Nuclease resistance and RNA affinity are key criteria in the search for opt
imal antisense nucleic acid modifications, but the origins of the various l
evels of resistance to nuclease degradation conferred by chemical modificat
ion of DNA and RNA are currently not understood. The 2'-O-aminopropyl (AP)-
RNA modification displays the highest nuclease resistance among all phospho
diester-based analogues and its RNA binding affinity surpasses that of phos
phorothioate DNA by 1 degrees C per modified residue. We found that oligode
oxynucleotides containing AP-RNA residues at their 3' ends competitively in
hibit the degradation of single-stranded DNA. by the Escherichia coli Kleno
w fragment (KF) 3'-5' exonuclease and snake venom phosphodiesterase. To she
d light on the origins of nuclease resistance brought about by the AP modif
ication, we determined the crystal structure of an A-form DNA duplex with A
P-RNA modifications at 1.6-Angstrom resolution. In addition, the crystal st
ructures of complexes between short DNA fragments carrying AP-RNA modificat
ions and wild-type KF were determined at resolutions between 2.2 and 3.0 An
gstrom and compared with the structure of the complex between oligo(dT) and
the D355A/E357A 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.