Molecular crowding regulates the structural switch of the DNA G-quadruplex

Almost all biochemical reactions in vitro have been investigated through numerous experiments conducted in dilute solutions containing low concentrations of solutes. However, biomacromolecules such as nucleic acids, proteins, and polysaccharides are designed to function and/or form their native structures in a living cell containing high concentrations of biomacromolecules, substrates, cofactors, salts, and so on. In the present study, we have demonstrated quantitatively the effect of molecular crowding on structures and stabilities of the G-quadruplex of d(G(4)T(4)G(4)). Molecular crowding with poly(ethylene glycol) (PEG) induced a structural transition from the antiparallel to the parallel G-quadruplex of d(G(4)T(4)G(4)), while molecular crowding with polycations did not alter the structure of the antiparallel G-quadruplex. The binding constants of putrescine, one of the polycations, for d(G(4)T(4)G(4)) in the absence and presence of Na(+) are calculated to be 277 and 2.5 M(-)(1), respectively. This indicates that the polycations coordinate to d(G(4)T(4)G(4)) with electrostatic interactions. The thermodynamic parameters of the antiparallel G-quadruplex formation under the crowding and noncrowding conditions induced by putrescine were also estimated. The stability of the antiparallel G-quadruplex decreased (-DeltaG degrees (25) decreased from 28 to 22 kcal mol(-)(1)) with molecular crowding by putrescine. Also, enthalpy and entropy changes in the structural formation under crowding and noncrowding conditions clearly showed that destabilization was entropy-driven. These quantitative parameters indicated that both the volume excluded by PEG and chemical interactions such as electrostatic interaction with solute polycations are critical for determining how molecular crowding affects the structure and stability of highly ordered DNA structures.     

Effect of molecular crowding on DNA polymerase activity

Live cells contain high concentrations of macromolecules, but almost all experimental biochemical data have been generated from dilute solutions that do not reflect conditions in vivo. To understand biomolecular behavior in vivo, properties studied in vitro are extrapolated to conditions in vivo; however, the molecular conditions within live cells are inherently crowded. The present study investigates the effect of molecular crowding on DNA polymerase activity using polyethylene glycol PEG of various molecular weights as a crowding agent. Polymerase activity assays under various conditions demonstrated that the activities of T7 and Taq DNA polymerases depend on the molecular weight and concentration of the crowding agent. Furthermore, equilibrium and kinetic analyses demonstrated that the binding affinity and catalytic activity of the polymerase increase and decrease, respectively, with increasing PEG concentrations. Based on quantitative parameters of the polymerase reactions, we improved the efficiency of PCR amplification under conditions of molecular crowding. These results suggest that quantitative measurements of biomolecular structure and function are useful for understanding the behavior of biomolecules in vivo and for biotechnology applications in vitro.     

Atomic force microscopy imaging of DNA under macromolecular crowding conditions

Studying the influence of macromolecular crowding at high ionic strengths on assemblies of biomolecules is of particular interest because these are standard intracellular conditions. However, up to now, no techniques offer the possibility of studying the effect of molecular crowding at the single molecule scale and at high resolution. We present a method to observe double-strand DNA under macromolecular crowding conditions on a flat mica surface by atomic force microscope. By using high concentrations of monovalent salt ([NaCl] > 100 mM), we promote DNA adsorption onto NiCl 2 pretreated muscovite mica. It therefore allows analysis of DNA conformational changes and DNA compaction induced by polyethylene glycol (PEG), a neutral crowding agent, at physiological concentrations of monovalent salt.     

Peptide nucleic acids on microarrays and other biosensors

The analysis of biomolecules using microarrays and other biosensors has a significant role in molecular biotechnology, and will become even more important in the future as a versatile tool for research and diagnostics. For many applications, the synthetic DNA mimic peptide nucleic acid (PNA) could be advantageous as a probe molecule, owing to its unique physicochemical and biochemical properties. PNA exhibits superior hybridization characteristics and improved chemical and enzymatic stability relative to nucleic acids. Furthermore, its different molecular structure enables new modes of detection, especially procedures that avoid the introduction of a label. In our opinion, all of these factors contribute significantly toward the establishment of faster and more reliable analytical processes and opens new fields of application.     

