From quadruplex to helix and back: meta-stable states can move through a cycle of conformational changes.

Strand displacement cycles can be driven by sequential addition of short oligonucleotide sequences. Successive inter- and intra-molecular interactions based on the rules of Watson-Crick base pairing allow us to design self-assembling molecular systems with predictable folding pathways and conformational changes. Here we present a particular strand displacement cycle that starts from a tethered quadruplex-forming sequence from the human telomere repeat (T(2)AG(3))(4) that forms a G-quartet within a stem-loop structure. Adding an almost matching single strand converts the four-stranded section into a defective double helix. This is the first step of the cycle. The subsequent addition of a "fuel strand" removes the single strand from the loop sequence in favor of a perfect double helix. This displacement frees the hairpin-loop to go back to its initial state. Analysis of this cycle, that resembles an enzyme-substrate pathway as far as the initial state will be regained at the end of the cycle, advances our understanding of the interchanges between meta-stable states that underlie some fundamental steps in molecular biology, and allow for the construction of nano-molecular machines.

[A calorimetric study of the triple helix formation: interaction of poly(C) - guanosine - protonated poly(C)].

The triple helix formation of poly(C) - guanosine - poly(C+) was investigated by the help of an LKB scanning micro-calorimeter. The existence of the triple helix could also be shown by recording the melting curves. The ultraviolet absorption at different wave lengths namely 275 nm, 260 nm, and 245 nm was plotted as a function of the temperature. Furthermore formation of the triple helix was shown by plotting the ultraviolet absorption at 245 nm during the increasing addition of guanosine solution to a fixed amount of poly(C) in the solution. Finally the formation of the triple helix was demonstrated by plotting the ultraviolet absorption at 245 nm of a certain mixture of the components while the pH value of the solution was continuously lowered. All these methods show that the monomer interacts with the polymer double helix to form a triple helix. The calorimetric measurements show that the reaction enthalpy is concentration dependent. Above a threshold concentration a rapid increase of the reaction enthalpy is observed. This increase occurs in a very narrow concentration interval. Above this interval a final value of the reaction enthalpy is reached. The amount of the reaction enthalpy for the interaction of guanosine with poly(C) - poly(C+) double helix is 5.5 Kcal (mol base triplet)-1.

Thermodynamic values of the helix-coil transition of DNA in the presence of quaternary ammonium salt.

The knowledge of the enthalpy and entropy of the helix-coil transition in DNA is necessary for the understanding of the stabilization of its native conformation in solution. Reported here is the transition temperature Tm, the transition enthalpy deltaH, determined with the help of an adiabatic scanning calorimeter, the transition entropy deltaS and the breadth of the helix-coil transition as a function of tetramethyl and tetraethyl ammonium chloride concentration.

Calorimetric measurements of the transition enthalpy of DNA in aqueous urea solutions.

The helix-coil equilibrium of DNA is delicately affected by the nature of the solvent. In this investigation the helical secondary structure was destabilized by an increasing concentration of urea. We found a linear dependence of the transition enthalpy deltaH on the urea concentration for calf thymus DNA as well as for salmon sperm DNA.

Energetics of a stable intramolecular DNA triple helix formation.

We have designed and synthesized by conventional chemical techniques a 38mer oligonucleotide consisting of a 5'd(Pu)10d(C)4d(Py)10d(T)4d(Py)10(3') sequence. This oligonucleotide assumes a randomly coiled conformation at pH 12. At pH 8.0 a hairpin helix forms between its 5' purine decamer sequence and the consecutive pyrimidine decamer leaving the second pyrimidine decamer as a dangling disordered 3' extension. On reducing the pH to 4.5 this second pyrimidine decamer folds back onto the major groove of the hairpin helix resulting in an intramolecular triple-stranded stem-loop structure. We have used a variety of biochemical (gel mobility, P1 nuclease digestion) and biophysical (ultraviolet light and circular dichroism spectroscopy, fluorimetry, microcalorimetry) techniques to characterize the different conformers, their stability and the folding pathway into an intramolecular triple helix. The thermodynamic properties of this intramolecular triple strand in 100 mM-Na+ are: tm, 71 degrees C; delta HvH, 119.4(+/- 11.9) kcal mol-1; delta Hcal, 121.9 (+/- 6.1) kcal mol-1 at pH 4.5; those of the hairpin are: tm, 63 degrees C; delta HvH, 71.7(+/- 4.0) kcal mol-1; delta Hcal, 69.9(+/- 3.5) kcal mol-1 at pH 8.0. At intermediate pH values, the triplex to coil transition breaks up into its component triplex to hairpin and hairpin to coil transitions with thermodynamic properties: tm, 41 degrees C; delta HvH, 58.7(+/- 4.2) kcal mol-1; delta Hcal, 39.8(+/- 2.0) kcal mol-1; and tm, 63 degrees C; delta HvH, 71.7(+/- 4.0) kcal mol-1; delta Hcal, 69.6(+/- 3.5) kcal mol-1 at pH 6.7.

