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Oligonucleotide

From Wikipedia, the free encyclopedia

Oligonucleotidesare shortDNAorRNAmolecules,oligomers,that have a wide range of applications ingenetic testing,research,andforensics.Commonly made in the laboratory bysolid-phase chemical synthesis,[1]these small fragments of nucleic acids can be manufactured as single-stranded molecules with any user-specified sequence, and so are vital forartificial gene synthesis,polymerase chain reaction(PCR),DNA sequencing,molecular cloningand asmolecular probes.In nature, oligonucleotides are usually found as small RNA molecules that function in the regulation of gene expression (e.g.microRNA),[2]or are degradation intermediates derived from the breakdown of larger nucleic acid molecules.

Oligonucleotides are characterized by thesequenceofnucleotideresidues that make up the entire molecule. The length of the oligonucleotide is usually denoted by "-mer"(fromGreekmeros,"part" ). For example, an oligonucleotide of six nucleotides (nt) is a hexamer, while one of 25 nt would usually be called a "25-mer". Oligonucleotides readily bind, in a sequence-specific manner, to their respectivecomplementaryoligonucleotides, DNA, or RNA to formduplexesor, less often, hybrids of a higher order. This basic property serves as a foundation for the use of oligonucleotides asprobesfor detecting specific sequences of DNA or RNA. Examples of procedures that use oligonucleotides includeDNA microarrays,Southern blots,ASO analysis,[3]fluorescent in situ hybridization(FISH),PCR,and the synthesis of artificial genes.

Oligonucleotides are composed of2'-deoxyribonucleotides(oligodeoxyribonucleotides), which can be modified at the backbone or on the 2' sugar position to achieve different pharmacological effects. These modifications give new properties to the oligonucleotides and make them a key element inantisense therapy.[4][5]

Synthesis

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Oligonucleotides are chemically synthesized using building blocks, protectedphosphoramiditesof natural or chemically modifiednucleosidesor, to a lesser extent, of non-nucleosidic compounds. The oligonucleotide chain assembly proceeds in the 3' to 5' direction by following a routine procedure referred to as a "synthetic cycle". Completion of a single synthetic cycle results in the addition of one nucleotide residue to the growing chain. A less than 100% yield of each synthetic step and the occurrence of side reactions set practical limits of the efficiency of the process. In general, oligonucleotide sequences are usually short (13–25 nucleotides long).[6]The maximum length of synthetic oligonucleotides hardly exceeds 200 nucleotide residues.HPLCand other methods can be used to isolate products with the desired sequence.[citation needed]

Chemical modifications

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Creating chemically stable short oligonucleotides was the earliest challenge in developing ASO therapies. Naturally occurring oligonucleotides are easily degraded by nucleases, an enzyme that cleaves nucleotides and is ample in every cell type.[7]Short oligonucleotide sequences also have weak intrinsic binding affinities, which contributes to their degradation in vivo.[8]

Backbone modifications

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Nucleosideorganothiophosphate(PS) analogs of nucleotides give oligonucleotides some beneficial properties. Key beneficial properties that PS backbones give nucleotides arediastereomeridentification of each nucleotide and the ability to easily follow reactions involving the phosphorothioate nucleotides, which is useful in oligonucleotide synthesis.[9]PS backbone modifications to oligonucleotides protects them against unwanted degradation by enzymes.[10]Modifying the nucleotide backbone is widely used because it can be achieved with relative ease and accuracy on most nucleotides.[9]Fluorescent modifications on 5' and 3' end of oligonucleotides was reported to evaluate the oligonucleotides structures, dynamics and interactions with respect to environment.[11]

Sugar ring modifications

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Another modification that is useful for medical applications of oligonucleotides is2' sugar modifications.Modifying the 2' position sugar increases the effectiveness of oligonucleotides by enhancing the target binding capabilities of oligonucleotides, specifically inantisense oligonucleotides therapies.[8]They also decrease non specific protein binding, increasing the accuracy of targeting specific proteins.[8]Two of the most commonly used modifications are 2'-O-methyl and the 2'-O-methoxyethyl.[8]Fluorescent modifications on the nucleobase was also reported.[11]

Antisense oligonucleotides

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Antisense oligonucleotides (ASO) are single strands of DNA or RNA that are complementary to a chosen sequence.[6]In the case ofantisense RNAthey preventprotein translationof certainmessenger RNAstrands by binding to them, in a process calledhybridization.[12]Antisense oligonucleotides can be used to target a specific, complementary (coding ornon-coding) RNA. If binding takes place this hybrid can be degraded by the enzymeRNase H.[12]RNase H is an enzyme that hydrolyzes RNA, and when used in an antisense oligonucleotide application results in 80-95% down-regulation of mRNA expression.[6]