Fragment molecular orbital calculations for biomolecules

Exploring biomolecule behavior, such as proteins and nucleic acids, using quantum mechanical theory can identify many life science phenomena from first principles. Fragment molecular orbital (FMO) calculations of whole single particles of biomolecules can determine the electronic state of the interior and surface of molecules and explore molecular recognition mechanisms based on intermolecular and intramolecular interactions. In this review, we summarized the current state of FMO calculations in drug discovery, virology, and structural biology, as well as recent developments from data science.     

The structural stability and catalytic activity of DNA and RNA oligonucleotides in the presence of organic solvents

Organic solvents and apolar media are used in the studies of nucleic acids to modify the conformation and function of nucleic acids, to improve solubility of hydrophobic ligands, to construct molecular scaffolds for organic synthesis, and to study molecular crowding effects. Understanding how organic solvents affect nucleic acid interactions and identifying the factors that dominate solvent effects are important for the creation of oligonucleotide-based technologies. This review describes the structural and catalytic properties of DNA and RNA oligonucleotides in organic solutions and in aqueous solutions with organic cosolvents. There are several possible mechanisms underlying the effects of organic solvents on nucleic acid interactions. The reported results emphasize the significance of the osmotic pressure effect and the dielectric constant effect in addition to specific interactions with nucleic acid strands. This review will serve as a guide for the selection of solvent systems based on the purpose of the nucleic acid-based experiments.     

Negative detection of biomolecules separated in polyacrylamide electrophoresis gels

Here we describe the protocols for negative or reverse detection of proteins, nucleic acids and lipopolysaccharides separated in polyacrylamide electrophoresis gels. These protocols are based on the selective synthesis and precipitation of a white imidazole-zinc complex in the gel, which is absent from those zones where biomolecules are located. These methods are highly sensitive (1-10 ng of biomolecules per band), very cheap as they use inexpensive, common laboratory reagents (imidazole and a Zn II salt), rapid (less than 20 min after gel washing), robust and simple (two steps). Reverse-stained biomolecules are reversibly fixed in the gel. After brief incubation in a zinc chelating agent, biomolecules can be recovered from the gel with the same efficiency as from unstained gels. In consequence, they are procedures of choice for micropreparative applications. References covering typical applications are included.     

A rapid assay for affinity and kinetics of molecular interactions with nucleic acids

The Differential Radial Capillary Action of Ligand Assay (DRaCALA) allows detection of protein interactions with low-molecular weight ligands based on separation of the protein-ligand complex by differential capillary action. Here, we present an application of DRaCALA to the study of nucleic acid-protein interactions using the Escherichia coli cyclic AMP receptor protein (CRP). CRP bound in DRaCALA specifically to (32)P-labeled oligonucleotides containing the consensus CRP binding site, but not to oligonucleotides with point mutations known to abrogate binding. Affinity and kinetic studies using DRaCALA yielded a dissociation constant and dissociation rate similar to previously reported values. Because DRaCALA is not subject to ligand size restrictions, whole plasmids with a single CRP-binding site were used as probes, yielding similar results. DNA can also function as an easily labeled carrier molecule for a conjugated ligand. Sequestration of biotinylated nucleic acids by streptavidin allowed nucleic acids to take the place of the protein as the immobile binding partner. Therefore, any molecular interactions involving nucleic acids can be tested. We demonstrate this principle utilizing a bacterial riboswitch that binds cyclic-di-guanosine monophosphate. DRaCALA is a flexible and complementary approach to other biochemical methods for rapid and accurate measurements of affinity and kinetics at near-equilibrium conditions.     

Volumetric properties of nucleic acids

Volumetric studies can yield useful new information on a myriad of intra- and intermolecular interactions that stabilize nucleic acid structures. In particular, appropriately designed volumetric measurements can characterize the conformation-dependent hydration properties of nucleic acids as a function of solution conditions, including temperature, pressure, ionic strength, pH, and cosolvent concentration. We have started to accumulate a substantial database on volumetric properties of DNA and RNA, as well as on related low molecular weight model compounds. This database already has provided unique insights into the molecular origins of various nucleic acid recognition processes, including helix-to-coil and helix-to-helix conformational transitions, as well as drug-DNA interactions. In this article, we review recent progress in volumetric investigations of nucleic acids, emphasizing how these data can be used to gain insight into intra-and intermolecular interactions, including hydration properties. Throughout this review, we underscore the importance of volume and compressibility data for characterizing the hydration properties of nucleic acids and their constituents. We also describe how such volumetric data can be interpreted at the molecular level to yield a better understanding of the role that hydration can play in modulating the stability and recognition of nucleic acids.     

