Atomistic structure of the SARS-CoV-2 pseudoknot in solution from SAXS-driven molecular dynamics.

SARS-CoV-2 depends on -1 programmed ribosomal frameshifting (-1 PRF) to express proteins essential for its replication. The RNA pseudoknot stimulating -1 PRF is thus an attractive drug target. However, the structural models of this pseudoknot obtained from cryo-EM and crystallography differ in some important features, leaving the pseudoknot structure unclear. We measured the solution structure of the pseudoknot using small-angle X-ray scattering (SAXS). The measured profile did not agree with profiles computed from the previously solved structures. Beginning with each of these solved structures, we used the SAXS data to direct all atom molecular dynamics (MD) simulations to improve the agreement in profiles. In all cases, this refinement resulted in a bent conformation that more closely resembled the cryo-EM structures than the crystal structure. Applying the same approach to a point mutant abolishing -1 PRF revealed a notably more bent structure with reoriented helices. This work clarifies the dynamic structures of the SARS-CoV-2 pseudoknot in solution.

Ionic Covalent Organic Framework as a Dual Functional Sensor for Temperature and Humidity.

Visual sensing of humidity and temperature by solids plays an important role in the everyday life and in industrial processes. Due to their hydrophobic nature, most covalent organic framework (COF) sensors often exhibit poor optical response when exposed to moisture. To overcome this challenge, the optical response is set out to improve, to moisture by incorporating H-bonding ionic functionalities into the COF network. A highly sensitive COF, consisting of guanidinium and diformylpyridine linkers (TG-DFP), capable of detecting changes in temperature and moisture content is fabricated. The hydrophilic nature of the framework enables enhanced water uptake, allowing the trapped water molecules to form a large number of hydrogen bonds. Despite the presence of non-emissive building blocks, the H-bonds restrict internal bond rotation within the COF, leading to reversible fluorescence and solid-state optical hydrochromism in response to relative humidity and temperature.

Mechanism of Cationic Lipid Induced DNA Condensation: Lipid-DNA Coordination and Divalent Cation Charge Fluctuations.

The condensation of nucleic acids by lipids is a widespread phenomenon in biology with crucial implications for drug delivery. However, the mechanisms of DNA assembly in lipid bilayers remain insufficiently understood due to challenges in measuring and assessing each component's contribution in the lipid-DNA-cation system. This study uses all-atom molecular dynamics simulations to investigate DNA condensation in cationic lipid bilayers. Our exhaustive exploration of the thermodynamic factors reveals unique roles for phospholipid head groups and cations. We observed that bridging cations between lipid and DNA drastically reduce charges, while mobile magnesium cations "ping-ponging" between double strands create charge fluctuations. While the first factor stabilizes the DNA-lipid complex, the latter creates attractive forces to induce the spontaneous condensation of DNAs. This novel mechanism not only sheds light on the current data regarding cationic lipid-induced DNA condensation but also provides potential design strategies for creating efficient gene delivery vectors for drug delivery.

Conformational dynamics during misincorporation and mismatch extension defined using a DNA polymerase with a fluorescent artificial amino acid.

High-fidelity DNA polymerases select the correct nucleotide over the structurally similar incorrect nucleotides with extremely high specificity while maintaining fast rates of incorporation. Previous analysis revealed the conformational dynamics and complete kinetic pathway governing correct nucleotide incorporation using a high-fidelity DNA polymerase variant containing a fluorescent unnatural amino acid. Here we extend this analysis to investigate the kinetics of nucleotide misincorporation and mismatch extension. We report the specificity constants for all possible misincorporations and characterize the conformational dynamics of the enzyme during misincorporation and mismatch extension. We present free energy profiles based on the kinetic measurements and discuss the effect of different steps on specificity. During mismatch incorporation and subsequent extension with the correct nucleotide, the rates of the conformational change and chemistry are both greatly reduced. The nucleotide dissociation rate, however, increases to exceed the rate of chemistry. To investigate the structural basis for discrimination against mismatched nucleotides, we performed all atom molecular dynamics simulations on complexes with either the correct or mismatched nucleotide bound at the polymerase active site. The simulations suggest that the closed form of the enzyme with a mismatch bound is greatly destabilized due to weaker interactions with active site residues, nonideal base pairing, and a large increase in the distance from the 3'-OH group of the primer strand to the α-phosphate of the incoming nucleotide, explaining the reduced rates of misincorporation. The observed kinetic and structural mechanisms governing nucleotide misincorporation reveal the general principles likely applicable to other high-fidelity DNA polymerases.

