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...