ProNAB: database for binding affinities of protein-nucleic acid complexes and their mutants.

Protein-nucleic acid interactions are involved in various biological processes such as gene expression, replication, transcription, translation and packaging. The binding affinities of protein-DNA and protein-RNA complexes are important for elucidating the mechanism of protein-nucleic acid recognition. Although experimental data on binding affinity are reported abundantly in the literature, no well-curated database is currently available for protein-nucleic acid binding affinity. We have developed a database, ProNAB, which contains more than 20 000 experimental data for the binding affinities of protein-DNA and protein-RNA complexes. Each entry provides comprehensive information on sequence and structural features of a protein, nucleic acid and its complex, experimental conditions, thermodynamic parameters such as dissociation constant (Kd), binding free energy (ΔG) and change in binding free energy upon mutation (ΔΔG), and literature information. ProNAB is cross-linked with GenBank, UniProt, PDB, ProThermDB, PROSITE, DisProt and Pubmed. It provides a user-friendly web interface with options for search, display, sorting, visualization, download and upload the data. ProNAB is freely available at https://web.iitm.ac.in/bioinfo2/pronab/ and it has potential applications such as understanding the factors influencing the affinity, development of prediction tools, binding affinity change upon mutation and design complexes with the desired affinity.

Supramolecular Architectures of Nucleic Acid/Peptide Hybrids.

Supramolecular architectures that are built artificially from biomolecules, such as nucleic acids or peptides, with structural hierarchical orders ranging from the molecular to nano-scales have attracted increased attention in molecular science research fields. The engineering of nanostructures with such biomolecule-based supramolecular architectures could offer an opportunity for the development of biocompatible supramolecular (nano)materials. In this review, we highlighted a variety of supramolecular architectures that were assembled from both nucleic acids and peptides through the non-covalent interactions between them or the covalently conjugated molecular hybrids between them.

Solidifying framework nucleic acids with silica.

Soft matter can serve as a template to guide the growth of inorganic components with well-controlled structural features. However, the limited design space of conventional organic and biomolecular templates restricts the complexity and accuracy of templated growth. In past decades, the blossoming of structural DNA nanotechnology has provided us with a large reservoir of delicate-framework nucleic acids with design precision down to a single base. Here, we describe a DNA origami silicification (DOS) approach for generating complex silica composite nanomaterials. By utilizing modified silica sol-gel chemistry, pre-hydrolyzed silica precursor clusters can be uniformly coated onto the surface of DNA frameworks; thus, user-defined DNA-silica hybrid materials with ~3-nm precision can be achieved. More importantly, this method is applicable to various 1D, 2D and 3D DNA frameworks that range from 10 to >1,000 nm. Compared to pure DNA scaffolds, a tenfold increase in the Young's modulus (E modulus) of these composites was observed, owing to their soft inner core and solid silica shell. We further demonstrate the use of solidified DNA frameworks to create 3D metal plasmonic devices. This protocol provides a platform for synthesizing inorganic materials with unprecedented complexity and tailored structural properties. The whole protocol takes ~10 d to complete.

Tetrahedral Framework Nucleic Acids Promote Corneal Epithelial Wound Healing in Vitro and in Vivo.

Poor post-traumatic wound healing can affect the normal function of damaged tissues and organs. For example, poor healing of corneal epithelial injuries may lead to permanent visual impairment. It is of great importance to find a therapeutic way to promote wound closure. Tetrahedral framework nucleic acids (tFNAs) are new promising nanomaterials, which can affect the biological behavior of cells. In the experiment, corneal wound healing is used as an example to explore the effect of tFNAs on wound healing. Results show that the proliferation and migration of human corneal epithelial cells are enhanced by exposure to tFNAs in vitro, possibly relevant to the activation of P38 and ERK1/2 signaling pathway. An animal model of corneal alkali burn is established to further identify the facilitation effect of tFNAs on corneal wound healing in vivo. Clinical evaluations and histological analyses show that tFNAs can improve the corneal transparency and accelerate the re-epithelialization of wounds. Both in vitro and in vivo experiments show that tFNAs can play a positive role in corneal epithelial wound healing.

Biophysical Techniques in Structural Biology.

