Stability and structural features of the duplexes containing nucleoside analogues with a fixed N-type conformation, 2′-O,4′-C-methyleneribonucleosides

Obika, Satoshi; Nanbu, Daishu; Hari, Yoshiyuki; Andoh, Jun-ichi; Morio, Ken‐ichiro; Doi, Takefumi; Imanishi, Takeshi

doi:10.1016/s0040-4039(98)01084-3

Cited by 640 publications

(490 citation statements)

References 14 publications

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“…1) contains a ribose ring which is locked by a O2'-C4'-methylene linkage, imposing conformational restriction to adopt an N-type sugar puckering. [2][3][4][5]10 Structural investigation of LNA oligonucleotides by NMR spectroscopy revealed their similarities with natural nucleic acid duplexes, and confirmed the RNA mimicking structures adopted by LNA. 11,12 LNA offers key properties needed for successful therapeutic exploitation of oligonucleotides, including (1) unprecedented binding affinity towards RNA (and DNA), (2) excellent base pairing specificity, (3) high bio-stability (resistance towards nucleolytic degradation, (4) low toxicity (at least for many LNA oligonucleotides) in animals and (5) convenient chemistry for manufacturing and modification.…”

Section: Introductionmentioning

confidence: 70%

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Locked nucleic acid as a novel class of therapeutic agents

Veedu¹,

Wengel²

2009

RNA Biology

112

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Section: Introductionmentioning

confidence: 70%

“…Among the numerous modifications known, LNA has shown broad usefulness within chemical biology. [2][3][4][5][6][7][8][9] LNA: Structural Features and Key Properties LNA ( Fig. 1) contains a ribose ring which is locked by a O2'-C4'-methylene linkage, imposing conformational restriction to adopt an N-type sugar puckering.…”

Section: Introductionmentioning

confidence: 99%

Locked nucleic acid as a novel class of therapeutic agents

Veedu¹,

Wengel²

2009

RNA Biology

112

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“…LNA nucleotides contain a modified sugar residue with an additional 2′-C,4′-C-oxymethylene linker that confines the ribose ring to the 3′-endo conformation (32,33). DNA:LNA duplexes demonstrate an increased stability and are more resistant to nucleases (34,35). We report that LNA-modified ssODNs (LMOs) yielded equal efficiencies in MMR-proficient and -deficient mESCs for the substitution of one to three bases.…”

Section: Significancementioning

confidence: 99%

LNA modification of single-stranded DNA oligonucleotides allows subtle gene modification in mismatch-repair-proficient cells

Ravesteyn

Dekker

Fish

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Synthetic single-stranded DNA oligonucleotides (ssODNs) can be used to generate subtle genetic modifications in eukaryotic and prokaryotic cells without the requirement for prior generation of DNA double-stranded breaks. However, DNA mismatch repair (MMR) suppresses the efficiency of gene modification by >100-fold. Here we present a commercially available ssODN design that evades MMR and enables subtle gene modification in MMR-proficient cells. The presence of locked nucleic acids (LNAs) in the ssODNs at mismatching bases, or also at directly adjacent bases, allowed 1-, 2-, or 3-bp substitutions in MMR-proficient mouse embryonic stem cells as effectively as in MMR-deficient cells. Additionally, in MMR-proficient Escherichia coli, LNA modification of the ssODNs enabled effective single-base-pair substitution. In vitro, LNA modification of mismatches precluded binding of purified E. coli MMR protein MutS. These findings make ssODN-directed gene modification particularly well suited for applications that require the evaluation of a large number of sequence variants with an easy selectable phenotype.subtle gene modification | DNA mismatch repair | locked nucleic acid | single-stranded oligonucleotides | embryonic stem cells S ince the rapid decline in genome sequencing costs and the accumulation of data from genome-wide association studies, thousands of single-nucleotide variations in disease-related genes have been identified. One approach to functionally investigate these variants of uncertain clinical significance is to introduce them into the endogenous gene of appropriate model systems that are readily accessible to phenotypic assessment. Recently developed protocols for subtle gene modification are based on templatedirected repair of DNA double-strand breaks (DSBs) that are site-specifically introduced by zinc-finger, TALE, or RNA-directed Cas9 nucleases (1). In particular, subtle mutations can effectively be introduced by single-stranded DNA oligonucleotides (ssODNs) carrying the modification of interest that serve as templates for the repair of CRISPR/Cas9-introduced DSBs (2). Several laboratories, including ours, have previously shown that ssODNs can also be used for subtle gene modification in the absence of DSBs. Although less efficient than nuclease-assisted gene modification, oligonucleotide-directed gene modification (also referred to as "oligo targeting") is attractive due to its lack of additional components, simplicity, and cost-effectiveness, especially in cases where the consequences of the planned modifications can be scored by a selectable or easily detectable phenotype.Different models have been proposed for the mechanism of oligo targeting (reviewed in ref.3). Substantial evidence has been obtained that gene modification takes place during DNA replication (4-7). In this model, the ssODN anneals to singlestranded DNA in the replication fork, where it can serve as a primer for DNA synthesis by replicative polymerases (8). This process thus physically incorporates the ssODN and delivers the mutat...

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“…113,114 In LNA the sugar ring is conformationally locked by a O2 0 -C4 0 methylene linkage to adopt N-type sugar puckering. [135][136][137] Toward developing LNA-modified aptamers, Veedu et al reported the enzymatic recognition capabilities of LNA nucleotides using DNA and RNA polymerases. [138][139][140][141][142][143][144] In 2013, Kuwahara and co-workers www.tandfonline.comreported an LNA (BNA) aptamer against thrombin using capillary electrophoresis-based SELEX (CE-SELEX) method.…”

Section: Chemically Modified Aptamer-oligonucleotide Chimeramentioning

confidence: 99%