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出典(authority):フリー百科事典『ウィキペディア（Wikipedia）』「2014/07/25 22:01:27」(JST)

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Oligonucleotides are short, single-stranded DNA or RNA molecules that have a wide range of applications in genetic testing, research, and forensics. Commonly made in the laboratory by solid-phase chemical synthesis, these small bits of nucleic acids can be manufactured with any user-specified sequence, and so are vital for artificial gene synthesis, polymerase chain reaction (PCR), DNA sequencing, library construction and as molecular probes. In nature, oligonucleotides are usually found as small RNA molecules that function in the regulation of gene expression (e.g. microRNA), or are degradation intermediates derived from the breakdown of larger nucleic acid molecules.

Oligonucleotides are characterized by the sequence of nucleotide residues that make up the entire molecule. The length of the oligonucleotide is usually denoted by "-mer" (from Greek meros, "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 respective complementary oligonucleotides, DNA, or RNA to form duplexes or, less often, hybrids of a higher order. This basic property serves as a foundation for the use of oligonucleotides as probes for detecting DNA or RNA. Examples of procedures that use oligonucleotides include DNA microarrays, Southern blots, ASO analysis, fluorescent in situ hybridization (FISH), and the synthesis of artificial genes. Oligonucleotides are also indispensable elements in antisense therapy.

Oligonucleotides composed of 2'-deoxyribonucleotides (oligodeoxyribonucleotides) are fragments of DNA and are often used in the polymerase chain reaction, a procedure that can greatly amplify almost any small amount of DNA. There, the oligonucleotide is referred to as a primer, allowing DNA polymerase to extend the oligonucleotide and replicate the complementary strand.

Synthesis

Oligonucleotides are chemically synthesized using building blocks, protected phosphoramidites of natural or chemically modified nucleosides or, to a lesser extent, of non-nucleosidic compounds. The oligonucleotide chain assembly proceeds in the direction from 3'- to 5'-terminus 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 so that the maximum length of synthetic oligonucleotides hardly exceeds 200 nucleotide residues. HPLC and other methods can be used to isolate products with the desired sequence.

Antisense oligonucleotides

Antisense oligonucleotides are single strands of DNA or RNA that are complementary to a chosen sequence. In the case of antisense RNA they prevent protein translation of certain messenger RNA strands by binding to them. Antisense DNA can be used to target a specific, complementary (coding or non-coding) RNA. If binding takes place this DNA/RNA hybrid can be degraded by the enzyme RNase H. The use of morpholino-antisense oligonucleotides for gene knockdowns in vertebrates, which is now a standard technique in developmental biology and is used to study altered gene expression and gene function, was first developed by Janet Heasman using Xenopus. ^[1]

DNA microarray

One subtype of DNA microarrays can be described as substrates (nylon, glass, etc.) to which oligonucleotides have been bound at high density. There are a number of applications of DNA microarrays within the life sciences.

Mass spectrometry analysis

A mixture of 5-methoxysalicylic acid and spermine can be used as a matrix for oligonucleotides analysis in MALDI mass spectrometry.^[2]

External links

RNAi Atlas: a database of RNAi libraries and their target analysis results
physorg.com | Genetic source of muscular dystrophy neutralized

References

^ Beta-catenin signaling activity dissected in the early Xenopus embryo: a novel antisense approach. Heasman J, Kofron M, Wylie C. Dev. Biol. 2000. 222, 124-34
^ Distler, AM; Allison, J (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. doi:10.1016/S1044-0305(01)00212-4. PMID 11322192.

Pierce (2005). GENETICS: A Conceptual Approach.
Weiss, B., ed. (1997). Antisense Oligodeoxynucleotides and Antisense RNA : Novel Pharmacological and Therapeutic Agents. Boca Raton, FL: CRC Press.
Weiss, B; Davidkova, G; Zhou, LW (1999). "Antisense RNA gene therapy for studying and modulating biological processes". Cellular and molecular life sciences : CMLS 55 (3): 334–58. PMID 10228554.
Hartwell, L. H., Hood, L., Goldberg, M. L., Reynolds, A. E., Silver, L. M., and Veres, R. C. (2008). Genetics from Genes to Genomes (3rd ed.). p. G-14.
Spingler, Bernhard (2012). "Chapter 3. Metal-Ion-Promoted Conformational Changes of Oligonucleotides". In Astrid Sigel, Helmut Sigel and Roland K. O. Sigel. Interplay between Metal Ions and Nucleic Acids. Metal Ions in Life Sciences 10. Springer. pp. 103–118. doi:10.1007/978-94-007-2172-2_3.

