Characterization of fully 2'-modified oligoribonucleotide hetero- and homoduplex hybridization and nuclease sensitivity

Cummins, Lendell L.; Owens, Stephen R.; Risen, Lisa M.; Lesnik, Elena A.; Freier, Sue; McGee, Danny P. C.; Guinosso, Charles J.; Cook, P. Dan

doi:10.1093/nar/23.11.2019

Cited by 341 publications

(293 citation statements)

References 18 publications

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“…In our system, the HBD forms a relatively rigid secondary structure stabilized by four disulfide bonds. In addition, the secondary structure of the aptamer is thermodynamically stabilized by the 2Јfluoro modifications (8), which greatly enhance helix formation because of their strong preference for the 3Јendo sugar pucker (30). Therefore, this VEGF aptamer may be more structured in the free state than most other RNA-based aptamers.…”

Section: The Local Structure Of the Hbd-aptamer Complex Is Conserved mentioning

confidence: 99%

A therapeutic aptamer inhibits angiogenesis by specifically targeting the heparin binding domain of VEGF ₁₆₅

Lee

Canny²,

Erkenez³

et al. 2005

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

213

170

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Aptamers recognize their targets with extraordinary affinity and specificity. The aptamer-based therapeutic, Macugen, is derived from a modified 2 fluoro pyrimidine RNA inhibitor to vascular endothelial growth factor (VEGF) and is now being used to treat the wet form of age-related macular degeneration. This VEGF 165 aptamer binds specifically to the VEGF165 isoform, a dimeric protein with a receptor-binding domain and a heparin-binding domain (HBD). To understand the molecular recognition between VEGF and this aptamer, binding experiments were used to show that the HBD contributes the majority of binding energy in the VEGF 165-aptamer complex. A tissue culture-based competition assay demonstrated that the HBD effectively competes with VEGF165 for aptamer binding in vivo. Comparison of NMR spectra revealed that structural features of the smaller HBD-aptamer complex are present in the full-length VEGF 164-aptamer complex. These data show that the HBD provides the binding site for the aptamer and is the primary determinant for the affinity and specificity in the VEGF 165-aptamer complex.age-related macular degeneration ͉ Macugen ͉ RNA ͉ NMR

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Section: The Local Structure Of the Hbd-aptamer Complex Is Conserved mentioning

confidence: 99%

A therapeutic aptamer inhibits angiogenesis by specifically targeting the heparin binding domain of VEGF ₁₆₅

Lee

Canny²,

Erkenez³

et al. 2005

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

213

170

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“…BR13-11M, which contains chemically modified nucleotides at all locations, except for the two nucleotides bracketing the bond cleaved in BR13, was attacked at the solitary nonprotected phosphodiester bond, indicating that extensive 2Ј-O-methyl modification does not prevent ribonucleolytic cleavage at nonmodified bonds (see ref. 43). …”

Section: Rne Cleaves Oligoribonucleotides Containing Specific Sequencmentioning

confidence: 99%

The catalytic domain of RNase E shows inherent 3′ to 5′ directionality in cleavage site selection

