2‘-<i>O</i>-Aminopropyl Ribonucleotides:  A Zwitterionic Modification That Enhances the Exonuclease Resistance and Biological Activity of Antisense Oligonucleotides

Griffey, Richard H.; Monia, Brett P.; Cummins, Lendell L.; Freier, Susan M.; Greig, Michael J.; Guinosso, Charles J.; Lesnik, Elena A.; Manalili, Sherilynn; Mohan, V.; Owens, Steven; Ross, Bruce S.; Sasmor, Henri; Wancewicz, Ed; Weiler, K.; Wheeler, Patrick; Cook, P. Dan

doi:10.1021/jm950937o

Cited by 129 publications

(112 citation statements)

References 23 publications

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“…The sugar conformation was C3Ј-endo,2Ј-exo and the underlying duplex showed only slight distortion (33). This substitution also conferred high nuclease resistance on the TFO (32,34). Although the AE TFOs were not tested in bioassays their properties would seem consistent with activity in cell-based assays.…”

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confidence: 99%

Targeted Gene Knockout by 2′-O-Aminoethyl Modified Triplex Forming Oligonucleotides

Puri¹,

Majumdar²,

Cuenoud³

et al. 2001

Journal of Biological Chemistry

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2؉ . Once formed they were also more stable in "physiological" buffer, with the greatest affinity and stability displayed by the TFO with all but one residue in the AE format. However, TFOs with lesser amounts of the AE modification formed the most stable triplexes in vivo, and showed the highest HPRT gene knockout activity. We conclude that the AE modification can enhance the biological activity of pyrimidine TFOs, but that extensive substitution is deleterious.

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confidence: 99%

Targeted Gene Knockout by 2′-O-Aminoethyl Modified Triplex Forming Oligonucleotides

Puri¹,

Majumdar²,

Cuenoud³

et al. 2001

Journal of Biological Chemistry

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“…The 2Ј-modified class of compounds also has proven to be valuable because of its enhanced nuclease resistance (4). Examples are the 2Ј-O-alkoxyalkyl compounds (8)(9)(10)(11) and 2Ј-O-aminopropyl (AP) RNA (4,12) (Fig. 1).…”

mentioning

confidence: 99%

“…However, the AP modification leads to much better protection than the 2Ј-O-propyl one, despite similar lengths of the substituents. ODNs containing short stretches of AP-RNA at their 3Ј ends display extraordinary resistance against degradation by 3Ј exonucleases (12) (Fig. 2).…”

