Thermodynamic description of oligonucleotide self-association in DNA concatamer structures

A scheme of equilibrium formation of concatamers by two different oligonucleotides has been considered. It is shown that in the general case, the dependence of the concentration of oligonucleotide components on temperature cannot be found in analytical form. Therefore, it is impossible to find the thermodynamic parameters of concatamer formation (ΔH ⁰, ΔS ⁰) and melting temperatures by analyzing the profiles of thermal denaturation of oligonucleotide complexes. An algorithm for numerical solution of implicit dependences has been developed. A number of approaches are considered that simplify the analysis of heat denaturation curves for concatamer complexes. It is shown that the dependence of the efficiency of concatamerization on temperature can be described analytically when duplex fragments have close stability and there is no cooperativity at the oligonucleotide junction. In this case, the dependence of melting temperature on thermodynamic parameters and oligonucleotide concentration has the same form as in the case of the duplex structure formed by a pair of non-self-complementary oligonucleotides. The ability of various model approaches to describe the experimental curves of concatamer heat denaturation is evaluated. For concatamer structures used as signal amplifiers in DNA hybridization analysis, a function is introduced that shows the relative contribution of a concatamer of given length to the magnitude of signal amplification. The dependence of the maximum of this function on the concentration of oligonucleotides, the thermodynamic characteristics of their complexes, and temperature has been determined. It is shown by the gel retardation assay that the function of the length distribution of concatamers qualitatively correlates with the experimental dependences.     

Predicting common foldings of homologous RNAs.

A new approach is proposed for determining common RNA secondary structures within a set of homologous RNAs. The approach is a combination of phylogenetic and thermodynamic methods which is based on the prediction of optimal and suboptimal secondary structures, topological similarity searches and phylogenetic comparative analysis. The optimal and suboptimal RNA secondary structures are predicted by energy minimization. Structural comparison of the predicted RNA secondary structures is used to find conserved structures that are topologically similar in all these homologous RNAs. The validity of the conserved structural elements found is then checked by phylogenetic comparison of the sequences. This procedure is used to predict common structures of ribonuclease P (RNAase P) RNAs.     

Common structures of the 5' non-coding RNA in enteroviruses and rhinoviruses. Thermodynamical stability and statistical significance

A total of 4051 suboptimal secondary structures are predicted by folding the 5' non-coding region of ten polioviruses, five human rhinoviruses and three coxsackieviruses using our new suboptimal folding algorithm for the prediction of both optimal and suboptimal RNA secondary structures. A comparative analysis of these RNA secondary structures reveals the conservation of common secondary structure that can be supported by phylogenetic data. The thermodynamic stability and statistical significance of these predicted, conserved helical elements are assessed and significant structure motifs in the 5' non-coding region are proposed. The possible roles of these structure motifs in the virus life cycle are discussed.     

Cucumber mosaic virus-associated RNA 5. VI. Characterization and denaturation-renaturation behavior of the double-stranded form.

The double-stranded form of cucumber mosaic virus-associated RNA 5 has been purified and further characterized. Its molecular weight determined by sedimentation equilibrium is 2.15 . 10(5). The buoyant density calculated from its symmetrical distribution in Cs2SO4, following isopycnic ultracentrifugation, is 1.615 g/cm3. The sedimentation rate of double-stranded cucumber mosaic virus-associated RNA 5 is slightly greater than that of cucumber mosaic virus-associated RNA 5; its electrophoretic mobility in polyacrylamide gel (2.4%) is less than that of cucumber mosaic virus-associated RNA 5. By the above standards the double-stranded cucumber mosaic virus-associated RNA 5 preparations used were found to be nomogeneous in size as well as density. Thermal denaturation monitored by means of ultraviolet light absorption produced multitransitional denaturation profiles. The average melting temperature (Tm) was 88 degrees C in 0.1 x SSC. Monotransitional denaturation profiles and slightly higher Tm values were obtained when resistance against ribonuclease digestion was measured. These denaturation experiments and other propertied led to the conclusion that double-stranded cucumber mosaic virus-associated RNA 5 and the double-stranded form of peanut stunt virus-associated RNA 5 are small double-stranded nucleic acids with several homostable base-pair regions, characterized by distinct G + C contents and Tm values.     

