Thermodynamics of RNA−RNA Duplexes with 2- or 4-Thiouridines:  Implications for Antisense Design and Targeting a Group I Intron

Testa, Stephen M.; Disney, Matthew D.; Turner, Douglas H.; Kierzek, Ryszard

doi:10.1021/bi991187d

Cited by 120 publications

(156 citation statements)

References 54 publications

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“…In most cases of the series 1-15 we did not observe remarkable fluctuations in Tm or DG in comparison to the reference G2 duplex (16). These differences were much smaller than those reported earlier for shorter, 4-6-bp RNA duplexes (Luyten and Herdewijn 1998;Testa et al 1999), probably due to the negligible contribution of one or two modified bases to the 19-bp duplex stability. Nonetheless, in some cases observed changes were meaningful.…”

Section: Hybridization Studiescontrasting

confidence: 69%

“…As has been already demonstrated, the first two nucleosides, due to the preferred C39-endo conformation of the ribose ring (Agris et al 1992;Davis et al 1998), improve the thermodynamic stability of the s 2 U- (Kumar and Davis 1997;Luyten and Herdewijn 1998;Testa et al 1999;Shohda et al 2000) and the C-containing doublestranded RNA helices . Moreover, the s 2 U unit, when present in the anticodon sequence of transfer RNA, enhances its specificity due to preferred base pairing with A and restricted wobble base pairing with G (Agris et al 1992).…”

Section: Introductionmentioning

confidence: 71%

“…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).…”

Section: Chemically Modified Sirnasmentioning

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

128

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: Hybridization Studiescontrasting

confidence: 69%

Section: Introductionmentioning

confidence: 71%

Section: Chemically Modified Sirnasmentioning

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

128

126

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“…Thermodynamic data indicate that 2-thio-U (sU) forms a stronger base pair with A than with standard U by ∼1 kcal/mol, and the G:sU wobble pair is slightly destabilized relative to G:U within RNA duplexes (15). Similarly, in DNA duplexes, the A:sT base pair is stabilized relative to A:T, and the G: sT wobble pair is significantly destabilized relative to G:T (16,17).…”

Section: -Thio-t Replacement Of T Enhances Copying Of Correct Reactimentioning

confidence: 99%

Fast and accurate nonenzymatic copying of an RNA-like synthetic genetic polymer

Zhang

Blain

Zielińska

et al. 2013

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

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Recent advances suggest that it may be possible to construct simple artificial cells from two subsystems: a self-replicating cell membrane and a self-replicating genetic polymer. Although multiple pathways for the growth and division of model protocell membranes have been characterized, no self-replicating genetic material is yet available. Nonenzymatic template-directed synthesis of RNA with activated ribonucleotide monomers has led to the copying of short RNA templates; however, these reactions are generally slow (taking days to weeks) and highly error prone. N 3′ -P 5′ -linked phosphoramidate DNA (3′-NP-DNA) is similar to RNA in its overall duplex structure, and is attractive as an alternative to RNA because the high reactivity of its corresponding monomers allows rapid and efficient copying of all four nucleobases on homopolymeric RNA and DNA templates. Here we show that both homopolymeric and mixed-sequence 3′-NP-DNA templates can be copied into complementary 3′-NP-DNA sequences. G:T and A:C wobble pairing leads to a high error rate, but the modified nucleoside 2-thiothymidine suppresses wobble pairing. We show that the 2-thiothymidine modification increases both polymerization rate and fidelity in the copying of a 3′-NP-DNA template into a complementary strand of 3′-NP-DNA. Our results suggest that 3′-NP-DNA has the potential to serve as the genetic material of artificial biological systems.origin of life | nonenzymatic primer extension | artificial genetic systems | nucleotide modifications | mismatch T he phosphoramidate nucleic acids are of particular interest as potential genetic materials for artificial life-forms because of their potential for replication by the nonenzymatic polymerization of amino-sugar nucleotides. Because of the greater nucleophilicity of the amino group relative to the 3′-hydroxyl group of ribo-and deoxyribo-nucleotides, amino-sugar nucleotides exhibit more rapid spontaneous polymerization. Obviating the requirement for a polymerase greatly simplifies the task of creating and assembling the components of an artificial cell, and thus of constructing simple living systems from inanimate materials. We and others have therefore explored the synthesis of a variety of phosphoramidatelinked nucleic acids, their corresponding amino-sugar monomers, and the characterization of nonenzymatic template-directed primer extension reactions in these systems (1-11). Among these systems, we have examined 2′-amino versions of the acyclic glycerol nucleic acid (5), 2′-amino-2′,3′-dideoxyribonucleic acid (4, 6), and 3′-amino-2′,3′-dideoxyribonucleic acid (7).The structural simplicity of the acyclic sugar-phosphate nucleic acid backbones has made them attractive targets for study. Indeed, an acyclic nucleotide consisting of a glycerol-phosphate backbone linked to a formylated nucleobase (12) was among the first of such nucleic acids to be chemically synthesized, but incorporation of this nucleotide into oligomers caused a severe loss of duplex stability. Much later, the glycerol nucleic acids, in which...