Counterion association with native and denatured nucleic acids: an experimental approach.

The melting temperature of the poly(dA) . poly(dT) double helix is exquisitely sensitive to salt concentration, and the helix-to-coil transition is sharp. Modern calorimetric instrumentation allows this transition to be detected and characterized with high precision at extremely low duplex concentrations. We have taken advantage of these properties to show that this duplex can be used as a sensitive probe to detect and to characterize the influence of other solutes on solution properties. We demonstrate how the temperature associated with poly(dA) . poly(dT) melting can be used to define the change in bulk solution cation concentration imparted by the presence of other duplex and triplex solutes, in both their native and denatured states. We use this information to critically evaluate features of counterion condensation theory, as well as to illustrate "crosstalk" between different, non-contacting solute molecules. Specifically, we probe the melting of a synthetic homopolymer, poly(dA) . poly(dT), in the presence of excess genomic salmon sperm DNA, or in the presence of one of two synthetic RNA polymers (the poly(rA) . poly(rU) duplex or the poly(rU) . poly(rA) . poly(rU) triplex). We find that these additions cause a shift in the melting temperature of poly(dA) . poly(dT), which is proportional to the concentration of the added polymer and dependent on its conformational state (B versus A, native versus denatured, and triplex versus duplex). To a first approximation, the magnitude of the observed tm shift does not depend significantly on whether the added polymer is RNA or DNA, but it does depend on the number of strands making up the helix of the added polymer. We ascribe the observed changes in melting temperature of poly(dA) . poly(dT) to the increase in ionic strength of the bulk solution brought about by the presence of the added nucleic acid and its associated counterions. We refer to this communication between non-contacting biopolymers in solution as solvent-mediated crosstalk. By comparison with a known standard curve of tm versus log[Na+] for poly(dA) . poly(dT), we estimate the magnitude of the apparent change in ionic strength resulting from the presence of the bulk nucleic acid, and we compare these results with predictions from theory. We find that current theoretical considerations correctly predict the direction of the t(m) shift (the melting temperature increases), while overestimating its magnitude. Specifically, we observe an apparent increase in ionic strength equal to 5% of the concentration of the added duplex DNA or RNA (in mol phosphate), and an additional apparent increase of about 9.5 % of the nucleic acid concentration (mol phosphate) upon denaturation of the added DNA or RNA, yielding a total apparent increase of 14.5 %. For the poly(rU) . poly(rA) . poly(rU) triplex, the total apparent increase in ionic strength corresponds to about 13.6% of the amount of added triplex (moles phosphate). The effect we observe is due to coupled equilibria between the solute molecules mediated by modulations in cation concentration induced by the presence and/or the transition of one of the solute molecules. We note that our results are general, so one can use a different solute probe sensitive to proton binding to characterize subtle changes in solution pH induced by the presence of another solute in solution. We discuss some of the broader implications of these measurements/results in terms of nucleic acid melting in multicomponent systems, in terms of probing counterion environments, and in terms of potential regulatory mechanisms.

Energetics of strand-displacement reactions in triple helices: a spectroscopic study.

