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Project Progress

Our primary goal is to synthesize Alternative Nucleic Acids (ANAs) to attempt the optimization of polymer structure subject to the constraints of prebiotic availability, template-directed reproduction, replication conservative mutation, and fitness. We have identified ANAs by taking small steps in “structure-space” away from RNA (the best model for a molecule bearing features both universal and unique to life) that may avoid some of the problems inherent in fulfillment of the aforementioned constraints. Specifically, the Switzer laboratory is examining ANAs with novel perturbations to (i) base-pairing domains and (ii) formal charges. These studies will help to define chemical parameters for molecular evolution. Furthermore, our work addresses whether nucleic acid-like molecules are sufficient to enable the origin of life and what limitations exist for life elsewhere in the universe based on a single biopolymer (eg. RNA) rather than multiple biopolymers (DNA, RNA, proteins, carbohydrates). Accomplishments by our group for the past year are highlighted below.

1. Discovery of a DNA Pentaplex (Chaput, J. C., Switzer, C. “A DNA Pentaplex Incorporating Nucleobase Quintets,” Proc. Natl. Acad. Sci., U.S.A., (1999) 96, 10614-10619). In the past year we have discovered a new structural form of DNA -â?? a pentaplex. In particular, our group has found that DNA bearing the purine component of a non-standard Watson-Crick base-pair (the iso-G:iso-C base-pair, used in past work to expand the genetic code) can form five-stranded helices via nucleobase quintets in the presence of monovalent cations, such as cesium ions. Not only does the DNA pentaplex go beyond the previous four-strand limit for a DNA helical assembly, it also raises the possibility further expanding DNA helical structures to hexaplexes or beyond. By way of background, peptides, in contrast to nucleic acids, routinely give structures with high degrees of strand association. The greater tendency seen for peptide strands to aggregate as compared to nucleic acids is on the one hand consistent with the gross physical properties of the two biopolymers — nucleic acids bear high negative charge densities for which repulsive coulombic forces must be balanced, and this is more difficult to do as more strands are combined. Peptides generally have diminished charge densities relative to nucleic acids. However, our work demonstrates DNA’s previous inability to combine beyond a tetraplex was not due to a general molecular property (eg., the repeating negative charge) but rather a result of a particular molecular feature (the positioning of functional groups on nucleobases). Our work further demonstrates that features common to DNA found in terrestrial genomes impose artificial limits on DNA structural behavior — limits that may not pertain to nucleic acid-like polymers either on the prebiotic Earth or extraterrestrially. The increase in nucleic acid structural diversity conferred by the pentaplex (and possibly even higher order structures) is sure to impact DNA’s fitness landscape with respect to both information carrier and potential as a catalyst. A goal of our laboratory is to gauge this impact.
See Five-Stranded DNA Complex Figure

2. Recombination with an Expanded Genetic Alphabet (Rice, K., Chaput, J. C., Cox, M. M., Switzer, C. “RecA Protein Promotes Strand Exchange with DNA Substrates Containing Isoguanine and 5-Methyl Isocytosine” Biochemistry, (2000), in press). Recombination mechanisms have the potential of imposing serious limitations on the fitness of nucleobases beyond those presently found in terrestrial genomes. This is true since selective advantages for organisms that utilize recombination competent genetic materials include an additional means for DNA repair, and the capacity for sexual, as opposed to asexual, reproduction. The Escherichia coli RecA protein pairs homologous DNA molecules and promotes DNA strand exchange and recombination in vitro. As a model for recombination, we have examined DNA strand exchange between a 70 nucleotide single-stranded DNA fragment and a 40 base-pair double-stranded DNA fragment, in which all G and C residues (at 18 positions distributed throughout the 40 bp exchanged region) were replaced with the non-standard, isomeric nucleotides iso-G and iso-C. It is found that the nonstandard oligonucleotides are substrates for the RecA protein, permitting DNA strand exchange in vitro at a rate and efficiency comparable to exchange with normal DNA substrates. This observation provides an expanded experimental basis for discussions of potential roles for iG and iC in a genetic code.