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xDNA: Toward A New Genetic System with Expanded Size

This project addresses the question of whether other genetic systems beside the natural one might be functional. Inspired by the early work of Leonard three decades ago, we have designed and synthesized new DNA bases that are larger than the natural ones by the addition of a benzene ring. Significantly, all the expanded-size nucleobases are fluorescent. We have shown that these can be assembled into large, and very stable, DNA-like helices. Recent work has shown that xDNA base pairs can be synthesized by polymerase enzymes, and especially exciting is our finding that they can function in a living cell, retaining the ability to encode sequence and even code for amino acids.


Our Future Goals:

• Find and develop new polymerase enzymes that can replicate long strands of xDNA

• Develop biological tools that exploit the fluorescence of xDNA and its high helix stability

• Explore the activity of xDNA in bacterial and eukaryotic cells

• Synthesize and study xRNA – a new large-sized RNA analog

• Study nonbiological applications of xDNA as a molecular wire and in nanostructures

• Synthesize more extreme forms of expanded DNA




A. R. Hernandez, L. W. Peterson, and E. T. Kool*, "Steric Restrictions of RISC in RNA Interference Identified with Size-Expanded RNA Nucleobases," ACS Chem. Biol., 2012, ASAP.


A. T. Krueger, L. W. Peterson, J. Chelliserry, D. J. Kleinbaum and E. T. Kool,* "Encoding Phenotype in Bacteria with an Alternative Genetic Set," J. Am. Chem. Soc., 2011, 133, 18447-18451.


A. R. Hernandez and E. T. Kool,* "The Components of xRNA: Synthesis and Fluorescence of a Full Genetic Set of Size-Expanded Ribonucleosides," Org. Lett., 2011, 13, 676-679.


S. K. Jarchow-Choy, A. T. Krueger, H. Liu, J. Gao, E. T. Kool. "Fluorescent xDNA Nucleotides as Efficient Substrates for a Template-independent Polymerase," Nucleic Acids Res., 2010, 39, 1586-1594.


H. Lu, A. T. Krueger, J. Gao, H. Liu, E. T. Kool. "Toward a Designed Genetic System with Biochemical Function: Polymerase Synthesis of Single and Multiple Size-Expanded DNA Base Pairs," Org. Biomol. Chem., 2010, 8, 2704-2710.


H. Lu, S. R. Lynch, A. H. F. Lee, E. T. Kool. "Structure and Replication of yDNA: A Novel Genetic Set Widened by Benzo-Homologation," ChemBioChem, 2009, 10, 2530-2538.


A. T. Krueger, E. T. Kool. "Redesigning the Architecture of the DNA Base Pair: Toward Biochemical and Biological Function of New Genetic Sets," Chem. Biol. 2009, 16, 242-248.


J. C. Delaney, J. Gao, H. Liu, N. Shrivastav, J. M. Essigmann, E. T. Kool. "Efficient Replication Bypass of Size-Expanded DNA Base Pairs in Bacterial Cells," Angew. Chem. Int. Ed. 2009, 48, 4524-4527.


A. T. Krueger, H. Lu, T. Hojland, H. Liu, J. Gao, E. T. Kool, "Toward the Replication of xDNA, a Size-expanded Unnatural Genetic System," Nucleic Acids Symp. Ser. 2008, 52, 455-456.


J. Chelliserrykattil, H. Lu, A. H. F. Lee, E. T. Kool. "Polymerase Amplification, Cloning, and Gene Expression of Benzohomolgous "yDNA" Base Pairs," ChemBioChem. 2008, 9, 2976-2980.


A. T. Krueger, E. T. Kool. "Fluorescence of Size-expanded DNA Bases: Reporting on DNA Sequence and Structure with an Unnatural Genetic Set" J. Am. Chem. Soc. 2008, 130, 3989-3999.


A. T. Krueger, H. Lu, A. H. F. Lee, E. T. Kool. "Synthesis and Properties of Size-Expanded DNAs: Toward Designed, Functional Genetic Systems" Acc. Chem. Res. 2006, 40, 41-50.


A. H. F. Lee, E. T. Kool. "Exploring the Limits of DNA Size: Naphtho-Homologated DNA Bases and Pairs" J. Am. Chem. Soc. 2006, 128, 9219-9230.


J. Gao, H. Lu, E. T. Kool. "Completion of the Eight-base Genetic System, xDNA, and its Interaction with the Natural Genetic System" Angew. Chem. Int. Ed. 2005, 44, 3118-3122.


A. H. F. Lee, E. T. Kool. "A New Four-Base Genetic Helix, yDNA, Composed of Widened Benzopyrimidine-Purine Pairs" J. Am. Chem. Soc. 2005, 127, 3332-3338.


A. H. F. Lee, E. T. Kool. "Novel Benzopyrimidines as Widened Analogues of DNA Bases" J. Org. Chem. 2005, 70, 132-140.


H. Liu, J. Gao, E. T. Kool. "Helix-Forming Properties of Size-Expanded DNA, an Alternate Four-Base Genetic Form" J. Am. Chem. Soc. 2005, 127, 1396-1402.


H. Liu, J. Gao, E. T. Kool. "Size-Expanded Analogues of dG and dC: Synthesis and Pairing Properties in DNA" J. Org. Chem. 2005, 70, 639-647.


H. Lu, K. He, E. T. Kool. "yDNA: A New Geometry for Size-Expanded Base Pairs" Angew. Chem. Int. Ed. Engl. 2004, 43, 5834-5836.


J. Gao, H. Liu, E. T. Kool. "Expanded Size Bases in Naturally Sized DNA: Evaluation of Steric Effects in Watson-Crick Pairing" J. Am. Chem. Soc. 2004, 126, 11826-11831.


H. Liu, S. R. Lynch, E. T. Kool. "Solution Structure of xDNA: A paired Genetic Helix with Increased Diameter" J. Am. Chem. Soc. 2004, 126, 6900-6905.


H. Liu, J. Gao, D. Saito, L. Maynard, E. T. Kool. "Toward a New Genetic System with Expanded Dimensions: Size-Expanded Analogs of Deoxyadenosine and Thymidine" J. Am. Chem. Soc. 2004, 126, 1102-1109.


H. Liu, J. Gao, S. Lynch, L. Maynard, D. Saito, E. T. Kool. "A Four-base Paired Genetic Helix with Expanded Size" Science 2003, 302, 868-871.