According to the Watson-Crick model, the sequence of bases in one strand of a double-stranded DNA (dsDNA) may be entirely arbitrary, but the sequence in the second strand is fixed by complementary base pairing. Each pyrimidine base forms a hydrogen-bonded base pair with only one of the two purine bases: C forms a base pair with G, and T pairs with A.

Opening image: G·C base pair. All four base pairs have the same overall dimensions. The distance separating the backbone C1′ atoms, shown in purple, is very important. In the "ideal" Watson-Crick model this distance is 10.85 Å for all four base pairs. However, observed values range from 10.4 to 10.9 Å. the distance between this pair.

G·C base pairs are joined by three hydrogen bonds.

hydrogen bonds. All three hydrogen bonds have nearly perfect geometry. Remember that in a strong hydrogen bond the donor X–H bond points directly at the lone pair orbital of the acceptor. Is this true for the two N–H···O hydrogen bonds in the G·C base pair shown here? For the N–H···N hydrogen bond?

the lone pair orbitals of the hydrogen bond acceptors.

Although G·C base pairs are generally planar, of G·C base pairs does occur. The model shown has a a propeller twist of about 20°. spacefill. More severe twist in G·C base pairs is unlikely owing to the strong cooperative interaction of the three hydrogen bonds.

alternate views of an A·T base pair. An ideal A·T base pair is planar and joined by two hydrogen bonds. Is that true for this A·T base pair?

Let's look at three A·T base pairs from a DNA double helix whose structure in solution has been determined by NMR analysis.

Note the variation in the distances between the C1′ atoms of the sugars on opposite strands of the A·T base pairs.

None of A·T base pairs in this double helix are planar. spacefill.

stacked base pairs are stabilized by π-π and van der Waals interactions. Close packing is maintained regardless of the geometry of an individual base pair or its orientation with respect to the helix axis.

Difluorotoluene (DFT) Mimics Thymine

In the '90s, Guckian, Krugh & Kool designed a series of experiments with difluorotoluene (DFT), a nonpolar isosteric analog of thymine. If dFTP, the 5′-deoxynucleoside triphosphate analog of dTTP, is added to a reaction mixture containing dATP, dGTP, dCTP, DNA polymerase, and a DNA template, then adenine in the template strand encodes the insertion of dFTP with the same accuracy as seen with dTTP. Likewise, fluorine in a template strand encodes the insertion of dATP. These results indicate that hydrogen bonding in A·T base pairs is not as important as previously thought.

A·T vs. A·DFT base pair. The fluorine (F) atoms in DFT are shown in light green. Note that fluorine cannot form hydrogen bonds. Consequently, the steric clash of the C–H proton with the ring nitrogen atom of adenine forces fluorine to rotate away from adenine. If you toggle between the two pairs you will notice that the C1′ atoms (shown in purple) are closer in the A·DFT base pair than in the A·T base pair (10.58 vs. 10.34 Å).

A 14-mer DNA duplex with a single A·F base pair in place of an A·T base pair. Substituting difluorotoluene for thymine causes negligible distortion in the helix.

exploded view. Note that the adenine paired with fluorine forms a weak out-of-plane to the thymine of the A·T base pair directly below the A·DFT base pair.

The fact that difluorotoluene codes efficiently and specifically for adenine in DNA replication strongly suggests that shape complementarity -- not hydrogen-bonding -- is the crucial aspect in nucleic acid structure and recognition. Even if this point of view is not shared, it has to be recognized that hydrogen-bonding in base pairs is only one force relevant to nucleic acid structures. Other interactions, especially base stacking, are important.

Summary

Flexibility of stacked base pairs is larger than has been assumed before.
Nonplanar base pairs are allowed.
A·T base pairs are more flexible than G·C base pairs.
A·T base pairs frequently have only one hydrogen bond.
Steric and other influences make Watson-Crick geometry the preferred mode of base pairing in double helices.
Bases can pair in a number of ways in addition to Watson-Crick geometry. Non-Watson-Crick base pairs are often found in RNA double helices.