DNA triple helices offer exciting new perspectives toward oligonucleotide-directed inhibition of gene expression. Purine and GT triplexes appear to be the most promising motifs for stable binding under physiological conditions compared to the pyrimidine motif, which forms at relatively low pH. There are, however, very little data available for comparison of the relative stabilities of the different classes of triplexes under identical conditions. We, therefore, designed a model system which allowed us to set up a competition between the oligonucleotides of the purine and pyrimidine motifs targeting the same Watson-Crick duplex. Several conclusions may be drawn: (i) a weak hypochromism at 260 nm is associated with purine triplex formation; (ii) delta H degree of GA, GT and TC triplex formation (at pH 7.0) was calculated as -0.1, -2.5 and -6.1 kcal/mol per base triplet, respectively. This unexpectedly low delta H degree for the purine triple helix formation implies that its delta G degree is nearly temperature-independent and it explains why these triplexes may still be observed at high temperatures. In contrast, the pyrimidine triplex is strongly favoured at lower temperatures; (iii) as a consequence, in a system where two third-strands compete for triplex formation, displacement of the GA or GT strand by a pyrimidine strand may be observed at neutral pH upon lowering the temperature. This original purine-to-pyrimidine triplex conversion shows a significant hypochromism at 260 nm and a hyperchromism at 295 nm which is similar to the duplex-to-triplex conversion in the pyrimidine motif. Further evidence for this triplex-to-triplex conversion is provided by mung bean-nuclease foot-printing assay.

Conformational stability of pGEX-expressed Schistosoma japonicum glutathione S-transferase: a detoxification enzyme and fusion-protein affinity tag.

A glutathione S-transferase (Sj26GST) from Schistosoma japonicum, which functions in the parasite's Phase II detoxification pathway, is expressed by the Pharmacia pGEX-2T plasmid and is used widely as a fusion-protein affinity tag. It contains all 217 residues of Sj26GST and an additional 9-residue peptide linker with a thrombin cleavage site at its C-terminus. Size-exclusion HPLC (SEC-HPLC) and SDS-PAGE studies indicate that purification of the homodimeric protein under nonreducing conditions results in the reversible formation of significant amounts of 160-kDa and larger aggregates without a loss in catalytic activity. The basis for oxidative aggregation can be ascribed to the high degree of exposure of the four cysteine residues per subunit. The conformational stability of the dimeric protein was studied by urea- and temperature-induced unfolding techniques. Fluorescence-spectroscopy, SEC-HPLC, urea- and temperature-gradient gel electrophoresis, differential scanning microcalorimetry, and enzyme activity were employed to monitor structural and functional changes. The unfolding data indicate the absence of thermodynamically stable intermediates and that the unfolding/refolding transition is a two-state process involving folded native dimer and unfolded monomer. The stability of the protein was found to be dependent on its concentration, with a delta G degree (H2O) = 26.0 +/- 1.7 kcal/mol. The strong relationship observed between the m-value and the size of the protein indicates that the amount of protein surface area exposed to solvent upon unfolding is the major structural determinant for the dependence of the protein's free energy of unfolding on urea concentration. Thermograms obtained by differential scanning microcalorimetry also fitted a two-state unfolding transition model with values of delta Cp = 7,440 J/mol per K, delta H = 950.4 kJ/mol, and delta S = 1,484 J/mol.

Thermodynamics of left-handed helix formation.

The thermodynamics of right- and left-handed helix formation by poly[d(G-C)] X poly[d(G-C)] and by poly-(dG-m5dC) X poly(dG-m5dC) were measured spectrophotometrically and calorimetrically. From the spectrophotometric measurements the thermal stabilities of the alternative helical conformations were evaluated as a function of counterion concentration. From the calorimetric measurements the enthalpies of either right-handed or left-handed helix formation were determined. The corresponding experimental delta H values are -8.6 and -11.2 kcal/mol base pairs for the two conformations in poly[dG-C)] X poly[d(G-C)], and -9.0 and -12.7 kcal/mol base pairs, respectively, for poly(dG-m5dC) X poly(dG-m5dC).

A novel nucleosome assembly procedure (with a little help from pectin).

