| Popis: |
DNA conformation, in particular its supercoiling, plays an important structural and functional role in gene accessibility as well as in DNA condensation. Enzyme driven changes of DNA plasmids between their linear, circular and supercoiled conformations control the level of condensation and DNA distal-site interactions. Much effort has been made to quantify the branched supercoiled state of DNA to understand its ubiquitous contribution to many biological functions, such as packaging, transcription, replication etc. Nanopore technology has proven to be an excellent label-free single-molecule method to investigate the conformations of the translocating DNA in terms of the current pulse readout. In this paper, we present a comprehensive study to detect different branched-supercoils on individual plasmid DNA molecules. Using a detailed event charge deficit (ECD) analysis of the translocating molecules, we reveal, for the first time, the distributions in size and the position of the plectoneme branches on the supercoiled plasmid. Additionally, this analysis also gives an independent measure of the effective nanopore length. Finally, we use our nanopore platform for measurement of enzyme-dependent linearization of these branched-supercoiled plasmids. By simultaneous measurement of both single-molecule DNA supercoiled conformations and enzyme-dependent bulk conformational changes, we establish nanopore sensing as a promising platform for an in-depth understanding of the structural landscapes of supercoiled DNA to decipher its functional role in different biological processes. |
