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Unnatural Base Design and Characterization
| The structure of duplex DNA is based on the complementary Watson-Crick hydrogen bonding patterns of adenine with thymine (dA:dT base pair) and guanine with cytosine (dG:dC base pair). Unnatural base pairs that adopt hydrogen bond interactions have shown thermodynamic orthogonality (e.g. iso-C:iso-G, κ:X), but each of these unnatural bases has a stable tautomeric form whose hydrogen bond donor and acceptor pattern is compatible with one of the natural nucleotides, and could lead to mispairing. Since hydrophobic interactions are very strong forces in aqueous biological systems, the unnatural pairs based on hydrophobicity could provide long term storage of genetic information and a unique opportunity to study molecular recognition in the context of the DNA replication. In addition, the development of an unnatural base pair will offer insights into the complex mechanism of DNA replication. Finally, a third base would expand the genetic alphabet for a wide variety of in vitro biotechnology applications, and would also lay the foundation for the in vivo expansion of an organism’s genetic code. |
In an effort to develop an orthogonal third base pair, more than 90 hydrophobic bases have been designed, synthesized as the triphosphate and phosphoramidite, and characterized. A few of these bases are shown in the slideshow at the top of the page. A number ‘first generation’ self-pairs and hetero-pairs have been identified with promising properties. These base pairs are shown below. |
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| Remarkably, we have found that neither H-bonding nor large aromatic surface area is required for base pair stability in duplex DNA or polymerase mediated replication. Thus, while our early efforts focused mostly on nucleobase analogs with large aromatic surface area, our more recent efforts have all focused on pairs formed between suitably substituted benzene rings. For example, the 3-fluorobenzene (3FB) self-pair shown above is now the most efficiently and selectively replicated unnatural base pair known. The 3FB triphosphate is inserted opposite 3FB in the template, and the nascent primer terminus extended with rates and selectivities that are beginning to rival those of natural synthesis. We have recently solved both the NMR structure (in collaboration with B. Geierstanger, GNF) and the X-ray structure (in collaboration with G. Spraggon, GNF) of DNA containing the 3FB self pair. While the structures of the mispairs are also currently under study, the structure of the self-pair contains hints as to why it is so efficiently replicated by DNA polymerases, and also offers hints about how to further optimize the self-pair. We are confident that additional rounds of synthetic chemistry and biochemical characterization will result in the further optimization of 3FB and the first truly realistic unnatural base pair candidate. For example, we have already found that the addition of judiciously placed nitrogen atoms or methyl and cyano groups, have a profound affect on base pair stability and replication. We are also initiating the study of RNA polymerase-mediated transcription of the 3FB self-pair, which is the next step in information storage and retreval. Lastly, we are actively pursuing the directed evolution of polymerases that are tailored to better recognize our unnatural base pairs. Click here to read more about this aspect of the project. |
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