Over the past six decades, steadily increasing progress in the application of the principles and techniques of the physical sciences to the study of biological systems has led to remarkable insights into the molecular basis of life. Of particular significance has been the way in which the determination of the structures and dynamical properties of proteins and nucleic acids has so often led directly to a profound understanding of the nature and mechanism of their functional roles. The increasing number and power of experimental and theoretical techniques that can be applied successfully to living systems is now ushering in a new era of structural biology that is leading to fundamentally new information about the maintenance of health, the origins of disease, and the development of effective strategies for therapeutic intervention. This article provides a brief overview of some of the most powerful biophysical methods in use today, along with references that provide more detailed information about recent applications of each of them. In addition, this article acts as an introduction to four authoritative reviews in this volume. The first shows the ways that a multiplicity of biophysical methods can be combined with computational techniques to define the architectures of complex biological systems, such as those involving weak interactions within ensembles of molecular components. The second illustrates one aspect of this general approach by describing how recent advances in mass spectrometry, particularly in combination with other techniques, can generate fundamentally new insights into the properties of membrane proteins and their functional interactions with lipid molecules. The third reviewdemonstrates the increasing power of rapidly evolving diffraction techniques, employing the very short bursts of X-rays of extremely high intensity that are now accessible as a result of the construction of free-electron lasers, in particular to carry out time-resolved studies of biochemical reactions. The fourth describes in detail the application of such approaches to probe the mechanism of the light-induced changes associated with bacteriorhodopsin's ability to convert light energy into chemical energy.

Preparing Frozen-Hydrated Protein-Nucleic Acid Assemblies for High-Resolution Cryo-EM Imaging.

High-resolution image acquisition and structure determination by cryo-electron microscopy is becoming increasingly streamlined. Preparing electron-microscopy grids of suitable quality remains, however, a critical bottleneck. Strategies to achieve particle monodispersity, optimal sample concentration and suitable ice thickness can vary from specimen to specimen. In this book chapter we describe our protocols for negative-stain grid and cryo-grid preparation, which we apply to studying protein-nucleic acid complexes.

Structure and Composition Define Immunorecognition of Nucleic Acid Nanoparticles.

Nucleic acid nanoparticles (NANPs) have evolved as a new class of therapeutics with the potential to detect and treat diseases. Despite tremendous advancements in NANP development, their immunotoxicity, one of the major impediments in clinical translation of traditional therapeutic nucleic acids (TNAs), has never been fully characterized. Here, we describe the first systematically studied immunological recognition of 25 representative RNA and DNA NANPs selected to have different design principles and physicochemical properties. We discover that, unlike traditional TNAs, NANPs used without a delivery carrier are immunoquiescent. We show that interferons (IFNs) are the key cytokines triggered by NANPs after their internalization by phagocytic cells, which agrees with predictions based on the experiences with TNAs. However, in addition to type I IFNs, type III IFNs also serve as reliable biomarkers of NANPs, which is usually not characteristic of TNAs. We show that overall immunostimulation relies on NANP shapes, connectivities, and compositions. We demonstrate that, like with traditional TNAs, plasmacytoid dendritic cells serve as the primary interferon producers among all peripheral blood mononuclear cells treated with NANPs, and scavenger receptor-mediated uptake and endosomal Toll-like receptor signaling are essential for NANP immunorecognition. The TLR involvement, however, is different from that expected for traditional TNA recognition. Based on these results, we suggest that NANP technology may serve as a prototype of auxiliary molecular language for communication with the immune system and the modulation of immune responses.

Homology threading to generate RNA polymerase structures.

Homology threading is a powerful technology for generating structural models based on homologous structures. Here we use threading to generate four complex RNA polymerase models. The models appear to be as useful as x-ray crystal structures or cryo-electron microscopy structures to support research projects.

How to Analyze and Present SAS Data for Publication.

SAS is a powerful technique to investigate oligomeric state and domain organization of macromolecules, e.g. proteins and nucleic acids, under physiological, functional and even time resolved conditions. However, reconstructing three dimensional structures from SAS data is inherently ambiguous, as no information about orientation and phase is available. In addition experimental artifacts such as radiation damage, concentration effects and incorrect background subtraction can hinder the interpretation of even lead to wrong results. In this chapter, explanations on how to analyze data and how to assess and minimize the influence of experimental artifacts on the data. Furthermore, guidelines on how to present the resulting data and models to demonstrate the data supports the conclusion being made and that it is not biased by artifacts, will be given.

Designing and Performing Biological Solution Small-Angle Neutron Scattering Contrast Variation Experiments on Multi-component Assemblies.

