Macromolecular recognition, such as sequence-specific recognition, occurring between proteins and DNA sequences, is critical to gene expression and to the development of organisms. One way to view this recognition phenomenon is that the DNA target sequence and the recognizing protein contain a set of complementary and specifically placed functional groups capable of forming (i) hydrogen-bonding interactions, (ii) ionic interactions and (iii) hydrophobic interactions. Probing these weak contacts between proteins and nucleic acids in such sequence-specific complexes, and understanding the contributions of such contacts to overall complex stability, will involve the use of isosteric nucleoside analogues employed to introduce incremental changes into the DNA recognition site, by what amounts to "atomic mutagenesis" of the functional group character. In this approach, a DNA target sequence is prepared in which one or more functional groups have been excised. If the lost function group is involved in a critical interaction that contributes to the recognition phenomenon, then an incremental response in the ability to form that complex should result. Differences in binding effectiveness can be energetically quantitated such that the contribution of a single interaction arising from a single function group can be assessed for its contribution to sequence-specific binding. Correspondingly, if the functional group is not involved in a critical intermolecular interaction, then no change in complex stability should be observed. Multiple substitutions into a given recognition site will determine whether such effects are additive or cooperative. The project focuses on the functional groups present in the DNA minor groove and a variety of analogue residues are described that maintain normal Watson-Crick hydrogen bonding but alter the nature of the functional groups in the minor groove. The project involves the synthesis of specific analogue nucleosides, their incorporation into DNA sequences and the use of those incrementally modified sequences in binding assays to probe the details of sequence-specific recognition.
Broader impacts: This project provides excellent opportunities for broad training of students and post-doctoral fellows who will conduct research at the interface of organic chemistry, biophysics, and biochemistry. In addition, a non-survey course for non-scientists will be developed. This course will cover topics drawn from popular press and, in part, from concepts described in this project. Finally, the project will continue to involve outreach to children through a series of scientific demonstrations that capture their imaginations as well as teach a lesson on the difference between science and what they perceive as magic.