Analysis of thermal melting curves

T(m) is defined as Temperature of melting or, more accurately, as temperature of midtransition. This term is often used for nucleic acids (DNA and RNA, oligonucleotides and polynucleotides). A thermal denaturation experiment determines the stability of the secondary structure of a DNA or RNA and aids in the choice of the sequences for antisense oligomers or PCR primers. Beyond a simple numerical value (the T(m)), a thermal denaturation experiment, in which the folded fraction of a structure is plotted vs. temperature, yields important thermodynamic information. We present the classic problems encountered during these experiments and try to demonstrate that a number of useful pieces of information can be extracted from these experimental curves.     

Dynamics and interactions of viroids

Viroids are single stranded circular RNA molecules of 120,000 daltons which are pathogens of certain higher plants and replicate autonomously in the host cell. Virusoids are similar to viroids in respect to size and circularity but do replicate only as a part of a larger plant virus. The structure and structural transitions have been investigated by thermodynamic, kinetic and hydrodynamic methods and have been compared to results from calculations of the most favorable native structures and the denaturation process. The algorithm of Zuker et al. was modified for the application to circular nucleic acids. For viroids the calculations confirm our earlier theoretical and experimental results about the extended native structure and the highly cooperative transition into a branched structure. Virusoids, although described in the literature as viroid-like, show less base pairing, branching in the native secondary structure, and only low cooperativity during denaturation. They resemble more closely the properties of random sequences with length, G:C content, and circularity as in viroids but sequences generated by a computer. The comparison of viroids, virusoids and circular RNA of random sequences underlines the uniqueness of viroid structure. The interactions of viroids with dye and oligonucleotide-ligands and with RNA-polymerase II from wheat germ, which enzyme replicates viroids in vitro, has been studied in order to correlate viroid structure and its ability for specific interactions. Specificity of the interactions may be interpreted on the basis of the neighbourhood of double stranded and single stranded regions. In the host cell viroids are localized in the cell nucleus; they may be detected as free nucleic acids and in high molecular weight complexes together with other RNA and proteins.     

In silico single strand melting curve: a new approach to identify nucleic acid polymorphisms in Totiviridae

Background

The PCR technique and its variations have been increasingly used in the clinical laboratory and recent advances in this field generated new higher resolution techniques based on nucleic acid denaturation dynamics. The principle of these new molecular tools is based on the comparison of melting profiles, after denaturation of a DNA double strand. Until now, the secondary structure of single-stranded nucleic acids has not been exploited to develop identification systems based on PCR. To test the potential of single-strand RNA denaturation as a new alternative to detect specific nucleic acid variations, sequences from viruses of the Totiviridae family were compared using a new in silico melting curve approach. This family comprises double-stranded RNA virus, with a genome constituted by two ORFs, ORF1 and ORF2, which encodes the capsid/RNA binding proteins and an RNA-dependent RNA polymerase (RdRp), respectively.

Results

A phylogenetic tree based on RdRp amino acid sequences was constructed, and eight monophyletic groups were defined. Alignments of RdRp RNA sequences from each group were screened to identify RNA regions with conserved secondary structure. One region in the second half of ORF2 was identified and individually modeled using the RNAfold tool. Afterwards, each DNA or RNA sequence was denatured in silico using the softwares MELTSIM and RNAheat that generate melting curves considering the denaturation of a double stranded DNA and single stranded RNA, respectively. The same groups identified in the RdRp phylogenetic tree were retrieved by a clustering analysis of the melting curves data obtained from RNAheat. Moreover, the same approach was used to successfully discriminate different variants of Trichomonas vaginalis virus, which was not possible by the visual comparison of the double stranded melting curves generated by MELTSIM.

