Structure of the Trinucleotide D-acp<sup>3</sup>U-A with Coordinated Mg<sup>2+</sup>Demonstrates that Modified Nucleosides Contribute to Regional Conformations of RNA

Stuart, J.W.; Basti, Mufeed M.; Smith, Wanda S.; Forrest, Brian; Guenther, Richard; Sierzputowska-Gracz, Hanna; Nawrot, Barbara; Małkiewicz, Andrzej; Agris, Paul F.

doi:10.1080/07328319608002031

Cited by 21 publications

(31 citation statements)

References 26 publications

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“…We assumed that the introduction of the C and s 2 U units, due to their well-documented positive influence on the A-type RNA helix structure (Kumar and Davis 1997;Davis et al 1998;Testa et al 1999;Diop-Frimpong et al 2005), would enhance siRNA duplex stability and silencing activity. In contrast, dihydrouridine disturbs the structure of a single-stranded RNA (Dalluge et al 1996;Stuart et al 1996). According to our best knowledge there have been no data demonstrating the influence of dihydrouridine on the structure of double-stranded RNA, nor any detailed reports on the influence of the rare nucleobases, used in the present investigation, on the silencing activity of siRNA duplexes.…”

Section: Chemically Modified Sirnasmentioning

confidence: 58%

“…Such a change was expected since, as mentioned above, a D nucleoside destabilizes the RNA structure (Dalluge et al 1996;Stuart et al 1996). Therefore, its insertion in the central part of the siRNA duplex has significant structural consequences.…”

Section: Hybridization Studiesmentioning

confidence: 99%

“…The decreased stability of the sG2/ aD10 duplex (7) was expected to be due to the presence of a nonaromatic nucleobase disrupting base stacking. Moreover, dihydrouridine preferentially adopts the B-DNA type of sugar ring conformation (C29-endo) and thus causes pronounced changes in the A-type RNA structure (Westhof et al 1988;Stuart et al 1996;Davis 1998). Even less stability was observed for the sG2/aW9 duplex (10), originating from the stronger structural perturbation of base stacking with the Watson-Crick base pairs situated at both sides of the wobble mismatch (Mizuno and Sundaralingam 1978;Varani and McClain 2000).…”

Section: Hybridization Studiesmentioning

confidence: 99%

“…Introduction of the s 2 U unit into the siRNA structure also seems to be profitable due to increased hydrophobicity of sulfur-modified molecules which might exhibit elevated cellular uptake (Cummins et al 1995). The dihydrouridine unit destabilizes the RNA structure as it prefers the C29-endo ribose ring conformation and induces the B-DNA-type sugar ring conformation (Stuart et al 1996). In addition, the D nucleoside contains a nonaromatic base ring that excludes stabilizing base stacking with neighboring nucleotides within the RNA strand (Westhof et al 1988;Davis 1998).…”

Section: Introductionmentioning

confidence: 99%

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Effect of base modifications on structure, thermodynamic stability, and gene silencing activity of short interfering RNA

Sipa

Sochacka

Kaźmierczak-Barańska

et al. 2007

RNA

129

126

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A series of nucleobase-modified siRNA duplexes containing ''rare'' nucleosides, 2-thiouridine (s 2 U), pseudouridine (C), and dihydrouridine (D), were evaluated for their thermodynamic stability and gene silencing activity. The duplexes with modified units at terminal positions exhibited similar stability as the nonmodified reference. Introduction of the s 2 U or C units into the central part of the antisense strand resulted in duplexes with higher melting temperatures (Tm). In contrary, D unit similarly like wobble base pair led to the less stable duplexes (DTm 3.9 and 6.6°C, respectively). Gene-silencing activity of siRNA duplexes directed toward enhanced green fluorescent protein or beta-site APP cleaving enzyme was tested in a dual fluorescence assay. The duplexes with s 2 U and C units at their 39-ends and with a D unit at their 59-ends (with respect to the guide strands) were the most potent gene expression inhibitors. Duplexes with s 2 U and C units at their 59-ends were by 50% less active than the nonmodified counterpart. Those containing a D unit or wobble base pair in the central domain had the lowest Tm, disturbed the A-type helical structure, and had more than three times lower activity than their nonmodified congener. Activity of siRNA containing the wobble base pair could be rescued by placing the thio-nucleoside at the position 39-adjacent to the mutation site. Thermally stable siRNA molecules containing several s 2 U units in the antisense strand were biologically as potent as their native counterparts. The present results provide a new chemical tool for modulation of siRNA gene-silencing activity.