Evaluation of weak interactions of proteins and organic cations with DNA duplex structures

Molecular interactions and reactions in living cells occur with high background concentrations of organic compounds including proteins. Uncharged water-soluble polymers are commonly used cosolutes in studies on molecular crowding, and most studies argue about the effects of intracellular crowding based on results obtained using polymer cosolutes. Further investigations using protein crowders and organic cations are important in understanding the effects of cellular environments on nucleic acids with negatively charged surfaces. We assessed the effects of using model globular proteins, serum proteins, histone proteins, structurally flexible polypeptides, di- and polyamines, and uncharged polymers. Thermal stability analysis of DNA oligonucleotide structures revealed that unlike conventional polymer cosolutes, basic globular proteins (lysozyme and cytochrome c) at high concentrations stabilized long internal and bulge loop structures but not fully matched duplexes. The selective stabilization of long loop structures suggests preferential binding to unpaired nucleotides in loops through weak electrostatic interactions. Furthermore, the ability of the proteins to stabilize the loop structures was enhanced under macromolecular crowding conditions. Remarkably, the effects of basic proteins on the stability of fully matched duplexes were dissimilar to those of basic amino-acid-rich polypeptides and polyamines. This study provides new insights into the interaction of nucleic acid structures with organic cations.     

Enzyme activity in the crowded milieu

The cytosol of a cell is a concentrated milieu of a variety of different molecules, including small molecules (salts and metabolites) and macromolecules such as nucleic acids, polysaccharides, proteins and large macromolecular complexes. Macromolecular crowding in the cytosolic environment is proposed to influence various properties of proteins, including substrate binding affinity and enzymatic activity. Here we chose to use the synthetic crowding agent Ficoll, which is commonly used to mimic cytosolic crowding conditions to study the crowding effect on the catalytic properties of glycolytic enzymes, namely phosphoglycerate kinase, glyceraldehyde 3-phosphate dehydrogenase, and acylphosphatase. We determined the kinetic parameters of these enzymes in the absence and in the presence of the crowding agent. We found that the Michaelis constant, K(m), and the catalytic turnover number, k(cat), of these enzymes are not perturbed by the presence of the crowding agent Ficoll. Our results support earlier findings which suggested that the Michaelis constant of certain enzymes evolved in consonance with the substrate concentration in the cell to allow effective enzyme function in bidirectional pathways. This conclusion is further supported by the analysis of nine other enzymes for which the K(m) values in the presence and absence of crowding agents have been measured.     

An inventory of the bacterial macromolecular components and their spatial organization

Formerly regarded as small 'bags' of nucleic acids with randomly diffusing enzymes, bacteria are organized by a sophisticated and tightly regulated molecular machinery. Here, we review qualitative and quantitative data on the intracellular organization of bacteria and provide a detailed inventory of macromolecular structures such as the divisome, the degradosome and the bacterial 'nucleolus'. We discuss how these metabolically active structures manage the spatial organization of the cell and how macromolecular crowding influences them. We present for the first time a visualization program, lifeexplorer, that can be used to study the interplay between metabolism and spatial organization of a prokaryotic cell.     

Nucleic acids as a basis for creating biosensors

Here we present a brief conception of biosensors. Structural peculiarities and properties of single- and double-stranded nucleic acids that are to be taken into account when creating biosensors on the basis of these biomolecules are considered. On the example of two biologically active compounds a possibility is shown for constructing biosensors on the basis of liquid-crystalline dispersions of low molecular mass DNA and on the basis of liquid-crystalline DNA dispersions immobilized due to their inclusion into the synthetic polymeric matrix.     

Cellular nucleic acid binding protein binds G-rich single-stranded nucleic acids and may function as a nucleic acid chaperone

