Real-time single-molecule studies of the motions of DNA polymerase fingers illuminate DNA synthesis mechanisms

DNA polymerases maintain genomic integrity by copying DNA with high fidelity. A conformational change important for fidelity is the motion of the polymerase fingers subdomain from an open to a closed conformation upon binding of a complementary nucleotide. We previously employed intraprotein single-molecule FRET on diffusing molecules to observe fingers conformations in polymerase-DNA complexes. Here, we used the same FRET ruler on surface-immobilized complexes to observe fingers-opening and closing of individual polymerase molecules in real time. Our results revealed the presence of intrinsic dynamics in the binary complex, characterized by slow fingers-closing and fast fingers-opening. When binary complexes were incubated with increasing concentrations of complementary nucleotide, the fingers-closing rate increased, strongly supporting an induced-fit model for nucleotide recognition. Meanwhile, the opening rate in ternary complexes with complementary nucleotide was 6 s-1, much slower than either fingers closing or the rate-limiting step in the forward direction; this rate balance ensures that, after nucleotide binding and fingers-closing, nucleotide incorporation is overwhelmingly likely to occur. Our results for ternary complexes with a non-complementary dNTP confirmed the presence of a state corresponding to partially closed fingers and suggested a radically different rate balance regarding fingers transitions, which allows polymerase to achieve high fidelity.</p

Self-healing dynamic bond-based rubbers: understanding the mechanisms in ionomeric elastomer model systems

While it is traditionally accepted that the chain interactions responsible for the elastic response in an elastomeric network are ideally permanent and instantaneously active, the ongoing investigation of self-healing materials reveals that the introduction of self-healing properties into elastomers requires high mechanical integrity under dynamic load conditions, while on long timescales (or at extended temperatures), the chain and bond dynamics must allow for an intrinsic repair of micro cracks occurring during operation and aging. Based on an acrylate-based amorphous ionomer model system with pendant carboxylate groups allowing the systematic variation of the composition and the nature of the counter ion, we demonstrate the interrelation between the morphological, thermal, and mechanical properties, and identify the prerequisites and tools for property adjustment and optimization of self-healing efficiency. While the ion fraction is directly related to the effective network density and elastic performance, the crossover frequency between viscous and elastic behavior is influenced by the nature of the counter ion. In order to achieve reliable elastic response and optimal damage repair, the ion fraction in these systems should be in the range of 5 mol% and the chain dynamics should be appropriate to allow for excellent self-healing behavior at moderate healing temperatures

Complex coacervate core micelles with spectroscopic labels for diffusometric probing of biopolymer networks

\u3cp\u3eWe present the design, preparation, and characterization of two types of complex coacervate core micelles (C3Ms) with cross-linked cores and spectroscopic labels and demonstrate their use as diffusional probes to investigate the microstructure of percolating biopolymer networks. The first type consists of poly(allylamine hydrochloride) (PAH) and poly(ethylene oxide)-poly(methacrylic acid) (PEO-b-PMAA), labeled with ATTO 488 fluorescent dyes. We show that the size of these probes can be tuned by choosing the length of the PEO-PMAA chains. ATTO 488-labeled PEO\u3csub\u3e113\u3c/sub\u3e-PMAA\u3csub\u3e15\u3c/sub\u3e micelles are very bright with 18 dye molecules incorporated into their cores. The second type is a \u3csup\u3e19\u3c/sup\u3eF-labeled micelle, for which we used PAH and a \u3csup\u3e19\u3c/sup\u3eF-labeled diblock copolymer tailor-made from poly(ethylene oxide)-poly(acrylic acid) (mPEO\u3csub\u3e79\u3c/sub\u3e-b-PAA\u3csub\u3e14\u3c/sub\u3e). These micelles contain approximately 4 wt % of \u3csup\u3e19\u3c/sup\u3eF and can be detected by \u3csup\u3e19\u3c/sup\u3eF NMR. The \u3csup\u3e19\u3c/sup\u3eF labels are placed at the end of a small spacer to allow for the necessary rotational mobility. We used these ATTO- and \u3csup\u3e19\u3c/sup\u3eF-labeled micelles to probe the microstructures of a transient gel (xanthan gum) and a cross-linked, heterogeneous gel (κ-carrageenan). For the transient gel, sensitive optical diffusometry methods, including fluorescence correlation spectroscopy, fluorescence recovery after photobleaching, and super-resolution single nanoparticle tracking, allowed us to measure the diffusion coefficient in networks with increasing density. From these measurements, we determined the diameters of the constituent xanthan fibers. In the heterogeneous κ-carrageenan gels, bimodal nanoparticle diffusion was observed, which is a signpost of microstructural heterogeneity of the network.\u3c/p\u3

Complex Coacervate Core Micelles with Spectroscopic Labels for Diffusometric Probing of Biopolymer Networks

We present the design, preparation, and characterization of two types of complex coacervate core micelles (C3Ms) with cross-linked cores and spectroscopic labels and demonstrate their use as diffusional probes to investigate the microstructure of percolating biopolymer networks. The first type consists of poly­(allylamine hydrochloride) (PAH) and poly­(ethylene oxide)–poly­(methacrylic acid) (PEO-<i>b</i>-PMAA), labeled with ATTO 488 fluorescent dyes. We show that the size of these probes can be tuned by choosing the length of the PEO–PMAA chains. ATTO 488-labeled PEO₁₁₃–PMAA₁₅ micelles are very bright with 18 dye molecules incorporated into their cores. The second type is a ¹⁹F-labeled micelle, for which we used PAH and a ¹⁹F-labeled diblock copolymer tailor-made from poly­(ethylene oxide)–poly­(acrylic acid) (mPEO₇₉-<i>b</i>-PAA₁₄). These micelles contain approximately 4 wt % of ¹⁹F and can be detected by ¹⁹F NMR. The ¹⁹F labels are placed at the end of a small spacer to allow for the necessary rotational mobility. We used these ATTO- and ¹⁹F-labeled micelles to probe the microstructures of a transient gel (xanthan gum) and a cross-linked, heterogeneous gel (κ-carrageenan). For the transient gel, sensitive optical diffusometry methods, including fluorescence correlation spectroscopy, fluorescence recovery after photobleaching, and super-resolution single nanoparticle tracking, allowed us to measure the diffusion coefficient in networks with increasing density. From these measurements, we determined the diameters of the constituent xanthan fibers. In the heterogeneous κ-carrageenan gels, bimodal nanoparticle diffusion was observed, which is a signpost of microstructural heterogeneity of the network