Heat capacity change for ribonuclease A folding.

The change in heat capacity deltaCp for the folding of ribonuclease A was determined using differential scanning calorimetry and thermal denaturation curves. The methods gave equivalent results, deltaCp = 1.15+/-0.08 kcal mol(-1) K(-1). Estimates of the conformational stability of ribonuclease A based on these results from thermal unfolding are in good agreement with estimates from urea unfolding analyzed using the linear extrapolation method.     

Fractionation with ethanol of nucleic acids from viroid-infected plants

A combination of high salt and low ethanol concentration allowed the fractionation of nucleic acids extracted from viroid-infected leaves. By adding 0.4-0.5 vol of ethanol to 1 vol of a solution in 2 M LiCl of nucleic acids (containing mainly DNA, 4S, 5S, 7S, and viroid RNAs), 85% of the DNA and 75% of the 4S RNA remained in solution, from where they could be recovered by increasing the ethanol concentration, whereas almost all 5S, 7S, and viroid RNAs precipitated. When this process was repeated three times a 95% elimination of the initial DNA and 4S RNA was achieved. The method can be of special interest in viroid purification considering that DNA and 4S RNA are the most abundant contaminants in the starting solution of nucleic acids. It is suggested that the highly ordered secondary structure of viroid RNA may be responsible for its particular behavior in the ethanol fractionation of nucleic acids.     

Heat capacity changes associated with guanine quadruplex formation: an isothermal titration calorimetry study

This study addresses the temperature dependence of the enthalpy of formation for several unimolecular quadruplexes in the presence of excess monovalent salt. We examined a series of biologically significant guanine-rich DNA sequences: thrombin binding aptamer (TBA) (d(G(2)T(2)G(2)TGTG(2)T(2)G(2)), PS2.M, a catalytically active aptamer (d(GTG(3)TAG(3)CG(3)T(2)G(2))), and the human telomere repeat (HT) (d(AG(3)(T(2)AG(3))(3))). Using CD spectra and UV melting, we confirmed the presence of quadruplex structures and established the temperature range in which quadruplex conformation is stable. We then performed ITC experiments, adding DNA to a solution containing excess NaCl or KCl. In this approach, only several additions are made, and only the enthalpy of quadruplex formation is measured. This measurement was repeated at different temperatures to determine the temperature dependence of the enthalpy change accompanying quadruplex formation. To control for the effect of nonspecific salt interactions during DNA folding, we repeated the experiment by replacing the quadruplex-forming sequences with a similar but nonfolding sequence. Dilution enthalpies were also subtracted to obtain the final enthalpy value involving only the quadruplex folding process. For all sequences studied, quadruplex formation was exothermic but with an increasing magnitude with increasing temperature. These results are discussed in terms of the change in heat capacity associated with quadruplex formation.     

Choice of synonymous codons associated with protein folding

Bioinformatical studies suggest that additional information provided by nucleic acids is necessary to construct protein three-dimensional structures. We find underlying correlations between the contents of bases. All correlations occur at the third codon position of a gene sequence. Four inverse relationships are observed between u(3) and c(3), between a(3) and g(3), between u(3) and g(3), and between c(3) and a(3); and two positive relationships are apparent between u(3) and a(3), and between c(3) and g(3). Their correlation coefficients reach -0.92, -0.89, -0.83, -0.85, 0.83, and 0.66, respectively, for large proteins with multistate folding kinetics. The interconnection of bases can be ascribed to choice of synonymous codons associated with protein folding in vivo. In this study, the refolding rate constants of large proteins correlate with the contents of the third base, suggesting that there is underlying biochemical rationale of guiding protein folding in choosing synonymous codons.     

Some unusual nucleic acid bases are products of hydroxyl radical oxidation of DNA and RNA

There are over 100 modified bases and their derivatives found in RNA and DNA. For some of them, data concerning their properties, synthesis and roles in cellular metabolism are available, but for others the knowledge of their functions and biosynthetic pathways is rather limited. We have analysed the chemical structure of modified nucleosides of DNA and RNA considering mainly their putative synthetic routes. On this basis we suggest, that in addition to enzymatic biosynthetic pathways well established for some odd bases, many rare nucleosides can be recognised as products of random chemical reactions. We identify them as primary or secondary products of the reaction of nucleic acids with hydroxyl radicals, the most active oxidising agent in the cell.     

