The biology of DNA triplexes in the model genetic organisms C. elegans and Drosophila

Lead Research Organisation: University of Southampton
Department Name: School of Biological Sciences

Abstract

The genome sequencing projects have provided the raw material for the scientific community to investigate the genetic basis of the normal physiological function of cells and their dysfunction in disease. To pursue this, new experimental tools are also required, especially to modulate the expression of individual genes in any cell and to define the consequence of these interventions. Ultimately, there is also the hope that such approaches may be used in clinical situations e.g. in the treatment of genetic diseases and cancers. Several of these approaches employ synthetic oligonucleotides. These are molecules either resemble the native chromosomal DNA, i.e. the genetic code, or the RNA which carries the genetic signal inside the cell. A particularly powerful method to regulate gene expression uses molecules which are designed to bind to DNA. These are modified oligonucleotides, called triplex-forming oligonucleotides (TFOs), which bind to the double-stranded DNA to form a triplex structure which prevents the gene from being expressed. Furthermore, in some instances this triplex structure can induce permanent genetic mutations. There is considerable interest in developing these molecules as a mechanism for regulating gene expression. Despite this interest however, relatively little is known about the effects of TFOs on gene expression in intact organisms. This project has put together a team of chemists and biologists keen to address this problem. They will systematically test and characterise the effects of TFOs in the model genetic animals the nematode worm, C. elegans and the fruit fly, Drosophila. Using these animals as models has tremendous advantages, especially because there is already a wealth of information on genetic mutants on which to base these studies. Essentially the project will endeavour to mimic the phenotypes observed in genetic mutants by treating wild-type animals with TFOs. Importantly, the effect of the TFOs will also be carefully characterised so that information can be obtained on how effectively they regulate gene expression. The project will also provide information on other features of the effects of TFOs in a physiological environment which are currently unknown, such as how specific and persistent the effects are, particularly in the context of the developing organism during cell division and growth. The net result of this will be an important advance in the understanding of the mechanism of action of gene regulation by modified oligonucleotides. This will be benefit both those in the scientific community who are looking for new tools to interpret the functional meaning of the animal genome sequences, and also those who wish to exploit this technology for the treatment of human disease.

Technical Summary

Defining the function of genes requires specific and potent methods for regulating their expression both temporally and in a tissue specific manner. This can be achieved by a number of different methods, including those which generate genetic mutants and those which employ molecules which interfere with gene expression e.g. morpholinos or double-stranded RNA. Of the methods that have been developed the most notable is RNAi. The speed and enthusiasm with which this technique has been adopted by biologists seeking to define gene function is testament to the urgent need for new tools to manipulate gene expression. Here we propose to develop another approach for this in intact animals. This targets DNA and therefore has several theoretical advantages over techniques such as antisense and RNAi, which target RNA. It utilizes triplex-forming oligonucleotides (TFOs) which bind in the major groove of duplex DNA, forming specific hydrogen bond contacts with exposed groups on the base pairs, thereby generating DNA base triplets. Unlike RNAi, it does not require complex cellular machinery and should be similar for all cell types, from prokaryote to eukaryote, from invertebrate to mammals. Furthermore, there are fewer sites to target (only two for a diploid cell); it is not subject to up-regulation of the target gene; and finally and most importantly, covalent attachment of the third strand can lead to irreversible gene inactivation or targeted mutagenesis, which could provide a universal means for gene targeting in the germ line of any organism. This project is a collaboration between chemists who have developed TFOs that are suitable for use in physiological conditions, and a group of biologists who seek to exploit them for targeting specific genes in the model genetic animals C. elegans and Drosophila. Essentially, the work will involve experiments aimed at phenocopying well characterised genetic mutants by administering TFOs to wild-type animals. C. elegans and Drosophila offer the opportunity to test a number of different methods for delivering TFOs to cells in the intact animals and to provide an accurate read-out of the functional consequences. Thus we will provide information on the efficiency, penetrance and heritability of TFO-mediated regulation of gene expression. Furthermore, we will make use of transcriptome profiling in TFO treated animals to rigorously test for the specificity of TFO effects. The outcome of the study will be a much-needed definitive biological analysis of the actions of TFOs in intact animals. This will underpin future developments which aim to use these molecules either in the clinic, or as tools to define gene function.

Publications

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Description We have prepared novel nucleotide analogues which enable the formation of stable DNA triplexes under physiological conditions and which are stable against degradation by cellular nucleases. Among these analogues 5-dimethylaminopropargyl deoxyuridine was developed as a useful stabilising analogue, with a relatively simple and reliable synthesis. We have successfully targeted promoter sequences, which contain pyrimidine interruptions, with triplex forming oligonucleotides in vitro and show that these can inhibit in vitro transcription
Exploitation Route It the problems with cell delivery can be overcome then this will provide a useful method for selective gene knockdown and possibly for mutagenesis
Sectors Pharmaceuticals and Medical Biotechnology