DNA oligonucleotide synthesis, crystallization and data collection
The RNA sequences were examined initially, and unfortunately, satisfactory crystals and diffraction were not obtained. The Se
and ) has been used to facilitate the crystal growth and maintain the RNA-like conformation, since the 2′-SeMe functionality (2′-Se-facilitator) does not cause structure perturbation. (13
) Thus, we have synthesized 3a
dU)-ACAC-3′; molecular formula: C78
: measured 2505.6 (calculated 2505.8)}, 3b
dU)ACAC-3′; molecular formula: C78
: measured 2520.6 (calculated 2521.1)} and 3c
dU)ACAC-3′; molecular formula: C78
: measured 2568.6 (calculated 2569.0)}. The 5-OMe-, 5-SMe- and 5-SeMe-dU functionalities were introduced into the oligonucleotides by using the corresponding phosphoramidites synthesized from the modified nucleosides (28–30
). In addition to its facilitation of crystal growth, the 2′-SeMe functionality can drive the DNA sequences into A-form conformation and structure. Therefore, this RNA-mimic system can serve as a useful model for the structure studies of RNA/RNA interactions and duplexes, which are usually difficult to crystallize and offer high-resolution structures. As expected, these three modified oligonucleotides were crystallized in 3–4 days and with high-diffraction quality, when screening with the Nucleic Acid Mini-kit (Hampton Research). Since the crystallization buffer conditions may affect the crystal packing and molecular interaction, the same buffer for 3a
was selected for the crystallization. Furthermore, by the diffraction screening, buffer no. 7 of the kit (10% v/v 2-Methyl-2,4-pentanediol (MPD), 40
mM sodium cacodylate, pH 6.0, 12
mM spermine tetra-HCl, 80
mM potassium chloride and 20
mM magnesium chloride) was identified to give the highest resolutions for these three DNA structures. Several large crystals (up to 0.2
mm in size, A) from the same buffer were found and mounted for diffraction data collection on X-ray beamline. These DNA molecules crystallized in the same space group (P43
2) as the native. The detailed data collection and refinement statistics information for each sequence are presented in and .
X-ray data collection of 5-OMe, 5-SMe and 5-SeMe-containing DNAs [5′-G(SedU)G(mx5dU)ACAC-3′]
Refinement statistics of 5-OMe-, 5-SMe- and 5-SeMe-containing DNAs [5′-G(SedU)G(mx5dU)ACAC-3′]
Structure comparison of 5-OMe, 5-SMe and 5-SeMe oligonucleotides
As showed in the duplex structures ( and ), the structures of the native and the 5-MeO-modified DNA (3a
) are very similar. We have also observed previously that the 2′-Se-facilitator does not cause significant local and global structure perturbation (13
). The 5-MeO locates in the major groove of the A-form helix (B). Its methyl group points to the phosphate oxygen (O-Rp), instead of turning around and pointing to a larger space available in the major groove. Moreover, the electron density map of mo5
dU/dA pair (C) clearly shows that the distance between the methyl carbon and phosphate Rp-oxygen is 2.98
Å. Both the distance and geometry indicate H-bond formation between the 5-CH3
-O and 5′-PO4−
groups. The H-bond interactions between relatively acidic C-H protons and nearby oxygen or nitrogen atoms have been widely investigated in biological macromolecule systems (32
). Moreover, the B-factor of the 5′-phosphate of the 5-OMe-dU structure is indeed smaller than that of the 5-Me-dU (i.e. T) structure (PDB ID: 1Z7I). Thus, this unique nucleobase/backbone interaction can reduce the backbone dynamics, significantly rigidifying the local conformation. The 5-OMe modification does not drastically add rigidity to the entire RNA, which is probably expected for this site-specific small modification.
Figure 3. Crystal structure comparisons. The red and yellow balls represent the oxygen and selenium atoms of the modified moieties, respectively. (A) The superimposed duplex structures of 5′-G(SedU)G(mo5dU)ACAC-3′ (3a; in red; PDB ID: 3LTR; 1.30Å (more ...)