Xanthogenate nucleic acid isolation from cultured and environmental cyanobacteria

The isolation of high-quality nucleic acids from cyanobacterial strains, in particular environmental isolates, has proven far from trivial. We present novel techniques for the extraction of high molecular weight DNA and RNA from a range of cultured and environmental cyanobacteria, including stains belonging to the genera Microcystis , Lyngbya , Pseudanabaena , Aphanizomenon , Nodularia , Anabaena , and Nostoc , based on the use of the nontoxic polysaccharide solubilizing compound xanthogenate. These methods are rapid, require no enzymatic or mechanical cell disruption, and have been used to isolate both DNA and RNA free of enzyme inhibitors or nucleases. In addition, these procedures have proven critical in the molecular analysis of bloom-forming and other environmental cyanobacterial isolates. Finally, these techniques are of general microbiological utility for a diverse range of noncyanobacterial microorganisms, including Gram-positive and Gram-negative bacteria and the Archea.     

Circulating nucleic acids (CNAs) and cancer—A survey

It has been known for decades that it is possible to detect small amounts of extracellular nucleic acids in plasma and serum of healthy and diseased human beings. The unequivocal proof that part of these circulating nucleic acids (CNAs) is of tumor origin, initiated a surge of studies which confirmed and extended the original observations. In the past few years many experiments showed that tumor-associated alterations can be detected at the DNA and RNA level. At the DNA level the detection of point mutations, microsatellite alterations, chromosomal alterations, i.e. inversion and deletion, and hypermethylation of promoter sequences were demonstrated. At the RNA level the overexpression of tumor-associated genes was shown. These observations laid the foundation for the development of assays for an early detection of cancer as well as for other clinical means.     

Single-molecule fluorescence of nucleic acids

Less than a decade old, single-molecule fluorescence of nucleic acids has rapidly become an important tool in the arsenal of biological probes. A variety of novel approaches to investigate conformational dynamics, catalytic mechanisms, folding pathways and protein-nucleic-acid interactions have recently been devised for nucleic acids using this technique. Combined with biomechanical tools and ensemble measurements, single-molecule fluorescence methods extend our ability to observe and understand biomolecules and complex biological processes.     

Chirality as a tool in nucleic acid recognition: principles and relevance in biotechnology and in medicinal chemistry

The understanding of the interaction of chiral species with DNA or RNA is very important for the development of new tools in biology and of new drugs. Several cases in which chirality is a crucial point in determining the DNA binding mode are reviewed and discussed, with the aim of illustrating how chirality can be considered as a tool for improving the understanding of mechanisms and the effectiveness of nucleic acid recognition. The review is divided into two parts: the former describes examples of chiral species interacting with DNA: intercalators, metal complexes, and groove binders; the latter part is dedicated to chirality in DNA analogs, with discussion of phosphate stereochemistry and chirality of ribose substitutes, in particular of peptide nucleic acids (PNAs) for which a number of works have been published recently dealing with the effect of chirality in DNA recognition. The discussion is intended to show how enantiomeric recognition originates at the molecular level, by exploiting the enormous progresses recently achieved in the field of structural characterization of complexes formed by nucleic acid with their ligands by crystallographic and spectroscopic methods. Examples of application of the DNA binding molecules described and the role of chirality in DNA recognition relevant for biotechnology or medicinal chemistry are reported.     

Comments on selected aspects of nucleic acid electrostatics

Recent experimental, theoretical, and computational developments in the field of nucleic acid electrostatics have brought interesting concepts to the fore. The phosphate charge on the double helix apparently influences its structure. When the charge is neutralized asymmetrically, the resulting force imbalance drives bending toward the neutralized side. When the charge is uniformly neutralized, the force imbalance acts to buckle the helical axis, resulting in a compact tertiary conformation. Sharing of condensed counterions by single strands is a stabilizing factor for formation of the double helix. Sharing of condensed counterions by two double helices causes clustering of DNA and may be a factor in RNA folding. Support for these statements is reviewed.     

