High-Speed Imaging of DNA Submolecular Structure and Dynamics
Submitted by fdavid on 07 May, 2019.
SPONSORED BLOG - BRUKER
Over the past several decades, atomic force microscopy (AFM) has become the standard for high-resolution structural analysis of samples ranging from single molecules to complex macromolecular systems. Unlike other high-resolution imaging techniques, it does not require any special sample modification (except for surface deposition), and thus the risk of introducing artefacts during preparation is reduced. In recent years, there has been an increased demand for novel developments featuring high-speed AFM imaging (few frames/s), quantitative nanomechanical characterization of samples, and advanced feedback imaging modes for tweaking the maximum attainable resolution.
Bruker Nano Surfaces recently launched the new JPK NanoWizard® ULTRA Speed 2 with a 2nd generation Vortis™ controller. The instrument is a benchmark for correlative, high-speed BioAFM systems, enabling real-time, in-situ measurements with advanced optical microscopy set-ups.
SUBMOLECULAR RESOLUTION OF DNA DOUBLE HELIX
DNA has an inherent structure based on four nitrogenous organic bases, which are deposited along a sugar-phosphate backbone of the molecule (made up of five-carbon deoxyribose and phosphate molecules), thus comprising two alpha-helical strands, held together by hydrogen bonds. Following a certain complementarity code, the hydrophobic organic bases in the strands not only bind to one another, but also twist the double helix in such a way that their contact with water is minimised, thus defining the spiral staircase-like molecular structure of DNA. The classical structure of a Watson-Crick form of DNA (B-DNA), the right-handed double helix with a 3.4 nm repeat, comprised of a major and minor groove (2.2 and 1.2 nm, respectively), can be studied with ultra-sharp AFM tips.1 Here we show an example of a plasmid DNA (pUC19) deposited on a polycathionic substrate in liquid, resolved in amplitude-modulation AFM (see Figure 1).
Figure 1. Submolecular resolution of a pUC19 DNA strand, showing the complete 3.4 nm helical repeat.
DNA SUPERCOILING AND DEHYBRIDIZATION
DNA molecules, both in vivo and in vitro, can vary in length and form, typically being either knotted, looped, linear or ringed, which can give rise to topological differences. This results in variations of the classical 10.4 base pairs (bp) per 3.4 nm turn, and is referred to as supercoiling. Supercoiling is generally driven by a torsional stress around the strand axis. Various stages in the life cycle of an organism, species variations, compactization state, or interactions with molecules have also been known to affect the degree of DNA supercoiling (topology). Ideally, this is characterised by a rather conservative linking number Lk that, according to the Calugareanu-White-Fuller theorem, is the sum of the number of helical turns (twists, Tw) and the number of double-helical cross-overs (writhes, Wr). For an pUC19 DNA with 2686 bp (10.4 bp/turn), this gives an Lk of 258.2 In nature, this number has to be further corrected by an additional super-helical density component (σ= 2-9 supercoils per 100 Tw)3 resulting in a corrected Lk' (for σ=6) of 242 and a linking difference of 16.4 This effectively means that the pUC19 DNA circles can revert between two ground states carrying either mostly torsional (Tw, Wr) or predominantly bending energy (Tw, Wr[-16]). The images below show a pUC19 plasmid measured in liquid, exhibiting partial dehybridization regions, as an attempt to minimise the torsional energy of the super-coiled construct (see Figure 2).
Figure 2. Topography image of complete pUC19 sequences. The strand positions already undergoing dehybridization are labelled with arrows.
The active set-point of these measurements is very close to the cantilever free amplitude (very low damping is required to preserve the 1 nm sharp tips in Figure 1), to ensure that these phenomena are not tip-induced.
HIGH-SPEED IMAGING OF DNA BUBBLES
The appearance of the pUC19 DNA (see Figure 2) suggests that the structure is carrying a lot of torsional energy, which leads to a higher propensity for the formation of dehybridization bubbles, some of which were already induced along the double strand.
To get a deeper insight into the kinetics of the process, a region of fully hybridized DNA strands was subjected to high-speed imaging with 600 lines/s and an acquisition rate close to 9 frames/s (see Figure 3). Selected snapshots of the full 1252 frame sequence, allow the identification of three different regimes, which describe the kinetics of an individual DNA bubble, namely (a) dehybridization of the DNA double helix; (b) existence of a metastable phase, and (c) rezipping of the DNA bubble back to the double-helical strand backbone.
Figure 3. Selected frames from a high-speed AFM video describing the thermodynamic single strand fluctuations of a dehybridized DNA helix in liquid. The video features three different kinetic regimes – dehybridization, metastable phase and closing of the DNA bubble, which are 68, 243 and 1009 frames long respectively. For the full movie sequence follow the QR code:
The results here offer a unique insight into a fundamental dynamic process taking place in supercoiled DNA systems. Similar events taking place on the millisecond scale are impossible to resolve with a conventional AFM. The new, high-speed JPK NanoWizard ULTRA Speed 2 BioAFM from Bruker allows the investigation of sensitive and dynamic single molecule systems at very high temporal (~ 100 ms) and spatial resolution, as shown by the submolecular structure of the DNA double helix. This is merely one example of the numerous possibilities to perform sample analysis of dynamic biochemical and physical scenarios that are just waiting to be redefined with the new, easy-to-use set-up.
For more information, please visit www.jpk.com/products/atomic-force-microscopy/nanowizard-ultra-speed-2
1. Pyne A, Thompson R, Leung C, Roy D and Hoogenboom BW. Single-Molecule Reconstruction of Oligonucleotide Secondary Structure by Atomic Force Microscopy. Small 10, 3257–3261 (2014).
2. Adamcik J, Jeon JH, Karczewski KJ, Metzler R and Dietler G. Quantifying supercoiling-induced denaturation bubbles in DNA. Soft Matter 8, 8651-8658 (2012).
3. Mirkin, S. M. DNA Topology: Fundamentals. in Encyclopedia of Life Sciences (ed. John Wiley & Sons, Ltd) (2001).
4. Jeon JH, Adamcik J, Dietler G and Metzler R. Supercoiling Induces Denaturation Bubbles in Circular DNA. Physical Review Letters 105, 208101 (2010).