Single-molecule studies of dna supercoils

A DNA consists of two strands that are interwound with each other, thus forming the famous double helix structure that is intrinsically twisted in a right-handed fashion. When rotations are applied to a DNA molecule, it can form higher-order structures, called DNA supercoils or plectonemes – see Figure 1.

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Figure 1. A coiled loop structure (plectoneme) can be formed when the DNA is twisted.

In a cell, DNA is constantly accessed and processed by enzymes such as RNA-, DNA-polymerases, topoisomerases, and helicases (see Figure 2 for an example). Often, these enzymatic activities introduce torsional stress on the DNA, introducing or releasing plectonemes. These DNA supercoils, in turn, alter the activity of the DNA processing enzymes. Hence, understanding the structure and dynamics of DNA supercoil is prerequisite for comprehensive understanding of the enzymes and their biological functions.

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Figure 2. A protein acting on the DNA can generate or remove plectonemes in the local region of the DNA.

Despite its fundamental importance, DNA supercoils and the associated biological processes are poorly understood, mainly due to the lack of proper experimental tools to examine them. Our laboratory has been pioneering in development of the techniques to visualize the supercoiled DNA at single-molecule level. We developed two different methods: 1) a hybrid of side-pulling magnetic tweezers and fluorescence microscopy (van Loenhout et al, Science, 2012) and 2) Intercalation–induced Supercoiling of DNA (ISD) (Ganji et al, Nano Lett, 2016) – see Figure 3. ISD does not require any external manipulation of DNA to introduce supercoiling and is a high-throughput technique that allow to visualize the dynamics of multiple supercoiled DNA molecules.

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Figure 3. (A-D) Schematic diagrams of the Intercalation-induced Supercoiled DNA (ISD). DNA is stretched and immobilized on streptavidin coated surface. A plectoneme appears instantaneously due to binding of intercalating dye. (E) A fluorescence microscopy movie with multiple DNA molecules showing plectonemes as diffusing bright spots along DNA.

By using this powerful technique, we observed that plectonemes prefer to localize at mispaired ssDNA sites along the supercoiled DNA. Our current interests are to explore the basic properties of DNA supercoils to understand how plectoneme formation is affected and controlled by the DNA sequence and by local mechanical properties of the DNA. Furthermore, we are currently expanding ISD to study the interactions of proteins with supercoiled DNA. Finally, we plan to study supercoiling induced by RNA-polymerase, a powerful molecular motor which generates torque on DNA template during RNA transcription. We are also interested in other DNA-processing enzymes that generate, exploit, and remove DNA supercoils for their biological functions.

For further enquiries please contact the lab at c.dekker [at] tudelft.nl.

Further reading:

  1. T.J. van Loenhout, M.V. de Grunt, C. Dekker, “Dynamics of DNA Supercoils”, Science 338, p. 94 (2012)
  2. Ganji, S.H. Kim, J. van der Torre, E. Abbondanzieri, C. Dekker, “Intercalation-based single-molecule fluorescence assay to study DNA supercoil dynamics”, Nano Lett. 16, 4699 (2016)