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Chemical stabilization of unnatural nucleotide triphosphates for the in vivo expansion of the genetic alphabet

A.W. Feldman, V.T. Dien, F.E. Romesberg, J. Am. Chem. Soc. (2017) 139:2464–2467.
pubpic2017feldmanWe report the synthesis and evaluation of unnatural triphosphates with their β,γ-bridging oxygen replaced with a difluoromethylene moiety, yielding dNaMTPCF2 and dTPT3TPCF2. We find that although dNaMTPCF2 cannot support in vivo replication, likely due to poor polymerase recognition, dTPT3TPCF2 can, and moreover, its increased stability can contribute to increased UBP retention. The results demonstrate the promise of a chemical approach to SSO optimization.

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A semisynthetic organism engineered for the stable expansion of the genetic alphabet

Y. Zhang, B.M. Lamb, A.W. Feldman, A.X. Zhou, T. Lavergne, L. Li, F.E. Romesberg, Proc. Natl. Acad. Sci. USA (2017) 114: 1317–1322.
pubpic2017zhangWe describe an optimized SSO that is healthy, more autonomous than its predecessor, and able to store increased information indefinitely. The SSO constitutes a stable form of semisynthetic life and lays the foundation for efforts to impart life with new forms and functions. Read more in The Guardian, The Washington Post, TSRI News & Views, and The San Diego Union Tribune, or listen to an interview in This Way Up from Radio New Zealand.

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The expanding world of DNA and RNA

T. Chen, N. Hongdilokkul, Z. Liu, D. Thirunavukarasu, F.E. Romesberg, Curr. Op. Chem. Biol. (2016) 34:80–87.
pubpic2016bchenWe review nucleotide modifications, such as those to phosphate and sugar moieties that increase nuclease resistance or the range of activities possible, as well as whole nucleobase replacement that results in selective pairing and the creation of unnatural base pairs. Both in vitro and in vivo examples are discussed, including efforts to create semi-synthetic organisms with altered or expanded genetic alphabets.

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FRET characterization of complex conformational changes in a large 16S ribosomal RNA fragment site-specifically labeled using unnatural base pairs

T. Lavergne, R. Lamichhane, D.A. Malyshev, Z. Li, L. Li, E. Sperling, J.R. Williamson, D.P. Millar, F.E. Romesberg, ACS Chem. Biol. (2016) 11:1347-1353.
pubpic2016lavergneWe report the synthesis and evaluation of several unnatural ribotriphosphates bearing linkers that allow the chemoselective attachment of different functionalities. One unnatural base pair is used to dual label a 243-nt fragment of a 16S RNA with Cy3 and Cy5, which are then used to characterize conformational changes in the presence of ribosomal proteins.

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An expanded genetic alphabet

D.A. Malyshev, F.E. Romesberg, Angew. Chem. Int. Ed. (2015) 54:11930–11944.
pubpic2015malyshevNatural nucleic acids and the genetic information they encode are limited by the use of only four nucleotides that form two base pairs, (d)G-(d)C and d(A)-dT/U. In the past decade, three classes of unnatural base pairs have been developed to a high level of proof-of-concept. This review summarizes their development and the potentially revolutionary applications that they are now enabling.

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A semi-synthetic organism with an expanded genetic alphabet

D.A. Malyshev, K. Dhami, T. Lavergne, T. Chen, N. Dai, J.M. Foster, I.R. Corrêa, F.E. Romesberg, Nature (2014) 509:385-388.
pubpic2014malyshevWe demonstrate that E. coli can stably maintain a plasmid containing d5SICS–dNaM with only the addition of an exogenously expressed algal nucleotide triphosphate transporter. Read the associated News & Views or read more in The New York Times, The Atlantic, Wired, Science, Chemistry World, and The San Diego Union Tribune. This work was also recognized as one of the top 10 scientific achievements of 2014 by Science.