Solution small-angle neutron scattering (SANS) combined with contrast variation provides information about the size and shape of individual components of a multi-component biological assembly, as well as the spatial arrangements between the components. The large difference in the neutron scattering properties between hydrogen and deuterium is key to the method. Isotopic substitution of deuterium for some or all of the hydrogen in either the molecule or the solvent can greatly alter the scattering properties of the biological assembly, often with little or no change to its biochemical properties. Thus, SANS with contrast variation provides unique information not easily obtained using other experimental techniques.If used correctly, SANS with contrast variation is a powerful tool for determining the solution structure of multi-component biological assemblies. This chapter discusses the principles of SANS theory that are important for contrast variation, essential considerations for experiment design and execution, and the proper approach to data analysis and structure modeling. As sample quality is extremely important for a successful contrast variation experiment, sample issues that can affect the outcome of the experiment are discussed as well as procedures used to verify the sample quality. The described methodology is focused on two-component biological complexes. However, examples of its use for multi-component assemblies are also discussed.

SAS-Based Structural Modelling and Model Validation.

Small angle scattering of X-rays (SAXS) and neutrons (SANS) is a structural technique to study disordered systems with chaotic orientations of scattering inhomogeneities at low resolution. An important example of such systems are solutions of biological macromolecules. Rapid development in the methodology for solution scattering data interpretation and model building during the last two decades brought the analysis far beyond the determination of just few overall structural parameters (which was the only possibility in the past) and ensured SAS a firm position in the methods palette of the modern life sciences. The advances in the methodology include ab initio approaches for shape and domain structure restoration from scattering curves without a priori structural knowledge, classification and validation of the models, evaluation of potential ambiguity associated with the reconstruction. In rigid body and hybrid modelling approaches, solution scattering is synergistically used with other structural techniques utilizing the complementary information such as atomic models of the components, intramolecular contacts, subunits orientations etc. for the reconstruction of complex systems. The usual requirement of the sample monodispersity has been loosed recently and the technique can now address such systems as weakly bound oligomers and transient complexes. These state-of-the-art methods are described together with the examples of their applications and the possible ways of post-processing of the models.

Electron Cryomicroscopy of Viruses at Near-Atomic Resolutions.

Recently, dozens of virus structures have been solved to resolutions between 2.5 and 5.0 Å by means of electron cryomicroscopy. With these structures we are now firmly within the "atomic age" of electron cryomicroscopy, as these studies can reveal atomic details of protein and nucleic acid topology and interactions between specific residues. This improvement in resolution has been the result of direct electron detectors and image processing advances. Although enforcing symmetry facilitates reaching near-atomic resolution with fewer particle images, it unfortunately obscures some biologically interesting components of a virus. New approaches on relaxing symmetry and exploring structure dynamics and heterogeneity of viral assemblies have revealed important insights into genome packaging, virion assembly, cell entry, and other stages of the viral life cycle. In the future, novel methods will be required to reveal yet-unknown structural conformations of viruses, relevant to their biological activities. Ultimately, these results hold the promise of answering many unresolved questions linking structural diversity of viruses to their biological functions.

Many Ways to Derivatize Macromolecules and Their Crystals for Phasing.

Due to the availability of many macromolecular models in the Protein Data Bank, the majority of crystal structures are currently solved by molecular replacement. However, truly novel structures can only be solved by one of the versions of the special-atom method. The special atoms such as sulfur, phosphorus or metals could be naturally present in the macromolecules, or could be intentionally introduced in a derivatization process. The isomorphous and/or anomalous scattering of X-rays by these special atoms is then utilized for phasing. There are many ways to obtain potentially useful derivatives, ranging from the introduction of special atoms to proteins or nucleic acids by genetic engineering or by chemical synthesis, to soaking native crystals in solutions of appropriate compounds with heavy and/or anomalously scattering atoms. No approach guarantees the ultimate success and derivatization remains largely a trial-and-error process. In practice, however, there is a very good chance that one of a wide variety of the available procedures will lead to successful structure solution.

Imaging and structural studies of DNA-protein complexes and membrane ion channels.