Cellular nucleic acid binding protein (CNBP) is a small single-stranded nucleic acid binding protein made of seven Zn knuckles and an Arg-Gly rich box. CNBP is strikingly conserved among vertebrates and was reported to play broad-spectrum functions in eukaryotic cells biology. Neither its biological function nor its mechanisms of action were elucidated yet. The main goal of this work was to gain further insights into the CNBP biochemical and molecular features. We studied Bufo arenarum CNBP (bCNBP) binding to single-stranded nucleic acid probes representing the main reported CNBP putative targets. We report that, although bCNBP is able to bind RNA and single-stranded DNA (ssDNA) probes in vitro, it binds RNA as a preformed dimer whereas both monomer and dimer are able to bind to ssDNA. A systematic analysis of variant probes shows that the preferred bCNBP targets contain unpaired guanosine-rich stretches. These data expand the knowledge about CNBP binding stoichiometry and begins to dissect the main features of CNBP nucleic acid targets. Besides, we show that bCNBP presents a highly disordered predicted structure and promotes the annealing and melting of nucleic acids in vitro. These features are typical of proteins that function as nucleic acid chaperones. Based on these data, we propose that CNBP may function as a nucleic acid chaperone through binding, remodeling, and stabilizing nucleic acids secondary structures. This novel CNBP biochemical activity broadens the field of study about its biological function and may be the basis to understand the diverse ways in which CNBP controls gene expression.     

Detection of Peptidic Sequences in the Ancient Acidic Sediments of Río Tinto,Spain

Biomarkers are molecules that are produced by or can be associated with biological activities. They can be used as tracers that give us an idea of the ancient biological communities that produced them, the paleoenvironmental conditions where they lived, or the mechanism involved in their transformation and preservation. As a consequence, the preservation potential of molecules over time depends largely on their nature, but also on the conditions of the environment, which controls the decomposition kinetics. In this context, proteins and nucleic acids, which are biomolecules bearing biological information, are among the most labile molecules. In this research, we report the presence of short-chained peptides obtained from extracts of ferruginous sedimentary deposits that have been produced under the acidic and oxidizing solutions of Río Tinto, Spain. These preliminary results go against the paradigmatic idea that considers the acidic and oxidizing environments inappropriate for the preservation of molecular information.     

A survey of the methods for the characterization of microbial consortia and communities

A survey of the available literature on methods most frequently used for the identification and characterization of microbial strains, communities, or consortia is presented. The advantages and disadvantages of the various methodologies were examined from several perspectives including technical, economic (time and cost), and regulatory. The methods fall into 3 broad categories: molecular biological, biochemical, and microbiological. Molecular biological methods comprise a broad range of techniques that are based on the analysis and differentiation of microbial DNA. This class of methods possesses several distinct advantages. Unlike most other commonly used methods, which require the production of secondary materials via the manipulation of microbial growth, molecular biological methods recover and test their source materials (DNA) directly from the microbial cells themselves, without the requirement for culturing. This eliminates both the time required for growth and the biases associated with cultured growth, which is unavoidably and artificially selective. The recovered nucleic acid can be cloned and sequenced directly or subpopulations can be specifically amplified using polymerase chain reaction (PCR), and subsequently cloned and sequenced. PCR technology, used extensively in forensic science, provides researchers with the unique ability to detect nucleic acids (DNA and RNA) in minute amounts, by amplifying a single target molecule by more than a million-fold. Molecular methods are highly sensitive and allow for a high degree of specificity, which, coupled with the ability to separate similar but distinct DNA molecules, means that a great deal of information can be gleaned from even very complex microbial communities. Biochemical methods are composed of a more varied set of methodologies. These techniques share a reliance on gas chromatography and mass spectrometry to separate and precisely identify a range of biomolecules, or else investigate biochemical properties of key cellular biomolecules. Like the molecular biological methods, some biochemical methods such as lipid analyses are also independent of cultured growth. However, many of these techniques are only capable of producing a profile that is characteristic of the microbial community as a whole, providing no information about individual members of the community. A subset of these methodologies are used to derive taxonomic information from a community sample; these rely on the identification of key subspecies of biomolecules that differ slightly but characteristically between species, genera, and higher biological groupings. However, when the consortium is already growing in chemically defined media (as is often the case with commercial products), the rapidity and relatively low costs of these procedures can mitigate concerns related to culturing biases. Microbiological methods are the most varied and the least useful for characterizing microbial consortia. These methods rely on traditional tools (cell counting, selective growth, and microscopic examination) to provide more general characteristics of the community as a whole, or else to narrow down and identify only a small subset of the members of that community. As with many of the biochemical methods, some of the microbiological methods can fairly rapidly and inexpensively create a community profile, which can be used to compare 2 or more entire consortia. However, for taxonomic identification of individual members, microbiological methods are useful only to screen for the presence of a few key predetermined species, whose preferred growth conditions and morphological characteristics are well defined and reproducible.     

