T. Chen, F.E. Romesberg, J Am Chem Soc (2017) Published online July 17.
We 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.
D. Thirunavukarasu, T. Chen, Z. Liu, N. Hongdilokkul, F.E. Romesberg, J. Am. Chem. Soc. (Comm.) (2017) 139:2892–2895.
We 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.
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. Chen, N. Hongdilokkul, Z. Liu, R. Adhikary, S.S. Tsuen, F.E. Romesberg, Nat. Chem. (2016) 8:556-562.
We report the development of a polymerase evolution system and its use to evolve thermostable polymerases that efficiently interconvert C2′-OMe modified oligonucleotides and their DNA counterparts via “transcription” and “reverse transcription,” or more importantly, PCR amplify partially C2′-OMe or C2′-F modified oligonucleotides.
T. Chen, F.E. Romesberg, FEBS Lett. (2014) 588:219-229.
We review directed evolution methods used to tailor the properties and/or substrate repertoire of polymerases for different applications and provide a survey of the many polymerases that have been evolved.
A.M. Leconte, M.P. Patel, L.E. Sass, P. McInerney, M. Jarosz, L. Kung, J.L. Bowers, P.R. Buzby, J.W. Efcavitch, F.E. Romesberg, Angew. Chem. Int. Ed. (2010) 34:5921-5924.
We identified a mutant of Taq DNA polymerase that incorporates fluorophore-labeled substrates 50- to 400-fold more efficiently into scarred primers in solution and also demonstrates significantly improved performance under actual sequencing conditions.
A.M. Leconte , F.E. Romesberg, in Protein Engineering (2009) C. Köhrer and U.L. RajBhandary, Eds., Springer-Verlag:Berlin, 291-314.
We review screening and selection methods to identify polymerases with novel function.