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Single-Molecule Analysis by Biological Nanopores
Published in Shuo Huang, Single-Molecule Tools for Bioanalysis, 2022
An emerging technology named single-molecule picometer-resolution nanopore tweezers (SPRNT) [137] has been recently developed for real-time observation of enzyme kinetics. The core component of SPRNT is a geometrically sharp biological nanopore MspA, which had been previously developed for nanopore sequencing [60]. In SPRNT, a strand of synthetic DNA with a known sequence serves as a molecular ruler. When bound with a motor enzyme, such as a helicase or a DNA polymerase, the DNA is electrophoretically driven into an engineered MspA nanopore, activating the progression of the DNA with a translocation speed of 1–100 nt/s through the enzyme (Figure 1.6A) [60]. Owning to the high spatial resolution of MspA, the measured ionic current readout directly reveals the relative position of the enzyme in reference to the DNA ruler, and a ~40 pm and a submillisecond spatiotemporal resolution could be achieved simultaneously (Figure 1.6B) [80, 138]. However, with a requirement of a high processivity from the enzyme, SPRNT is in principle limited to DNA/RNA binding enzymes with a high binding affinity to their nucleic acid substrates.
Optical Tweezers
Published in Yuri L. Lyubchenko, An Introduction to Single Molecule Biophysics, 2017
The same geometry has been largely used to monitor DNA and RNA processing enzymes, such as the bacteriophage ϕ29 (Smith et al. 2001b; Chemla et al. 2005), T7 DNA polymerase (Wuite et al. 2000), RNA polymerases (Abbondanzieri et al. 2005; Fazal et al. 2015), helicases (Dumont et al. 2006; Garcia-Garcia et al. 2015), and the ribosome (Wen et al. 2008; Kaiser et al. 2011) (Figure 5.7c). In those experiments, the molecular motor is usually attached to one of the trapped beads and is linked to the second bead through a DNA or RNA tether. The processive movements of the motor along the nucleic acid tether shorten or lengthen it, so that the motor movements can be detected through bead movements (Figure 5.7c, top). When properly optimized, this assay allows resolving single base-pair stepping of RNA polymerase (Abbondanzieri et al. 2005), as illustrated in detail in Section 5.4, where we discuss spatial resolution in optical tweezers (Figure 5.7c, bottom). Moreover, in this assay, enzyme processivity can be efficiently analyzed under constant force by using force-clamp techniques that we describe in Section 5.3.4.
Biomimetic Nanowalkers
Published in Klaus D. Sattler, st Century Nanoscience – A Handbook, 2020
First, the present DNA nanowalkers already make possible some new technological applications that are not possible for the previous burning-bridge walkers, such as three-dimensional control of nanomotion with walker-rotor integrated systems [22,83] in nano/microrobotic platforms or assembly lines, artificial muscle (walkers of reduced processivity suffice). From a larger perspective, DNA nanowalkers are not only model systems but have clear potentials for real-world applications, thanks to the fast expanding DNA nanotechnology. DNA walkers can be readily integrated into large and sophisticated DNA platforms that offer a bridge towards more applications.
Polar residues lining the binding cleft of a Serratia marcescens family 18 chitinase position the substrate for attack and stabilize associative interactions
Published in Molecular Physics, 2019
Suvamay Jana, Anne Grethe Hamre, Vincent G. H. Eijsink, Morten Sørlie, Christina M. Payne
Apparent processivity (Papp) is mathematically defined as the number of catalytic events an enzyme performs divided by the number of times the enzyme acquires the substrate [59]. However, in practice, Papp can be regarded as an average number of catalytic acts (Ncatal) an enzyme performs per initiation of a processive run (Ninit) [8]. This processive ability can be measured by a number of different methods, depending on the substrate [33,59–61]. For chitinases, one way of measuring Papp is to determine the [(GlcNAc)2] / [GlcNAc] ratio. Since the smallest structural unit of chitin is a disaccharide, the product of repeated processive enzymatic actions will be dimers – (GlcNAc)2. Monomers, GlcNAc, originate from initial productive binding when the sugar in the non-reducing end occupies a subsite with an odd number. Hence, a high ratio of [(GlcNAc)2]/[GlcNAc] indicates a high degree of apparent processivity.