Conclusion

In silico analysis indicate that ssRNA melting curves are more informative than dsDNA melting curves. Furthermore, conserved RNA structures may be determined from analysis of individuals that are phylogenetically related, and these regions may be used to support the reconstitution of their phylogenetic groups. These findings are a robust basis for the development of in vitro systems to ssRNA melting curves detection.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-243) contains supplementary material, which is available to authorized users.     

Study of the structural properties of recombinant gamma-interferon by circular dichroism and differential scanning microcalorimetry]

Recombinant human gamma-interferon is dimeric in solution at pH 7-4 as revealed by analytical gel-filtration. It was shown by circular dichroism that decreasing pH to 5.0 does not affect the secondary and tertiary structures of gamma-interferon macromolecule. It was established that heat denaturation process of gamma-interferon obeys the two-state transition model and can be described as the first-order reversible reaction. Temperature dependence of the denaturation-renaturation rate constants was shown to be consistent with the Arrhenius law. The equilibrium value of the denaturation temperature was found. Effective enthalpy of denaturation was determined both by thermodynamic and kinetic approaches. The data obtained showed that in the pH range 7-4 the dimeric IFN-gamma structure may be considered as a single cooperative thermodynamic domain. Thus, it may be concluded that gamma-interferon dimerization is necessary for the existence of the corresponding tertiary structure of the macromolecule.     

Interpretation of thermograms of protein denaturation in non-equilibrium conditions

Data are presented concerning the effect of heating rate on the denaturation parameters of small and oligomeric globular proteins: Kunitz trypsin inhibitor from soybeans and 1,5-Ribulose Bisphosphate Carboxylase from tobacco leaves. Substantional dependence of denaturation temperature on the heating rate reflects non-equilibrium pattern of denaturation of these proteins under experimental conditions. To interpret these data a kinetic approach is proposed, which permits determination of equilibrium value of the denaturation temperature and of the constant of de- and renaturation rate. The conformation transitions in the proteins studied are shown to be relatively slow processes. Their rate is comparable to the velocity of temperature change in a calorimeter, which is the cause of non-equilibrium effects in a calorimetric experiment.     

Possible involvement of DNA-linked RNA in the initiation of Bacillus subtilis chromosome replication.

After thermal denaturation, an in vivo-labeled RNA was found in a temperature-sensitive initiation mutant of Bacillus subtilis (dna-37) associated with high-molecular-weight DNA. This RNA could be clearly distinguished from other RNA species by different techniques of separation, such as Sepharose 2B filtration, chromatography on nitrocellulose, and equilibrium centrifugation in density gradient. It was obtained even when HCHO was present during denaturation and chilling of nucleic acids and was still detected after a second denaturation as well as after incubation with proteinase K. Properties of the complex were not altered by prior treatment with RNase H. A control experiment using two samples of the complex treated either with pancreatic DNase or with pancreatic RNase, denatured together and centrifuged in the same density gradient, showed that no artifactual associations occur between the DNA and the RNA components of the complex. These results demonstrate that the DNA and RNA in the complex are associated by neither hydrogen bonds nor proteins, but are indicative of a DNA-RNA covalent linkage. In addition, during synchronous replication after a previous period at a nonpermissive temperature, DNA-linked RNA synthesis took place at specific times which coincided with the appearance of rifampin resistance of the first and the second replication cycles. A possible involvement of this RNA in the initiation of chromosome replication is discussed.     

