VEGF-A was also co-localized with SRSF1 in some, but not all central terminals

rns are quite sensitive to minor groove width and cleavage results with alternating AT DNA sequences are similar to those of B-form DNA sequences but not to those of A-tract DNA. X-ray and NMR structures of DNA duplexes with A-tracts have narrow minor grooves while alternating AT structures have been observed with both narrow and wide minor grooves and a variety of polymorphic structures. As part of an effort to develop new antiparasitic drugs to target the AT-rich mitochondrial, kDNA of kinetoplastid parasites, the structure-dependent global conformational changes induced by a number of minor groove targeting compounds were evaluated for both alternating AT and A-tract DNA sequences. PAGE results suggest that the compounds typically leave the curvature of intrinsically bent A-tract DNA nearly unchanged. In contrast, significant compound induced mobility decreases that correlate with bending distortions are observed PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19844094 for alternating AT sequences. Ligand induced changes in bending are an important new observation since such bending can interfere with gene expression and contribute to antiparasitic biological activity. DNA curvature can be due to a global feature of nucleic acids associated with the summation of small influences on the topology for individual base pair steps or it can be due to more dramatic effects such as local kinks in the helical conformation. A key unanswered question from the PAGE observations on mobility changes is what is the molecular basis of compound induced structural changes in alternating AT DNA A high resolution structure of the alternating AT sequence and its complex with a minor groove binder is a critical missing piece of current information in our understanding of local DNA microstructures. We have used NMR including residual dipolar coupling methods together with restrained MD simulations to determine the structures of the free DNA and complexes. Specific questions to answer are: is the gel mobility reduction actually due to DNA induced bending by minor groove complex formation; what are the specific changes in the DNA helical structure that cause the conformational purchase SB-590885 change that leads to the gel anomaly; and is the conformational change a local kink or a more global effect and what is its magnitude The results presented here answer these questions and take the field of local structural variations of DNA forward in an important new area. We have previously shown that minor groove binding ligands exhibit microscopic rearrangements on the millisecond time scale without dissociating off the DNA into the bulk solution. This rearrangement results in chemical exchange processes and NMR signals that greatly complicate structure determination. It is therefore Chembiochem. Author manuscript; available in PMC 2014 February 11. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Rettig et al. Page 3 necessary to explore solution conditions and minor groove binding ligands where exchange process are minimal. Netropsin exhibits the most favorable dynamic properties and was therefore chosen as a representative ligand to investigate induced bending in alternating AT DNA sequences. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Results and Discussion Gel Migration Studies In order to compare PAGE and NMR results, the same DNA sequence was used for both methods. The alternating AT sequence was evaluated in ligation ladder PAGE experiments with and without netropsin. The m