Results – Structure Calculations and Analysis

IT TO FUNCTION AS A UNIVERSAL BASE

2.7 Results – Structure Calculations and Analysis

2.7.1 A1 Glycosidic Bond Angle

The intense crosspeak of A1 H1’ to H8 in both UB and control oligonucleotides indicates the presence of a syn population. An approximate syn population of <25% was calculated from the observed NOE together with theoretical syn vs. anti H1’ to H8 distances. (Supplemental In-‐ formation Figure 3 and Calculation 1) Because this anomaly was found in both control and UB sequences, restraints were added to the calculations to lock the A1 base into an anti conforma-‐ tion to reflect the major population.

2.7.2 Structure Calculation

The UB containing oligonucleotide is highly restrained containing a total of 440 non-‐RDC restraints and 47 RDC giving a total of ~27 and ~24 restraints per residue for the NOESY + RDC and non-‐RDC structures respectively. (Table 2-‐2) Standard deviations of mass averaged R.M.S.D. values over the entire 10 ns restrained MD trajectories are 0.25 and 0.12 Å for the non-‐RDC and NOESY + RDC simulations respectively. The average heavy atom R.M.S.D. for the final bundle of structures is 0.37 Å for both the NOESY + RDC and non-‐RDC structures. (Figure 2-‐4) The structure with the lowest AMBER restraints violations was chosen as the final struc-‐

ture. Interestingly, CORMA Rx values slightly increased upon the implementation of RDC values,

yet both UB structures are still in excellent agreement with NOESY data as exhibited with total

CORMA RX_{values < 0.06. Total AMBER RDC violations were 5.63 kcal / mol for all restraints.}

structure containing only RDC restraints was calculated. This structure calculation did not use NOESY distance restraints, base pair angle restraint, sugar puckering restraints, or backbone torsion angle restraints. Resulting RDC penalties were minimal with a total AMBER violation of < 2 kcal / mol.

The control structure has slightly fewer restraints than the UB structure; it shows excel-‐

lent agreement with NOESY data with total CORMA RX_{values of 0.051 and 0.050 for the NOESY}

and NOESY + RDC structures respectively. AMBER RDC violations totaled 3.83 kcal / mol for all

restraint. Unlike the UB structure, RDC implementation did not drive up RX values establishing

that the observed increase for the UB structure is a result of the UB modification. An RDC only structure was not calculated for the control. (Table 2-‐2)

2.7.3 Structure Analysis

For both the non-‐RDC and NOESY + RDC UB structures, all bases including both the UB5 and T14 bases are intrahelical and the overall global geometry is B-‐type. (Figure 2-‐5 A) As ex-‐ pected from the imino proton data, the UB5:T14 base pair does not form a hydrogen bond in-‐ volving the T14 imino proton. Instead, the amino group of UB5 is hydrogen bonded to T14 O4 carbonyl. (Figure 2-‐12) This orientation of the UB5:T14 base pair is accommodated in the du-‐

plex via backbone perturbations, primarily the torsion angles around the UB (G4 ζ, UB5 ζ, G6 α,

and C16 α). This unusual base pairing results in a heightened stretch and opening helical pa-‐

rameters (1 Å and 27.8 ° respectively) in UB5:T14 base pair as well as an increase of 1.8 Å in the UB5:T14 anomeric carbon distance as compared to the control structure. This results in a dis-‐ placed backbone, distortions in the helical twist (G4-‐UB5 30.4°, UB5-‐G6 29.7°, G6-‐C7 41.2°),

and a slight bend in the global helical axis (total axis bend of 19 vs. 4° for the UB and control respectively) as compared to the control structure. (Figure 2-‐5 B and C) As a consequence of

the under-‐twisting and the unique N8_{UB base connectivity, the local stacking of G4-‐UB5-‐G6 is}

disrupted with G4 poorly stacked on top of UB5. (Figure 2-‐6 A)

For completeness, it is noted that the T2-‐G3 step has an apparent bend in both the UB and control structures. (Figure 2-‐7A, Supplemental Information Figure 4) This bend is primarily generated by a heightened positive roll of 12.9° in the T2-‐G3 step. The James group found simi-‐ lar results for T-‐G / A-‐C steps with similarly large positive rolls. (59) Intriguingly, when RDC re-‐ straints were incorporated, the T2-‐G3 bend was removed from both the UB and control struc-‐ tures.

2.7.4 RDC Implementation

Although the inclusion of RDC restraints with NOESY data did not result in a major con-‐ formational change for the core sequence of the UB structure (G4, UB5, G6, C13, T14, C15

heavy atom R.M.S.D. of 0.63 Å) (Figure 2-‐7B), the slight increase in total CORMA RX values for

the UB structure and not the control prompted the calculation of an RDC only structure. The resulting structure is similar to the NOESY + RDC results except for the UB5:T14 base pair. Here, the predicted base pair includes a hydrogen bond between the T14 imino proton and UB5 N1. (Figure 2-‐8 A, B, C)

2.7.5 Supercooled Aqueous NMR

Due to the ambiguous orientation of the UB5-‐T14 base pair, further probing of the imino proton spectra was conducted via supercooled aqueous NMR utilizing 1 mm capillaries. When temperatures were dropped to -‐10 °C, an additional imino proton peak begins to emerge at 14 ppm, suggestive of the formation of an additional A:T base pair. A NOESY spectrum at -‐12 °C was used to confirm the identity as T14 through the appearance of a cross peak between the new imino proton and an additional methyl resonance. (Figure 2-‐9) All observed imino proton resonances shifted as anticipated with decreasing temperatures indicating that an overall global conformation change in the oligonucleotide did not occur.

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