Substrate specificity and proposed structure of the proofreading complex of T7 DNA polymerase.

Faithful replication of genomic DNA by high-fidelity DNA polymerases is crucial for the survival of most living organisms. While high-fidelity DNA polymerases favor canonical base pairs over mismatches by a factor of ∼1 × 105, fidelity is further enhanced several orders of magnitude by a 3'-5' proofreading exonuclease that selectively removes mispaired bases in the primer strand. Despite the importance of proofreading to maintaining genome stability, it remains much less studied than the fidelity mechanisms employed at the polymerase active site. Here we characterize the substrate specificity for the proofreading exonuclease of a high-fidelity DNA polymerase by investigating the proofreading kinetics on various DNA substrates. The contribution of the exonuclease to net fidelity is a function of the kinetic partitioning between extension and excision. We show that while proofreading of a terminal mismatch is efficient, proofreading a mismatch buried by one or two correct bases is even more efficient. Because the polymerase stalls after incorporation of a mismatch and after incorporation of one or two correct bases on top of a mismatch, the net contribution of the exonuclease is a function of multiple opportunities to correct mistakes. We also characterize the exonuclease stereospecificity using phosphorothioate-modified DNA, provide a homology model for the DNA primer strand in the exonuclease active site, and propose a dynamic structural model for the transfer of DNA from the polymerase to the exonuclease active site based on MD simulations.

Oligomer Dynamics of LL-37 Truncated Fragments Probed by α-Hemolysin Pore and Molecular Simulations.

Oligomerization of antimicrobial peptides (AMPs) is critical in their effects on pathogens. LL-37 and its truncated fragments are widely investigated regarding their structures, antimicrobial activities, and application, such as developing new antibiotics. Due to the small size and weak intermolecular interactions of LL-37 fragments, it is still elusive to establish the relationship between oligomeric states and antimicrobial activities. Here, an α-hemolysin nanopore, mass spectrometry (MS), and molecular dynamic (MD) simulations are used to characterize the oligomeric states of two LL-37 fragments. Nanopore studies provide evidence of trapping events related to the oligomer formation and provide further details on their stabilities, which are confirmed by MS and MD simulations. Furthermore, simulation results reveal the molecular basis of oligomer dynamics and states of LL-37 fragments. This work provides unique insights into the relationship between the oligomer dynamics of AMPs and their antimicrobial activities at the single-molecule level. The study demonstrates how integrating methods allows deciphering single molecule level understanding from nanopore sensing approaches.

An exploration of machine learning models for the determination of reaction coordinates associated with conformational transitions.

Determining collective variables (CVs) for conformational transitions is crucial to understanding their dynamics and targeting them in enhanced sampling simulations. Often, CVs are proposed based on intuition or prior knowledge of a system. However, the problem of systematically determining a proper reaction coordinate (RC) for a specific process in terms of a set of putative CVs can be achieved using committor analysis (CA). Identifying essential degrees of freedom that govern such transitions using CA remains elusive because of the high dimensionality of the conformational space. Various schemes exist to leverage the power of machine learning (ML) to extract an RC from CA. Here, we extend these studies and compare the ability of 17 different ML schemes to identify accurate RCs associated with conformational transitions. We tested these methods on an alanine dipeptide in vacuum and on a sarcosine dipeptoid in an implicit solvent. Our comparison revealed that the light gradient boosting machine method outperforms other methods. In order to extract key features from the models, we employed Shapley Additive exPlanations analysis and compared its interpretation with the "feature importance" approach. For the alanine dipeptide, our methodology identifies ϕ and θ dihedrals as essential degrees of freedom in the C7ax to C7eq transition. For the sarcosine dipeptoid system, the dihedrals ψ and ω are the most important for the cisαD to transαD transition. We further argue that analysis of the full dynamical pathway, and not just endpoint states, is essential for identifying key degrees of freedom governing transitions.

Kinetic and thermodynamic analysis defines roles for two metal ions in DNA polymerase specificity and catalysis.