The acidic polysaccharide pectin (alpha-1,4-polygalacturonic acid) has been introduced as a nucleosome assembly facilitator as a substitute for polyglutamic acid. The pectin-assembled nucleosomes were indistinguishable from polyglutamic acid-assembled nucleosomes by thermal denaturation and DNAse I digestion. Pectin had two major advantages over polyglutamic acid-the yield of assembled cores was approximately 50% higher and the pectin could be easily removed after completion of the reassembly procedure by dialysis following pectinase cleavage.

A calorimetric study of polyguanylic acid at neutral pH.

The structure of polyguanylic acid (poly G) at neutral pH has been studied by optical and calorimetrical methods. It can be shown that diverging from earlier findings Poly G reversibly undergoes a cooperative thermal transition. Thermal denaturation curves are recorded at 253 nm as a function of the sodium ion concentration. The denaturation enthalpy of poly G in dilute aqueous solution is determined to 2.2 kcal/mole g. It is concluded, that the part of the ordered poly G structure, which gives rise to a temperature dependent cooperative transition, arises from stacking interactions of adjacent bases in the single strand.

Calorimetric studies of the interaction between DNA and poly-L-lysine.

The transition enthalpy deltaH of the helix-random coil transition of the DNA-polylysine complex was measured as a function of the peptide:nucleotide ratio by the help of an adiabatic scanning differential calorimeter. Furthermore the transition enthalpy of a complex with a specific peptide:nucleotide ratio was determined as a function of the cation concentration of the solution. Finally the reaction enthalpy of the interaction of polylysine with native and denatured DNA was measured with the help of a LKB batch calorimeter. From the results of the calorimetric measurements one can conclude that the transition enthalpy of the DNA-polylysine complexes is linearly dependent on the nucleotide: peptide ratio. The extrapolated value for the 1:1 complex is 14.4 kcal per mole base pairs.

Kinetics of the helix-coil transition of a polypeptide with non-ionic side groups, derived from ultrasonic relaxation measurements.

Ultrasonic absorption and velocity dispersion curves have been measured in the temperature induced helix-coil transition range of poly-N5-(3-hydroxypropyl)-L-glutamine in a methanol/water mixture. The results clearly reflect an effect due to the kinetics of the conformational conversion. A practically single relaxation time is observed which passes through a maximum when plotted versus the degree of transition. This maximum occurs at definitely less than 50% helix as predicted for by the theory for the comparatively short chain length involved here. The results are discussed in relation to previous theoretical and experimental findings.

Demonstration of G . U wobble base pairs by Raman and IR spectroscopy.

When guanine and uracil form hydrogen bonds in the pairing scheme first proposed by Crick one would expect that poly(A,G) will form an unperturbed double helix with poly U at room temperature in a dilute electrolyte solution (0.1 M NaCl). We have demonstrated by Raman- and IR-spectroscopy that the secondary structure of poly(A.G) . poly U is very similar to the structure of poly A . poly U; only the thermal stability of the double helix seems slightly lower than the stability of poly A . poly U, whereas the average helix length is unaffected by the dispersed G . U base pairs. From our input ratio of guanine and adenine we estimate that about every fourth base pair is a wobble pair.

Comparison of [(18)F]altanserin and [(18)F]deuteroaltanserin for PET imaging of serotonin(2A) receptors in baboon brain: pharmacological studies.

The regional distribution in brain, distribution volumes, and pharmacological specificity of the PET 5-HT(2A) receptor radiotracer [(18)F]deuteroaltanserin were evaluated and compared to those of its non-deuterated derivative [(18)F]altanserin. Both radiotracers were administered to baboons by bolus plus constant infusion and PET images were acquired up to 8 h. The time-activity curves for both tracers stabilized between 4 and 6 h. The ratio of total and free parent to metabolites was not significantly different between radiotracers; nevertheless, total cortical R(T) (equilibrium ratio of specific to nondisplaceable brain uptake) was significantly higher (34-78%) for [(18)F]deuteroaltanserin than for [(18)F]altanserin. In contrast, the binding potential (Bmax/K(D)) was similar between radiotracers. [(18)F]Deuteroaltanserin cortical activity was displaced by the 5-HT(2A) receptor antagonist SR 46349B but was not altered by changes in endogenous 5-HT induced by fenfluramine. These findings suggest that [(18)F]deuteroaltanserin is essentially equivalent to [(18)F]altanserin for 5-HT(2A) receptor imaging in the baboon.

