Polymerase Amplification, Cloning, and Gene Expression of Benzo‐Homologous “yDNA” Base Pairs
Abstract
A widened DNA base‐pair architecture is studied in an effort to explore the possibility of whether new genetic system designs might possess some of the functions of natural DNA. In the “yDNA” system, pairs are homologated by addition of a benzene ring, which yields (in the present study) benzopyrimidines that are correctly paired with purines. Here we report initial tests of ability of the benzopyrimidines yT and yC to store and transfer biochemical and biological information in vitro and in bacterial cells. In vitro primer extension studies with two polymerases showed that the enzymes could insert the correct nucleotides opposite these yDNA bases, but with low selectivity. PCR amplifications with a thermostable polymerase resulted in correct pairings in 15–20 % of the cases, and more successfully when yT or yC were situated within the primers. Segments of DNA containing one or two yDNA bases were then ligated into a plasmid and tested for their ability to successfully lead the expression of an active protein in vivo. Although active at only a fraction of the activity of fully natural DNA, the unnatural bases encoded the correct codon bases in the majority of cases when singly substituted, and yielded functioning green fluorescent protein. Although the activities with native polymerases are modest with these large base pairs, this is the first example of encoding protein in vivo by an unnatural DNA base pair architecture.
Number of times cited: 23
- Amit M. Jabgunde, Faten Jaziri, Omprakash Bande, Matheus Froeyen, Mikhail Abramov, Hoai Nguyen, Guy Schepers, Eveline Lescrinier, Vitor B. Pinheiro, Valérie Pezo, Philippe Marlière and Piet Herdewijn, Methylated Nucleobases: Synthesis and Evaluation for Base Pairing In Vitro and In Vivo, Chemistry – A European Journal, 24, 48, (12695-12707), (2018).
- Subhendu Sekhar Bag and Suman Kalyan Das, Design, Synthesis and Photophysical Property of a Doubly Widened Fused‐Triazolyl‐Phenanthrene Unnatural Nucleoside, ChemistrySelect, 2, 12, (3577-3583), (2017).
- Elena Eremeeva, Michail Abramov, Lia Margamuljana and Piet Herdewijn, Base‐Modified Nucleic Acids as a Powerful Tool for Synthetic Biology and Biotechnology, Chemistry – A European Journal, 23, 40, (9560-9576), (2017).
- Rania Abu El Asrar, Lia Margamuljana, Mikhail Abramov, Omprakash Bande, Stefano Agnello, Miyeon Jang and Piet Herdewijn, Enzymatic Incorporation of Modified Purine Nucleotides in DNA, ChemBioChem, 18, 24, (2408-2415), (2017).
- Izumi Okamura, Soyoung Park, Ryota Hiraga, Seigi Yamamoto and Hiroshi Sugiyama, Synthesis, Photophysical Properties, and Enzymatic Incorporation of an Emissive Thymidine Analogue, Chemistry Letters, 10.1246/cl.161024, 46, 2, (245-248), (2017).
- Elena Eremeeva, Michail Abramov, Lia Margamuljana, Jef Rozenski, Valerie Pezo, Philippe Marlière and Piet Herdewijn, Chemical Morphing of DNA Containing Four Noncanonical Bases, Angewandte Chemie International Edition, 55, 26, (7515-7519), (2016).
- Elena Eremeeva, Michail Abramov, Lia Margamuljana, Jef Rozenski, Valerie Pezo, Philippe Marlière and Piet Herdewijn, Chemical Morphing of DNA Containing Four Noncanonical Bases, Angewandte Chemie, 128, 26, (7641-7645), (2016).
- Masayasu Kuwahara, Kenta Hagiwara and Hiroaki Ozaki, Polymerase Reactions that Involve Modified Nucleotides, Modified Nucleic Acids in Biology and Medicine, 10.1007/978-3-319-34175-0_18, (429-453), (2016).