To further study the modification, we hypothesized initially that the distance between the methyl C and O-Rp may play a critical role. A larger atom from the same elemental family [such as selenium and sulfur (atomic radii: 1.16
Å and 1.04
Å, respectively) versus oxygen (atomic radius: 0.73
Å)] might present the methyl group closer to the 5′-phosphate and enhance the H-bond. Thus, we replaced the oxygen atom of the 5-O-Me group with a larger selenium or sulfur atom. However, we found that the single Se or S atom replacement disrupts the H-bond and causes local alterations, though the native and modified duplexes have very similar overall structures, which is consistent with the UV-melting study (Supplementary Table S2
). In the 5-SeMe or 5-SMe structure (circle 1 in B), the methyl group points to the major groove, instead of the 5′-phosphate backbone (B and C). Furthermore, the dihedral angle between the 5-CH3
-Se-C (or 5-CH3
-S-C) and uracil planes is ~95°, whereas the same dihedral angle in the 5-O-Me modification is approximately zero (0.3°). These observations indicate that the atomic size of the bridging atom is not the key factor for generating the H-bond, instead the electronic effects of 5-oxygen (such as electronegativity and conjugation with uracil) may play critical roles in the H-bond formation. We also attempt to directly compare the differences in rigidity between the OMe, SMe and SeMe RNAs. It is difficult because of the impacts of two factors: one is the H-bond formation in the case of OMe, and the other one is the atomic weights of sulfur and selenium atoms. The H-bond reduces the dynamics (i.e. increasing rigidity) and the higher atomic mass can also reduce the local dynamics. These two competing factors cause the difficulty to evaluate the rigidity order among these modified RNAs, even though we can directly compare the structures of the OMe, SMe, and SeMe RNAs.
Moreover, after the 5-selenium or -sulfur replacement, the 5′-phosphate group of the neighboring 5′-nucleotide (dG3, circle 2 in B) rotates ~110° about its C4′–C5′ bond (B and D), compared with the native and 5-O-Me modification. On the contrary, the 5-O-Me does not cause the backbone rotation (D), and the 5-O-Me modified and native nucleobases have virtually identical structures (E). The backbone rotation caused by the 5-Se-Me and 5-S-Me modifications is due to the alteration of the water networking surrounding the uracil 5-position, which in turn changes the water interactions in the major groove and with the phosphate backbone. Our crystal structures () reveal that two additional highly ordered water molecules (W1 and W2) are recruited to the major groove in the case of the 5-Se and 5-S modifications. The 5-Se or 5-S atom forms a H-bond with W1, considering that the distance between the 5-Se (or -S) atom and W1 is 3.42
Å, Se and S atoms are 0.43 and 0.31
Å, respectively, larger than O atom, and a typical H-bond length is 2.2–3.5
). W1 also forms a H-bond with W2, which subsequently forms H-bonds with W3 and the 5′-phosphate O-Rp of dG3. W4 and W5 remain at almost the same locations in the major groove. W2 is probably the direct cause of the backbone rotation.
Figure 4. Hydration pattern comparison of the major grooves of SedU2-dG3-dU4. The superimposed local structures of the 5-OMe, 5-SMe and 5-SeMe DNAs are colored as red, blue and yellow, respectively. The H-bonds are shown in red for the 5-OMe DNA, and blue for the (more ...)
The 5-Se-Me and 5-S-Me DNAs have virtually identical structures, and the distances between the methyl carbon and phosphate oxygen are 3.66
Å in both the cases ( and Supplementary Figure S1
), indicating no H-bond formation. The coplanar conformation of the 5-O-Me probably allows maximal conjugation between the oxygen atom and uracil aromaticity, which makes the methyl group most acidic. Unlike the 5-Se-Me and 5-S-Me modifications, the formed H-bond by the 5-O-Me prevents the water networking from disruption and retains the A-form helix structure without significant perturbation. Our research results may provide a plausible reason why oxygen (with proper electronic property) is chosen to modify the uracil 5-position in tRNAs, instead of selenium or sulfur.
Figure 5. Superimposed structures of the nucleotides. mo5dU4 of 3a [5′-G(SedU)G(mo5dU)ACAC-3′; in red], ms5dU4 of 3b (blue), mSe5dU4 of 3c (yellow), and the native T4 of 5′-G(SedU)GTACAC-3′ (PDB ID: 1Z7I; in cyan). The numbers in (more ...)