The use ofMorpholinoantisense oligonucleotides for gene knockdowns invertebrates,which is now a standard technique indevelopmental biologyand is used to study alteredgene expressionand gene function, was first developed by Janet Heasman usingXenopus.[13]FDA-approved Morpholino drugs includeeteplirsenandgolodirsen.The antisense oligonucleotides have also been used to inhibit influenza virus replication in cell lines.[14][15]

Neurodegenerative diseases that are a result of a single mutant protein are good targets for antisense oligonucleotide therapies because of their ability to target and modify very specific sequences of RNA with high selectivity.[3]Many genetic diseases includingHuntington's disease,Alzheimer's disease,Parkinson's disease,andamyotrophic lateral sclerosis(ALS) have been linked to DNA alterations that result in incorrect RNA sequences and result in mistranslated proteins that have a toxic physiological effect.[16]

Cell internalisation

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Cell uptake/internalisation still represents the biggest hurdle towards successful oligonucleotide (ON) therapeutics. A straightforward uptake, like for most small-molecule drugs, is hindered by the polyanionic backbone and the molecular size of ONs. The exact mechanisms of uptake and intracellular trafficking towards the place of action are still largely unclear. Moreover, small differences in ON structure/modification (vide supra) and difference in cell type leads to huge differences in uptake. It is believed that cell uptake occurs on different pathways after adsorption of ONs on the cell surface. Notably, studies show that most tissue culture cells readily take up ASOs (phosphorothiote linkage) in a non-productive way, meaning that no antisense effect is observed. In contrast to that conjugation of ASO with ligands recognised by G-coupled receptors leads to an increased productive uptake.[17]Next to that classification (non-productive vs. productive), cell internalisation mostly proceeds in an energy-dependant way (receptor mediated endocytosis) but energy-independent passive diffusion (gymnosis) may not be ruled out. After passing the cell membrane, ON therapeutics are encapsulated in earlyendosomeswhich are transported towards late endosomes which are ultimately fused withlysosomescontaining degrading enzymes at low pH.[18]To exert its therapeutic function, the ON needs to escape the endosome prior to its degradation. Currently there is no universal method to overcome the problems of delivery, cell uptake and endosomal escape, but there exist several approaches which are tailored to specific cells and their receptors.[19]

A conjugation of ON therapeutics to an entity responsible for cell recognition/uptake not only increases the uptake (vide supra) but is also believed to decrease the complexity of the cell uptake as mainly one (ideally known) mechanism is then involved.[18]This has been achieved with small molecule-ON conjugates for example bearing anN-acetyl galactosaminewhich targets receptors ofhepatocytes.[20]These conjugates are an excellent example for obtaining an increased cell uptake paired with targeted delivery as the corresponding receptors are overexpressed on the target cells leading to a targeted therapeutic (compare antibody-drug conjugates which exploit overexpressed receptors on cancer cells).[19]Another broadly used and heavily investigated entity for targeted delivery and increased cell uptake of oligonucleotides areantibodies.

Analytical techniques

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Chromatography

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Alkylamidescan be used aschromatographicstationary phases.[21]Those phases have been investigated for the separation of oligonucleotides.[22]Ion-pair reverse-phase high-performance liquid chromatography is used to separate and analyse the oligonucleotides after automated synthesis.[23]

Mass spectrometry

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A mixture of5-methoxysalicylic acidandsperminecan be used as a matrix for oligonucleotides analysis inMALDImass spectrometry.[24]ElectroSpray Ionization Mass Spectrometry (ESI-MS) is also a powerful tool to characterize the mass of oligonucleotides.[25]

DNA microarray

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DNA microarrays are a useful analytical application of oligonucleotides. Compared to standardcDNA microarrays,oligonucleotide based microarrays have more controlled specificity over hybridization, and the ability to measure the presence and prevalence of alternatively spliced orpolyadenylatedsequences.[26]One subtype of DNA microarrays can be described as substrates (nylon, glass, etc.) to which oligonucleotides have been bound at high density.[27]There are a number ofapplications of DNA microarrayswithin the life sciences.[citation needed]

See also

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  • Aptamers,oligonucleotides with important biological applications
  • Morpholinos,oligos with non-natural backbones, which do not activate RNase-H but can reduce gene expression or modify RNA splicing
  • Polymorphism,the appearance in a population of the same gene in multiple forms because of mutations; can often be tested with ASO probes
  • CpG Oligodeoxynucleotide,an ODN with immunostimulatory properties
  • Polypurine reverse-Hoogsteen hairpins,PPRHs, oligonucleotides that can bind either DNA or RNA and decrease gene expression.

