Using NMR and molecular dynamics to link structure and dynamics effects of the universal base 8-aza, 7-deaza, N8 linked adenosine analog

Abstract

A truly universal nucleobase enables a host of novel applications such as simplified templates for PCR primers, randomized sequencing and DNA based devices. A universal base must pair indiscriminately to each of the canonical bases with little or preferably no destabilization of the overall duplex. In reality, many candidates either destabilize the duplex or do not base pair indiscriminatingly. The novel base 8-aza-7-deazaadenine (pyrazolo[3,4-d]pyrimidin- 4-amine) N⁸-(2'deoxyribonucleoside), a deoxyadenosine analog (UB), pairs with each of the natural DNA bases with little sequence preference. We have utilized NMR complemented with molecular dynamic calculations to characterize the structure and dynamics of a UB incorporated into a DNA duplex. The UB participates in base stacking with little to no perturbation of the local structure yet forms an unusual base pair that samples multiple conformations. These local dynamics result in the complete disappearance of a single UB proton resonance under native conditions. Accommodation of the UB is additionally stabilized via heightened backbone conformational sampling. NMR combined with various computational techniques has allowed for a comprehensive characterization of both structural and dynamic effects of the UB in a DNA duplex and underlines that the UB as a strong candidate for universal base applications.

Figure 1.

UB schematic. Schematic of 8-aza-7-deazaadenine (pyrazolo[3,4-d]pyrimidin-4-amine) N8-(2′deoxyribonucleoside) (UB) in the “anti” orientation.

Figure 2.

Imino proton and ³¹P NMR spectra. (A) ¹H imino proton spectrum of the control and UB sequences at 7°C (top and bottom, respectively). Imino protons for residues G4 and G6 of the UB sequence (which flank UB5) are labeled in grey. (B) ³¹P spectra for the control and UB sequences at 22°C. Arrows and circles indicate the G6 and C15 phosphorous resonances, which exhibit significant perturbations in chemical shifts as compared to the control.

Figure 3.

UB5:T14 and A5:T14 base conformations. (A) Base pair for UB5:T14 (top) and the control A5:T14 (bottom) (both are from the solved rMD structures). Anomeric carbon distances are referenced. Sugar and backbone are in grey; base colors: blue = nitrogen, grey = carbon, red = oxygen, white = hydrogen. (B) Base stacking for G4 (yellow), UB5/A5 (red) and G6 (blue). UB sequence on left, control on right (both are from the solved rMD structures). (C) Predicted UB-T base pair hydrogen bonding pattern determined from previous thermal stability studies (Seela F. & Debelak, H., NAR, 2000).

Figure 4.

NOE connectivity and NMR spectra indicating unusual dynamics. (A) NOE pathways. Solid line signifies normal NOE contacts; dotted line = weak NOE, double line = unanticipated NOE contacts; OL = signal overlap. See text for details of unanticipated NOE contacts. (B) Supercooled aqueous NMR spectra of the imino proton region at −12°C showing the appearance of a ‘shoulder’ peak at 14 ppm. Inset: NOESY spectrum collected at −12°C showing crosspeaks between the imino proton region and methyl region. Arrow indicates the appearance of a new crosspeak. (C) ¹H-¹³C HSQC spectra focused on the adenosine C2–H2 region; top spectrum is collected at 57°C, bottom spectrum is at 19°C. (D) ¹H–³¹P HETCOR spectrum showing crosspeaks for H3′–P (4.6–5.1 ppm, ¹H) and P–H4′ (3.9–4.5 ppm, ¹H) (22°C). Circled are the weakened intensity for the UB5 H3′– G6 P and normal intensity G6 P–G6 H4′ crosspeaks.

Figure 5.

(A) G4 and UB5 backbone torsion angles versus MD trajectory time for rMD, MDtar and long term unconstrained MD simulations for the UB sequence. From top to bottom: G4 epsilon, G4 zeta, UB5 alpha, UB5 beta and UB5 gamma backbone torsion angles. From left to right: 9 ns of restrained MD, 1 ns of MD using time averaged restraints and 400 ns of unconstrained MD. Occurrences of populations A, B and C are labeled and separated with dashed lines in the MDtar graphs. For all graphs: X-axis is the trajectory time in ns and the Y-axis is torsion angle in degrees. Gauche− (g−) = −60° (i.e. 300°), gauche+ (g+) = +60° and trans (t) = 180°. (B) Scatter plots of torsion angles versus UB5 alpha torsion angle from the unconstrained MD trajectory. For all plots, the X axis is UB5 alpha torsion angle. Side projections are histograms for the respective torsion angle. Populations are outlined as follows: blue box = population A, orange box = population B, green box = population C.