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Molecules (Basel, Switzerland) Oct 2020In the last three decades, oligonucleotides have been extensively investigated as probes, molecular ligands and even catalysts within therapeutic and diagnostic... (Review)
Review
In the last three decades, oligonucleotides have been extensively investigated as probes, molecular ligands and even catalysts within therapeutic and diagnostic applications. The narrow chemical repertoire of natural nucleic acids, however, imposes restrictions on the functional scope of oligonucleotides. Initial efforts to overcome this deficiency in chemical diversity included conservative modifications to the sugar-phosphate backbone or the pendant base groups and resulted in enhanced in vivo performance. More importantly, later work involving other modifications led to the realization of new functional characteristics beyond initial intended therapeutic and diagnostic prospects. These results have inspired the exploration of increasingly exotic chemistries highly divergent from the canonical nucleic acid chemical structure that possess unnatural physiochemical properties. In this review, the authors highlight recent developments in modified oligonucleotides and the thrust towards designing novel nucleic acid-based ligands and catalysts with specifically engineered functions inaccessible to natural oligonucleotides.
Topics: Animals; Aptamers, Nucleotide; Base Pairing; Carbohydrates; Catalysis; Enzymes; Gene Editing; Humans; Ligands; Nucleic Acids; Oligonucleotides; Oligonucleotides, Antisense; Pentoses
PubMed: 33066073
DOI: 10.3390/molecules25204659 -
Organic & Biomolecular Chemistry Jul 2016With the advent of novel bioorthogonal reactions and "click" chemistry, an increasing number of strategies for the single labelling of proteins and oligonucleotides have... (Review)
Review
With the advent of novel bioorthogonal reactions and "click" chemistry, an increasing number of strategies for the single labelling of proteins and oligonucleotides have emerged. Whilst several methods exist for the site-selective introduction of a single chemical moiety, site-selective and bioorthogonal dual modification of biomolecules remains a challenge. The introduction of multiple modules enables a plethora of permutations and combinations and can generate a variety of bioconjuguates with many potential applications. From de novo approaches on oligomers to the post-translational functionalisation of proteins, this review will highlight the main strategies to dually modify biomolecules.
Topics: Click Chemistry; Models, Molecular; Oligonucleotides; Proteins
PubMed: 27278999
DOI: 10.1039/c6ob01010e -
Journal of Materials Chemistry. B Apr 2020Intracellular small ligands and biomacromolecules are playing crucial roles not only as executors but also as regulators. It is essential to develop tools to investigate... (Review)
Review
Intracellular small ligands and biomacromolecules are playing crucial roles not only as executors but also as regulators. It is essential to develop tools to investigate their dynamics to interrogate their functions and reflect the cellular status. Light-up RNA aptamers are RNA sequences that can bind with their cognate nonfluorescent fluorogens and greatly activate their fluorescence. The emergence of genetically encoded light-up RNA aptamers has provided fascinating tools for studying intracellular small ligands and biomacromolecules owing to their high fluorescence activation degree and facile programmability. Here we review the burgeoning field of light-up RNA aptamers. We first briefly introduce light-up RNA aptamers with a focus on the photophysical properties of the fluorogens. Then design strategies of genetically encoded light-up RNA aptamer based sensors including turn-on, signal amplification and ratiometric rationales are emphasized.
Topics: Aptamers, Nucleotide; Biosensing Techniques; Particle Size; Surface Properties
PubMed: 31984401
DOI: 10.1039/c9tb02668a -
Biosensors & Bioelectronics Oct 2023Following the recent pandemic and with the emergence of cell-free nucleic acids in liquid biopsies as promising biomarkers for a broad range of pathologies, there is an... (Review)
Review
Following the recent pandemic and with the emergence of cell-free nucleic acids in liquid biopsies as promising biomarkers for a broad range of pathologies, there is an increasing demand for a new generation of nucleic acid tests, with a particular focus on cost-effective, highly sensitive and specific biosensors. Easily miniaturized electrochemical sensors show the greatest promise and most typically rely on the chemical functionalization of conductive materials or electrodes with sequence-specific hybridization probes made of standard oligonucleotides (DNA or RNA) or synthetic analogues (e.g. Peptide Nucleic Acids or PNAs). The robustness of such sensors is mostly influenced by the ability to control the density and orientation of the probe at the surface of the electrode, making the chemistry used for this immobilization a key parameter. This exhaustive review will cover the various strategies to immobilize nucleic acid probes onto different solid electrode materials. Both physical and chemical immobilization techniques will be presented. Their applicability to specific electrode materials and surfaces will also be discussed as well as strategies for passivation of the electrode surface as a way of preventing electrode fouling and reducing nonspecific binding.