RNA structures with pseudo-knots: Graph-theoretical, combinatorial, and statistical properties

The secondary structures of nucleic acids form a particularly important class of contact structures. Many important RNA molecules, however, contain pseudo-knots, a structural feature that is excluded explicitly from the conventional definition of secondary structures. We propose here a generalization of secondary structures incorporating ‘non-nested’ pseudo-knots, which we call bi-secondary structures, and discuss measures for the complexity of more general contact structures based on their graph-theoretical properties. Bi-secondary structures are planar trivalent graphs that are characterized by special embedding properties. We derive exact upper bounds on their number (as a function of the chain length n) implying that there are fewer different structures than sequences. Computational results show that the number of bi-secondary structures grows approximately like 2.35ⁿ. Numerical studies based on kinetic folding and a simple extension of the standard energy model show that the global features of the sequence-structure map of RNA do not change when pseudo-knots are introduced into the secondary structure picture. We find a large fraction of neutral mutations and, in particular, networks of sequences that fold into the same shape. These neutral networks percolate through the entire sequence space.     

Dependence of purine 8C-H exchange on nucleic acid conformation and base-pairing geometry: a dynamic probe of DNA and RNA secondary structures

Laser Raman spectroscopy is employed as a probe of hydrogen isotope exchange in nucleic acids exhibiting different secondary structures. The rates for deuterium exchange of 8C-H groups in adenine (A), hypoxanthine (I), and guanine (G) residues of ribo- and deoxyribopolynucleotides are compared with corresponding rates of mononucleotides. In general, nucleic acid secondary structure significantly retards the rate of purine 8C-H exchange. Specifically, the exchange kinetics are strongly dependent on both the kind and amount of secondary structure. The retardation factor (R), defined as the quotient of rate constants for monomer and polymer exchanges, is greatest for the A-helix (9.5 ± 1), intermediate for the B-helix (2.8 ± 0.6), and smallest for the Z-helix (1.5), thus permitting the three authenticated DNA structures to be distinguished from one another using the Raman dynamic probe. Polyribonucleotide complexes of the A-helix family that contain the same backbone conformation and ribosyl pucker (C3′ - endo/anti) but that differ in the geometry of base pairing or number of helix strands are also clearly distinguished by their different deuterium exchange rates. The extraordinarily large retardation of 8C-H exchange (R > 200) that occurs in multihelical structures is attributed to hydrogen bonding by the purine 7N acceptor. These results indicate that 8C-H exchange may be exploited to detect Hoogsteen base pairing and possibly interactions of nucleic acid-binding proteins that involve the 7N site as hydrogen-bond acceptor. The feasibility of the method for evaluation of solvent penetration to encapsidated genomes of DNA and RNA viruses is considered. The present results also reveal a number of new vibrational band assignments for identification of DNA and RNA secondary structures from equilibrium Raman spectra.     

A dynamic programming algorithm for finding alternative RNA secondary structures.

Dynamic programming algorithms that predict RNA secondary structure by minimizing the free energy have had one important limitation. They were able to predict only one optimal structure. Given the uncertainties of the thermodynamic data and the effects of proteins and other environmental factors on structure, the optimal structure predicted by these methods may not have biological significance. We present a dynamic programming algorithm that can determine optimal and suboptimal secondary structures for an RNA. The power and utility of the method is demonstrated in the folding of the intervening sequence of the rRNA of Tetrahymena. By first identifying the major secondary structures corresponding to the lowest free energy minima, a secondary structure of possible biological significance is derived.     

Experimental Evidence That GNA and TNA Were Not Sequential Polymers in the Prebiotic Evolution of RNA

Systematic investigation into the chemical etiology of ribose has led to the discovery of glycerol nucleic acid (GNA) and threose nucleic acid (TNA) as possible progenitor candidates of RNA in the origins of life. Coupled with their chemical simplicity, polymers for both systems are capable of forming stable Watson-Crick antiparallel duplex structures with themselves and RNA, thereby providing a mechanism for the transfer of genetic information between successive genetic systems. Investigation into whether both polymers arose independently or descended from a common evolutionary pathway would provide additional constraints on models that describe the emergence of a hypothetical RNA world. Here we show by thermal denaturation that complementary GNA and TNA mixed sequence polymers are unable, even after prolonged incubation times, to adopt stable helical structures by intersystem cross-pairing. This experimental observation suggests that GNA and TNA, whose structures derive from one another, were not consecutive polymers in the same evolutionary pathway to RNA. Reviewing Editor: Dr. Niles Lehman     

Conserved features of Y RNAs revealed by automated phylogenetic secondary structure analysis.

