Structural Studies of LNA:RNA Duplexes by NMR: Conformations and Implications for RNase H Activity

Bondensgaard, Kent; Petersen, Michael; Singh, Sanjay K.; Rajwanshi, Vivek K.; Kumar, Ravindra; Wengel, Jesper; Jacobsen, Jens Peter

doi:10.1002/1521-3765(20000804)6:15<2687::aid-chem2687>3.0.co;2-u

Cited by 177 publications

(69 citation statements)

References 23 publications

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“…Commonly used LNA contains a methylene bridge connecting the 29 oxygen with the 49 carbon of the ribose ring. This bridge locks the ribose ring in the 39-endo conformation characteristic of RNA (Bondensgaard et al 2000;Braasch and Corey 2001). LNA has been shown to be compatible with siRNA intracellular machinery, preserving molecule integrity while offering several improvements that are relevant to the development of siRNA technology (Braasch et al 2003;Elmen et al 2005).…”

Section: -Sugar Modificationssupporting

confidence: 92%

Hydrophobization and bioconjugation for enhanced siRNA delivery and targeting

Paula¹,

Bentley²,

Mahato³

2007

RNA

204

162

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RNA interference (RNAi) is an evolutionarily conserved process by which double-stranded small interfering RNA (siRNA) induces sequence-specific, post-transcriptional gene silencing. Unlike other mRNA targeting strategies, RNAi takes advantage of the physiological gene silencing machinery. The potential use of siRNA as therapeutic agents has attracted great attention as a novel approach for treating severe and chronic diseases. RNAi can be achieved by either delivery of chemically synthesized siRNAs or endogenous expression of small hairpin RNA, siRNA, and microRNA (miRNA). However, the relatively high dose of siRNA required for gene silencing limits its therapeutic applications. This review discusses several strategies to improve therapeutic efficacy as well as to abrogate off-target effects and immunostimulation caused by siRNAs. There is an in-depth discussion on various issues related to the (1) mechanisms of RNAi, (2) methods of siRNA production, (3) barriers to RNAi-based therapies, (4) biodistribution, (5) design of siRNA molecules, (6) chemical modification and bioconjugation, (7) complex formation with lipids and polymers, (8) encapsulation into lipid particles, and (9) target specificity for enhanced therapeutic effectiveness.

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Section: -Sugar Modificationssupporting

confidence: 92%

Hydrophobization and bioconjugation for enhanced siRNA delivery and targeting

Paula¹,

Bentley²,

Mahato³

2007

RNA

204

162

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“…The effect of the northern-biased modifications on human RNase H1 activity was further supported by the LNAs, which are locked in the northern C3-endo pucker and resulted in slower cleavage rates when positioned at the DNA/MOE junctions (Figs. 2 and 8B) (Bondensgaard et al, 2000).…”

Section: Discussionmentioning

confidence: 99%

The Positional Influence of the Helical Geometry of the Heteroduplex Substrate on Human RNase H1 Catalysis

