed via cycling to generate genomic libraries up to 10 (5) individuals at each round for diversity generation. YOGE cycling alone or in combination with phenotypic selections or endonuclease-based negative genotypic selections can be used to generate modified alleles easily in yeast populations with high frequencies.
Topics: Genetic Engineering; Genome, Fungal; Oligonucleotides; Recombination, Genetic; Saccharomyces cerevisiae
PubMed: 24160921
DOI: 10.1021/sb400117c