- Daniel G. Singleton, Rohanah Hussain, Giuliano Siligardi, Pawan Kumar, Patrick J. Hrdlicka, Nina Berova and Eugen Stulz, Increased duplex stabilization in porphyrin-LNA zipper arrays with structure dependent exciton coupling, Org. Biomol. Chem., 10.1039/C5OB01681A, 14, 1, (149-157), (2016).
- Nicole A. Leal, Hyo-Joong Kim, Shuichi Hoshika, Myong-Jung Kim, Matthew A. Carrigan and Steven A. Benner, Transcription, Reverse Transcription, and Analysis of RNA Containing Artificial Genetic Components, ACS Synthetic Biology, 4, 4, (407), (2015).
- Haruka Otomo, Soyoung Park, Seigi Yamamoto and Hiroshi Sugiyama, Amplification of fluorescent DNA through enzymatic incorporation of a highly emissive deoxyguanosine analogue, RSC Adv., 4, 59, (31341), (2014).
- Lingjun Li, Mélissa Degardin, Thomas Lavergne, Denis A. Malyshev, Kirandeep Dhami, Phillip Ordoukhanian and Floyd E. Romesberg, Natural-like Replication of an Unnatural Base Pair for the Expansion of the Genetic Alphabet and Biotechnology Applications, Journal of the American Chemical Society, 136, 3, (826), (2014).
- Malte Winnacker and Eric T. Kool, Künstliche genetische Systeme bestehend aus vergrößerten Basenpaaren, Angewandte Chemie, 125, 48, (12728-12739), (2013).
- Malte Winnacker and Eric T. Kool, Artificial Genetic Sets Composed of Size‐Expanded Base Pairs, Angewandte Chemie International Edition, 52, 48, (12498-12508), (2013).
- Purshotam Sharma, Linda A. Lait and Stacey D. Wetmore, Exploring the limits of nucleobase expansion: computational design of naphthohomologated (xx-) purines and comparison to the natural and xDNA purines, Physical Chemistry Chemical Physics, 15, 37, (15538), (2013).
- Karin Betz, Denis A. Malyshev, Thomas Lavergne, Wolfram Welte, Kay Diederichs, Floyd E. Romesberg and Andreas Marx, Structural Insights into DNA Replication without Hydrogen Bonds, Journal of the American Chemical Society, 135, 49, (18637), (2013).
- Yin Nah Teo and Eric T. Kool, DNA-Multichromophore Systems, Chemical Reviews, 10.1021/cr100351g, 112, 7, (4221-4245), (2012).
- Vitor B Pinheiro and Philipp Holliger, The XNA world: progress towards replication and evolution of synthetic genetic polymers, Current Opinion in Chemical Biology, 10.1016/j.cbpa.2012.05.198, 16, 3-4, (245-252), (2012).
- Thomas J. Bandy, Ashley Brewer, Jonathan R. Burns, Gabriella Marth, ThaoNguyen Nguyen and Eugen Stulz, DNA as supramolecular scaffold for functional molecules: progress in DNA nanotechnology, Chem. Soc. Rev., 10.1039/B820255A, 40, 1, (138-148), (2011).
- Haige Lu, Stephen R. Lynch, Alex H. F. Lee and Eric T. Kool, Structure and Replication of yDNA: A Novel Genetic Set Widened by Benzo‐Homologation, ChemBioChem, 10, 15, (2530-2538), (2009).
- P. Sandin, G. Stengel, T. Ljungdahl, K. Borjesson, B. Macao and L. M. Wilhelmsson, Highly efficient incorporation of the fluorescent nucleotide analogs tC and tCO by Klenow fragment, Nucleic Acids Research, 37, 12, (3924), (2009).
- David Loakes and Philipp Holliger, Polymerase engineering: towards the encoded synthesis of unnatural biopolymers, Chemical Communications, 10.1039/b903307f, 31, (4619), (2009).
- Laura Albrecht, Katie Wilson and Stacey Wetmore, Computational Evaluation of Nucleotide Insertion Opposite Expanded and Widened DNA by the Translesion Synthesis Polymerase Dpo4, Molecules, 10.3390/molecules21070822, 21, 7, (822), (2016).
