xDNA

xDNA (also known as expanded DNA or benzo-homologated DNA) is a size-expanded nucleotide system synthesized from the fusion of a benzene ring and one of the four natural bases: adenine, guanine, cytosine, and thymine. This size expansion produces an 8 letter alphabet which has a larger information density by a factor of 2n compared to natural DNA's (often referred to as B-DNA in literature) 4 letter alphabet. As with normal base-pairing, A pairs with xT, C pairs with xG, G pairs with xC, and T pairs with xA. The double helix is thus 2.4Å wider than a natural double helix. While similar in structure to B-DNA, xDNA has unique absorption, fluorescence, and stacking properties. Initially synthesized as an enzyme probe by Nelson J. Leonard's group, benzo-homologated adenine was the first base synthesized. Later, Eric T. Kool's group finished synthesizing the remaining three expanded bases, eventually followed by yDNA ('wide' DNA), another benzo-homologated nucleotide system, and naphtho-homologated xxDNA and yyDNA. xDNA is more stable when compared to regular DNA when subjected to higher temperature, and while entire strands of xDNA, yDNA, xxDNA and yyDNA exist, they are currently difficult to synthesize and maintain. Experiments with xDNA provide new insight into the behavior of natural B-DNA. The extended bases xA, xC, xG, and xT are naturally fluorescent, and single strands composed of only extended bases can recognize and bind to single strands of natural DNA, making them useful tools for studying biological systems. xDNA is most commonly formed with base pairs between a natural and expanded nucleobase, however x-nucleobases can also be paired together. Current research supports xDNA as a viable genetic encoding system in the near future. The first nucleotide to be expanded was the purine adenine. Nelson J. Leonard and colleagues synthesized this original x-nucleotide, which was referred to as 'expanded adenine'. xA was used as a probe in the investigation of active sites of ATP-dependent enzymes, more specifically what modifications the substrate could take while still being functional. Almost two decades later, the other three bases were successfully expanded and later integrated into a double helix by Eric T. Kool and colleagues. Their goal was to create a synthetic genetic system which mimics and surpasses the functions of the natural genetic system, and to broaden the applications of DNA both in living cells and in experimental biochemistry. Once the expanded base set was created, the goal shifted to identifying or developing faithful replication enzymes and further optimizing the expanded DNA alphabet. In benzo-homologated purines (xA and xG), the benzene ring is bound to the nitrogenous base through nitrogen-carbon (N-C) bonds. Benzo-homologated pyrimidines are formed through carbon-carbon (C-C) bonds between the base and the benzene. Thus far, x-nucleobases have been added to strands of DNA using phosphoramidite derivatives, as traditional polymerases have been unsuccessful in synthesizing strands of xDNA. X-nucleotides are poor candidates as substrates for B-DNA polymerases as their size interferes with binding at the catalytic domain. Attempts at using template-independent enzymes have been successful as they have a reduced geometric constraint for substrates. Terminal deoxynucleotidyl transferase (TdT) has been used previously to synthesize strands of bases which have been bound to fluorophores. Using TdT, up to 30 monomers can be combined to form a double-helix of xDNA, however this oligomeric xDNA appears to inhibit its own extension beyond this length due to the overwhelming hydrogen bonding. In order to minimize inhibition, xDNA can be hybridized into a regular helix. For xDNA to be used as a substitute structure for information storage, it requires a reliable replication mechanism. Research into xDNA replication using a Klenow fragment from DNA Polymerase I shows that a natural base partner is selectively added in instances of single-nucleotide insertion. However, DNA Polymerase IV (Dpo4) has been able to successfully use xDNA for these types of insertions with high fidelity, making it a promising candidate for future research in extending replicates of xDNA. xDNA's mismatch sensitivity is similar to that of B-DNA. 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 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 xDNA's 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. Expanded nucleotides and their oligomeric helices share many properties with their natural B-DNA counterparts, including their pairing preference: A with T, C with G. The various differences in chemical properties between xDNA and B-DNA support the hypothesis that the benzene ring which expands x-nucleobases is not, in fact, chemically inert. xDNA is more hydrophobic than B-DNA, and also has a smaller HOMO-LUMO gap (distance between the highest occupied molecular orbital and lowest unoccupied molecular orbital) as a result of modified saturation. xDNA has higher melting temperatures than B-DNA (a mixed decamer of xA and T has a melting temperature of 55.6 °C, 34.3 °C higher than the same decamer of A and T), and exhibits an 'all-or-nothing' melting behaviour.