References

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  1. ^Yang J, Stolee JA, Jiang H, Xiao L, Kiesman WF, Antia FD, et al. (October 2018). "Solid-Phase Synthesis of Phosphorothioate Oligonucleotides Using Sulfurization Byproducts for in Situ Capping".The Journal of Organic Chemistry.83(19): 11577–11585.doi:10.1021/acs.joc.8b01553.PMID30179468.S2CID52157806.
  2. ^Qureshi A, Thakur N, Monga I, Thakur A, Kumar M (1 January 2014)."VIRmiRNA: a comprehensive resource for experimentally validated viral miRNAs and their targets".Database.2014:bau103.doi:10.1093/database/bau103.PMC4224276.PMID25380780.
  3. ^abMonga I, Qureshi A, Thakur N, Gupta AK, Kumar M (2017)."ASPsiRNA: A Resource of ASP-siRNAs Having Therapeutic Potential for Human Genetic Disorders and Algorithm for Prediction of Their Inhibitory Efficacy".G3: Genes, Genomes, Genetics.7(9): 2931–2943.doi:10.1534/g3.117.044024.PMC5592921.PMID28696921.
  4. ^Weiss, B., ed. (1997). Antisense Oligodeoxynucleotides and Antisense RNA: Novel Pharmacological and Therapeutic Agents. Boca Raton, Florida: CRC Press
  5. ^Weiss B, Davidkova G, Zhou LW (1999)."Antisense RNA gene therapy for studying and modulating biological processes".Cellular and Molecular Life Sciences.55(3): 334–58.doi:10.1007/s000180050296.PMC11146801.PMID10228554.S2CID9448271.
  6. ^abcDias N, Stein CA (March 2002)."Antisense oligonucleotides: basic concepts and mechanisms".Molecular Cancer Therapeutics.1(5): 347–55.PMID12489851.
  7. ^Frazier KS (January 2015). "Antisense oligonucleotide therapies: the promise and the challenges from a toxicologic pathologist's perspective".Toxicologic Pathology.43(1): 78–89.doi:10.1177/0192623314551840.PMID25385330.S2CID37981276.
  8. ^abcdDeVos SL, Miller TM (July 2013)."Antisense oligonucleotides: treating neurodegeneration at the level of RNA".Neurotherapeutics.10(3): 486–97.doi:10.1007/s13311-013-0194-5.PMC3701770.PMID23686823.
  9. ^abEckstein F (April 2000). "Phosphorothioate oligodeoxynucleotides: what is their origin and what is unique about them?".Antisense & Nucleic Acid Drug Development.10(2): 117–21.doi:10.1089/oli.1.2000.10.117.PMID10805163.
  10. ^Stein CA, Subasinghe C, Shinozuka K, Cohen JS (April 1988)."Physicochemical properties of phosphorothioate oligodeoxynucleotides".Nucleic Acids Research.16(8): 3209–21.doi:10.1093/nar/16.8.3209.PMC336489.PMID2836790.
  11. ^abMichel BY, Dziuba D, Benhida R, Demchenko AP, Burger A (2020)."Probing of Nucleic Acid Structures, Dynamics, and Interactions With Environment-Sensitive Fluorescent Labels".Frontiers in Chemistry.8:112.Bibcode:2020FrCh....8..112M.doi:10.3389/fchem.2020.00112.PMC7059644.PMID32181238.
  12. ^abCrooke ST (April 2017)."Molecular Mechanisms of Antisense Oligonucleotides".Nucleic Acid Therapeutics.27(2): 70–77.doi:10.1089/nat.2016.0656.PMC5372764.PMID28080221.
  13. ^Heasman J, Kofron M, Wylie C (June 2000)."Beta-catenin signaling activity dissected in the early Xenopus embryo: a novel antisense approach".Developmental Biology.222(1): 124–34.doi:10.1006/dbio.2000.9720.PMID10885751.
  14. ^Kumar P, Kumar B, Rajput R, Saxena L, Banerjea AC, Khanna M (November 2013). "Cross-protective effect of antisense oligonucleotide developed against the common 3' NCR of influenza A virus genome".Molecular Biotechnology.55(3): 203–11.doi:10.1007/s12033-013-9670-8.PMID23729285.S2CID24496875.
  15. ^Kumar B, Khanna M, Kumar P, Sood V, Vyas R, Banerjea AC (May 2012). "Nucleic acid-mediated cleavage of M1 gene of influenza A virus is significantly augmented by antisense molecules targeted to hybridize close to the cleavage site".Molecular Biotechnology.51(1): 27–36.doi:10.1007/s12033-011-9437-z.PMID21744034.S2CID45686564.