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Section: Chemically Modified Sirnasmentioning

confidence: 58%

Section: Hybridization Studiesmentioning

confidence: 99%

Section: Hybridization Studiesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Effect of base modifications on structure, thermodynamic stability, and gene silencing activity of short interfering RNA

Sipa

Sochacka

Kaźmierczak-Barańska

et al. 2007

RNA

129

126

View full text Add to dashboard Cite

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“…The effect of side chain substitution by the 3-amino-3-carboxy moiety at N-3 of uridine was studied by nuclear magnetic resonance spectroscopy and found to result in only a small influence on the C3Ј-endo/C2Ј-endo ribose conformer population (43). However, evidence from nuclear magnetic resonance spectroscopy that acp 3 U binds Mg 2ϩ in small oligonucleotides suggests a possible role for acp 3 U in the stabilization of regional RNA structure (44).…”

Section: Resultsmentioning

confidence: 99%

Identities and Phylogenetic Comparisons of Posttranscriptional Modifications in 16 S Ribosomal RNA from Haloferax volcanii

Kowalak¹,

Bruenger²,

Crain³

et al. 2000

Journal of Biological Chemistry

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Small subunit (16 S) rRNA from the archaeon Haloferax volcanii, for which sites of modification were previously reported, was examined using mass spectrometry. A census of all modified residues was taken by liquid chromatography/electrospray ionization-mass spectrometry analysis of a total nucleoside digest of the rRNA. Following rRNA hydrolysis by RNase T 1 , accurate molecular mass values of oligonucleotide products were measured using liquid chromatography/electrospray ionization-mass spectrometry and compared with values predicted from the corresponding gene sequence. Three modified nucleosides, distributed over four conserved sites in the decoding region of the molecule, were characterized: 3-(3-amino-3-carboxypropyl)uridine-966, N 6 -methyladenosine-1501, and N 6 ,N 6 -dimethyladenosine-1518 and -1519 (all Escherichia coli numbering). Nucleoside 3-(3-amino-3-carboxypropyl)uridine, previously unknown in rRNA, occurs at a highly conserved site of modification in all three evolutionary domains but for which no structural assignment in archaea has been previously reported. Nucleoside N 6 -methyladenosine, not previously placed in archaeal rRNAs, frequently occurs at the analogous location in eukaryotic small subunit rRNA but not in bacteria. H. volcanii small subunit rRNA appears to reflect the phenotypically low modification level in the Crenarchaeota kingdom and is the only cytoplasmic small subunit rRNA shown to lack pseudouridine.

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Preparation of Short Interfering RNA Containing the Modified Nucleosides 2‐Thiouridine, Pseudouridine, or Dihydrouridine

Nawrot

Sochacka

2009

CP Nucleic Acid Chemistry

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Modified uridine derivatives such as 2-thiouridine (s(2)U), pseudouridine (Psi), and dihydrouridine (D) are naturally existing nucleoside units identified in tRNA molecules. Recently, we have shown that such base-modified units introduced into functionally important sites of siRNA modulate thermodynamic stability of the duplex and its gene silencing activity. In this unit, we describe chemical synthesis of 3'-phosphoramidite derivatives of s(2)U and D units (the 3'-phosphoramidite derivative of Psi is commercially available), and their use for the synthesis of RNA oligonucleotides according to the routine phosphoramidite protocol. The only exception concerns the oxidation step with I(2)/pyridine/water which, if applied towards oligonucleotides containing s(2)U units, would lead to the loss of sulfur. Therefore, to avoid this side reaction, tert-butyl hydroperoxide is used as an oxidizing reagent. After the oligonucleotide chain assembly is completed, the resulting oligomer is deprotected under mild basic conditions (MeNH(2)/EtOH/DMSO) to avoid dihydrouracil ring opening, which is a reported side-reaction during the routine synthesis of dihydrouridine-containing RNA. Oligonucleotides modified with s(2)U, D, or Psi units are useful models for structure-function studies. Here, the procedure for preparation of siRNA duplexes is described.

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Structure of the Trinucleotide D-acp³U-A with Coordinated Mg²⁺Demonstrates that Modified Nucleosides Contribute to Regional Conformations of RNA

Cited by 21 publications

References 26 publications

Effect of base modifications on structure, thermodynamic stability, and gene silencing activity of short interfering RNA

Effect of base modifications on structure, thermodynamic stability, and gene silencing activity of short interfering RNA

Identities and Phylogenetic Comparisons of Posttranscriptional Modifications in 16 S Ribosomal RNA from Haloferax volcanii

Preparation of Short Interfering RNA Containing the Modified Nucleosides 2‐Thiouridine, Pseudouridine, or Dihydrouridine

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Structure of the Trinucleotide D-acp3U-A with Coordinated Mg2+Demonstrates that Modified Nucleosides Contribute to Regional Conformations of RNA

Cited by 21 publications

References 26 publications

Effect of base modifications on structure, thermodynamic stability, and gene silencing activity of short interfering RNA

Effect of base modifications on structure, thermodynamic stability, and gene silencing activity of short interfering RNA

Identities and Phylogenetic Comparisons of Posttranscriptional Modifications in 16 S Ribosomal RNA from Haloferax volcanii

Preparation of Short Interfering RNA Containing the Modified Nucleosides 2‐Thiouridine, Pseudouridine, or Dihydrouridine

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Structure of the Trinucleotide D-acp³U-A with Coordinated Mg²⁺Demonstrates that Modified Nucleosides Contribute to Regional Conformations of RNA