This proposal for research on "Sequence Specific Incorporation of Functionalized Bases into RNA" between Dr. Christopher Y. Switzer and Professor Steven A. Benner of the Eidgenoessische Technische Hochschule, Zurich, Switzerland, is sponsored by NSF. Ribonucleotides are involved in cell division, genetic regulation, viral growth, and tumor transformation. The recent discovery of catalytic RNA and its ability to serve as an RNA polymerase has resurrected early ideas that the first biological catalysts were RNA molecules. There is little information regarding the confor- mation of these molecules in solution. The investigation of RNA tertiary and quaternary structure in such activities as RNA splicing, tRNA maturation, and translation has been limited by the lack of flexibility available with existing RNA synthesis tech- nologies. Unnatural bases containing structural probes (photo- affinity labels, spin-labels), if incorporated at specific sites, would facilitate advances in these areas. A nucleotide base pair distinct from those of A/T(U) and G/C that is incorporated into RNA by a polymerase could allow such sequence-specific introduction of structural probes. The goal of the proposed work is to synthesize the iso-cytidine bases carrying functional groups at the 5-position, and to incorporate them enzymatically into RNA under the direction of a DNA template containing d-iso-G. The iso-C/iso-G base pair has a Watson-Crick hydrogen bonding pattern different from the A/T(U) and G/C base pairs. Because of this different hydrogen bonding pattern, a RNA polymerase should faithfully incorporate the iso-C derivatives opposite only to d-iso-G. These functionalized bases will assist in experiments to probe RNA tertiary and quaternary structure.