Exploring the role of chirality in nucleic acid recognition

The study of the base-pairing properties of nucleic acids with sugar moieties in the backbone belonging to the L-series (β-L-DNA, β-L-RNA, and their analogs) are reviewed. The major structural factors underlying the formation of stable heterochiral complexes obtained by incorporation of modified nucleotides into natural duplexes, or by hybridization between homochiral strands of opposite sense of chirality are highlighted. In addition, the perspective use of L-nucleic acids as candidates for various therapeutic applications, or as tools for both synthetic biology and etiology-oriented investigations on the structure and stereochemistry of natural nucleic acids is discussed.     

Age-dependent changes of nucleic acid labeling in different rat brain regions

The effects of aging on in vivo DNA and RNA labeling and on RNA content in various brain regions of 4-, 12-, and 24-month-old rats were investigated. No difference in [methyl-¹⁴C]thymidine incorporation into DNA of cerebral cortex and cerebelllum during aging was observed.The ratio of RNA/DNA content significantly decreased from 4 to 24 months of age in cerebral cortex, cerebellum and striatum. RNA labeling decreased by 15% in cerebral cortex of 24-month-old animals while in the other brain areas examined (cerebellum, hippocampus, hypothalamus, brainstem, striatum) did not change during aging.In the cerebral cortex, the ratio of the specific radioactivity of microsomal RNA to that of nuclear RNA, determined by in vivo experiments, was not affected by the aging process. A significant decrease of total, poly(A)+ RNA and poly(A)- RNA content was observed in the same brain area of 24-month-old rats compared to 4-month-old ones. Moreover, densitometric and radioactivity patterns obtained by gel electrophoresis of labeled RNA after in vitro experiments (tissue slices of cerebral cortex) showed a different ribosomal RNA processing during aging. In vivo chronic treatment with CDP-choline was able to increase RNA labeling in corpus striatum of 24-month-old animals.     

Synthesis and properties of peptide nucleic acid labeled at the N-terminus with HiLyte Fluor 488 fluorescent dye

Fluorescently labeled peptide nucleic acids (PNAs) are important tools in fundamental research and biomedical applications. However, synthesis of labeled PNAs, especially using modern and expensive dyes, is less explored than similar preparations of oligonucleotide dye conjugates. Herein, we present a simple procedure for labeling of the PNA N-terminus with HiLyte Fluor 488 as the last step of solid phase PNA synthesis. A minimum excess of 1.25 equiv of activated carboxylic acid achieved labeling yields close to 90% providing a good compromise between the price of dye and the yield of product and significant improvement over previous literature procedures. The HiLyte Fluor 488-labeled PNAs retained the RNA binding ability and in live cell fluorescence microscopy experiments were brighter and significantly more photostable than PNA labeled with carboxyfluorescein. In contrast to fluorescein-labeled PNA, the fluorescence of PNAs labeled with HiLyte Fluor 488 was independent of pH in the biologically relevant range of 5–8. The potential of HiLyte Fluor 488-labeling for studies of PNA cellular uptake and distribution was demonstrated in several cell lines.     

Amino acid substitutions affecting protein dynamics in eglin C do not affect heat capacity change upon unfolding

The heat capacity change upon unfolding (deltaC(p)) is a thermodynamic parameter that defines the temperature dependence of the thermodynamic stability of proteins; however, physical basis of the heat capacity change is not completely understood. Although empirical surface area-based calculations can predict heat capacity changes reasonably well, accumulating evidence suggests that changes in hydration of those surfaces is not the only parameter contributing to the observed heat capacity changes upon unfolding. Because packing density in the protein interior is similar to that observed in organic crystals, we hypothesized that changes in protein dynamics resulting in increased rigidity of the protein structure might contribute to the observed heat capacity change upon unfolding. Using differential scanning calorimetry we characterized the thermodynamic behavior of a serine protease inhibitor eglin C and two eglin C variants with altered native state dynamics, as determined by NMR. We found no evidence of changes in deltaC(p) in either of the variants, suggesting that changes in rigidity do not contribute to the heat capacity change upon unfolding in this model system.     

