In this proposal, experiments are described that will develop the synthesis and explore applications of synthetic oligonucleotides bearing non-natural tethered functionality. Such functionally tethered oligonucleotides (FTOs) have recently been used in other laboratories to effect complementary addressed DNA cleavage, detection and intercalative binding; however, no general strategy for their synthesis has yet been reported. Based on preliminary results obtained in this laboratory, a general FTO synthesis has been devised which incorporates the following features: (i) high chemical yield and ease of purification; (ii) access to a wide range of tether structures from a single precursor (for each nucleotide); (iii) access to both major and minor groove attachment positions; (iv) nominal modification of the conventional automated DNA synthesis procedure; and (v) minimal disruption of DNA structure or suitability as an enzymatic substrate. The present FTO synthetic approach has been demonstrated for tether attachment to 2'-deoxycytidine residues in the major groove of DNA. The experiments detailed herein will expand the scope of attachment sites to include 2-deoxyadenosine (dA) in the DNA major groove and 2'-deoxyguanosine (dG) in the minor groove. The ability to attach non-natural ligands in the minor groove will be particularly significant, since the ligand's presence would be transparent to the majority of DNA binding proteins. Several novel applications of FTOs will be examined. Small, switchable molecular devices will be engineered into oligonucleotides. These devices will be used to trap the DNA molecule in non-ground-state conformers that are of significant biological interest: supercoiled or kinked DNA, Z-DNA, and cruciforms. In addition, FrO technology will be used to develop a method for assaying sequence specific interaction of proteins with dA (major groove) and dG (minor groove). Neither of these interactions are able to be studied at present, and thus the proposed methodology will lead to a significant increase in the understand of molecular recognition in DNA protein complexes. The first demonstration of minor groove binding by a sequence specific DNA binding protein, recently reported, has greatly increased the need for methods to elucidate specific protein DNA contacts in the minor groove, for which no adequate method presently exists. The experiments proposed herein are the first in a long-term project aimed at using chemical technology to synthesize novel DNA structures, to elucidate the basis for molecular recognition in DNA binding molecules, and to target drug delivery to specific DNA sequences or structural motifs.
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