The proposed research involves understanding and manipulating the interplay of natural and artificial DNA-binding molecules. The long term objective of this research is to evaluate rationally-designed or affinity-selected compounds as modulators of DNA function by exploring the properties of such compounds at the molecular level. This strategy is intended to lay a foundation both for the eventual development of novel information-directed therapeutics, and for a broadened understanding of fundamental molecular genetic processes.
The specific aims of this proposal concern a new approach for artificial regulation of gene expression by nucleic acid ligands. These proposed regulatory ligands are unique in that they are composed entirely of nucleic acids (DNA, or ultimately, RNA) rather than polypeptides. This proposal describes the design and implementation of an artificial repressor/operator system that is to be functional in E. coli. This system will be based on inducible synthesis of a stable repressor RNA capable of recognizing a homopurine operator in double-helical DNA via triple-helix formation. Preliminary studies have shown the feasibility of this approach using an in vitro model in which oligonucleotide regulation has been conferred upon a bacteriophage T7 promoter. The project has four specific aims to extend these results: 1. Analysis of additional RNA oligomers as transcriptional repressors using the in vitro T7 RNA polymerase repression system described above. 2. Identification of other potential sequence-specific repressor RNAs using multiple cycles of operator affinity selection and amplification. 3. Cloning and inducible in vivo synthesis of repressor RNAs in stable expression cassettes for gene regulation experiments in E. coli. 4. Assembly of an in vivo assay system for monitoring expression of a reporter gene driven by a bacteriophage T7 promoter under the control of an inducible RNA repressor. This project is significant for two key reasons. First, design and implementation of a simple transcriptional control interaction based on nucleic acid ligands will offer a model for creation of similar interactions for therapeutic control of target genes in eukaryotes (after delivery of repressor RNAs using viral vectors). Second, the proposed approach may shed light on the fascinating possibility that certain cases of natural gene regulation could involve recognition of double-helical DNA by RNA.
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