Yin

Vickers

Cohen

2002

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

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RNase E, a multifunctional endoribonuclease of Escherichia coli, attacks substrates at highly specific sites. By using synthetic oligoribonucleotides containing repeats of identical target sequences protected from cleavage by 2 -O-methylated nucleotide substitutions at specific positions, we investigated how RNase E identifies its cleavage sites. We found that the RNase E catalytic domain (i.e., N-Rne) binds selectively to 5 -monophosphate RNA termini but has an inherent mode of cleavage in the 3 to 5 direction. Target sequences made uncleavable by the introduction of 2 -O-methylmodified nucleotides bind to RNase E and impede cleavages at normally susceptible sites located 5 to, but not 3 to, the protected target. Our results indicate that RNase E can identify cleavage sites by a 3 to 5 ''scanning'' mechanism and imply that anchoring of the enzyme to the 5 -monophosphorylated end of these substrates orients the enzyme for directional cleavages that occur in a processive or quasiprocessive mode. In contrast, we find that RNase G, which has extensive structural homology with and size similarity to N-Rne, and can functionally complement RNase E gene deletions when overexpressed, has a nondirectional and distributive mode of action.has a demonstrated role in the processing of ribosomal RNA (1, 2), the chemical degradation of bulk cellular RNA (3-7), the decay of specific regulatory, messenger, and structural RNAs (for recent reviews, see refs. 8 and 9), the control of plasmid DNA replication (10), and the removal of poly(A) tails from transcripts (11,12). RNase E cleaves preferentially at specific sites in single-strand RNA segments rich in AϩU nucleotides (13)(14)(15)(16)). An inherent mode of action involving entry of RNase E at the 5Ј end of substrates followed by a 5Ј to 3Ј wave of cleavages has been inferred from the greater in vivo stability of fragments at the 3Ј ends of certain RNA substrates (17-20) together with evidence that (i) the enzyme prefers a free 5Ј end for endonucleolytic cleavage (18,20), (ii) RNase E degradation efficiency is affected by 5Ј-phosphorylation in vivo (10) and in vitro (19,20), and (iii) 5Ј regions of secondary structure can impede cleavages in vivo (21). However, RNA degradation in vivo also can occur in the opposite direction (e.g., ref. 22).As RNase E cleavage of complex substrates potentially can be affected by differences in the affinity of the enzyme for target sites having different sequences (15, 16), by secondary structure (23), and in vivo also by the attachment of ribosomes (24-26) and͞or RNA-binding proteins (27), conclusions about the inherent mode of action of RNase E from studies of long natural substrates may be problematical. We wished to elucidate the mechanism of target-site selection by RNase E in the absence of these confounding factors; to do this, we designed, and chemically synthesized, oligoribonucleotide substrates that contain repeats of identical target sequences and are devoid of regions able to engage in base paring. We found that the RNase E catalytic...

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“…All the oligonucleotides were characterized by ESMS, and purities were assessed by HPLC and capillary gel electrophoresis. Table 1) (12,13,19). It is interesting to note that modifications with the possibility of extended gauche effects such as in 2′-O-MOE (Figure 2: D, 2′-O-FET; E, 2′-O-TFE; I, 2′-O-DMAOE; J, 2′-O-MAOE; and K, 2′-O-IME) exhibited higher Exonuclease Stability.…”

Section: Synthesis Of 2′-o-modified Nucleosides and Oligonucleotidesmentioning

confidence: 99%

“…It is interesting to note that modifications with the possibility of extended gauche effects such as in 2′-O-MOE (Figure 2: D, 2′-O-FET; E, 2′-O-TFE; I, 2′-O-DMAOE; J, 2′-O-MAOE; and K, 2′-O-IME) exhibited higher Exonuclease Stability. The exonuclease stability of the 2′-modified oligonucleotide O was evaluated using the snake venom phosphodiesterase (SVPD) assay (19) (Figure 3). The modifications in O were placed at the 3′-end, and all internucleosidic linkages were phosphodiesters.…”

Section: Synthesis Of 2′-o-modified Nucleosides and Oligonucleotidesmentioning

confidence: 99%

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Probing the Influence of Stereoelectronic Effects on the Biophysical Properties of Oligonucleotides: Comprehensive Analysis of the RNA Affinity, Nuclease Resistance, and Crystal Structure of Ten 2‘-O-Ribonucleic Acid Modifications^,

