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Polymerase chain transcription: exponential synthesis of RNA and modified RNA

T. Chen, F.E. Romesberg, J Am Chem Soc (2017) Published online July 17.
pubpic2017chenWe show that the evolved Stoffel fragment mutant, SFM4-3, efficiently transcribes RNA or 2′-F-modified RNA and that it also efficiently PCR amplifies oligonucleotides of mixed RNA and DNA composition. Using thermocycling and a novel DNA template, we demonstrate a polymerase chain transcription (PCT) reaction that results in the exponential production of orders of magnitude more RNA or modified RNA than is available by conventional transcription.

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Synthetic biology parts for the storage of increased genetic information in cells

S.E. Morris, A.W. Feldman, F.E. Romesberg, ACS Synth Biol (2017) Published online June 27.
pubpic2017MorrisWe 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.

Conformational heterogeneity and DNA recognition by the morphogen bicoid

R. Adhikary, Y.X. Tan, J. Liu, J. Zimmermann, M. Holcomb, C. Yvellez, P.E. Dawson, F.E. Romesberg, Biochemistry (2017) 56:2787–2793.
pubpic2017AdhikaryACS Editors’ Choice: We introduce C-D bonds within different structural elements in the Drosophila transcription factor bicoid, a homeodomain protein with morphogenic activity. C-D absorptions are characterized in the free and DNA-bound states, as well as during thermal denaturation. Residues in the DNA recognition helix, but not other helices, exhibit multiple conformations, and overall the data are consistent with the existence of multiple stable conformations in the folded state.

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Selection of 2′-fluoro-modified aptamers with optimized properties

D. Thirunavukarasu, T. Chen, Z. Liu, N. Hongdilokkul, F.E. Romesberg, J. Am. Chem. Soc. (Comm.) (2017) 139:2892–2895.
pubpic2017thirunavukarasuWe report the selection and characterization of 2′-F purine aptamers that bind human neutrophil elastase (HNE) with reasonable affinity. The 2′-F substituents are found to facilitate the selection of specific interactions, and we demonstrate than only a few can optimize properties far beyond simple nuclease resistance.

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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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Transparent window vibrational probes for the characterization of proteins with high structural and temporal resolution

R. Adhikary, J. Zimmermann, F.E. Romesberg, Chem. Rev. (2017) 117:1927–1969.
pubpic2017adhikaryWe review the strengths and weaknesses of the different transparent window vibrational probes, methods by which they may be site-specifically incorporated into peptides and proteins, and the physicochemical properties they may be used to study. These topics are put into context through four case studies focused on KSI, SH3, DHFR, and cyt c.