![]() ![]() 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. Replication įor xDNA to be used as a substitute structure for information storage, it requires a reliable replication mechanism. In order to minimize inhibition, xDNA can be hybridized into a regular helix. 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. Terminal deoxynucleotidyl transferase (TdT) has been used previously to synthesize strands of bases which have been bound to fluorophores. Attempts at using template-independent enzymes have been successful as they have a reduced geometric constraint for substrates. X-nucleotides are poor candidates as substrates for B-DNA polymerases as their size interferes with binding at the catalytic domain. 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. Benzo-homologated pyrimidines are formed through carbon-carbon (C-C) bonds between the base and the benzene. In benzo-homologated purines (xA and xG), the benzene ring is bound to the nitrogenous base through nitrogen-carbon (N-C) bonds. Once the expanded base set was created, the goal shifted to identifying or developing faithful replication enzymes and further optimizing the expanded DNA alphabet. ![]() 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. Almost two decades later, the other three bases were successfully expanded and later integrated into a double helix by Eric T. 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. Leonard and colleagues synthesized this original x-nucleotide, which was referred to as "expanded adenine". The first nucleotide to be expanded was the purine adenine. Current research supports xDNA as a viable genetic encoding system in the near future. xDNA is most commonly formed with base pairs between a natural and expanded nucleobase, however x-nucleobases can also be paired together. ![]() 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. Experiments with xDNA provide new insight into the behavior of natural B-DNA. 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. 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. Leonard's group, benzo-homologated adenine was the first base synthesized. Initially synthesized as an enzyme probe by Nelson J. While similar in structure to B-DNA, xDNA has unique absorption, fluorescence, and stacking properties. The double helix is thus 2.4 Å wider than a natural double helix. As with normal base-pairing, A pairs with xT, C pairs with xG, G pairs with xC, and T pairs with xA. This size expansion produces an 8 letter alphabet which has a larger information density by a factor of 2 n compared to natural DNA's (often referred to as B-DNA in literature) 4 letter alphabet. 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. ![]()
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