et al. 2005

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The syntheses of 10 new RNA 2′-O-modifications, their incorporation into oligonucleotides, and an evaluation of their properties such as RNA affinity and nuclease resistance relevant to antisense activity are presented. All modifications combined with the natural phosphate backbone lead to significant gains in terms of the stability of hybridization to RNA relative to the first-generation DNA phosphorothioates (PS-DNA). The nuclease resistance afforded in particular by the 2′-O-modifications carrying a positive charge surpasses that of PS-DNA. However, small electronegative 2′-O-substituents, while enhancing the RNA affinity, do not sufficiently protect against degradation by nucleases. Similarly, oligonucleotides containing 3′-terminal residues modified with the relatively large 2′-O-[2-(benzyloxy)ethyl] substituent are rapidly degraded by exonucleases, proving wrong the assumption that steric bulk will generally improve protection against nuclease digestion. To analyze the factors that contribute to the enhanced RNA affinity and nuclease resistance we determined crystal structures of self-complementary A-form DNA decamer duplexes containing single 2′-O-modified thymidines per strand. Conformational preorganization of substituents, favorable electrostatic interactions between substituent and sugar-phosphate backbone, and a stable water structure in the vicinity of the 2′-O-modification all appear to contribute to the improved RNA affinity. Close association of positively charged substituents and phosphate groups was observed in the structures with modifications that protect most effectively against nucleases. The promising properties exhibited by some of the analyzed 2′-O-modifications may warrant a more detailed evaluation of their potential for in vivo antisense applications. Chemical modification of RNA can also be expected to significantly improve the efficacy of small interfering RNAs (siRNA). Therefore, the 2′-O-modifications introduced here may benefit the development of RNAi therapeutics.The search for nucleic acid analogues with optimal properties for potential applications as antisense therapeutics has prompted the synthesis and biochemical characterization of hundreds of modifications over the past decade (1). The first-generation phosphorothioate oligodeoxynucleotides (PS-DNA) offer a number of advantages, such as ease of synthesis, nuclease resistance sufficient for parenteral administration, activation of RNase H for clearing the target RNA, and sufficiently large binding to cellular and serum proteins for uptake, absorption, and distribution (2). However, PS-DNAs also revealed limitations with regard to their use as antisense therapeutics because of their relatively low binding affinity to RNA (ca. 1°C < DNA per modified nucleotide), the inhibition of RNase H at high concentration, and their nonspecific binding to proteins. Moreover, PSDNAs do not penetrate the blood-brain barrier and they show poor oral bioavailability (3).Among the potential sites for chemical modification available i...

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Characterization of fully 2'-modified oligoribonucleotide hetero- and homoduplex hybridization and nuclease sensitivity

Cited by 341 publications

References 18 publications

A therapeutic aptamer inhibits angiogenesis by specifically targeting the heparin binding domain of VEGF ₁₆₅

A therapeutic aptamer inhibits angiogenesis by specifically targeting the heparin binding domain of VEGF ₁₆₅

The catalytic domain of RNase E shows inherent 3′ to 5′ directionality in cleavage site selection

Probing the Influence of Stereoelectronic Effects on the Biophysical Properties of Oligonucleotides: Comprehensive Analysis of the RNA Affinity, Nuclease Resistance, and Crystal Structure of Ten 2‘-O-Ribonucleic Acid Modifications^,

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Characterization of fully 2'-modified oligoribonucleotide hetero- and homoduplex hybridization and nuclease sensitivity

Cited by 341 publications

References 18 publications

A therapeutic aptamer inhibits angiogenesis by specifically targeting the heparin binding domain of VEGF 165

A therapeutic aptamer inhibits angiogenesis by specifically targeting the heparin binding domain of VEGF 165

The catalytic domain of RNase E shows inherent 3′ to 5′ directionality in cleavage site selection

Probing the Influence of Stereoelectronic Effects on the Biophysical Properties of Oligonucleotides: Comprehensive Analysis of the RNA Affinity, Nuclease Resistance, and Crystal Structure of Ten 2‘-O-Ribonucleic Acid Modifications,

Contact Info

Product

Resources

About

A therapeutic aptamer inhibits angiogenesis by specifically targeting the heparin binding domain of VEGF ₁₆₅

A therapeutic aptamer inhibits angiogenesis by specifically targeting the heparin binding domain of VEGF ₁₆₅

Probing the Influence of Stereoelectronic Effects on the Biophysical Properties of Oligonucleotides: Comprehensive Analysis of the RNA Affinity, Nuclease Resistance, and Crystal Structure of Ten 2‘-O-Ribonucleic Acid Modifications^,