Crystal structure, stability and Ago2 affinity of phosphorodithioate-modified RNAs

Department of Biochemistry, Vanderbilt Uni 37232, USA. E-mail: martin.egli@vanderbil 343-8070 AM Biotechnologies LLC, 12521 Gulf Free xianbin.yang@thioaptamer.com; Fax: +1-83 Department of Bioorganic Chemistry, Ce Studies, Polish Academy of Sciences, Lodz, P Sigma Life Science, 9186 Six Pines, The Wo † Electronic supplementary information including crystallographic experiments illustrations of EphA2 siRNA (Fig. S1), s duplexes (Fig. S2), CD spectra (Fig. S3 structures of Ago2–siRNA and PAZ–si 10.1039/c4ra10986d Cite this: RSC Adv., 2014, 4, 64901

To gain insight into the conformational consequences of PS2 modication and potential changes as a result of combining it with the ribose 2 0 -O-methyl substitution, we determined crystal structures of the RNA duplexes (CGC PS2 GAAUUAGCG) 2 (C PS2 G-RNA), (CGCGA MePS2 AUUAGCG) 2 (A MePS2 A-RNA), and (CGCGAAUUA MePS2 GCG) 2 (A MePS2 G-RNA) at resolutions of 1.19, 1.18 and 1.13 Å, respectively.‡ The structures were phased by molecular replacement, using the previously determined structure of the native RNA 16 as search model [ESI (Table S1 †)].
The structures of the PS2-modied RNA duplexes are isomorphous with that of the native RNA. 16Thus, the duplex is located on a crystallographic dyad and a single dodecamer strand constitutes the asymmetric unit.Residues are numbered 1-12 and those in the symmetry-related strand are numbered 1 # -12 # (Fig. 2).Superimposition of the C PS2 G and native RNA duplexes demonstrates their virtually identical conformations (Fig. S2 †).In particular PS2 modication does not trigger any changes in the ribose conformation and the backbone torsion angles.The most obvious deviation concerns the P-S bond lengths in the phosphorodithioate moiety (ca.1.94 Å) compared to the P-O bonds in the native phosphate (ca.1.60 Å) (Fig. S2 †).
In the A MePS2 A-and A MePS2 G-RNA duplexes, 2 0 -O-methyl groups are directed into the minor groove, a result of the familiar ap orientation around the C2 0 -O2 0 bond (Fig. 3). 17,18herefore, neither the PS2 modication alone nor the combination of the PS2 and 2 0 -O-methyl modications appear to have a notable effect on the conformation of A-form RNA judging from the crystallographic results.
However, the 2 0 -O-methyl modication can offset the slight thermodynamic destabilization caused by the PS2 moiety compared with the native RNA duplex. 10We determined the melting temperatures (T m ) of siRNAs containing two to six MePS2 modications and found that they exhibit thermal    stabilities that are comparable to that of the unmodied RNA (PO2) (Table 1).
To assess potential conformational consequences of the MePS2 modication in solution, we measured the circular dichroism (CD) spectra of the siRNA duplexes listed in Table 1.All spectra were closely similar to the spectrum of the native duplex and are consistent with the typical A-type structure (a maximum of the positive Cotton effect at 268 nm and a crossover point at 240 nm; Fig. S3 †).
The lack of an obvious change in both the conformation and stability of MePS2-RNA relative to native RNA points to another cause of the favorable therapeutic properties of this modication. 15To better understand the increased Ago2 affinity of siRNA containing MePS2-modied sense strands (Fig. S1 †), we built models of PAZ-siRNA complexes † based on the crystal structure of a short RNA fragment featuring a 3 0 two-residue overhang bound to the human Ago2 PAZ domain. 19This domain is most likely the only region of Ago2 affected by MePS2 modication near the siRNA 3 0 -end (Fig. S4 †).Comparison of the complexes with siRNA and MePS2-siRNA reveals formation of a hydrophobic patch in the latter, that involves both the 2 0 -O-methyl and PS2 moieties as well as methionine and cysteine side chains from the PAZ domain (Fig. 4).The hypothesis that increased hydrophobicity conferred by the MePS2 modication plays a key role in the stronger binding to Ago2 and its enhanced efficacy is supported by the relative Ago2 affinities of siRNAs containing no modication, PS, MePS, PS2 or MePS2 (Table 2).

Conclusions
The MePS2 modication in the backbone region adjacent to the 3 0 -dTdT overhang of the siRNA sense strand triggers a strongly enhanced affinity to the RISC Ago2 slicer and silencing of proteins that confer chemoresistance in ovarian cancer cell lines and tumors. 15This nding is surprising given the fact that the antisense siRNA strand mediates cleavage of the target RNA at the Ago2 active site and indicates that chemical modication of the sense strand can play a key role in siRNA loading and efficacy.
We demonstrate here that MePS2 modication does not affect RNA conformation and stability.Instead the increased Ago2 affinity is likely the result of favourable hydrophobic interactions with the PAZ domain mediated by both the 2 0 -Omethyl and PS2 moieties.This conclusion is consistent with the inability of PS2 sulphur atoms to form H-bonds with water molecules lining the RNA backbone in the crystal structure (Fig. 5).
Both steric and electronic factors can contribute to the increased nuclease resistance afforded by a modication. 18,20,21he bulkier sulphur atoms in the PS2 moiety along with the increased hydrophobicity relative to PO2 might exclude nonthiophilic metal ions (e.g.Mg 2+ ) from an exonuclease active site or result in suboptimal positioning of the water nucleophile for attack at the phosphate group.

Fig. 2
Fig. 2 Crystal structure of the C PS2 G-RNA duplex.(A) View across the major and minor grooves.Fourier Fo-Fc difference electron density (2.5s level) around O1P and O2P of G4 and G16 (indicating the presence of sulfur) is depicted as a green meshwork, terminal residues are labeled, and water molecules are red spheres.(B) Close-up view of residue G4 with difference density around O1P and O2P before incorporation of sulfur.(C) View into the central major groove with sulfur atoms colored in yellow.

Fig. 3
Fig. 3 Close-up views of the MePS2-modified backbones in the (A) A MePS2 A-and (B) A MePS2 G duplexes.Fourier (2Fo-Fc) electron density (1.2s level) is depicted as a green meshwork, and 2 0 -O-methyl carbon and sulfur atoms are highlighted in cyan and yellow, respectively.

Fig. 4
Fig. 4 Models of the interactions between the human Ago2 PAZ domain and the (A) MePS2-modified and (B) native siRNA sense strands.Only the 3 0 -terminal five residues of the RNA are shown (n-4 to n) and the TT-overhang is visible on the upper right in both panels.The formation of a hydrophobic patch between the Met-273 and Cys-270 side chains, methoxy moiety of G n-2 and PS2 of T n-1 is highlighted with a dashed circle in panel A. PS2 groups, sulfur atoms (yellow) and 2 0 -O-methyl carbons (cyan) are depicted in ball-and-stick mode and hydrogen atoms are white spheres of smaller radius.Note the significantly more polar environment of the phosphate (His, 2Â Tyr, Lys) between overhanging dTs, visible on the right-hand side of the two panels.

Fig. 5
Fig.5The PS2 moiety disrupts H-bonding interactions in the water chain bridging adjacent O2P atoms in the major groove.

Table 1
Melting temperatures T m of native and MePS2-siRNAs

Table 2
Human Ago2 binding affinities of EphA2 siRNAs with various