Y RNAs are small 'cytoplasmic' RNAs which are components of the Ro ribonucleoprotein (RNP) complex. The core of this complex, which is found in the cell nuclei of higher eukaryotes as well as the cytoplasm, is composed of a complex between the 60 kDa Ro protein and Y RNAs. Human cells contain four distinct Y RNAs (Y1, Y3, Y4 and Y5), while other eukaryotes contain a variable number of Y RNA homologues. When detected in a particular species, the Ro RNP has been present in every cell type within that particular organism. This characteristic, along with its high conservation among vertebrates, suggests an important function for Ro RNP in cellular metabolism; however, this function has not yet been definitively elucidated. In order to identify conserved features of Y RNA sequences and structures which may be directly involved in Ro RNP function, a phylogenetic comparative analysis of Y RNAs has been performed. Sequences of Y RNA homologues from five vertebrate species have been obtained and, together with previously published Y RNA sequences, used to predict Y RNA secondary structures. A novel RNA secondary structure comparison algorithm, the suboptimal RNA analysis program, has been developed and used in conjunction with available algorithms to find phylogenetically conserved secondary structure models for YI, Y3 and Y4 RNAs. Short, conserved sequences within the Y RNAs have been identified and are invariant among vertebrates, consistent with a direct role for Y RNAs in Ro function. A subset of these are located wholly or partially in looped regions in the Y3 and Y4 RNA predicted model structures, in accord with the possibility that these Y RNAs base pair with other cellular nucleic acids or are sites of interaction between the Ro RNP and other macromolecules.     

Structural transitions of bovine serum albumin detected during scanning calorimetry

The calorimetry studies of temperature dependence of bovine serum albumin heat capacity for the temperature interval 20-150 degrees C and pH value varying from 4 to 9 were carried out. It is shown that in the pH range considered four types of denaturation curves differing in quantity and temperatures of endothermic pikes exist. The different types of the melting curves correspond to different types of protein domain structure. At 110-120 degrees C and pH greater than or equal to 5.6 the high temperature maximum is shown to exist. The later is supposed to be due not to BSA denaturation process but to cooperative destruction of postdenaturation remnants of the secondary protein structure.     

Sequence- and structural-selective nucleic acid binding revealed by the melting of mixtures

A simple method for the detection of sequence- and structural-selective ligand binding to nucleic acids is described. The method is based on the commonly used thermal denaturation method in which ligand binding is registered as an elevation in the nucleic acid melting temperature (T_m). The method can be extended to yield a new, higher -throughput, assay by the simple expediency of melting designed mixtures of polynucleotides (or oligonucleotides) with different sequences or structures of interest. Upon addition of ligand to such mixtures at low molar ratios, the T_m is shifted only for the nucleic acid containing the preferred sequence or structure. Proof of principle of the assay is provided using first a mixture of polynucleotides with different sequences and, second, with a mixture containing DNA, RNA and two types of DNA:RNA hybrid structures. Netropsin, ethidium, daunorubicin and actinomycin, ligands with known sequence preferences, were used to illustrate the method. The applicability of the approach to oligonucleotide systems is illustrated by the use of simple ternary and binary mixtures of defined sequence deoxyoligonucleotides challenged by the bisanthracycline WP631. The simple mixtures described here provide proof of principle of the assay and pave the way for the development of more sophisticated mixtures for rapidly screening the selectivity of new nucleic acid binding compounds.     