Magnesium ions play a critical role in catalysis by many enzymes and contribute to the fidelity of DNA polymerases through a two-metal ion mechanism. However, specificity is a kinetic phenomenon and the roles of Mg2+ ions in each step in the catalysis have not been resolved. We first examined the roles of Mg2+ by kinetic analysis of single nucleotide incorporation catalyzed by HIV reverse transcriptase. We show that Mg.dNTP binding induces an enzyme conformational change at a rate that is independent of free Mg2+ concentration. Subsequently, the second Mg2+ binds to the closed state of the enzyme-DNA-Mg.dNTP complex (Kd = 3.7 mM) to facilitate catalysis. Weak binding of the catalytic Mg2+ contributes to fidelity by sampling the correctly aligned substrate without perturbing the equilibrium for nucleotide binding at physiological Mg2+ concentrations. An increase of the Mg2+ concentration from 0.25 to 10 mM increases nucleotide specificity (kcat/Km) 12-fold largely by increasing the rate of the chemistry relative to the rate of nucleotide release. Mg2+ binds very weakly (Kd ≤ 37 mM) to the open state of the enzyme. Analysis of published crystal structures showed that HIV reverse transcriptase binds only two metal ions prior to incorporation of a correct base pair. Molecular dynamics simulations support the two-metal ion mechanism and the kinetic data indicating weak binding of the catalytic Mg2+. Molecular dynamics simulations also revealed the importance of the divalent cation cloud surrounding exposed phosphates on the DNA. These results enlighten the roles of the two metal ions in the specificity of DNA polymerases.

Structure-guided DNA-DNA attraction mediated by divalent cations.

Probing the role of surface structure in electrostatic interactions, we report the first observation of sequence-dependent dsDNA condensation by divalent alkaline earth metal cations. Disparate behaviors were found between two repeating sequences with 100% AT content, a poly(A)-poly(T) duplex (AA-TT) and a poly(AT)-poly(TA) duplex (AT-TA). While AT-TA exhibits non-distinguishable behaviors from random-sequence genomic DNA, AA-TT condenses in all alkaline earth metal ions. We characterized these interactions experimentally and investigated the underlying principles using computer simulations. Both experiments and simulations demonstrate that AA-TT condensation is driven by non-specific ion-DNA interactions. Detailed analyses reveal sequence-enhanced major groove binding (SEGB) of point-charged alkali ions as the major difference between AA-TT and AT-TA, which originates from the continuous and close stacking of nucleobase partial charges. These SEGB cations elicit attraction via spatial juxtaposition with the phosphate backbone of neighboring helices, resulting in an azimuthal angular shift between apposing helices. Our study thus presents a distinct mechanism in which, sequence-directed surface motifs act with cations non-specifically to enact sequence-dependent behaviors. This physical insight allows a renewed understanding of the role of repeating sequences in genome organization and regulation and offers a facile approach for DNA technology to control the assembly process of nanostructures.

Taming the Topology of Calix[4]arene-Based 2D-Covalent Organic Frameworks: Interpenetrated vs Noninterpenetrated Frameworks and Their Selective Removal of Cationic Dyes.

A bowl-shaped calix[4]arene with its exciting host-guest chemistry is a versatile supramolecular building block for the synthesis of distinct coordination cages or metal-organic frameworks. However, its utility in the synthesis of crystalline covalent organic frameworks (COFs) remains challenging, presumably due to its conformational flexibility. Here, we report the synthesis of a periodic 2D extended organic network of calix[4]arenes joined by a linear benzidine linker via dynamic imine bonds. By tuning the interaction among neighboring calixarene units through varying the concentration in the reaction mixture, we show the selective formation of interpenetrated (CX4-BD-1) and non-interpenetrated (CX4-BD-2) frameworks. The cone-shaped calixarene moiety in the structural backbone allows for the interweaving of two neighboring layers in CX4-BD-1, making it a unique example of interpenetrated 2D layers. Due to the high negative surface charge from calixarene units, both COFs have shown high performance in charge-selective dye removal and an exceptional selectivity for cationic dyes irrespective of their molecular size. The charge distribution of the COFs and the resulting selectivity for the cationic dyes were further investigated using computational methods.

Revealing the distinct folding phases of an RNA three-helix junction.

