IT TO FUNCTION AS A UNIVERSAL BASE
2.6 Results – NMR Structural Data
The DNA duplex chosen for the NMR structural studies is: 1 9
5’ ATGGXGCTC
TACCTCGAG 5’
18 10
where X is the UB; for a control sequence, X is dA. NMR assignments for 1H, 13C, and 31P were
completed using standard 1D and 2D NMR method. (21, 22)
2.6.1 Glycosidic Bond Orientation
Because of the unique attachment of the UB, the glycosidic bond orientation must be defined. Adenosine anti and syn conformations place either the H8 or N3 atoms on the endo face of the sugar ring. For the UB, a similar convention is used where the “anti” and “syn” ori-‐ entations place the H7 or N9 atoms on the endo side of the sugar respectively, which yield quite different H7-‐H1’ distances of 3.8 Å for “anti” and 2.5 Å for “syn”. This distance was used to characterize the glycosidic bond torsion angle. (Figure 2-‐1 B)
Energy minimization calculations at the HF 6-‐31G level (Spartan’04) on a single UB nu-‐ cleoside indicate two stable conformations representing the “syn” and “anti” glycosidic bond torsion angles with the “syn” conformation most stable. Previous studies of single UB nucleo-‐
tides reveal that the base is perpendicular to the sugar and in the “syn” type conformation. (12)
2.6.2 Glycosidic Bond Orientation in the Duplex
A low mixing time NOESY experiment (75 ms) was used to estimate distance between the UB H7 – H1’ protons. Under low mixing times, spin diffusion is minimized thus allowing for approximate distance calculations. Using the cytosine H5-‐H6 crosspeak intensity with a dis-‐ tance of 2.46 Å as a reference, a distance of 2.3 Å was calculated for UB5 H7 – H1’ protons. This establishes the base orientation as “syn”. This result was verified with UB5 H7 to H2’, H2’’, and H3’ distances. Higher mixing time data in which the MARDIGRAS program was used to resolve spin diffusion effects also supports this conformation. All residues surrounding the UB are in anti conformations.
2.6.3 Overall Helical Geometry
NOESY base to base and base to sugar contacts determine the overall helical structure of a DNA duplex. All anticipated NOE base to sugar H1’ contacts were identified, the only ex-‐ ception being a reduction in intensity between the G4 base H8 to UB5 sugar H1’ protons. All base to base, H2’, H2’’, and H3’ pathways are all accounted for with the exception of the base to base and base to H3’ pathways for G4 to UB5. (Figure 2-‐2 A) The NOE intensities are consis-‐ tent with an overall B type helix. In addition, several unique crosspeaks were observed: G6 sugar H1’ and H3’ to the UB5 base H7 proton as well as UB5 sugar H1’ to G6 sugar H3’ and H4’. (Figures 2-‐2 A, B) These contacts, with the G4 H8 to UB5 H1’ NOE, confirm an intrahelical orien-‐ tation of the UB base.
2.6.4 Base Pairing
Imino proton NMR was used to detect and assign the base pairs. All imino proton reso-‐ nances were in anticipated chemical shifts indicative of A:T and G:C Watson-‐Crick base pairing. The T14 imino proton could not be observed. Of note, the bases flanking the UB5 (G4 and G6) exhibit sharp line shapes. (Figure 2-‐2 C)
In contrast with adenosines, the UB5 amino group could not be identified in H2O NOESY
spectra. All other anticipated imino / amino pathways were detected. A weak NOESY cross-‐ peak between G4’s imino proton and the UB5 base H7 was identified which confirms the UB5 intrahelical orientation.
2.6.5 Chemical Shift Perturbations
Absolute values of differences in chemical shifts (|UB – control| = Δ ppm) for various sugar and base protons as well as phosphorus resonances were calculated. As shown in Figure 2-‐3 A and 2-‐3 B, the largest chemical shift perturbations for both the sugar and base protons were localized to areas around the UB5:T14 base pair. Sugar protons in T14 contained the larg-‐ est overall chemical shifts (Σ Δ ppm of 0.675) and largest changes in individual proton reso-‐ nances (H1’ and H2’2 of 0.319 and 0.241 Δ ppm respectively). (Figure 2-‐3 A) Base proton chemical shift changes were mainly localized to C15. (Figure 2-‐3 B)
As compared to the control sequence, 31P chemical shifts for UB5-‐P-‐G6 and T14-‐P-‐C15
are shifted 0.52 and -‐0.56 ppm from their control values. Other perturbations are localized to the sequence around the UB5:T14 base pair. (Figure 2-‐3 C)
2.6.6 Deoxyribose Conformation
Using a graphical method, fraction south (fS) sugar puckering values were calculated
from the individual coupling constants JH1’-‐H2’1 and JH1’-‐H2’2 as well as sum of couplings ΣJH1’, ΣJH3’,
ΣJH2’1, and ΣJH2’2. (35) The results from this analysis place the UB5 sugar in a strong S conforma-‐
tion. All other residues are predominately in an S conformation with the exception of C7 which was ~50% FS. Due to overlap, FS for G3 was estimated from the JH2’1-‐H3’, JH2’2-‐H3’, and JH1’-‐H2’1 cou-‐
pling patterns, which estimates it to be predominately S. Due to severe overlap, the conforma-‐ tions of the 3’ terminal C9 and T18 sugars could not be determined. For the control sequence, all residues measured were found to be in S conformation.
2.6.7 Backbone Conformation
Experimentally determined epsilon torsion angles from ctNOESY experiments did not reveal a unique perturbation for any of the residues. All residues, including UB5-‐P-‐G6 and T14-‐ P-‐C15, fall within canonical B-‐type ranges.
1H – 31P HETCOR experiments interestingly yield a weak intensity for the H3’ to 31P peak
for the UB5-‐p-‐G6 sequence. Because of the non-‐perturbed epsilon values, this is suggestive of dynamics. (Supplemental Information Figure 1 and 2)