  16. ^Smith RA, Miller TM, Yamanaka K, Monia BP, Condon TP, Hung G, et al. (August 2006)."Antisense oligonucleotide therapy for neurodegenerative disease".The Journal of Clinical Investigation.116(8): 2290–6.doi:10.1172/JCI25424.PMC1518790.PMID16878173.
  17. ^Ming, Xin; Alam, Md Rowshon; Fisher, Michael; Yan, Yongjun; Chen, Xiaoyuan; Juliano, Rudolph L. (2010-06-15)."Intracellular delivery of an antisense oligonucleotide via endocytosis of a G protein-coupled receptor".Nucleic Acids Research.38(19): 6567–6576.doi:10.1093/nar/gkq534.ISSN1362-4962.PMC2965246.PMID20551131.
  18. ^abHawner, Manuel; Ducho, Christian (2020-12-16)."Cellular Targeting of Oligonucleotides by Conjugation with Small Molecules".Molecules.25(24): 5963.doi:10.3390/molecules25245963.ISSN1420-3049.PMC7766908.PMID33339365.
  19. ^abCrooke, S. T. (2017)."Cellular uptake and trafficking of antisense oligonucleotides".Nat. Biotechnol.35(3): 230–237.doi:10.1038/nbt.3779.PMID28244996.S2CID1049452.
  20. ^Prakash, Thazha P.; Graham, Mark J.; Yu, Jinghua; Carty, Rick; Low, Audrey; Chappell, Alfred; Schmidt, Karsten; Zhao, Chenguang; Aghajan, Mariam; Murray, Heather F.; Riney, Stan; Booten, Sheri L.; Murray, Susan F.; Gaus, Hans; Crosby, Jeff (July 2014)."Targeted delivery of antisense oligonucleotides to hepatocytes using triantennary N-acetyl galactosamine improves potency 10-fold in mice".Nucleic Acids Research.42(13): 8796–8807.doi:10.1093/nar/gku531.ISSN1362-4962.PMC4117763.PMID24992960.
  21. ^Buszewski B, Kasturi P, Gilpin RK, Gangoda ME, Jaroniec M (August 1994). "Chromatographic and related studies of alkylamide phases".Chromatographia.39(3–4): 155–61.doi:10.1007/BF02274494.S2CID97825477.
  22. ^Buszewski B, Safaei Z, Studzińska S (January 2015)."Analysis of oligonucleotides by liquid chromatography with alkylamide stationary phase".Open Chemistry.13(1).doi:10.1515/chem-2015-0141.
  23. ^Gilar, M.; Fountain, K. J.; Budman, Y.; Neue, U. D.; Yardley, K. R.; Rainville, P. D.; Russell Rj, 2nd; Gebler, J. C. (2002-06-07)."Ion-pair reversed-phase high-performance liquid chromatography analysis of oligonucleotides:: Retention prediction".Journal of Chromatography A.958(1–2): 167–182.doi:10.1016/S0021-9673(02)00306-0.ISSN0021-9673.PMID12134814.{{cite journal}}:CS1 maint: numeric names: authors list (link)
  24. ^Distler AM, Allison J (April 2001). "5-Methoxysalicylic acid and spermine: a new matrix for the matrix-assisted laser desorption/ionization mass spectrometry analysis of oligonucleotides".Journal of the American Society for Mass Spectrometry.12(4): 456–62.Bibcode:2001JASMS..12..456D.doi:10.1016/S1044-0305(01)00212-4.PMID11322192.S2CID18280663.
  25. ^Shah S, Friedman SH (March 2008). "An ESI-MS method for characterization of native and modified oligonucleotides used for RNA interference and other biological applications".Nature Protocols.3(3): 351–6.doi:10.1038/nprot.2007.535.PMID18323805.S2CID2093309.
  26. ^Relógio A, Schwager C, Richter A, Ansorge W, Valcárcel J (June 2002)."Optimization of oligonucleotide-based DNA microarrays".Nucleic Acids Research.30(11): 51e–51.doi:10.1093/nar/30.11.e51.PMC117213.PMID12034852.
  27. ^Gong P, Harbers GM, Grainger DW (April 2006). "Multi-technique comparison of immobilized and hybridized oligonucleotide surface density on commercial amine-reactive microarray slides".Analytical Chemistry.78(7): 2342–51.doi:10.1021/ac051812m.PMID16579618.

Further reading

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  • Spingler B (January 2012). "Chapter 3. Metal-Ion-Promoted Conformational Changes of Oligonucleotides". In Sigel A, Sigel H, Sigel RK (eds.).Interplay between metal ions and nucleic acids.Vol. 10. Springer Science & Business Media. pp. 103–118.doi:10.1007/978-94-007-2172-2_3.PMID22210336.{{cite book}}:|journal=ignored (help)
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