Stacking of the nucleotides in a double helix is a major determinant of the helixs stability. With the added surface area and hydrogen available for bonding, stacking potential for the nucleobases increases with the addition of a benzene spacer. By increasing the separation between the nitrogenous bases and either sugar-phosphate backbone, the helixs stacking energy is less variable and therefore more stable. The energies for natural nucleobase pairs vary from 18 to 52 kJ/mol. This variance is only 14–40 kJ/mol for xDNA.
Due to an increased overlap between and expanded strand of DNA and its neighbouring strand, there are greater interstrand interactions in expanded and mixed helices, resulting in a significant increase in the helixs stability. xDNA has enhanced stacking abilities resultant from changes in inter- and intrastrand hydrogen bonding that arise from the addition of a benzene spacer, but expanding the bases does not alter hydrogens contribution to the stability of the duplex. These stacking abilities are exploited by helices consisting of both xDNA and B-DNA in order to optimize the strength of the helix. Increased stacking is seen most prominently in strands consisting only of A and xA and T and xT, as T-xA has stronger stacking interactions than T-A.
The energy resultant from pyrimidines ranges from 30 to 49 kJ/mol. The range for purines is between 40-58kJ/mol. By replacing one nucleotide in a double-helix with an expanded nucleotide, the strength of the stacking interactions increases by 50%. Expanding both nucleotides results in a 90% increase in stacking strength. While xG has an overall negative effect on the binding strength of the helix, the other three expanded bases outweigh this with their positive effects. The change in energy caused by expanding the bases is mostly dependent on the rotation of the bond about the nucleobases' centers of mass, and center of mass stacking interactions improve the stacking potential of the helix. Because the size-expanded bases widen the helix, it is more thermally stable with a higher melting temperature.