Correspondence between anomalous m- and DeltaCp-values in protein folding

Proteins folding according to a classical two-state system characteristically show V-shaped chevron plots. We have previously interpreted the symmetrically curved chevron plot of the protein U1A as denaturant-dependent movements in the position of the transition state ensemble (TSE). S6, a structural analog of U1A, shows a classical V-shaped chevron plot indicative of straightforward two-state kinetics, but the mutant LA30 has a curved unfolding limb, which is most consistent with TSE mobility. The kinetic m-values (derivatives of the rate constants with respect to denaturant concentration) in themselves depend on denaturant concentration. To obtain complementary information about putative mobile TSEs, we have carried out a thermodynamic analysis of the three proteins, based on data for refolding and unfolding over the range 10 degrees C to 70 degrees C. The data at all temperatures can be fitted to two-state model systems. Importantly, for all three proteins the activation heat capacities are, within error, identical to the heat capacities measured in independent experiments under equilibrium conditions. Although the equilibrium heat capacities are essentially invariant with regard to denaturant concentration, the activation heat capacities, similar to the structurally equivalent kinetic m-values, show marked denaturant dependence. Furthermore, the values of beta++ at different denaturant concentrations measured by m-values and by heat capacity values are very similar. These observations are consistent with significant transition state movements within the framework of two-state folding. The basis for TSE movement appears to be enthalpic rather than entropic, suggesting that the binding energy of denaturant-protein interactions is a major determinant of the response of energy landscape contours to changing environments.     

The folding pathway of the genomic hepatitis delta virus ribozyme is dominated by slow folding of the pseudoknots

Hepatitis delta virus (HDV) replicates by a double rolling-circle mechanism that requires self-cleavage by closely related genomic and antigenomic versions of a ribozyme. We have previously shown that the uncleaved genomic ribozyme is subject to a variety of alternative (Alt) pairings. Sequence upstream of the ribozyme can regulate self-cleavage activity by formation of an Alt 1 ribozyme-containing structure that severely inhibits self-cleavage, or a P(-1) self-structure that permits rapid self-cleavage. Here, we test three other alternative pairings: Alt P1, Alt 2, and Alt 3. Alt P1 and Alt 3 contain primarily ribozyme-ribozyme interactions, while Alt 2 involves ribozyme-flanking sequence interaction. A number of single and double mutant ribozymes were prepared to increase or decrease the stability of the alternative pairings, and rates of self-cleavage were determined. Results of these experiments were consistent with the existence of the proposed alternative pairings and their ability to inhibit the overall rate of native ribozyme folding. Local misfolds are treated as internal equilibrium constants in a binding polynomial that modulates the intrinsic rate of cleavage. This model of equilibrium effects of misfolds should be general and apply to other ribozymes. All of the alternative pairings sequester a pseudoknot-forming strand. Folding of ribozymes containing Alt P1 and Alt 2 was accelerated by urea as long as the native ribozyme fold was sufficiently stable, while folding of mutants in which both of these alternative pairings had been removed were not stimulated by urea. This behavior suggests that the pseudoknots form by capture of an unfolded or appropriately rearranged alternative pairing by its complementary native strand. Fast-folding mutants were prepared by either weakening alternative pairings or by strengthening native pairings. A kinetic model was developed that accommodates these features and explains the position of the rate-limiting step for the G11C mutant. Implications of these results for structural and dynamic studies of the uncleaved HDV ribozyme are discussed.     

Sequence-based study of two related proteins with different folding behaviors

Z(SPA-1) is an engineered protein that binds to its parent, the three-helix-bundle Z domain of staphylococcal protein A. Uncomplexed Z(SPA-1) shows a reduced helix content and a melting behavior that is less cooperative, compared with the wild-type Z domain. Here we show that the difference in folding behavior between these two sequences can be partly understood in terms of an off-lattice model with 5-6 atoms per amino acid and a minimalistic potential, in which folding is driven by backbone hydrogen bonding and effective hydrophobic attraction.     

Out-of-plane vibration modes of nucleic acid bases

The out-of-plane vibration modes of uracil, cytosine and their deuterated and methylated derivatives such as 1,5-dimethyluracil (1-methylthymine), I-methylcytosine, 5-methylcytosine and 1,5-dimethylcytosine have been computed. The calculated wave-numbers have been compared to the published Raman peak and infrared band positions observed for solid or aqueous samples. The calculations have been carried out on a non-redundant set of symmetrical coordinates and a valence force field has been used. Some characteristic modes located between 750 and 800 cm^-1 found in the infrared spectra of 2-deoxycytidine, 2-deoxythymidine 5-monophosphate and polynucleotides containing cytosine and thymine bases can be interpreted from the calculated results on 1-methylthymine and 1-methylcytosine.