UpToDate Contents

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4. 小細胞肺癌の治療に対する実験的アプローチ experimental approaches to treatment for small cell lung cancer
5. デュシェンヌ型およびベッカー型筋ジストロフィーの治療 treatment of duchenne and becker muscular dystrophy

English Journal

Gold-nanobeacons for gene therapy: evaluation of genotoxicity, cell toxicity and proteome profiling analysis.

Conde J1, Larguinho M, Cordeiro A, Raposo LR, Costa PM, Santos S, Diniz MS, Fernandes AR, Baptista PV.Author information 1CIGMH, DCV, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa , Caparica , Portugal.AbstractAntisense therapy is a powerful tool for post-transcriptional gene silencing suitable for down-regulating target genes associated to disease. Gold nanoparticles have been described as effective intracellular delivery vehicles for antisense oligonucleotides providing increased protection against nucleases and targeting capability via simple surface modification. We constructed an antisense gold-nanobeacon consisting of a stem-looped oligonucleotide double-labelled with 3'-Cy3 and 5'-Thiol-C6 and tested for the effective blocking of gene expression in colorectal cancer cells. Due to the beacon conformation, gene silencing was directly detected as fluorescence increases with hybridisation to target, which can be used to assess the level of silencing. Moreover, this system was extensively evaluated for the genotoxic, cytotoxic and proteomic effects of gold-nanobeacon exposure to cancer cells. The exposure was evaluated by two-dimensional protein electrophoresis followed by mass spectrometry to perform a proteomic profile and 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay, glutathione-S-transferase assay, micronucleus test and comet assay to assess the genotoxicity. This integrated toxicology evaluation showed that the proposed nanotheranostics strategy does not exhibit significant toxicity, which is extremely relevant when translating into in vivo systems.
Nanotoxicology.Nanotoxicology.2014 Aug;8(5):521-32. doi: 10.3109/17435390.2013.802821. Epub 2013 May 28.
Antisense therapy is a powerful tool for post-transcriptional gene silencing suitable for down-regulating target genes associated to disease. Gold nanoparticles have been described as effective intracellular delivery vehicles for antisense oligonucleotides providing increased protection against nucl
PMID 23642008

GPR30 mediates estrogen rapid signaling and neuroprotection.

Tang H1, Zhang Q2, Yang L3, Dong Y2, Khan M2, Yang F3, Brann DW4, Wang R5.Author information 1Institute of Molecular Medicine and Genetics, Medical College of Georgia, Georgia Regents University, 1120 15th Street, Augusta, GA 30912, USA; Neurobiology Institute, Medical Research Center, Hebei United University, Tangshan 063000, China.2Institute of Molecular Medicine and Genetics, Medical College of Georgia, Georgia Regents University, 1120 15th Street, Augusta, GA 30912, USA.3Neurobiology Institute, Medical Research Center, Hebei United University, Tangshan 063000, China.4Institute of Molecular Medicine and Genetics, Medical College of Georgia, Georgia Regents University, 1120 15th Street, Augusta, GA 30912, USA. Electronic address: dbrann@gru.edu.5Institute of Molecular Medicine and Genetics, Medical College of Georgia, Georgia Regents University, 1120 15th Street, Augusta, GA 30912, USA; Neurobiology Institute, Medical Research Center, Hebei United University, Tangshan 063000, China. Electronic address: minruiwang@gmail.com.AbstractG-protein-coupled estrogen receptor-30 (GPR30), also known as G-protein estrogen receptor-1 (GPER1), is a putative extranuclear estrogen receptor whose precise functions in the brain are poorly understood. Studies using exogenous administration of the GPR30 agonist, G1 suggests that GPR30 may have a neuroprotective role in cerebral ischemia. However, the physiological role of GPR30 in mediating estrogen (E2)-induced neuroprotection in cerebral ischemia remains unclear. Also unclear is whether GPR30 has a role in mediating rapid signaling by E2 after cerebral ischemia, which is thought to underlie its neuroprotective actions. To address these deficits in our knowledge, the current study examined the effect of antisense oligonucleotide (AS) knockdown of GPR30 in the hippocampal CA1 region upon E2-BSA-induced neuroprotection and rapid kinase signaling in a rat model of global cerebral ischemia (GCI). Immunohistochemistry demonstrated that GPR30 is strongly expressed in the hippocampal CA1 region and dentate gyrus, with less expression in the CA3 region. E2-BSA exerted robust neuroprotection of hippocampal CA1 neurons against GCI, an effect abrogated by AS knockdown of GPR30. Missense control oligonucleotides had no effect upon E2-BSA-induced neuroprotection, indicating specificity of the effect. The GPR30 agonist, G1 also exerted significant neuroprotection against GCI. E2-BSA and G1 also rapidly enhanced activation of the prosurvival kinases, Akt and ERK, while decreasing proapototic JNK activation. Importantly, AS knockdown of GPR30 markedly attenuated these rapid kinase signaling effects of E2-BSA. As a whole, the studies provide evidence of an important role of GPR30 in mediating the rapid signaling and neuroprotective actions of E2 in the hippocampus.
Molecular and cellular endocrinology.Mol Cell Endocrinol.2014 Apr 25;387(1-2):52-8. doi: 10.1016/j.mce.2014.01.024. Epub 2014 Mar 1.
G-protein-coupled estrogen receptor-30 (GPR30), also known as G-protein estrogen receptor-1 (GPER1), is a putative extranuclear estrogen receptor whose precise functions in the brain are poorly understood. Studies using exogenous administration of the GPR30 agonist, G1 suggests that GPR30 may have a
PMID 24594140

