Aqueous Solution Derivatization of Unprotected RNA/DNA

Orgel, Leslie

Abstract

The primary objective of this research program is to develop simple, efficient, specific procedures for covalently crosslinking protein transcription factors to the short double-stranded DNA sequences to which they bind. We plan to use these procedures to control transcription in eukaryotic cells by decoying transcription factors, so we will develop DNA sequences that are resistant to nuclease digestion, and crosslinking reagents that can function in the intranuclear environment. We hope that this approach will, in the long run, lead to the synthesis of DNA- crosslinker adducts that can be used in whole animals and can, therefore, find a place in medicine. In much of our work we will use DNA sequences that fold into hairpin and dumbbell structures with the enhancer-binding sequence present in the double-helical stem. Such sequences bind enhancer proteins with undiminished affinity but do not undergo strand separation. The dumbbell sequences are also completely resistant to exonuclease digestion in serum. Hairpins and dumbbells, therefore, should prove superior to short double- stranded DNA's as decoys. Crosslinking reagents that we plan to use include a variety of metal complexes and a number of commercially available bifunctional alkylating and acylating agents. Usually we will attach the crosslinkers to DNA via modified nucleotides. The resulting adducts will first be used to crosslink to protein in vitro. Then the most effective adducts will be tested as transcription inhibitors in eukaryotic cells using a chloramphenicol acetyl transferase (CAT) assay system. The systems that we plan to study include the CREB-CRE system, the Jun-TRE system and the Zif268 protein with its consensus binding site. A second objective of our program is to develop methods for immobilizing polynucleotides stereospecifically on metal or mineral surfaces. This will facilitate studies of nucleic acid structure by electron tunnelling microscopy and atomic force microscopy. We will insert several functional groups into the nucleic acid in positions that can simultaneously contact a planar surface, for example, that of metallic gold. The tight attachment of modified nucleotides to gold should hold the nucleic acid in a fixed position and orientation on the surface.