Intercalation-Based Single-Molecule Fluorescence Assay To Study DNA Supercoil Dynamics

Abstract

DNA supercoiling crucially affects cellular processes such as DNA replication, gene expression, and chromatin organization. However, mechanistic understanding of DNA supercoiling and the related DNA-processing enzymes has remained limited, mainly due to the lack of convenient experimental tools to probe these phenomena. Here, we report a novel high-throughput single-molecule assay for real-time visualization of supercoiled DNA molecules, named ISD (Intercalation-induced Supercoiling of DNA). We use an intercalating dye to induce supercoiling of surface-attached DNA molecules as well as to visualize coiled-loop structures (i.e., plectonemes) formed on DNA. The technique is solely based on epifluorescence microscopy and requires no mechanical manipulation of the DNA molecules. This new assay allows to track positions and sizes of individual plectonemes and characterize their position-dependent dynamics such as nucleation, termination, and diffusion. We describe the ISD technique and demonstrate its potential by establishing that plectonemes are pinned to a local 10-nucleotide long mispaired sequence along a double-stranded DNA molecule.

Coarse-Grained Modelling of DNA Plectoneme Pinning in the Presence of Base-Pair Mismatches

“…Defects in duplex DNA can arise from base-pair mismatches 18,25 , damaged 26 or modified bases 27 , locally melted DNA bubbles 28 , a protein introduced kink in the DNA 29 . An elastic rod with a defect that locally decreases the bending rigidity, when subjected to torsional stress, would buckle at the defect 30 .…”

Supercoiling DNA Locates Mismatches

“…[7] demonstrated that energy is minimized by the colocalization of plectoneme end-loops and regions of unpaired bases as “tip bubbles.” Ganji et al . [8] used an intercalator based method for creating fluorescent plectonemes and showed a preference for plectoneme pinning at the location of a 10 bp mismatch.…”

Bioinspired Polymeric Helical and Superhelical Microfibers via Microfluidic Spinning

“…On the other hand, superhelical structure, which is a hierarchical structure in which a helix is further folded to produce a more ordered helix, is another higher order and more sophisticated structure from natural creatures, especially in the closed‐circular DNA systems. Superhelical structures also play important roles in a number of biological processes, such as compacting DNA and storing energy for essential biological functions including muscle contraction, metabolism, transcription, molecular chaperons, membrane channels, and chromatin organization . Reported creations of synthetic superhelical fibers were mainly based on the self‐assembly of chiral/achiral molecules or π‐conjugated polymers by carefully adjusting the subtle interplays of various noncovalent interactions .…”

“…Superhelical structures also play important roles in a number of biological processes, such as compacting DNA and storing energy for essential biological functions including muscle contraction, metabolism, transcription, molecular chaperons, membrane channels, and chromatin organization. [25][26][27][28] Reported creations of synthetic superhelical fibers were mainly based on the self-assembly of chiral/achiral molecules or π-conjugated polymers by carefully adjusting the subtle interplays of various noncovalent interactions. [29][30][31][32] And thus, precise molecular design and complicated synthesis procedure were often required.…”