Biomolecular simulations under realistic macroscopic salt conditions, bioRxiv, 2017-11-30

Biomolecular simulations are typically performed in an aqueous environment where the number of ions remains fixed for the duration of the simulation, generally with either a minimally neutralizing ion environment or a number of salt pairs intended to match the macroscopic salt concentration. In contrast, real biomolecules experience local ion environments where the salt concentration is dynamic and may differ from bulk. The degree of salt concentration variability and average deviation from the macroscopic concentration remains, as yet, unknown. Here, we describe the theory and implementation of a Monte Carlo osmostat that can be added to explicit solvent molecular dynamics or Monte Carlo simulations to sample from a semigrand canonical ensemble in which the number of salt pairs fluctuates dynamically during the simulation. The osmostat reproduce the correct equilibrium statistics for a simulation volume that can exchange ions with a large reservoir at a defined macroscopic salt concentration. To achieve useful Monte Carlo acceptance rates, the method makes use of nonequilibrium candidate Monte Carlo (NCMC) moves in which monovalent ions and water molecules are alchemically transmuted using short nonequilibrium trajectories, with a modified Metropolis-Hastings criterion ensuring correct equilibrium statistics for an (Δ𝜇, 𝑁, 𝑝, 𝑇) ensemble. We demonstrate how typical protein (DHFR and the tyrosine kinase Src) and nucleic acid (Drew-Dickerson B-DNA dodecamer) systems exhibit salt concentration distributions that significantly differ from fixed-salt bulk simulations and display fluctuations that are on the same order of magnitude as the average.

biorxiv biophysics 0-100-users 2017

CRISPR-Cas12a target binding unleashes single-stranded DNase activity, bioRxiv, 2017-11-30

AbstractCRISPR-Cas12a (Cpf1) proteins are RNA-guided DNA targeting enzymes that bind and cut DNA as components of bacterial adaptive immune systems. Like CRISPR-Cas9, Cas12a can be used as a powerful genome editing tool based on its ability to induce genetic changes in cells at sites of double-stranded DNA (dsDNA) cuts. Here we show that RNA-guided DNA binding unleashes robust, non-specific single-stranded DNA (ssDNA) cleavage activity in Cas12a sufficient to completely degrade both linear and circular ssDNA molecules within minutes. This activity, catalyzed by the same active site responsible for site-specific dsDNA cutting, indiscriminately shreds ssDNA with rapid multiple-turnover cleavage kinetics. Activation of ssDNA cutting requires faithful recognition of a DNA target sequence matching the 20-nucleotide guide RNA sequence with specificity sufficient to distinguish between closely related viral serotypes. We find that target-dependent ssDNA degradation, not observed for CRISPR-Cas9 enzymes, is a fundamental property of type V CRISPR-Cas12 proteins, revealing a fascinating parallel with the RNA-triggered general RNase activity of the type VI CRISPR-Cas13 enzymes.One Sentence SummaryCas12a (Cpf1) and related type V CRISPR interference proteins possess non-specific, single-stranded DNase activity upon activation by guide RNA-dependent DNA binding.

biorxiv biochemistry 0-100-users 2017

 

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