An extra dimension in nucleic acid sequence recognition

In recent years the human genome has been the subject of intense study and the sequence of all 3 billion units of information (bases) is now known. Genomic DNA is a double helix consisting of two complementary strands held together by Watson-Crick base pairs (AT and GC), and the ability to recognise specific DNA sequences forms the basis of molecular biology, molecular genetics and diagnostics. The conventional approach to DNA sequence recognition is to probe one of the two DNA strands with an oligonucleotide (short single strand of DNA) which will only bind to an equivalent length of DNA with an exactly complementary base sequence. This is an efficient process in vitro as it is easy to denature the DNA duplex by heat, thereby allowing an oligonucleotide probe to bind to one of the strands. In theory the ability to interfere with the biological function of DNA would be a very powerful means of killing viruses and curing certain diseases such as cancer. A method of achieving this objective (antisense technology) has recently emerged. This technology relies upon a chemically synthesised piece of DNA binding to mRNA and preventing protein synthesis. Antisense can be used to inhibit the synthesis of essential viral proteins and important proteins in cell proliferation (e.g. kinases). The first antisense drugs are now coming into the clinic but progress has been limited, partly because the target for antisense therapy (mRNA) is produced in large quantities in vivo (for some proteins there are as many as 25,000 mRNA precursors in a single cell) and feedback mechanisms exits to increase mRNA production if it falls below a certain level. Therefore it is almost impossible to completely inhibit mRNA. The situation at the level of the gene, however, is totally different; there are only 2 copies of each gene and even in the case of genes that occur in tandem, only a handful of copies exist. Directly switching off genes by an external agent is an extremely attractive proposition and in principle it could be achieved by blocking the double helix so that the proteins that interact with DNA can no longer function. This would prevent replication and transcription, the mechanisms by which DNA is copied and the RNA messengers (mRNA) control the synthesis of proteins. The difficulty lies in developing a chemical agent that can bind tightly to a specific region of duplex DNA in the presence of the entire human genome. If such an agent could be developed it would have profound implications in molecular biology, diagnostics and medicine. The antisense approach is of no use here as it is not possible to separate the two strands of genomic DNA in vivo to allow the antisense oligonucleotide to bind. However, Nature offers us clues to solving the problem. It has been known for some time that a third strand of natural DNA can fit into the major groove of the DNA duplex and bind in a sequence-specific manner, provided that one of the two strands of the duplex is purine-rich. The recognition rules are simple; a third strand thymine recognizes A.T and a third strand cytosine recognises G.C. Unfortunately, recognition of A.T and G.C is not sufficient, we must also have a means of recognising T.A. and C.G. In addition, the stability of triplexes at physiological pH is too low to be of value in any practical applications. Natural bases are simply not good enough and the triplex approach will only become feasible if four chemically modified bases can be developed for incorporation into triplex forming oligonucleotides to enable the sequence specific recognition of DNA duplexes. This is the aim of our research and we have four first generation base analogues. The next stage is to refine these molecules to produce a practical working system and evaluate them in diagnostic and biomedical applications.

In theory the ability to interfere with the biological function of DNA is a powerful means of killing viruses and curing genetic diseases. Antisense technology has recently emerged as a means of achieving the first of these objectives. It relies upon chemically synthesised DNA binding to mRNA and preventing protein synthesis. Progress has been limited, partly because the target for antisense therapy (mRNA) is produced in large quantities and feedback mechanisms exist to maintain its production. The situation at the level of the gene, however, is totally different. Directly switching off genes by an external agent could be achieved by blocking the double helix to prevent replication and transcription. The challenge lies in developing a chemical agent that can bind tightly to a specific region of duplex DNA in the presence of the entire genome. It is known that a third strand of natural DNA can fit into the major groove of the DNA duplex and bind in a sequence-specific manner. A third strand thymine recognises A.T. and a third strand cytosine recognises G.C. However, we must also have a means of recognising T.A and C.G. Also, the stability of triplexes at physiological pH is too low, so natural bases are not good enough. Our aim is to develop four chemically modified bases for use in triplexes and we currently have four first generation base analogues. In this project we will refine these molecules, determine the thermodynamic and kinetic properties of the new analogues, produce a practical working system and evaluate this in several diagnostic and biomedical applications.