Differential stability of 2'F-ANA*RNA and ANA*RNA hybrid duplexes: roles of structure, pseudohydrogen bonding, hydration, ion uptake and flexibility

Abstract

Hybrids of RNA with arabinonucleic acids 2'F-ANA and ANA have very similar structures but strikingly different thermal stabilities. We now present a thorough study combining NMR and other biophysical methods together with state-of-the-art theoretical calculations on a fully modified 10-mer hybrid duplex. Comparison between the solution structure of 2'F-ANA*RNA and ANA*RNA hybrids indicates that the increased binding affinity of 2'F-ANA is related to several subtle differences, most importantly a favorable pseudohydrogen bond (2'F-purine H8) which contrasts with unfavorable 2'-OH-nucleobase steric interactions in the case of ANA. While both 2'F-ANA and ANA strands maintained conformations in the southern/eastern sugar pucker range, the 2'F-ANA strand's structure was more compatible with the A-like structure of a hybrid duplex. No dramatic differences are found in terms of relative hydration for the two hybrids, but the ANA*RNA duplex showed lower uptake of counterions than its 2'F-ANA*RNA counterpart. Finally, while the two hybrid duplexes are of similar rigidities, 2'F-ANA single strands may be more suitably preorganized for duplex formation. Thus the dramatically increased stability of 2'F-ANA*RNA and ANA*RNA duplexes is caused by differences in at least four areas, of which structure and pseudohydrogen bonding are the most important.

Figure 1.

(A) Structures of ANA and 2′F-ANA in comparison with DNA and RNA. (B) Numbering scheme of the 10-mer hybrid duplexes used for this study. X indicates the DNA, 2′F-ANA or ANA strand. The corresponding hybrids are named DR, FR and AR, respectively. Thymines are replaced by uracils in the ANA strand. (Use of T instead of U does explain some of the increased duplex stability of the 2′F-ANA-containing hybrid. However, comparison of 2′F-ANA and ANA-containing hybrids where neither strand includes T/U and studies where both 2′F-ANA and ANA contained T confirm that the 2′F-ANA-containing hybrids are still significantly more stable than their ANA-containing counterparts.) (6,7).

Figure 2.

Melting of AR (ANA•RNA), DR (DNA•RNA), DD (DNA•DNA), RR (RNA•RNA) and FR (2′F-ANA•RNA), as followed by observing the change in A₂₆₀ of duplex samples upon heating from 5 to 65°C. Duplexes were 20 µM in phosphate buffer (140 mM KCl, 5 mM Na₂HPO₄, 1 mM MgCl₂, pH 7.2) Data derived from UV melting experiments such as these are given in Table 1. Starting values of A₂₆₀ were 0.89 ± 0.04.

Figure 3.

Heteronuclear ¹⁹F–¹H correlation HOESY spectrum of FR in D₂O. Sequential assignment pathway between intra-residual and sequential ¹⁹F–¹H₆/¹H₈ cross-peaks is shown. Strong sequential ¹⁹F(T)–H8(A/G) cross-peaks (at TA and TG steps) are indicated with arrows.

Figure 4.

(A) Superposition of the 10 refined structures of FR (left) and AR (right). Stereoscopic views of the average structures of the three hybrids FR (B), AR (C) and DR (D). Blue and magenta indicate different strands. Fluorine atoms are shown in green.

Figure 5.

(A) Values of minor groove width along the sequence of the three hybrids, and (B) view of the hybrids from their minor groove side. Standard A-form and B-form duplexes are shown for comparison.

Figure 6.

Details of the thermodynamic cycle in MD/TI free energy calculations.

Figure 7.

Details of X2′–H8 interactions in FR and AR. Left, the structure of the FR duplex, showing distances between F2′ and aromatic H6/H8 protons. These distances correlate well with cross-peak intensity of the ¹⁹F – ¹H HOESY spectra shown in Figure 3. Right, the same region of the AR duplex showing unfavorable steric interactions with 2′-OH.