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confidence: 99%

Structural origins of the exonuclease resistance of a zwitterionic RNA

Teplova

Wallace

Tereshko

et al. 1999

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

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Nuclease resistance and RNA affinity are key criteria in the search for optimal antisense nucleic acid modifications, but the origins of the various levels of resistance to nuclease degradation conferred by chemical modification of DNA and RNA are currently not understood. The 2-O-aminopropyl (AP)-RNA modification displays the highest nuclease resistance among all phosphodiester-based analogues and its RNA binding affinity surpasses that of phosphorothioate DNA by 1°C per modified residue. We found that oligodeoxynucleotides containing AP-RNA residues at their 3 ends competitively inhibit the degradation of single-stranded DNA by the Escherichia coli Klenow fragment (KF) 3-5 exonuclease and snake venom phosphodiesterase. To shed light on the origins of nuclease resistance brought about by the AP modification, we determined the crystal structure of an A-form DNA duplex with AP-RNA modifications at 1.6-Å resolution. In addition, the crystal structures of complexes between short DNA fragments carrying AP-RNA modifications and wild-type KF were determined at resolutions between 2.2 and 3.0 Å and compared with the structure of the complex between oligo(dT) and the D355A͞E357A KF mutant. The structural models suggest that interference of the positively charged 2-O-substituent with the metal ion binding site B of the exonuclease allows AP-RNA to effectively slow down degradation.antisense ͉ cationic oligonucleotides ͉ exonuclease inhibitor ͉ hydration ͉ x-ray crystallography S econd-generation oligonucleotide analogues for use as antisense (AS) therapeutics are now emerging (1, 2). Two improvements over the first-generation DNA phosphorothioates (PS-DNA) are of particular importance. First, several of the modifications confer increased affinity toward target RNA. Second and perhaps more importantly, they have increased ability to evade nuclease degradation. In the quest to achieve both goals, a large number of compounds with modifications located in the backbone, sugar, or base portion of the nucleic acid framework were introduced (3). Among the sites available for modification, the 2Ј position of the deoxyribose sugar has shown particular promise (4). Attachment of electron withdrawing moieties at the 2Ј carbon shifts the conformational equilibrium of the sugar toward the C3Ј-endo pucker, thus preorganizing the AS oligonucleotide for binding to the RNA target (5-7). The 2Ј-modified class of compounds also has proven to be valuable because of its enhanced nuclease resistance (4). Examples are the 2Ј-O-alkoxyalkyl compounds (8-11) and 2Ј-O-aminopropyl (AP) RNA (4, 12) (Fig. 1).Protection of AS oligodeoxynucleotides (ODNs) against 3Ј exonucleases appears to be of particular concern in view of practical applications in vivo or in cell cultures (9). The nuclease resistance conferred by simple 2Ј-O-alkyl substituents is correlated with the length of the alkyl chain (3,13,14). Longer and more bulky substituents lead to improved protection and the resistance of the 2Ј-O-pentyl modification is similar to that of PS-DNA. Howeve...

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“…1 H, 13 C, 31 P NMR spectra were recorded with a Bruker DRX 300 MHz spectrometer; 19 F NMR spectra were measured with a Bruker Avance II + 600 MHz spectrometer equipped with a TCI Prodigy Cryo-Probe. The chemical shifts are referenced to the residual proton signal of the deuterated solvents: CDCl 3 ( 1 H: δ = 7.26 ppm, 13 C: δ = 77.1 ppm), [D 6 ]DMSO ( 1 H: δ = 2.5 ppm, 13 C: δ = 39.5 ppm), 31 P shifts are relative to external 85% phosphoric acid, 19 F shifts are relative to external CCl 3 F. 1 Hand 13 C-assignments were based on COSY and HSQC experiments. Compound 3 (1.0 g, 2.28 mmol) was coevaporated with pyridine (6 mL) and subsequently dissolved in anhydrous pyridine (7 mL).…”

Section: Experimental Section Generalmentioning

confidence: 99%

An Unconventional Acid‐Labile Nucleobase Protection Concept for Guanosine Phosphoramidites in RNA Solid‐Phase Synthesis

Jud

Micura

2017

Chemistry A European J

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We present an innovative O 6 -tert-butyl/N 2 -tert-butyloxycarbonyl protection concept for guanosine (G) phosphoramidites. This concept is advantageous for 2′-modified G building blocks because of very efficient synthetic access when compared with existing routes that usually employ O 6 -(4-nitrophenyl)ethyl/N 2 -acyl protection or that start from 2-aminoadenosine involving enzymatic transformation into guanosine later on in the synthetic path. The new phosphoramidites are fully compatible with 2′-O-tBDMS or TOM phosphoramidites in standard RNA solid-phase synthesis and deprotection, and provide excellent quality of tailored RNAs for the growing range of applications in RNA biophysics, biochemistry, and biology.

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2‘-O-Aminopropyl Ribonucleotides: A Zwitterionic Modification That Enhances the Exonuclease Resistance and Biological Activity of Antisense Oligonucleotides

Cited by 129 publications

References 23 publications

Targeted Gene Knockout by 2′-O-Aminoethyl Modified Triplex Forming Oligonucleotides

Targeted Gene Knockout by 2′-O-Aminoethyl Modified Triplex Forming Oligonucleotides

Structural origins of the exonuclease resistance of a zwitterionic RNA

An Unconventional Acid‐Labile Nucleobase Protection Concept for Guanosine Phosphoramidites in RNA Solid‐Phase Synthesis

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