Project: Research project

Project Details


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
Effective start/end date5/1/934/30/98