Functional Group Interactions in DNA - Protein Recognition

Boston College, Chestnut Hill MA

Investigators

Abstract

This project applies the tools of organic chemistry to prepare specifically altered DNA sequences, either by total chemical synthesis, or by synthesis of modified DNA subunits (nucleotides) that can then be linked together by enzymes called "DNA polymerases" to obtain DNA molecules modified at desired locations. A variety of biophysical tools will be applied to study the effects of these modifications on DNA structure and thermodynamic stability, and the abilities of proteins (TBP, trp repressor) and minor groove binding ligands (DAPI, Hoechst 33258) to bind these modified DNA duplexes. Intellectual Merit: This project will focus on two areas of modification: (i) First, minor groove functional groups in A-T rich regions will be probed by synthesizing analogues such as 3-deazaadenine or 3-deaza-3- methyladenine that are expected be disrupt binding of water molecules in the minor groove. These derivatives will be studied to assess the contribution of such water interactions to DNA structure and stability. A 3-deaza-3-hydroxymethyladenine derivative will synthesized as a modified adenosine residue with a built-in structural water analogue (hydroxymethyl group). By mimicking the water structure, studies employing this analogue are expected to provide insight into DNA helix stabilization by water groove binding. Related analogues will also provide new types of labeling sites to introduce reporter groups to the minor groove. (ii) Second, a series of pre-organized analogues will be synthesized in which the nucleobase and carbohydrate moieties are fused into a single rigid structure that maintains the conformation adopted upon duplex formation. These modifications are expected to reduce DNA single-strand flexibility, thus pre-organizing it to promote helix formation and increase helix stability. Determination of the thermodynamic parameters for duplex formation will quantify the differences between native and analogue sequences. The process of designing, synthesizing, and testing new analogues to effect specific changes in DNA structure and properties will deepen our understanding of the behavior of this all important biopolymer. The students working on the project will be exposed to all aspects of the process. They will take part in the design and preparation of the requisite modified nucleoside monomers, perform the DNA synthesis and purification, and then study the properties of the analogue DNA sequence. Broader Impacts: (1) The modified nucleotides and DNA analogues prepared during the course of the proposed work will be made available to researchers in related fields to study the effects of these modifications on various biological processes involving DNA. (2) The PI will recruit undergraduates, especially women and members of underrepresented groups to participate in research on this project. The university is making available two to three "Lin Fellowships" (named after a former chairman, Jeong-Long Lin) for summer-support students from underrepresented groups to work in the PI's laboratory. (3) Other outreach activities planned by PI involve relatively young children who have little to no exposure to chemistry. An outreach program to 5th grade students in Needham, MA is planned to bring the excitement of chemistry to those just beginning to think about science.