In bio-imaging by electron microscopy, damage of the sample and limited contrast are the two main hurdles for reaching high image quality. We extend a new preparation method based on nanofabrication and super-hydrophobicity to the imaging and structural studies of nucleic acids, nucleic acid-protein complexes (DNA/Rad51 repair protein complex) and neuronal ion channels (gap-junction, K+ and GABAA channels) as paradigms of biological significance and increasing complexity. The preparation method is based on the liquid phase and is compatible with physiological conditions. Only in the very last stage, samples are dried for TEM analysis. Conventional TEM and high-resolution TEM (HRTEM) were used to achieve a resolution of 3.3 and 1.5 Å, respectively. The EM dataset quality allows the determination of relevant structural and metrological information on the DNA structure, DNA-protein interactions and ion channels, allowing the identification of specific macromolecules and their structure.

Characterization of Nucleic Acid Compaction with Histone-Mimic Nanoparticles through All-Atom Molecular Dynamics.

The development of nucleic acid (NA) based nanotechnology applications rely on the efficient packaging of DNA and RNA. However, the atomic details of NA-nanoparticle binding remains to be comprehensively characterized. Here, we examined how nanoparticle and solvent properties affect NA compaction. Our large-scale, all-atom simulations of ligand-functionalized gold nanoparticle (NP) binding to double stranded NAs as a function of NP charge and solution salt concentration reveal different responses of RNA and DNA to cationic NPs. We demonstrate that the ability of a nanoparticle to bend DNA is directly correlated with the NPs charge and ligand corona shape, where more than 50% charge neutralization and spherical shape of the NP ligand corona ensured the DNA compaction. However, NP with 100% charge neutralization is needed to bend DNA almost as efficiently as the histone octamer. For RNA in 0.1 M NaCl, even the most highly charged nanoparticles are not capable of causing bending due to charged ligand end groups binding internally to the major groove of RNA. We show that RNA compaction can only be achieved through a combination of highly charged nanoparticles with low salt concentration. Upon interactions with highly charged NPs, DNA bends through periodic variation in groove widths and depths, whereas RNA bends through expansion of the major groove.

Analyses of nuclear proteins and nucleic acid structures using atomic force microscopy.

Since the inception of atomic force microscopy (AFM) in 1986, the value of this technology for exploring the structure and biophysical properties of a variety of biological samples has been increasingly recognized. AFM provides the opportunity to both image samples at nanometer resolution and also measure the forces on the surface of the sample. Here, we describe a variety of methods for studying nuclear samples including single nucleic acid molecules, higher-order chromatin structures, the nucleolus, and the nucleus. Protocols to prepare nucleic acids, nucleic acid-protein complexes, reconstituted chromatin, the cell nucleus, and the nucleolus are included, as well as protocols describing how to prepare the AFM substrate and the AFM tip. Finally, we describe how to perform conventional imaging, high-speed imaging, recognition imaging, force spectroscopy, and nanoindentation experiments.

Self-assembly of nucleic acids, silk and hybrid materials thereof.

Top-down approaches based on etching techniques have almost reached their limits in terms of dimension. Therefore, novel assembly strategies and types of nanomaterials are required to allow technological advances. Self-assembly processes independent of external energy sources and unlimited in dimensional scaling have become a very promising approach. Here,we highlight recent developments in self-assembled DNA-polymer, silk-polymer and silk-DNA hybrids as promising materials with biotic and abiotic moieties for constructing complex hierarchical materials in 'bottom-up' approaches. DNA block copolymers assemble into nanostructures typically exposing a DNA corona which allows functionalization, labeling and higher levels of organization due to its specific addressable recognition properties. In contrast, self-assembly of natural silk proteins as well as their recombinant variants yields mechanically stable ß-sheet rich nanostructures. The combination of silk with abiotic polymers gains hybrid materials with new functionalities. Together, the precision of DNA hybridization and robustness of silk fibrillar structures combine in novel conjugates enable processing of higher-order structures with nanoscale architecture and programmable functions.

PET-FCS: probing rapid structural fluctuations of proteins and nucleic acids by single-molecule fluorescence quenching.

Quenching of organic fluorophores by aromatic amino acids and DNA nucleotides with expelled electron donating properties allows the study of conformational dynamics of biomolecules. Efficient fluorescence quenching via photoinduced electron transfer (PET) requires van der Waals contact and can be used as reporter for structural fluctuations at the 1-nm scale in proteins, peptides, and nucleic acids. The combination of PET with fluorescence correlation spectroscopy (FCS) establishes a powerful method (PET-FCS) to study equilibrium dynamics at the single-molecule level on time scales from nano- to milliseconds. We delineate the fundamentals of PET-based fluorescence quenching, reporter engineering, instrumental and experimental design, and provide examples.