S.E. Morris, A.W. Feldman, F.E. Romesberg, ACS Synth Biol (2017) Published online June 27.
We report a steady-state kinetic characterization of the rate with which the Klenow fragment of E. coli DNA polymerase I synthesizes the dNaM-dTPT3 UBP and its mispairs in a variety of sequence contexts. The data demonstrate that dNaMTP and dTPT3TP are well optimized and standardized parts for the expansion of the genetic alphabet.
A.W. Feldman, V.T. Dien, F.E. Romesberg, J. Am. Chem. Soc. (2017) 139:2464–2467.
We 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.
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.
We 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.
T. Chen, N. Hongdilokkul, Z. Liu, D. Thirunavukarasu, F.E. Romesberg, Curr. Op. Chem. Biol. (2016) 34:80–87.
We 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.
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.
We 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.
D.A. Malyshev, F.E. Romesberg, Angew. Chem. Int. Ed. (2015) 54:11930–11944.
Natural 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.
K. Dhami, D.A. Malyshev, P. Ordoukhanian, T. Kubelka, M. Hocek, F.E. Romesberg, Nucleic Acids Res (2014) 42:10235-10244.
We report a PCR-based screen to identify promising unnatural base pairs from a library of 111 unnatural nucleotides (~6,000 candidate base pairs).