Remarkable new insight has emerged into the biological role of RNA in cells. RNA folding and dynamics enable many of these newly discovered functions, calling for an understanding of RNA self-assembly and conformational dynamics. Because RNAs pass through multiple structures as they fold, an ensemble perspective is required to visualize the flow through fleetingly populated sets of states. Here, we combine microfluidic mixing technology and small angle X-ray scattering (SAXS) to measure the Mg-induced folding of a small RNA domain, the tP5abc three helix junction. Our measurements are interpreted using ensemble optimization to select atomically detailed structures that recapitulate each experimental curve. Structural ensembles, derived at key stages in both time-resolved studies and equilibrium titrations, reproduce the features of known intermediates, and more importantly, offer a powerful new structural perspective on the time-progression of folding. Distinct collapse phases along the pathway appear to be orchestrated by specific interactions with Mg ions. These key interactions subsequently direct motions of the backbone that position the partners of tertiary contacts for later bonding, and demonstrate a remarkable synergy between Mg and RNA across numerous time-scales.

Cryo_fit: Democratization of flexible fitting for cryo-EM.

Cryo-electron microscopy (cryo-EM) is becoming a method of choice for describing native conformations of biomolecular complexes at high resolution. The rapid growth of cryo-EM in recent years has created a high demand for automated solutions, both in hardware and software. Flexible fitting of atomic models to three-dimensional (3D) cryo-EM reconstructions by molecular dynamics (MD) simulation is a popular technique but often requires technical expertise in computer simulation. This work introduces cryo_fit, a package for the automatic flexible fitting of atomic models in cryo-EM maps using MD simulation. The package is integrated with the Phenix software suite. The module was designed to automate the multiple steps of MD simulation in a reproducible manner, as well as facilitate refinement and validation through Phenix. Through the use of cryo_fit, scientists with little experience in MD simulation can produce high quality atomic models automatically and better exploit the potential of cryo-EM.

Tracking fluctuation hotspots on the yeast ribosome through the elongation cycle.

Chemical modification was used to quantitatively determine the flexibility of nearly the entire rRNA component of the yeast ribosome through 8 discrete stages of translational elongation, revealing novel observations at the gross and fine-scales. These include (i) the bulk transfer of energy through the intersubunit bridges from the large to the small subunit after peptidyltransfer, (ii) differences in the interaction of the sarcin ricin loop with the two elongation factors and (iii) networked information exchange pathways that may functionally facilitate intra- and intersubunit coordination, including the 5.8S rRNA. These analyses reveal hot spots of fluctuations that set the stage for large-scale conformational changes essential for translocation and enable the first molecular dynamics simulation of an 80S complex. Comprehensive datasets of rRNA base flexibilities provide a unique resource to the structural biology community that can be computationally mined to complement ongoing research toward the goal of understanding the dynamic ribosome.

Redox-Responsive Covalent Organic Nanosheets from Viologens and Calix[4]arene for Iodine and Toxic Dye Capture.

Owing to their chemical and thermal stabilities, high uptake capacities, and easy recyclability, covalent organic polymers (COPs) have shown promise as pollutant sponges. Herein, we describe the use of diazo coupling to synthesize two cationic COPs, COP1++ and COP2++ , that incorporate a viologen-based molecular switch and an organic macrocycle, calix[4]arene. The COPs form nanosheets that have height profiles of 6.00 nm and 8.00 nm, respectively, based on AFM measurements. The sheets remain morphologically intact upon one- or two-electron reductions of their viologen subunits. MD simulations of the COPs containing dicationic viologens indicate that the calix[4]arenes adopt a partial cone conformation and that, in height, the individual 2D polymer layers are 5.48 Šin COP1++ and 5.65 Šin COP2++ , which, together with the AFM measurements, suggests that the nanosheets are composed of 11 and 14 layers, respectively. Whether their viologens are in dicationic, radical cationic, or neutral form, the COPs exhibit high affinity for iodine, reaching up to 200 % mass increase when exposed to iodine vapor at 70 °C, which makes the materials among the best-performing nanosheets for iodine capture reported in the literature. In addition, the COPs effectively remove Congo red from solution in the pH range of 2-10, reaching nearly 100 % removal within 15 minutes at acidic pH.

The in vitro selection world.

Through iterative cycles of selection, amplification, and mutagenesis, in vitro selection provides the ability to isolate molecules of desired properties and function from large pools (libraries) of random molecules with as many as 10(16) distinct species. This review, in recognition of a quarter of century of scientific discoveries made through in vitro selection, starts with a brief overview of the method and its history. It further covers recent developments in in vitro selection with a focus on tools that enhance the capabilities of in vitro selection and its expansion from being purely a nucleic acids selection to that of polypeptides and proteins. In addition, we cover how next generation sequencing and modern biological computational tools are being used to complement in vitro selection experiments. On the very least, sequencing and computational tools can translate the large volume of information associated with in vitro selection experiments to manageable, analyzable, and exploitable information. Finally, in vivo selection is briefly compared and contrasted to in vitro selection to highlight the unique capabilities of each method.

