CH391L/S14/Non-canonical Nucleotides

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Non-canonical nucleobases are small heterocyclic molecules that function similar to the natural nucleobases in DNA and RNA by base-pairing through hydrogen bounding, hydrophobic interactions, or other means. These base-pairing capabilities are essential in functioning like the natural base pairs. Non-canonical nucleobases are used, or proposed to use for, for a variety of purposes, including improving the theoretical efficiency of DNA computing, researching the evolution of life, and containing synthetic life. Several research groups are working on non-canonical nucleobases, and utilizing them in different ways. Despite advances, there are several significant obstacles to implementing non-canonical nucleobases in vivo.


History and Examples

A collection of several non-canonical base pairs that have been developed[1]

Original Proposal and Experimental Validation

In 1962, it was proposed that a third possible DNA base-pair might occur in nature, the iso-C/Iso-G pairing. The iso-C/iso-G pairing remains unobserved in nature, though several other non-Watson-Crick base-pairing events have been seen, including DNA triple helix and the G-quadruplex. In 1989 the Benner group demonstrated that the proposed iso-C/iso-G pair could be successfully maintained by natural DNA replication machinary when supplemented with synthetic iso-C/iso-G dNTPs. The Benner incorporation of iso-C/iso-G was enzymatically incorporated from a template containing iso-C using an E. coli DNA polymerase fragment[2]. Additionally, it was shown that T7 RNA polymerase was capable of transcribing iso-C/iso-G as a third set of base pairs with a template DNA sequencing containing iso-C and iso-G.

Since the original paper demonstrating the propagation of non-canonical bases in DNA and RNA, several labs have published other base pairs that are capable of preservation in DNA and RNA.


The base-pairing patterns of xDNA, showing the expanded bases pairing with natural bases.
xDNA is a is an expanded DNA set with the four nature nucleobases pairing with four size expanded nucleobases. The four size expanded nucleobases are natural nucleobases with an additional benzene ring, so there exists adenine (A), thymine (T), cytosine (C), and guanine (G), and there exists xA, xT, xC, and xG. In xDNA, a natural base pairs with an extended base, so A will pair with xT, and G will pair with xC. This pairing preserve the natural hydrogen bonding formations, but widens the helix throughout the entire DNA molecule by 2.4 Angstroms[3]. xDNA is more thermal stable then natural DNA, and can base pair with natural RNA and DNA of up to four bases in length quite well, but as then length approaches eight base pairs or longer, xDNA does not hybridize with DNA or RNA. This would make xDNA orthogonal to DNA, while still preserving some of the features, and would greatly expand the code by doubling the available bases. Additionally, xDNA regions of up to four base pairs have been preserved in vivo in plasmids carried by E. coli [3].

Hirao Bases

Ichiro Hirao at RIKEN and TagCyx biotechnologies have developed a set of base pairs build around Ds and a variety of pairing options, initially Pa, but subsequently published base pairing with Pn and Px. The Hirao bases are efficiently propagated in normal PCR conditions[4].
The Hirao base pairs, Pn and Px are capable of replication using PCR without modification, with DsTPs and PxTPs
. Hirao used his bases in aptamer selections, and pulled out aptamers with 100 fold improvement affinity for their targets compared to the previously published aptamers for the target molecules(VEGF-165 and interferon-gamma)[5].


Most work in the field is focused on expanding the number of non-canonical bases available. Some work is focused on expanding the implementation of non-canonical bases, especially in the field of life sciences, and trying to make these systems work in vivo as well as in vitro. Despite moderate success, no huge advances have recently been published, with the largest non-canonical base pair use in vivo at four bases long. Steven Benner is working on implementing non-canonical bases into cells genomes, and has stated that the solutions to the problems might be more difficult than he had imagined, and that the work had lead to "on God damn problem after another."


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  1. Error fetching PMID 19318205: [Kool2009]
  2. Error fetching PMID 1688644: [Benner1990]
  3. Error fetching PMID 21981660: [Kruger2012]
  4. Error fetching PMID 18776457: [Hirao2008]
  5. Error fetching PMID 23563318: [Hirao2013]
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