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 1H 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.

The Properties of Locked Methanocarba Nucleosides in Biochemistry, Biotechnology, and Medicinal Chemistry

“…It may be surmised that the S-modified nucleotides actually do stabilize a B-like helix, and that the more hydrated environment associated with this type of helix requires a larger enthalpic change upon melting. Although the sequence of the DD is self-complimentary and, under certain conditions, has a tendency to form hairpin structures, there was no indication of these folds in any of the ODN species studied with the S-modified nucleotides [46].…”

“…In the A-form, sugar puckering is closely clustered around a 3 0 -endo (N) conformation, while the puckering distribution in the standard B-form is more diffuse around the C2 0 -endo (S) conformation. Because the locking of the conformation can be achieved with a rigid bicyclo [3.1.0]hexane pseudosugar, the embedded cyclopentane ring can be secured into either a 2 0 -exo ( 2 0 E, N) or 3 0 -exo ( 3 0 E, S), as shown previously in Figure 12 on conformation should restrict the degree of flexibility of the sugar-phosphate backbone, and are expected to influence the biophysical and biological properties of the duplex [46].…”

“…Since the furanose rings of the native DD dodecamer (CGCGAATTCGCG) are puckered almost exclusively in the standard B-type (S) conformation, it was anticipated that these substitutions would either stabilize or disrupt the local/global structure accordingly. Results from circular dichroism (CD), nuclear magnetic resonance (NMR) and differential scanning calorimetry (DSC) measurements have provided a descriptive picture of the effect of either modified nucleoside on duplex formation and stability [46]. The incorporation of N-locked analogues only minimally perturbed the DD system with regard to duplex formation and thermodynamics.…”

“…Replacing the middle thymidine nucleotides with S-locked analogues led to a more complex equilibrium of species rather than the idealized assumption of a stable all-B-DNA duplex in a manner that was predicted a priori [46]. Interpreting the data regarding the effects of incorporating the S-locked analogues is more difficult, especially in light of the concentration dependence of the aforementioned equilibrium.…”

The Conformationally Constrained N-Methanocarba-dT Analogue Adopts an Unexpected C4′-exo Sugar Pucker in the Structure of a DNA Hairpin

“…Moreover, neither NMR nor CD experiments provided any indication for the presence of a hairpin in solution. 24–26 Modeling of methoxy groups at C2' of N -MCdT7 and -T8 results in several short distances between 2'-oxygen and methyl carbon and backbone and base atoms (Figure 5). Therefore, it is unlikely that a DDD with rU(T) or 2'- O Me-rU(T) nucleotides at positions 7 and 8 would adopt the hairpin conformation observed here for the N -MCdT-modified dodecamer.…”