Position of premature termination codons determines susceptibility of hERG mutations to nonsense-mediated mRNA decay in long QT syndrome.

Gong Q1, Stump MR1, Zhou Z2.Author information 1Knight Cardiovascular Institute, Oregon Health & Science University, Portland, OR, USA.2Knight Cardiovascular Institute, Oregon Health & Science University, Portland, OR, USA. Electronic address: zhouzh@ohsu.edu.AbstractThe degradation of human ether-a-go-go-related gene (hERG, KCNH2) transcripts containing premature termination codon (PTC) mutations by nonsense-mediated mRNA decay (NMD) is an important mechanism of long QT syndrome type 2 (LQT2). The mechanisms governing the recognition of PTC-containing hERG transcripts as NMD substrates have not been established. We used a minigene system to study two frameshift mutations, R1032Gfs*25 and D1037Rfs*82. R1032Gfs*25 introduces a PTC in exon 14, whereas D1037Rfs*82 causes a PTC in the last exon (exon 15). We showed that R1032Gfs*25, but not D1037Rfs*82, reduced the level of mutant mRNA compared to the wild-type minigene in an NMD-dependent manner. The deletion of intron 14 prevented degradation of R1032Gfs*25 mRNA indicating that a downstream intron is required for NMD. The recognition and elimination of PTC-containing transcripts by NMD required that the mutation be positioned >54-60nt upstream of the 3'-most exon-exon junction. Finally, we used a full-length hERG splicing-competent construct to show that inhibition of downstream intron splicing by antisense morpholino oligonucleotides inhibited NMD and rescued the functional expression of a third LQT2 mutation, Y1078*. The present study defines the positional requirements for the susceptibility of LQT2 mutations to NMD and posits that the majority of reported LQT2 nonsense and frameshift mutations are potential targets of NMD.
Gene.Gene.2014 Apr 15;539(2):190-7. doi: 10.1016/j.gene.2014.02.012. Epub 2014 Feb 13.
The degradation of human ether-a-go-go-related gene (hERG, KCNH2) transcripts containing premature termination codon (PTC) mutations by nonsense-mediated mRNA decay (NMD) is an important mechanism of long QT syndrome type 2 (LQT2). The mechanisms governing the recognition of PTC-containing hERG tran
PMID 24530480

Japanese Journal

脊髄性筋萎縮症の最近のスプライシング病態,治療研究

佐橋健太郎,祖父江元
Brain and nerve : 神経研究の進歩 66(12), 1471-1480, 2014-12
NAID 40020289406

遺伝子の損傷診断・発現抑制・デリバリーを統合的に機能化する人工核酸の開発

中川治
YAKUGAKU ZASSHI 134(12), 1319-1329, 2014
… Artificial nucleic acids have recently been widely used with their properties optimized for various technologies such as the inhibition of gene expression (antisense/antigene strategies, RNA interference) and genetic diagnosis (single nucleotide polymorphism (SNP), damaged nucleobase). … however, there is no crucial tool for oligonucleotide therapeutics at present. …
NAID 130004712984

1P-001 穿刺中間体とアンチセンスDNAを用いた細菌の遺伝子発現抑制技術の開発(遺伝子工学,一般講演)

満留裕也,吉田ナオト
日本生物工学会大会講演要旨集 65, 18, 2013-08-25
NAID 110009737381

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リンク元	「アンチセンスオリゴ」「antisense oligodeoxynucleotide」「アンチセンスオリゴヌクレオチド」
関連記事	「antisense」「oligonucleotide」