Structural origins of the exonuclease resistance of a zwitterionic RNA

Abstract

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...

Enhanced splice correction by 3′, 5′-serinol and 2′-(ω-O-methylserinol) guarded OMe-RNA/DNA mixmers in cells

“…24 The amino pendant group in the minor grove as in 2′-O-(2-aminoethyl)-substituent was earlier found to be responsible for displacing the essential cations in the hydrolytic enzyme binding site, thus inhibiting the enzyme activity. 25 As expected, when (2′-R-AMP) modification was introduced in DNA oligomers, the AONs were found to be as good as 2′-MOE oligomers in terms of efficiency of duplex formation, along with much higher resistance to enzymatic degradation compared with 2′-MOE oligomers. 24 In this article, we now present the synthesis of protected-(2-amino-1,3-dihydroxypropyl) monomer unit from l-serine, as a universal serinol cap to the oligomers at 3′, 5′-ends and the 2′-O-R-AMP-ribothymidine monomer to increase the enzymatic resistance of 2′-OMe RNA without disturbing the efficacy of duplex formation.…”

Inhibition of Klenow DNA polymerase and poly(A)-specific ribonuclease by aminoglycosides

“…In this report, we demonstrate that aminoglycosides inhibit the catalytic activity of two metalloenzymes, Klenow pol and PARN+ We propose that the aminoglycoside binds the enzymes in the vicinity of their active sites and thereby inhibits their catalytic activities+ In analogy with the inhibition of ribozyme activity by aminoglycosides (see Rogers et al+, 1996;Hermann & Westhof, 1998;Hoch et al+, 1998;Walter et al+, 1999;Mikkelsen et al+, 1999Mikkelsen et al+, , 2001, and references therein) we suggest that the inhibition is caused by the aminoglycoside distorting the active site and/or displacing functionally important divalent metal ions+ The kinetic data showing that neomycin B is a mixed noncompetitive inhibitor of both enzymes (Fig+ 3) is compatible with this model of inhibition+ Further support is provided by the observation that neomycin B perturbed the iron-mediated hydroxyl radical cleavage reactions in the vicinity of the active sites of both enzymes (Fig+ 4)+ Finally, the kinetic evidence that the neomycin B inhibition of PARN was released in a competitive manner by Mg(II) ions (Fig+ 3) is in direct line with this proposal+ It has previously been suggested that neomycin B inhibits DNA polymerase I (Lazarus & Kitron, 1973) and DNase I of E. coli (Woegerbauer et al+, 2000) by binding to the DNA substrates+ However, our results provide no (II) ions compared to the amount of aminoglycoside required to inhibit PARN (e+g+, the Mg(II) ion concentration was more than 1,000-fold higher than the K i value for neomycin B) argues that the chelating properties of aminoglycosides were not causing the inhibition+ Taken together we favor a model of inhibition where the aminoglycoside binds the active site of the enzyme and thereby interferes with its function+ Furthermore, it is plausible that the aminoglycoside binding site overlaps with the binding sites for essential divalent metal ions and that aminoglycoside binding displaces functionally important metal ions+ This model of inhibition resembles models for inhibition of ribozyme activities by aminoglycosides (see Hermann & Westhof, 1998;Hoch et al+, 1998;Mikkelsen et al+, 1999Mikkelsen et al+, , 2001, and references therein) and is analogous to how the amino group of 29-O-aminopropyl substituted RNA directly interferes with divalent metal ion binding at the exonuclease active site of Klenow pol (Teplova et al+, 1999)+…”

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

“…That TFO displayed a dramatic enhancement of target affinity and stability of the resultant triplex. AE-02 contained 6 contiguous 2Ј-AE residues at the 3Ј end and was designed with the expectation that this organization would confer enhanced binding as well as resistance to 3Ј-exonuclease activity (48). The AE-03 TFO retained the motif of a 3Ј patch of AE combined with a 5Ј patch.…”