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“…On comparison, the hydrogen bond between N1 of A and N3-H of s 2 U (Figure 2A, left) is only indirectly affected, rendering its stability similar to that of the U:A pair (Figure 2B, left). In summary, in the absence of the sulfur modification, the more electronegative oxygen in the 2-position of U will readily form a hydrogen bond with the hydrogen on N1 of G. This makes the U:G interaction more similar to the U:A interaction (Figure 2B) compared with the relative stability of the s 2 U:G and s 2 U:A pairs (Figure 2A) [(15) and cited references]. In addition to conferring pairing specificity, the s 2 U modification also restricts uridine dynamics by locking the nucleoside in the anti , C3′- endo conformation (16,17).…”

Section: Introductionmentioning

confidence: 99%

Stabilizing contributions of sulfur-modified nucleotides: crystal structure of a DNA duplex with 2'-O-[2-(methoxy)ethyl]-2-thiothymidines

Diop-Frimpong

Prakash²,

Rajeev³

et al. 2005

Nucleic Acids Research

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Substitution of oxygen atoms by sulfur at various locations in the nucleic acid framework has led to analogs such as the DNA phosphorothioates and 4′-thio RNA. The phosphorothioates are excellent mimics of DNA, exhibit increased resistance to nuclease degradation compared with the natural counterpart, and have been widely used as first-generation antisense nucleic acid analogs for applications in vitro and in vivo. The 4′-thio RNA analog exhibits significantly enhanced RNA affinity compared with RNA, and shows potential for incorporation into siRNAs. 2-Thiouridine (s2U) and 5-methyl-2-thiouridine (m5s2U) are natural nucleotide analogs. s2U in tRNA confers greater specificity of codon–anticodon interactions by discriminating more strongly between A and G compared with U. 2-Thio modification preorganizes the ribose and 2′-deoxyribose sugars for a C3′-endo conformation, and stabilizes heteroduplexes composed of modified DNA and complementary RNA. Combination of the 2-thio and sugar 2′-O-modifications has been demonstrated to boost both thermodynamic stability and nuclease resistance. Using the 2′-O-[2-(methoxy)ethyl]-2-thiothymidine (m5s2Umoe) analog, we have investigated the consequences of the replacement of the 2-oxygen by sulfur for base-pair geometry and duplex conformation. The crystal structure of the A-form DNA duplex with sequence GCGTAT*ACGC (T* = m5s2Umoe) was determined at high resolution and compared with the structure of the corresponding duplex with T* = m5Umoe. Notable changes as a result of the incorporation of sulfur concern the base-pair parameter ‘opening’, an improvement of stacking in the vicinity of modified nucleotides as measured by base overlap, and a van der Waals interaction between sulfur atoms from adjacent m5s2Umoe residues in the minor groove. The structural data indicate only minor adjustments in the water structure as a result of the presence of sulfur. The observed small structural perturbations combined with the favorable consequences for pairing stability and nuclease resistance (when combined with 2′-O-modification) render 2-thiouracil-modified RNA a promising candidate for applications in RNAi.

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Thermodynamics of RNA−RNA Duplexes with 2- or 4-Thiouridines: Implications for Antisense Design and Targeting a Group I Intron

Cited by 120 publications

References 54 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

Fast and accurate nonenzymatic copying of an RNA-like synthetic genetic polymer

Stabilizing contributions of sulfur-modified nucleotides: crystal structure of a DNA duplex with 2'-O-[2-(methoxy)ethyl]-2-thiothymidines

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