RNA secondary structures of increasing complexity are probed combining single molecule stretching experiments and stochastic unfolding/refolding simulations. We find that force-induced unfolding pathways cannot usually be interpretated by solely invoking successive openings of native helices. Indeed, typical force-extension responses of complex RNA molecules are largely shaped by stretching-induced, long-lived intermediates including non-native helices. This is first shown for a set of generic structural motifs found in larger RNA structures, and then for Escherichia coli's 1540-base long 16S ribosomal RNA, which exhibits a surprisingly well-structured and reproducible unfolding pathway under mechanical stretching. Using out-of-equilibrium stochastic simulations, we demonstrate that these experimental results reflect the slow relaxation of RNA structural rearrangements. Hence, micromanipulations of single RNA molecules probe both their native structures and long-lived intermediates, so-called "kinetic traps", thereby capturing -at the single molecular level- the hallmark of RNA folding/unfolding dynamics.
We review the basic concepts and tools for mechanically unzipping RNA hairpins using force spectroscopy. By pulling apart the ends of an RNA molecule using optical tweezers, it is possible to measure the folding free energy at varying experimental conditions. Energy measurements permit us to characterize the thermodynamics of RNA hybridization (base pairing and stacking), the dynamics of the formation of native and kinetic (intermediates and misfolded) molecular states, and interactions with metallic ions. This paper introduces basic concepts and reviews recent developments related to RNA forc…
Accurate knowledge of RNA hybridization is essential for understanding RNA structure and function. Here we mechanically unzip and rezip a 2-kbp RNA hairpin and derive the 10 nearest-neighbor base pair (NNBP) RNA free energies in sodium and magnesium with 0.1 kcal/mol precision using optical tweezers. Notably, force-distance curves (FDCs) exhibit strong irreversible effects with hysteresis and several intermediates, precluding the extraction of the NNBP energies with currently available methods. The combination of a suitable RNA synthesis with a tailored pulling protocol allowed us to obtain th…
RNA function crucially depends on its structure. Thermodynamic models currently used for secondary structure prediction rely on computing the partition function of folding ensembles, and can thus estimate minimum free-energy structures and ensemble populations. These models sometimes fail in identifying native structures unless complemented by auxiliary experimental data. Here, we build a set of models that combine thermodynamic parameters, chemical probing data (DMS, SHAPE), and co-evolutionary data (Direct Coupling Analysis, DCA) through a network that outputs perturbations to the ensemble f…
Nano-scale mechanical probing of supported lipid bilayers with atomic force microscopy
我们目前的理论和实验的力-距离曲线$F(z_0)$的原子力显微镜（AFM）针尖（半径$R$）压痕支持的流体双层（厚度$2d$）。对于实际条件，力由双层的面积压缩性模量$κ_A$主导，并且在极好的近似下由$F= πκ_A R z_0^2/(2d-z_0)^2$给出。我们的模型很好地描述了三组分脂质双层中共存的液体有序区和液体无序区的AFM力曲线，并提供了与文献值一致的$κ_A$。液体有序相具有类似于我们通过氢键断裂建模的屈服响应。
Molecular dynamics simulations are often used to provide feedback in the design workflow of DNA nanostructures. However, even with coarse-grained models, convergence of distributions from unbiased simulation is slow, limiting applications to equilibrium structural properties. Given the increasing interest in dynamic, reconfigurable, and deformable devices, methods that enable efficient quantification of large ranges of motion, conformational transitions, and mechanical deformation are critically needed. Metadynamics is an automated biasing technique that enables the rapid acquisition of molecu…
The oxidative damage of DNA is a compelling issue in molecular biophysics as it plays a vital role in the epigenetic control of gene expression and is believed to be associated with mutagenesis, carcinogenesis, and ageing. To understand the microscopic structural changes in physical properties of DNA and the resulting influence on its function due to oxidative damage of its nucleotide bases, we have conducted all-atom molecular dynamic simulations of double-stranded DNA (dsDNA) with its guanine bases being oxidized. The guanine bases are more prone to oxidative damage due to the lowest value o…
In a diversity of physiological contexts, eukaryotic cells adhere to an extracellular matrix (ECM), a disordered network with complex nonlinear mechanics. Such cells can perform mechanosensation: using local force probing they can measure and respond to their substrate's mechanical properties. It remains unclear, however, how the mechanical complexity of the ECM at the cellular scale impacts mechanosensation. Here, we investigate the physical limits of mechanosensation imposed by the inherent structural disorder and nonlinear elastic response of the ECM. Using a theoretical framework for disor…
The folding of biological macromolecules is a fundamental process of which we lack a full comprehension. Mostly studied in proteins and RNA, single-stranded DNA (ssDNA) also folds, at physiological salt conditions, by forming non-specific secondary structures that are difficult to characterize with biophysical techniques. Here we present a helix-coil model for secondary structure formation, where ssDNA bases are organized in two different types of domains (compact and free). The model contains two parameters: the energy gain per base in a compact domain, $ε$, and the cooperativity related to t…
