DNA molecules must be tightly packaged to fit inside cells;simultaneously, DNA must be accessible to protein complexes in order to be replicated, transcribed or repaired. Thus, the method of packaging for a certain DNA fragment is likely to vary with the type of genes that fragment contains (e.g. with transcription frequency), and with location of the fragment relative to the origin(s) of replication. However, we do not know details of the link between the structure of a particular chromosome fragment and the information content of that fragment. This is a basic issue for molecular biology, as variations in packaging of a certain gene likely affect transcription frequency, and thus serve as a mode of transcriptional control. It is also relevant to human health, since a number of human diseases have been linked to abnormal chromatin structure or to impaired chromatin remodeling abilities. The details of the structure of specific fragments of native chromosomes are not well known in any organism, mainly because of a lack of techniques available to study the structure of specific parts of the genome. To rectify this deficiency, we propose to develop a new technique that will allow isolation of specific chromosomal fragments and testing of their structure. To do this, we will first develop methods to isolate and immobilize DNA fragments in a sequence-specific manner using labeled nucleic-acid analogs, such as PNA and LNA. Once such methods have been developed, we will work to purify and isolate specific chromatin fragments from a model organism, and isolate them within a single-molecule device that measures biomolecular elasticity- elasticity is a direct and quantitative probe of structure. We will test the robustness and specificity of the protocol, and generate data demonstrating that our approach is broadly applicable to the study of chromatin structure.
DNA in the cell is stored in a highly compact, folded structure. Multiple human diseases have been linked to abnormalities in this structure around specific genes;however, we lack a clear microscopic picture of chromatin structure. Here, we propose to develop a novel technique that will permit direct study of the structure of chromatin in the vicinity of a given piece of DNA.
|Berezney, John P; Saleh, Omar A (2014) Locked nucleic acid oligomers as handles for single molecule manipulation. Nucleic Acids Res 42:e150|