Structural and binding study of modified siRNAs with the Argonaute 2 PAZ domain by NMR spectroscopy

By using high-resolution NMR spectroscopy, the structures of a natural short interfering RNA (siRNA) and of several altritol nucleic acid (ANA)-modified siRNAs were determined. The interaction of modified siRNAs with the PAZ domain of the Argonaute 2 protein of Drosophila melanogaster was also studied. The structures show that the modified siRNA duplexes (ANA/RNA) adopt a geometry very similar to the naturally occurring A-type siRNA duplex. All ribose residues, except for the 3' overhang, show 3'-endo conformation. The six-membered altritol sugar in ANA occurs in a chair conformation with the nucleobase in an axial position. In all siRNA duplexes, two overhanging nucleotides at the 3' end enhance the stability of the first neighboring base pair by a stacking interaction. The first overhanging nucleotide has a rather fixed position, whereas the second overhanging nucleotide shows larger flexibility. NMR binding studies of the PAZ domain with ANA-modified siRNAs demonstrate that modifications in the double-stranded region of the antisense strand have some small effects on the binding affinity as compared with the unmodified siRNA. Modification of the 3' overhang with thymidine (dTdT) residues shows a sixfold increase in the binding affinity compared with the unmodified siRNA (relative binding affinity of 17% compared with dTdT-modified overhang), whereas modification of the 3' overhang with ANA largely decreases the binding affinity.