Certain intrinsically difficult-to-replicate DNA sequences are associated with replication stress, which can lead to cancer-causing mutations. Extensive bioinformatics studies of the mammalian genome identified a variety of such sequences, many of which constitute centrally positioned DNA tandem repeats. This work focuses on one of them, (CAGAGG)n. Based on a variety of biophysical techniques including circular dichroism (CD), UV-vis, 1H NMR, AUC, and gel electrophoresis, we suggest that (CAGAGG)n folds into an unknown structure that does not fall into any currently known category of DNA motifs (e.g. duplex, triplex, guanine quadruplex, i-motif, etc). Inspired by the similarities in CD spectra with a non-canonical DNA structure from Plavec’s laboratory, we hypothesize that the (CAGAGG)n repeats form stacked planar G-C-G-C elements (either a tetrad or adjacent base pairs) connected by AGAG loops in an overall antiparallel conformation (Fig. 1). In addition to extensive biophysical verification of this model, our current effort has been to crystallize this sequence in order to solve its 3D structure via X-ray crystallography. We have pursued a wide array of approaches to establish a suitable oligonucleotide for crystallization, including crystallizing constructs of various lengths and frame shifts and co-crystallizing with nondisruptive and stabilizing ligands. Recently, we have designed small targeted modifications to the repeat sequence in order to increase its stability and to encourage favorable crystal packing. We used PAGE gel electrophoresis and CD spectroscopy to verify viability of new constructs for crystallization and their structural consistency with the native sequences. Crystallization results will be discussed.

Efforts towards the crystal structure of a noncanonical DNA repeat implicated in cancer

254th National Meeting (2017)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.