Representation of DNA sequences by use of helical wheels to identify pattern formation with protein binding potential.

Similar to the protein segments with helical potential the three-dimensional structures of DNA sequences can be represented by a two-dimensional projection along the axis of the double helix. We will call this projection according to Schiffer and Edmundson "helical wheel". In the first place the wheels are employed here as graphical restatements of known sequences which represent binding sites of restriction-modification enzymes. Specific recognition of these sites has to be based on sequence related multi contact interactions of weak bonds. Because of the spatial and directional characteristics of H-bonds we will utilize the pattern formation of H-bond donor--and acceptor groups protruding into the major groove of the helix to identify the particular sequences. The characteristics of these patterns can be readily visualized and compared by examination of these wheels.

Codification and evolution of experimentally observed specific recognition sites for restriction enzymes on DNA.

The list of published restriction endonucleases along with their substrates provides an excellent data base for the evaluation of the evolution and codification of the key elements for specific recognition sites on the DNA. In this paper the considerations will be limited to palindromic tetramer-, pentamer-, and hexamer-sequences. It is basically assumed that each base pair within these sequences has to be recognized by directionally unique bidentate hydrogen bonds either within the plane of the base pair or by bridging the appropriate H-bond donor/acceptor groups of the neighbouring bases of the same strand. Thus sequence specificity is mediated by twelve (eight) H-bonds, originating from the protein recognition modules. Besides a pronounced preference for GC base pairs expressed by their high frequency in the most abundant sequences, serving the need of maximal thermodynamic stability of the double helical substrates, it can also be shown that the stacking of consecutive bases within the recognition site sequences plays a major role in shaping the particular DNA/protein interface. Finally it will be demonstrated that the full set of sequences discussed in this paper can readily be derived by stepwise expanding the vocabulary of three simple tetrameric sequences by inserting single base pairs into the centre of a minimal sequence, thus creating all the published pentameric restriction sites, or by inserting/adding two GC base pairs in a palindromic way, thus creating the known multiplicity of hexameric sites.

Genes leave their characteristic marks on the melting profile of plasmid DNAs.

High resolution melting profiles of four linearised plasmids (pUC9, pGV403, pHP2, and pBR322) were recorded by means of UV absorbance vs. temperature scanning. The set of transitions obtained for each plasmid are compared to each other and to the transitions obtained for their excised particular antibiotica resistance gene. It can be shown that each gene leaves a characteristic mark on the melting profile of its parental plasmid.

Development of patient-specific hematopoietic stem and progenitor cell grafts from pluripotent stem cells, in vitro.

Pluripotent stem cells hold great promise for future applications in many areas of regenerative medicine. Their defining property of differentiation towards any of the three germ layers and all derivatives thereof, including somatic stem cells, explains the special interest of the biomedical community in this cell type. In this review, we focus on the current state of directed differentiation of pluripotent stem cells towards hematopoietic stem cells (HSCs). HSCs are especially interesting because they are the longest known and, thus, most intensively investigated somatic stem cells. They were the first stem cells successfully used for regenerative purposes in clinical human medicine, namely in bone marrow transplantation, and also the first stem cells to be genetically altered for the first successful gene therapy trial in humans. However, because of the technical difficulties associated with this rare type of cell, such as the current incapability of prospective isolation, in vitro expansion and gene repair by homologous recombination, there is great interest in using pluripotent stem cells, such as Embryonic Stem (ES-) cells, as a source for generating and genetically altering HSCs, ex vivo. This has been hampered by ethical concerns associated with the use of human ES-cells. However, since Shinya Yamanaka´s successful attempts to reprogram somatic cells of mice and men to an ES-cell like state, so-called induced pluripotent stem (iPS) cells, this field of research has experienced a huge boost. In this brief review, we will reflect on the status quo of directed hematopoietic differentiation of human and mouse pluripotent stem cells.