Topics: Nucleic Acids; Biosensing Techniques; DNA; Peptide Nucleic Acids; Oligonucleotides; Electrodes; Electrochemical Techniques; Nucleic Acid Hybridization
PubMed: 37406480
DOI: 10.1016/j.bios.2023.115440 -
Nucleic Acid Therapeutics Dec 2014Phosphorothioates have found their usefulness in the general area of oligonucleotide therapeutic applications. Initially this modification was introduced into the... (Review)
Review
Phosphorothioates have found their usefulness in the general area of oligonucleotide therapeutic applications. Initially this modification was introduced into the antisense methodology because of the nuclease resistance of the phosphorothioate linkage in comparison with that of the phosphate linkage. However, as experimental data accumulated, it was detected that this chemical modification also facilitates cellular uptake and bioavailibity in vivo. Thus, today the majority of therapeutic oligonucleotides contain this modification. This review will discuss the historical development of this modification and present some of its chemical properties where they differ from those of the phosphate group. The antisense application will be discussed in the original context with cleavage of the target mRNA, but other target RNAs such as microRNAs and long noncoding RNAs will also be covered. It continues with applications where the target RNA should not be cleaved. A brief presentation of decoy oligonucleotides will be included, as well as some miscellaneous applications. Cellular uptake is a crucial step for oligonucleotides to reach their target and will be briefly reviewed. Lastly, a most surprising recent observation is the presence of phosphorothioate groups in bacterial DNA where functions still remain to be fully determined.
Topics: Animals; History, 20th Century; Humans; Mice; Oligonucleotides, Antisense; Phosphorothioate Oligonucleotides; Protein Binding; Rabbits; Transcription Factors
PubMed: 25353652
DOI: 10.1089/nat.2014.0506 -
Current Protocols Feb 2023Phosphorodiamidate morpholino oligonucleotides (PMOs) are a successful class of antisense reagents that efficiently modulate gene expression. Because PMOs do not follow...
Phosphorodiamidate morpholino oligonucleotides (PMOs) are a successful class of antisense reagents that efficiently modulate gene expression. Because PMOs do not follow standard phosphoramidite chemistry, optimized synthetic protocols for these compounds are relatively scarce in the literature. This paper presents detailed protocols for synthesizing full-length PMOs using chlorophosphoramidate chemistry by manual solid-phase synthesis. We first describe the synthesis of Fmoc-protected morpholino hydroxyl monomers, and the corresponding chlorophosphoramidate monomers, from commercially available protected ribonucleosides. The new Fmoc chemistry necessitates the use of a milder base, such as N-ethylmorpholine (NEM), and coupling reagent, such as 5-(ethylthio)-1H-tetrazole (ETT), which are also tolerated for acid-sensitive trityl chemistry. These chlorophosphoramidate monomers are then employed for PMO synthesis in a manual solid-phase procedure using four sequential steps. The synthetic cycle for each nucleotide incorporation consists of (a) deblocking of the 3'-N protecting group using an acidic deblocking cocktail for trityl and base deblocking for Fmoc, (b) neutralization, (c) coupling in the presence of ETT and NEM, and (d) capping of the unreacted morpholine ring-amine. The method uses safe, stable, and inexpensive reagents, and the process is expected to be scalable. After full-length PMO synthesis and ammonia-mediated cleavage from the solid support and deprotection, a range of PMOs with different lengths can be obtained conveniently and efficiently with reproducible good yields. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of the novel Fmoc-protected morpholino monomers Basic Protocol 2: Synthesis of the phosphorylating reagent (N,N-dimethylphosphoramic dichloride) required for chlorophosphoramidate monomer synthesis Basic Protocol 3: Synthesis of chlorophosphoramidate monomers of Fmoc-protected morpholino monomers Basic Protocol 4: Solution-phase standardization of dimer and trimer PMO synthesis using Fmoc chemistry Basic Protocol 5: Solid-phase synthesis, purification, and characterization of full-length (25-mer) no-tail PMO using both trityl and Fmoc chemistry.
Topics: Morpholinos; Oligonucleotides, Antisense
PubMed: 36802170
DOI: 10.1002/cpz1.686 -
Biomedical Chromatography : BMC Jul 2021As a new molecular recognition element, oligonucleotide aptamer not only has higher affinity and specificity to target molecules, but also has the advantages of wide... (Review)
Review
As a new molecular recognition element, oligonucleotide aptamer not only has higher affinity and specificity to target molecules, but also has the advantages of wide recognition range, in vitro synthesis and chemical stability compared with conventional antibodies. Since a kind of screening method termed systematic evolution of ligands by exponential enrichment (SELEX) was reported, scientists have extensively researched the methodology of how to highly and efficiently screen out aptamers from a library consisting of a large number of random oligonucleotides. Certainly capillary electrophoresis-based screening methodologies, including nonequilibrium capillary electrophoresis of equilibrium mixtures, equilibrium capillary electrophoresis of equilibrium mixtures, non-SELEX, ideal-filter capillary electrophoresis, capillary transient isotachophoresis, etc., are revolutionary. Compared with conventional SELEX, these capillary electrophoresis-based methodologies show incomparable advantages such as the single-round screening of aptamers and increased successful screening rate. Methodology studies on the screening process of aptamers are comprehensively reviewed.
