A method for assaying DNA flexibility

•The sequence dependence of the mechanical properties of DNA can regulate DNA-deforming biological processes.•Loop-seq is a recent next-generation sequencing based method to measure DNA bendability in high throughput.•Here we present a rapid, low-throughput variant of the loop-seq protocol that does not involve next-generation sequencing.•We validate the assay against earlier loop-seq measurements.•We measure the binding site of bacterial IHF as being unusually rigid and comment on its functional relevance. The transcription, replication, packaging, and repair of genetic information ubiquitously involves DNA:protein interactions and other biological processes that require local mechanical distortions of DNA. The energetics of such DNA-deforming processes are thus dependent on the local mechanical properties of DNA such as bendability or torsional rigidity. Such properties, in turn, depend on sequence, making it possible for sequence to regulate diverse biological processes by controlling the local mechanical properties of DNA. A deeper understanding of how such a “mechanical code” can encode broad regulatory information has historically been hampered by the absence of technology to measure in high throughput how local DNA mechanics varies with sequence along large regions of the genome. This was overcome in a recently developed technique called loop-seq. Here we describe a variant of the loop-seq protocol, that permits making rapid flexibility measurements in low-throughput, without the need for next-generation sequencing. We use our method to validate a previous prediction about how the binding site for the bacterial transcription factor Integration Host Factor (IHF) might serve as a rigid roadblock, preventing efficient enhancer-promoter contacts in IHF site containing promoters in E. coli, which can be relieved by IHF binding.