Development of a sensitive and rapid nucleic acid assay with tetraphenyl porphyrinatoiron chloride by a resonance light scattering technique.

Based on the interaction between nucleic acids and tetraphenyl porphyrinatoiron chloride (FeTPPCl), a novel method for the determination of nucleic acids at the nanogram level has been developed. Under the optimum conditions, the weak resonance light scattering (RLS) intensity of FeTPPCl was greatly enhanced by nucleic acids and the enhanced RLS intensity was proportional to the concentration of nucleic acids in the range 0.02-2.8 microg/mL for calf thymus DNA, 0.05-3.3 microg/mL for fish sperm DNA and 0.07-4.5 microg/mL for yeast RNA. The detection limits (3sigma) were 2.9 ng/mL for calf thymus DNA, 3.9 ng/mL for fish sperm DNA and 5.2 ng/mL for yeast RNA. Almost no interference could be observed from proteins, nucleosides and most of the metal ions. The proposed method showed good reliability, sensitivity, rapidity and reproducibility when applied to the determination of nucleic acids in synthetic and biochemical samples. The time savings make this method suitable for large routine analyses.     

FRETmatrix: a general methodology for the simulation and analysis of FRET in nucleic acids

Förster resonance energy transfer (FRET) is a technique commonly used to unravel the structure and conformational changes of biomolecules being vital for all living organisms. Typically, FRET is performed using dyes attached externally to nucleic acids through a linker that complicates quantitative interpretation of experiments because of dye diffusion and reorientation. Here, we report a versatile, general methodology for the simulation and analysis of FRET in nucleic acids, and demonstrate its particular power for modelling FRET between probes possessing limited diffusional and rotational freedom, such as our recently developed nucleobase analogue FRET pairs (base–base FRET). These probes are positioned inside the DNA/RNA structures as a replacement for one of the natural bases, thus, providing unique control of their position and orientation and the advantage of reporting from inside sites of interest. In demonstration studies, not requiring molecular dynamics modelling, we obtain previously inaccessible insight into the orientation and nanosecond dynamics of the bases inside double-stranded DNA, and we reconstruct high resolution 3D structures of kinked DNA. The reported methodology is accompanied by a freely available software package, FRETmatrix, for the design and analysis of FRET in nucleic acid containing systems.     

Equilibrium Sampling for Biomolecules under Mechanical Tension

In the studies of force-induced conformational transitions of biomolecules, the large timescale difference from experiments presents the challenge of obtaining convergent sampling for molecular dynamics simulations. To circumvent this fundamental problem, an approach combining the replica-exchange method and umbrella sampling (REM-US) was developed to simulate mechanical stretching of biomolecules under equilibrium conditions. Equilibrium properties of conformational transitions can be obtained directly from simulations without further assumptions. To test the performance, we carried out REM-US simulations of atomic force microscope (AFM) stretching and relaxing measurements on the polysaccharide pustulan, a (1→6)-β-D-glucan, which undergoes well-characterized rotameric transitions in the backbone bonds. With significantly enhanced sampling convergence and efficiency, the REM-US approach closely reproduced the equilibrium force-extension curves measured in AFM experiments. Consistent with the reversibility in the AFM measurements, the new approach generated identical force-extension curves in both stretching and relaxing simulations—an outcome not reported in previous studies, proving that equilibrium conditions were achieved in the simulations. REM-US may provide a robust approach to modeling of mechanical stretching on polysaccharides and even nucleic acids.     

Sulfur-containing Nucleic Acids: Synthesis,Chemical Reactions in Supramolecular Biopolymer Complexes,Biological Applications

The chemical behavior of sulfur-containing oligonucleotides and their reactivity in self-assembled nucleic acids (NA) and specific NA–protein complexes is considered. Reviewed are postsynthetic approaches that allow introducing sulfur-containing linkages at preselected positions of the sugar-phosphate backbone of DNA and between neighboring nucleobases, to incorporate disulfide bridges between complementary strands of double- and triple-stranded DNAs, in large catalytic RNA, etc. Special reference is given to the site-specific chemical modifications as a tool for elucidating the structure, folding, and function of biomolecules. Structure-directed chemical reactions are shown to be helpful in detecting point mutations in DNA, targeting the modifications on specific positions of NA, probing the molecular recognition in protein–DNA interfaces, studying the conformational dynamics of nucleic acids, and discriminating between different folding models.