Sequence specific recognition and cleavage of double helical DNA

Abstract

Deoxyribonucleic acid (DNA) is the stuff of our genes. There are four bases (A, T, G, C) possible for each nucleotide position on each strand of the DNA double helix. Human DNA consists of 3 x 10^9 base pairs. Within these molecules contains the information; what makes humans grow, what makes humans different, and what makes humans human. Within the constraints of the A,T and G,C complementary nature of double helical DNA, for a binding site size of n base pairs there are (4^n)/2 distinguishable sequences for odd n, and (4^n)/2 + (4^(n/2))/2 for even n. Ten years ago we embarked on a program to understand the chemical principles that would make possible the sequence specific recognition of DNA uniquely at the chromosome level (one site in 10^8 base pairs). This requires specific recognition at the ≥ 15 base pair level. In order to analyze the large numbers of potential binding sites on DNA for synthetic sequence specific DNA binding molecules, we realized that the construction of molecules with two separate structural and functional domains: sequence specific recognition and non-specific cleavage of DNA would allow the powerful methods for separating DNA fragments such as gel electrophoresis to be brought to bear on the DNA recognition analysis. Indeed, the design of synthetic sequence specific DNA binding molecules has advanced in recent years due to analytical techniques such as footprinting and affinity cleaving which allow rapid and precise analyses of hundreds of potential DNA binding sites to nucleotide resolution on sequencing gels (Dervan, 1986). Our group has focused on the construction of molecules that bind in the minor and major groove of DNA with incrementally increasing sequence specificity as a first step toward defining the chemical principles for creating specificity at the ≥ 15 base pair level (Dervan, 1986). The tools of chemical synthesis are used in combination with nucleic acid techniques. This may lead to new reagents for mapping chromosomes, diagnosis of disease states at the level of DNA, and novel chemotherapeutic strategies such as artificial repressors for inactivation of these genes. This lecture is a brief report on progress in design of sequence specific DNA binding molecules.

Additional details