Similar to natural bases, x-nucleotides selectively assemble into a duplex-structure resembling B-DNA. xDNA was originally synthesized by incorporating a benzene ring into the nitrogenous base. However, other expanded bases have been able to incorporate thiophene and [[Benzothiophene|benzo[b]thiophene]] as well. xDNA and yDNA use benzene rings to widen the bases and are thus termed "benzo-homologated". Another form of expanded nucleobases known as yyDNA incorporate naphthalene into the base and are "naptho-homologated". xDNA has a rise of 3.2Å and a twist of 32°, significantly smaller than B-DNA, which has a rise of 3.3Å and a twist of 34.2° xDNA nucleotides can occur on both strands—either alone (known as "doubly expanded DNA") or mixed with natural bases—or exclusively on one strand or the other. Similar to B-DNA, xDNA can recognize and bind complementary single-stranded DNA or RNA sequences.
Duplexes formed from xDNA are similar to natural duplexes aside from the distance between the two sugar-phosphate backbones. xDNA helices have a greater number of base pairs per turn of the helix as a result of a reduced distance between neighbour nucleotides. NMR spectra report that xDNA helices are anti-parallel, right-handed and take an anti conformation around the glycosidic bond, with a C2-endo sugar pucker. Helices created from xDNA are more likely to take a B-helix over an A-helix conformation, and have an increased major groove width by 6.5Å (where the backbones are farthest apart) and decreased minor groove width by 5.5Å (where the backbones are closest together) compared to B-DNA. Altering groove width affects the xDNAs ability to associate with DNA-binding proteins, but as long as the expanded nucleotides are exclusive to one strand, recognition sites are sufficiently similar to B-DNA to allow bonding of transcription factors and small polyamide molecules. Mixed helices present the possibility of recognizing the four expanded bases using other DNA-binding molecules.