Measurement of the Length Dependence of DNA Cyclization using Next Generation Sequencing

Abstract

DNA flexibility is important both for fundamental biophysics and also because DNA flexibility affects DNA packaging and the regulation of gene expression through DNA looping. Historically DNA flexibility has been studied with experiments ranging from biochemical ring closure or DNA looping experiments to AFM, crystallography, and tethered particle microscopy. Even so, the flexibility of DNA in vitro and in vivo remains controversial. We have constructed a library of DNA molecules ranging from 125 to 225 base pairs, via ligation of pools of synthetic DNA of different lengths and PCR, yielding 1023 distinct sequences. The design incorporated barcoding for redundant identification of each molecule, allowing for a ligation reaction to be performed on the entire library in the same reaction mixture. Two different DNA concentrations were used to promote either unimolecular cyclization or bimolecular ligation and thereby explore a wide range of cyclization efficiencies (J factors). A portion of each reaction mixture was treated with BAL-31 to destroy non-cyclized molecules. All products were linearized by restriction digestion and Illumina indices were added to the four mixtures of cyclized and non-cyclized products. The initial library and all of the reaction mixtures were sequenced in a single Illumina MiSeq 300 base pair paired end run. From the roughly 30 million sequence reads obtained, we expect to extract J factors for each of the 1023 molecules in the library, giving an overlapping set of J factors as a function of length. The analysis of the length dependence of ring closure will be presented. We believe this data set will be valuable for improving course grain modeling of DNA. The same methodology should be applicable to DNA loops anchored by proteins.