Lima¹,

Rose²,

Nichols³

et al. 2006

Mol Pharmacol

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In a companion study published in this issue (p. 83), we showed that chimeric substrates containing 2Ј-methoxyethyl (MOE) nucleotides inhibited human RNase H1 activity. In this study, we prepared chimeric substrates containing a central DNA region with flanking northern-biased MOE nucleotides hybridized to complementary RNA. Conformationally biased and flexible modified nucleotides were positioned at the junctions between the DNA and MOE residues of the chimeric substrates to modulate the effects of the MOE residues on human RNase H1 activity. The strong northern-biased locked-nucleic acid modification exacerbated the negative effects of the MOE modifications resulting in slower human RNase H1 cleavage rates. Enhanced cleavage rates were observed for the eastern-biased 2Ј-ara-fluorothymidine and bulge inducing N-methylthymidine modifications positioned at the 5Ј-DNA/3Ј-MOE junction as well as the southern-biased 2Ј-methylthiothymidine and conformationally flexible tetrafluoroindole (TFI) modifications positioned at the 5Ј-MOE/3Ј-DNA junction. The heterocycle of the ribonucleotide opposing the TFI deoxyribonucleotide had no effect on the human RNase H1 activity, whereas nucleotide substitutions adjacent the TFI significantly affected the cleavage rate. Mismatch base pair(s) exhibited similar effects on human RNase H1 activity as the TFI modifications. The effects of the TFI modification and mismatch base pair(s) on human RNase H1 activity were influenced by the position of the modification relative to the nucleotides interacting with the catalytic site of the enzyme rather than the juxtaposition of the modification to the MOE residues. Finally, these results provide a method for enhancing the human RNase H1 activity of chimeric antisense oligonucleotides (ASO) as well as the design of more potent ASO drugs.Human RNase H1 has been shown to play a dominant role in the activity of DNA-like antisense oligonucleotides . In several cell lines, the human RNase H1 protein was both overexpressed and reduced using DNA-like antisense oligonucleotides (ASOs) and small interfering RNAs targeting the mRNA of the enzyme. The effects of these manipulations on the potencies of a number of DNA-like ASOs to several different target RNAs showed that increasing the level and activity of human RNase H1 increased the potency of the ASOs and reducing the level of the enzyme resulted in a commensurate reduction in the potency of the ASOs . Moreover, overexpression of human RNase H1 in mouse liver increased the potency of a DNA-like ASO targeting Fas after intravenous administration.We have demonstrated that human RNase H1 is composed of an RNA-binding domain, a spacer region, and a catalytic domain . Once bound to the heteroduplex substrate, the RNA binding and the catalytic domains are separated by approximately one helical turn; the RNA binding domain is positioned 3Ј on the RNA relative to the catalytic domain (Lima et al., 2003). The RNA-binding domain is responsible for the strong positional preference for cleavage exhibited...

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“…The fact that LNA structurally mimics the C3-endo sugar conformation that characterizes RNA (47), one wonders whether the designed gapmers activate a RNA interference -mediated silencing mechanism of the BCR/ABL oncogene. In fact, the designed LNA-DNA gapmers suppress the expression of BCR/ABL as efficiently as siRNA does (32 -35).…”

Section: Discussionmentioning

confidence: 99%

Antisense locked nucleic acids efficiently suppress BCR/ABL and induce cell growth decline and apoptosis in leukemic cells

Rapozzi

Cogoi²,

Xodo³

2006

Molecular Cancer Therapeutics

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Chronic myeloid leukemia (CML) develops when a hematopoietic stem cell acquires the Philadelphia chromosome carrying the BCR/ABL fusion gene. This gives the transformed cells a proliferative advantage over normal hematopoietic cells. Silencing the BCR/ABL oncogene by treatment with specific drugs remains an important therapeutic goal. In this work, we used locked nucleic acid (LNA) -modified oligonucleotides to silence BCR/ABL and reduce CML cell proliferation, as these oligonucleotides are resistant to nucleases and exhibit an exceptional affinity for cognate RNA. The anti-BCR/ABL oligonucleotides were designed as LNA-DNA gapmers, consisting of end blocks of 3/4 LNA monomers and a central DNA stretch of 13/14 deoxyribonucleotides. The gapmers were complementary to the b2a2 and b3a2 mRNA junctions with which they form hybrid duplexes that have melting temperatures of 79°C and 75°C, respectively, in a 20 mmol/L NaCl-buffered (pH 7.4) solution. Like DNA, the designed LNA-DNA gapmers were capable of activating RNase H and promote cleavage of the target b2a2 and b3a2 BCR/ABL mRNAs. The treatment of CML cells with junction-specific antisense gapmers resulted in a strong and specific reduction of the levels of BCR/ABL transcripts (f20% of control) and protein p210 BCR/ABL

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Structural Studies of LNA:RNA Duplexes by NMR: Conformations and Implications for RNase H Activity

Cited by 177 publications

References 23 publications

Hydrophobization and bioconjugation for enhanced siRNA delivery and targeting

Hydrophobization and bioconjugation for enhanced siRNA delivery and targeting

The Positional Influence of the Helical Geometry of the Heteroduplex Substrate on Human RNase H1 Catalysis

Antisense locked nucleic acids efficiently suppress BCR/ABL and induce cell growth decline and apoptosis in leukemic cells

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