Biophysical studies of DNA modified with conformationally constrained nucleotides: comparison of 2'-exo (north) and 3'-exo (south) 'locked' templates

Abstract

The biophysical properties of oligodeoxyribonucleotides (ODNs) selectively modified with conformationally 'locked' bicyclo[3.1.0]hexane pseudosugars (Maier,M.A., Choi,Y., Gaus,H., Barchi,J.J. Jr, Marquez,V.E., Manoharan,M. (2004) Synthesis and characterization of oligonucleotides containing conformationally constrained bicyclo[3.1.0]hexane pseudosugar analogs Nucleic Acids Res., 32, 3642-3650) have been studied by various techniques. Six separate synthetic ODNs based on the Dickerson Drew dodecamer sequence (CGCGAAT*T*CGCG) were examined where each one (or both) of the thymidines (T*) were substituted with a bicyclic pseudosugar locked in either a North (2'-exo) or South (3'-exo) ring pucker. Circular dichroism spectroscopy, differential scanning calorimetry and (1)H NMR spectroscopy were used to examine the duplex stability and conformational properties of the ODNs. Replacement of one or both thymidines with North-locked sugars (RNA-like) into the dodecamer did not greatly affect duplex formation or melt temperatures but distinct differences in thermodynamic parameters were observed. In contrast, incorporation of South-locked sugar derivatives that were predicted to stabilize this standard B-DNA, had the unexpected effect of causing a conformational equilibrium between different duplex forms at specific strand and salt concentrations. Our data and those of others suggest that although DNA can tolerate modifications with RNA-like (North) nucleotides, a more complicated spectrum of changes emerges with modifications restricted to South (DNA-like) puckers.

Figure 1.

Pseudorotational cycle for nucleoside furanose ring puckers. Ring puckers for standard nucleosides are typically either in the ‘Northern’ or ‘Southern’ hemispheres (illustrated by blue cones). Depictions of the N and S conventions along with the structure of an LNA monomer are shown. The pseudorotational positions for the N-MCT and S-MCT analogs are shown on the left half of the wheel.

Figure 2.

Structures used in this study. The bicyclo [3.1.0] hexane nucleosides that were incorporated into the DNA are shown along with an illustration and nomenclature of the extra protons of these systems (in red).

Figure 3.

CD spectra of S-MCT- (A) and N-MCT- (B) modified DNAs at 25°C, at 100 mM Na⁺, pH 7.0 (phosphate buffer).

Figure 4.

(A) Enthalpy–entropy compensation plot for the ODNs in Figure 2. (B) Bar plot of ΔH_C, TΔS_c and ΔG₂₅. Each bar shows the comparison of the three thermodynamic parameters. The three bars in each cluster represent (from left to right) the data for the labeled ODN in 0.1, 0.5 and 1.0 M Na⁺

Figure 5.

DSC data for the native DD, T7^N, T8^N and T7^NT8^N ODNs at 0.1 M Na⁺ (black), 0.5 M Na⁺ (red), 1.0 M Na⁺ (blue), pH 7.0 (phosphate buffer). Exact conditions are given in the experimental section.

Figure 6.

DSC data for the native DD, T7^S, T8^S and T7^ST8^S ODNs at 0.1 M Na⁺ (black), 0.5 M Na⁺ (red), 1.0 M Na⁺ (blue), pH 7.0 (phosphate buffer). Exact conditions are given in the experimental section.

Figure 7.

NMR spectra of the imino ¹H region of the N-MCT modified oligos at various temperatures. The assignments are as follows: filled diamond T8, filled square T7, filled triangle G2, circle G4, star G10 and double dagger, G12.

Figure 8.

NMR spectra of the imino ¹H region of the S-MCT modified oligos at various temperatures. The assignments are as follows: filled diamond T8, filled square T7, filled triangle G2, filled circle G4, star G10 and double dagger, G12.

Figure 9.

Changes in the chemical shift of the imino protons between the N-MCT- and S-MCT- modified ODN species and the native DD at 5°C (A) and at 25°C (B).

Figure 10.

Changes in the chemical shift of select non-exchangeable protons between the N-MCT- (A) and S-MCT- (B) modified ODN species and the native DD at 25°C.

Figure 11.

1D NMR spectra of (A) T7^S and (B) T7^ST8^S imino regions at 200 μM strand concentration. Additional peaks that were not observed in 1–2 mM solutions are indicated with arrows.

Figure 12.

Side-by-side view of (A) T7^ST8^S (with residues labeled) and (B) the native dodecamer to illustrate the position and potential contacts of the additional atoms in the bicyclo[3.1.0]hexane systems compared to standard furanose rings. Only residues G4-T8 (3′-5′) and C9-A5 (5′-3′) are displayed for clarity. Backbone atoms are cyan; H5′, H5′′ of selected residues are dark blue; carbon and hydrogen atoms of the cyclopropane ring are shown in red and green, respectively.