Furthermore, we performed the computation study of the uracils derivatized with the 5-OMe, 5-SMe and 5-SeMe modifications in order to understand the orientation of these 5- functionalities. The computation study indicates that the 5-O-Me, 5-S-Me, and 5-Se-Me moieties form the dihedral angles of 60.2°, 62.7° and 68.7° with the uracil ring, respectively (). The theoretical calculation suggests that the out-of-plane conformation is intrinsically favorable to minimize the energy levels of the modified uracils. In the determined crystal structures, the 5-Se-Me and 5-S-Me moieties still prefer the out-of-plane conformation with increased dihedral angles (95°). However, the 5-O-Me moiety forms a small dihedral angle (0.3°) and prefers the in-the-plane conformation. The H-bond formation alters the intrinsic orientation of the 5-OMe, whereas this H-bond is not observed in the 5-SeMe or 5-SMe modification. It is clear that the 5-O makes the methyl hydrogen more acidic than 5-S and 5-Se, and facilitates the H-bond formation in the A-form duplex. Moreover, our high-resolution structure indicates that the coplanar conformation of the 5-OMe with the uracil base enhances the base stacking interaction with the 5′-nucleobase adjacent to the mo5dU. The stabilization gained from the better stacking and H-bonding interactions compensates the destabilization from maintaining the unfavorable coplanar conformation of the 5-OMe, which is consistent with the computation study.
The calculated geometries of the 5-modifications in (A) N-Me-5-OMe-uracil; (B) N-Me-5-SMe-uracil and (C) N-Me-5-SeMe-uracil.
Since the 5-O can make the methyl hydrogen more acidic than 5-S and 5-Se, it is consistent with the H-bond formation observed in the crystal structure containing the 5-OMe modification. Moreover, our high-resolution structure indeed indicates that the coplanar conformation of the 5-OMe with the uracil base enhances the base stacking interaction with the nucleobases adjacent to the mo5
dU. The stabilization gained from the better stacking and H-onding interactions is used to compensate the energy required for the unfavorable coplanar conformation, which is indicated by the computation study. According to the theoretical research, the out-of-plane conformation is more stable, thus the 5-SMe and 5-SeMe maintain the out-of-plane conformation, since they cannot form the H-bonding interaction to compensate the coplanar conformation requirement. Furthermore, the melting temperature (T
m) data in Supplementary Table S2
indicate that the overall stabilities of the non-modified and 5-OMe duplexes are virtually identical. The similar stability indicates that the H-bond and stacking of the 5-OMe-U overcome the instability caused by the coplanar conformation of the 5-OMe relative to the uracil base. The structural data is consistent with the results of thermostability and computation studies. On the basis of the computational simulation, the out-of-plane conformation is more stable for all three modifications, including the 5-OMe, 5-SMe and 5-SeMe, which suggests the conjugation is probably less important for the orientation of the 5-OMe relative to the uracil plane. The H-bonding is most likely the major player determining the coplanar conformation of the 5-OMe relative to the uracil base. Consequently, the 5-OMe modification does not affect overall U-pairing stability.
Our studies indicate that the 5-Me group orientation and the H-bond formation are unique features of the 5-O-CH3
modification in A-form helix. The rigidified local structure and conformation may facilitate the base recognition in tRNA–mRNA interaction and translation. So far, the 5-OMe-U modification has only been found on 34 position of tRNA, which is the first nucleotide in anticodon and pairs with the third nucleobase in codon. It was reported that the mnm5 (5-CH2
) modification reduces the flexibility of the anticodon and contributes to ‘pre-organize’ the anticodon into an A-form structure ready to interact with the codon (9
). Similarly, by forming the H-bond, the 5-OMe functionality can make the anticodon pre-organized for interacting with codon. Moreover, it was reported (8
) that the reading efficiency of tRNA containing the 5-OMe-U modification (UGA) is higher than that of its non-modified counterpart (i.e. tRNA containing native UGA) when reading UCU and UCG codons (the wobble reading). Our observations of the H-bond formation and the local rigidification shed new light on the roles of the 5-OMe in anticodon/codon interaction and mRNA reading function. Although the context of the sequence is different from tRNA, the formation of the duplexes, including A-form duplex, is sequence-independent, as long as the sequences are complementary to each other. Our observation reveals rather general H-bond formation of the OMe modification in the A-form duplex. Therefore, our crystal structure study suggests that the H-bond may form when the OMe-modified tRNA encounters the condon in mRNA.