Topics: Animals; Aptamers, Nucleotide; Electrophoresis, Capillary; Humans; Mice; Oligonucleotides; SELEX Aptamer Technique
PubMed: 33660332
DOI: 10.1002/bmc.5109 -
Proceedings of the National Academy of... Dec 2018Abundant and essential motifs, such as phosphate-binding loops (P-loops), are presumed to be the seeds of modern enzymes. The Walker-A P-loop is absolutely essential in...
Abundant and essential motifs, such as phosphate-binding loops (P-loops), are presumed to be the seeds of modern enzymes. The Walker-A P-loop is absolutely essential in modern NTPase enzymes, in mediating binding, and transfer of the terminal phosphate groups of NTPs. However, NTPase function depends on many additional active-site residues placed throughout the protein's scaffold. Can motifs such as P-loops confer function in a simpler context? We applied a phylogenetic analysis that yielded a sequence logo of the putative ancestral Walker-A P-loop element: a β-strand connected to an α-helix via the P-loop. Computational design incorporated this element into de novo designed β-α repeat proteins with relatively few sequence modifications. We obtained soluble, stable proteins that unlike modern P-loop NTPases bound ATP in a magnesium-independent manner. Foremost, these simple P-loop proteins avidly bound polynucleotides, RNA, and single-strand DNA, and mutations in the P-loop's key residues abolished binding. Binding appears to be facilitated by the structural plasticity of these proteins, including quaternary structure polymorphism that promotes a combined action of multiple P-loops. Accordingly, oligomerization enabled a 55-aa protein carrying a single P-loop to confer avid polynucleotide binding. Overall, our results show that the P-loop Walker-A motif can be implemented in small and simple β-α repeat proteins, primarily as a polynucleotide binding motif.
Topics: Adenosine Triphosphate; Amino Acid Sequence; Binding Sites; Catalytic Domain; DNA; Evolution, Molecular; Magnesium; Models, Molecular; Mutation; Nucleoside-Triphosphatase; Phosphates; Phylogeny; Polynucleotides; Protein Binding; Protein Conformation; Protein Interaction Domains and Motifs; Proteins; RNA; RNA-Binding Proteins; Sequence Alignment; Sequence Homology, Amino Acid
PubMed: 30504143
DOI: 10.1073/pnas.1812400115 -
Biochimie Nov 2018Systematic evolution of ligand by exponential enrichment (SELEX) is an efficient method used to isolate high-affinity single stranded oligonucleotides from a large... (Review)
Review
Systematic evolution of ligand by exponential enrichment (SELEX) is an efficient method used to isolate high-affinity single stranded oligonucleotides from a large random sequence pool. These SELEX-derived oligonucleotides named aptamer, can be selected against a broad spectrum of target molecules including proteins, cells, microorganisms and chemical compounds. Like antibodies, aptamers have a great potential in interacting with and binding to their targets through structural recognition and are therefore called "chemical antibodies". However, aptamers offer advantages over antibodies including smaller size, better tissue penetration, higher thermal stability, lower immunogenicity, easier production, lower cost of synthesis and facilitated conjugation or modification with different functional moieties. Thus, aptamers represent an attractive substitution for protein antibodies in the fields of biomarker discovery, diagnosis, imaging and targeted therapy. Enormous interest in aptamer technology triggered the development of SELEX that has underwent numerous modifications since its introduction in 1990. This review will discuss the recent advances in SELEX methods and their advantages and limitations. Aptamer applications are also briefly outlined in this review.
Topics: Animals; Aptamers, Nucleotide; Humans; SELEX Aptamer Technique
PubMed: 30193856
DOI: 10.1016/j.biochi.2018.09.001 -
Experimental Cell Research Feb 2023Cancer is responsible for about one in six deaths in the world. Conventional cancer treatments like chemotherapy, radiotherapy, and surgery are associated with drug... (Review)
Review
Cancer is responsible for about one in six deaths in the world. Conventional cancer treatments like chemotherapy, radiotherapy, and surgery are associated with drug poisoning and poor prognosis. Thanks to advances in RNA delivery and target selection, new cancer medicines are now conceivable to improve the quality of life and extend the lives of cancer patients. Antisense oligonucleotides (ASOs) and siRNAs are the most important tools in RNA therapies. Locked Nucleic Acids (LNAs) are one of the newest RNA analogs, exhibiting more affinity to binding, sequence specificity, thermal stability, and nuclease resistance due to their unique properties. Assays using LNA are also used in molecular diagnostic methods and provide accurate and rapid mutation detection that improves specificity and sensitivity. This study aims to review the special properties of LNA oligonucleotides that make them safe and effective antisense drugs for cancer treatment by controlling gene expression. Following that, we go over all of the molecular detection methods and cancer treatment antisense tactics that are possible with LNA technology.
Topics: Humans; Quality of Life; Oligonucleotides; Oligonucleotides, Antisense; RNA; Nucleic Acid Conformation; Neoplasms
PubMed: 36521777
DOI: 10.1016/j.yexcr.2022.113442