Polyelectrolyte properties of single stranded DNA measured using SAXS and single-molecule FRET: Beyond the wormlike chain model.

Nucleic acids are highly charged polyelectrolytes that interact strongly with salt ions. Rigid, base-paired regions are successfully described with wormlike chain models, but nonbase-paired single stranded regions have fundamentally different polymer properties because of their greater flexibility. Recently, attention has turned to single stranded nucleic acids due to the growing recognition of their biological importance, as well as the availability of sophisticated experimental techniques sensitive to the conformation of individual molecules. We investigate polyelectrolyte properties of poly(dT), an important and widely studied model system for flexible single stranded nucleic acids, in physiologically important mixed mono- and divalent salt. We report measurements of the form factor and interparticle interactions using SAXS, end-to-end distances using smFRET, and number of excess ions using ASAXS. We present a coarse-grained model that accounts for flexibility, excluded volume, and electrostatic interactions in these systems. Predictions of the model are validated against experiment. We also discuss the state of all-atom, explicit solvent molecular dynamics simulations of poly(dT), the next step in understanding the complexities of ion interactions with these highly charged and flexible polymers.

Sequence-Dependent Orientational Coupling and Electrostatic Attraction in Cation-Mediated DNA-DNA Interactions.

Condensation of DNA is vital for its biological functions and controlled nucleic acid assemblies. However, the mechanisms of DNA condensation are not fully understood due to the inability of experiments to access cation distributions and the complex interplay of energetic and entropic forces during assembly. By constructing free energy surfaces using exhaustive sampling and detailed analysis of cation distributions, we elucidate the mechanism of DNA condensation in different salt conditions and with different DNA sequences. We found that DNA condensation is facilitated by the correlated dynamics of the localized cations at the grooves of DNA helices. These dynamics are strongly dependent on the salt conditions and DNA sequences. In the presence of magnesium ions, major groove binding facilitates attraction. In contrast, in the presence of polyvalent cations, minor groove binding serves to create charge patterns, leading to condensation. Our findings present a novel advancement in the field and have broad implications for understanding and controlling nucleic acid complexes in vivo and in vitro.

Light-driven self-assembly of spiropyran-functionalized covalent organic framework.

Controlling the number of molecular switches and their relative positioning within porous materials is critical to their functionality and properties. The proximity of many molecular switches to one another can hinder or completely suppress their response. Herein, a synthetic strategy involving mixed linkers is used to control the distribution of spiropyran-functionalized linkers in a covalent organic framework (COF). The COF contains a spiropyran in each pore which exhibits excellent reversible photoswitching behavior to its merocyanine form in the solid state in response to UV/Vis light. The spiro-COF possesses an urchin-shaped morphology and exhibits a morphological transition to 2D nanosheets and vesicles in solution upon UV light irradiation. The merocyanine-equipped COFs are extremely stable and possess a more ordered structure with enhanced photoluminescence. This approach to modulating structural isomerization in the solid state is used to develop inkless printing media, while the photomediated polarity change is used for water harvesting applications.

RNA and its ionic cloud: solution scattering experiments and atomically detailed simulations.

RNA molecules play critical roles in many cellular processes. Traditionally viewed as genetic messengers, RNA molecules were recently discovered to have diverse functions related to gene regulation and expression. RNA also has great potential as a therapeutic and a tool for further investigation of gene regulation. Metal ions are an integral part of RNA structure and should be considered in any experimental or theoretical study of RNA. Here, we report a multidisciplinary approach that combines anomalous small-angle x-ray scattering and molecular-dynamics (MD) simulations with explicit solvent and ions around RNA. From experiment and simulation results, we find excellent agreement in the number and distribution of excess monovalent and divalent ions around a short RNA duplex. Although similar agreement can be obtained from a continuum description of the solvent and mobile ions (by solving the Poisson-Boltzmann equation and accounting for finite ion size), the use of MD is easily extended to flexible RNA systems with thermal fluctuations. Therefore, we also model a short RNA pseudoknot and find good agreement between the MD results and the experimentally derived solution structures. Surprisingly, both deviate from crystal structure predictions. These favorable comparisons of experiment and simulations encourage work on RNA in all-atom dynamic models.