Dynamics and Interactions of Viroids

Abstract

Viroids are single stranded circular RNA molecules of 120 000 dal tons which are pathogens of certain higher plants and replicate autonomously in the host cell. Virusoids are similar to viroids in respect to size and circularity but do replicate only as a part of a larger plant virus. The structure and structural transitions have been investigated by thermodynamic, kinetic and hydrodynamic methods and have been compared to results from calculations of the most favorable native structures and the denaturation process. The algorithm of Zuker et al. was modified for the application to circular nucleic acids.

For viroids the calculations confirm our earlier theoretical and experimental results about the extended native structure and the highly cooperative transition into a branched structure. Virusoids, although described in the literature as viroid-like, show less base pairing, branching in the native secondary structure, and only low cooperativity during denaturation. They resemble more closely the properties of random sequences with length, G:C content, and circularity as in viroids but sequences generated by a computer. The comparison of viroids, virusoids and circular RNA of random sequences underlines the uniqueness of viroid structure.

The interactions of viroids with dye and oligonucleotide-ligands and with RNA-polymerase II from wheat germ, which enzyme replicates viroids in vitro, has been studied in order to correlate viroid structure and its ability for specific interactions. Specificity of the interactions may be interpreted on the basis of the neighbourhood of double stranded and single stranded regions. In the host cell viroids are localized in the cell nucleus; they may be detected as free nucleic acids and in high molecular weight complexes together with other RNA and proteins.     

Relationships Between Genomic G+C Content,RNA Secondary Structures,and Optimal Growth Temperature in Prokaryotes

G:C pairs are more stable than A:T pairs because they have an additional hydrogen bond. This has led to many studies on the correlation between the guanine+cytosine (G+C) content of nucleic acids and temperature over the last 20 years. We collected the optimal growth temperatures (T_opt) and the G+C contents of genomic DNA; 23S, 16S, and 5S ribosomal RNAs; and transfer RNAs for 764 prokaryotic species. No correlation was found between genomic G+C content and T_opt, but there were striking correlations between the G+C content of ribosomal and transfer RNA stems and T_opt. Two explanations have been proposed—neutral evolution and selection pressure—for the approximate equalities of G and C (respectively, A and T) contents within each strand of DNA molecules. Our results do not support the notion that selection pressure induces complementary oligonucleotides in close proximity and therefore numerous secondary structures in prokaryotic DNA, as the genomic G+C content does not behave in the same way as that of folded RNA with respect to optimal growth temperature. Received: 25 September 1996 / Accepted: 21 January 1997     

Length dependence of RNA-RNA annealing

The association of complementary nucleic acids can be described by a second order rate constant k. For extended molecules, including complex nucleic acids, values of k were shown to be proportional to the square root of the chain length L of the shorter nucleic acid strand at temperatures between tm and tm-30 degrees C. For homopolymers this is true over a wider temperature range. Below temperatures of tm-30 degrees C, annealing rate constants may sharply decrease due to the formation of intramolecular structures. It seems to be reasonable to assume that the formation of intramolecular structures of nucleic acids reduces the density of nucleation sites for annealing and, thereby, lowers the rates of association. Here, we examined the relationship between RNA chain length and the kinetics of RNA-RNA annealing at physiological ionic strength and temperature. We used a complete sequence space derived from chloramphenicol acetyltransferase (cat) sequences to average over all structures for each given length. For groups of progressively longer antisense RNA species and a 800 nucleotides long complementary RNA, the observed annealing rate constants kobs were measured in vitro. The structure-averaged values for kobs of RNA-RNA annealing were not related to the square root of the chain length. Instead, they were found to be proportional to 10(alphaL) (alpha=0.0017). Here, a theoretical model is suggested in which the observed length dependence is mainly influenced by ionic interactions between complementary RNA strands. The observed length dependence has substantial implications for the biological behavior of long-chain complementary RNA including the design of antisense RNA. The efficacy of antisense RNA in living cells is known to be related to annealing kinetics in vitro. Thus, on a statistical basis and independent of individual structures, long-chain rather than short-chain antisense RNA should lead to stronger inhibition.