Antisense oligonucleotides made of 2'‐<i>O</i>‐alkylRNA: their properties and applications in RNA biochemistry

Lamond, Angus I.; Sproat, Brian S.

doi:10.1016/0014-5793(93)81427-2

Cited by 105 publications

(58 citation statements)

References 37 publications

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“…This result excludes the possibility that the growth inhibition upon si-tRF1001 transfection is due entirely to a reduction in the pre-tRNA-Ser-TGA or an off-target activity of the siRNA duplex. Instead of a plain oligoribonucleotide that did not rescue in our experimental condition, we used 29-O-methyl derivatives, because this modification is known to make the oligoribonucleotide chemically stable and resistant to cellular nucleases (for review, see Lamond and Sproat 1993).…”

Section: Trf-1001 Is Required For Cell Proliferationmentioning

confidence: 99%

A novel class of small RNAs: tRNA-derived RNA fragments (tRFs)

Lee¹,

Shibata²,

Malhotra³

et al. 2009

Genes Dev.

960

1,248

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New types of small RNAs distinct from microRNAs (miRNAs) are progressively being discovered in various organisms. In order to discover such novel small RNAs, a library of 17-to 26-base-long RNAs was created from prostate cancer cell lines and sequenced by ultra-high-throughput sequencing. A significant number of the sequences are derived from precise processing at the 59 or 39 end of mature or precursor tRNAs to form three series of tRFs (tRNA-derived RNA fragments): the tRF-5, tRF-3, and tRF-1 series. These sequences constitute a class of short RNAs that are second most abundant to miRNAs. Northern hybridization, quantitative RT-PCR, and splinted ligation assays independently measured the levels of at least 17 tRFs. To demonstrate the biological importance of tRFs, we further investigated tRF-1001, derived from the 39 end of a Ser-TGA tRNA precursor transcript that is not retained in the mature tRNA. tRF-1001 is expressed highly in a wide range of cancer cell lines but much less in tissues, and its expression in cell lines was tightly correlated with cell proliferation. siRNAmediated knockdown of tRF-1001 impaired cell proliferation with the specific accumulation of cells in G2, phenotypes that were reversed specifically by cointroducing a synthetic 29-O-methyl tRF-1001 oligoribonucleotide resistant to the siRNA. tRF-1001 is generated in the cytoplasm by tRNA 39-endonuclease ELAC2, a prostate cancer susceptibility gene. Our data suggest that tRFs are not random by-products of tRNA degradation or biogenesis, but an abundant and novel class of short RNAs with precise sequence structure that have specific expression patterns and specific biological roles.[Keywords: Small RNA; tRNA; deep sequencing; cancer cell proliferation] Supplemental material is available at http://www.genesdev.org.

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Section: Trf-1001 Is Required For Cell Proliferationmentioning

confidence: 99%

A novel class of small RNAs: tRNA-derived RNA fragments (tRFs)

Lee¹,

Shibata²,

Malhotra³

et al. 2009

Genes Dev.

960

1,248

View full text Add to dashboard Cite

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“…They were purified in one step by HPLC on a reverse phase column eluted with an acetonitrile gradient (10-50%o) in 100 mM ammonium acetate buffer pH 7.2. 2'-O-alkyl RNA-phosphodiester DNA sandwich oligonucleotides were synthesized on an Applied Biosystems synthesizer model 380B using protected 2'-O-alkylribonucleoside-3'-O-phosphoramidite monomers (6) (21).…”

Section: Oligonucleotide Synthesis and Analysismentioning

confidence: 99%

“…Methylphosphonate oligonucleotides bind weakly to complementary RNA sequences, compared to unmodified oligonucleotides, whereas 2'-O-methyl derivatives bind more strongly (6,26,27). We compared the binding of sandwich oligomers or of ODNI, to the perfect RNA target (35-55) and to a mismatched one corresponding to the (390) site of the 0-globin mRNA.…”

Section: Stability Of Synthetic Heteroduplexesmentioning

confidence: 99%

“…In our case, the sequence of the (390) RNA site on the ,3-globin message shows the limit of the first possibility: as mismatches are close to the ends of the duplexes, shortening the antisense oligonucleotide will lead to a perfect hybrid with P-globin mRNA as well as with a-mRNA. Therefore, we prepared sandwich antisense oligonucleotides with bulkier groups on the 2' position as this leads to a decreased affinity for the complementary sequence (6). Indeed 2'-O-allyl (A) and 2'-O-butyl (B) analogues of ODN2 gave lower Tm values than M-ODN2 with both the complementary sequence RNA (35-55) and with the mismatched one (Table 1).…”

Section: Effects Of Other 2'-o-alkyl Sandwichesmentioning

confidence: 99%

“…Several modifications, introduced into oligomers to reduce their sensitivity to nucleases, impaired their ability to elicit RNase H activity. This is the case with methylphosphonates (3,4), phosphoramidates (5), 2'-O-alkyl RNA (6) and alpha analogues (4,7,8). Composite oligonucleotides made of differently modified (or unmodified) parts were prepared that aimed at combining different properties within a single molecule (9)(10)(11).…”

Section: Introductionmentioning

confidence: 99%

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RNase H is responsible for the non-specific inhibition ofin vitrotranslation by 2′-O-alkyl chimeric oligonucleotides: high affinity or selectivity, a dilemma to design antisense oligomers

Larrouy¹,

Boiziau²,

Sproat

et al. 1995

Nucl Acids Res

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Ribonuclease H (RNase H) which recognizes and cleaves the RNA strand of mismatched RNA-DNA heteroduplexes can induce non-specific effects of antisense oligonucleotides. In a previous paper [Lar-rouy et a!. (1992), Gene, 121,[189][190][191][192][193][194], we demonstrated that ODN1, a phosphodiester 1 5mer targeted to the AUG initiation region of a-globin mRNA, inhibited non-specifically 3-globin synthesis in wheat germ extract due to RNase H-mediated cleavage of f-globin mRNA. Specificity was restored by using MP-ODN2, a methylphosphonate-phosphodiester sandwich analogue of ODNI, which limited RNase H activity on non-perfect hybrids. We report here that 2'-O-alkyl RNA-phosphodiester DNA sandwich analogues of ODN1, with the same phosphodiester window as MP-ODN2, are non-specific inhibitors of globin synthesis in wheat germ extract, whatever the substituent (methyl, allyl or butyl) on the 2'-OH. These sandwich oligomers induced the cleavage of non-target f-globin RNA sites, similarly to the unmodified parent oligomer ODN1. This is likely due to the increased affinity of 2'-O-alkyl-ODN2 chimeric oligomers for both fully and partly complementary RNA, compared to MP-ODN2. In contrast, the fully modified 2'-O-methyl analogue of ODN1 was a very effective and highly specific antisense sequence. This was ascribed to its inability (i) to induce RNA cleavage by RNase H and (ii) to physically prevent the elongation of the polypeptide chain.

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Chemical Stability of Nucleic Acid–Derived Drugs

Pogocki

Schöneich

2000

Journal of Pharmaceutical Sciences

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Antisense oligonucleotides made of 2'‐O‐alkylRNA: their properties and applications in RNA biochemistry

Cited by 105 publications

References 37 publications

A novel class of small RNAs: tRNA-derived RNA fragments (tRFs)

A novel class of small RNAs: tRNA-derived RNA fragments (tRFs)

RNase H is responsible for the non-specific inhibition ofin vitrotranslation by 2′-O-alkyl chimeric oligonucleotides: high affinity or selectivity, a dilemma to design antisense oligomers

Chemical Stability of Nucleic Acid–Derived Drugs

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