| Name | Concetta |
| Surname | Giancola |
| Institution | University of Naples – Federico II |
| concetta.giancola@unina.it | |
| Address | Department of Pharmacy, University of Napoli Federico II, Via D. Montesano 49, 80131, Napoli, Italy; InterUniversity Center for Research in Neurosciences, University of Campania "Luigi Vanvitelli", Napoli, Italy |
The stability of a 16-mer DNA triple helix containing a 3-N(ferrocenemethyl)-thymidine residue in the third strand has been investigated in comparison with the unmodified triplex of the same sequence. A complete physico-chemical characterization of the two triple helices on changing the pH by means of calorimetry, circular dichroism and molecular modeling is therefore reported. The thermodynamic parameters were obtained in the pH range 5.5-7.2 by differential scanning calorimetry (DSC). For both triplexes the T(m) and Delta H degrees (T(m)) values increase on decreasing the pH. In the pH range 7.2-6.0 the triplex containing the ferrocenemethyl nucleoside is less stable than the unmodified one, whereas the modified triplex becomes more stable at pH 5.5. Such difference in stability at each pH value is overwhelmingly enthalpic in origin. CD spectra show conformational changes on decreasing the pH for both the triplexes. By spectroscopic pH titration the apparent pK(a) values of the cytosines in the two triplexes could be estimated, with the cytosines in the TFO containing the ferrocenemethyl residue having lower apparent pK(a) values. These results are consistent with the calorimetric data, showing a decrease of the thermodynamic parameters in the pH range 7.2-6.0 and an increase at pH 5.5 for the ferrocenylated triplex with respect to the unmodified one. The thermodynamic and spectroscopic data are also discussed in relation to molecular models.
Telomeric guanine-rich sequence can adopt quadruplex structures that are important for their biological role in chromosomal stabilisation. G quartets are characterised by the cyclic hydrogen bonding of four guanine bases in a coplanar arrangement and their stability is ion-dependent. In this work we compare the stability of [d(TGGGT)](4) and [d(T*GGGT)](4) quadruplexes. The last one contains a modified thymine, where the hydroxyl group substitutes one hydrogen atom of the methyl group of the thymine in the [d(TGGGT)](4) sequence. We used a combination of spectroscopic, calorimetric and computational techniques to characterise the G-quadruplex formation. NMR and CD spectra of [d(T*GGGT)](4) were characteristic of parallel-stranded, tetramolecular quadruplex. CD and DSC melting experiments reveal that [d(T*GGGT)](4) is less stable that unmodified quadruplex. Molecular models suggest possible explanation for the observed behaviour.
In this paper we report a thermodynamic characterisation of stability and melting behaviour of four different triple helices at pH 6.0. The target duplex consists of 16 base pairs in alternate sequence of the type 5'-(purine)(m)(pyrimidine)(m)-3'. The four triplexes are formed by targeting the 16-mer duplex with an all pyrimidine 16-mer or 15-mer or 14-mer third strand. The 16-mer oligonucleotide contains a 3'-3' phosphodiester junction and corresponding triplex was named 16-mer P. The 14-mer oligonucleotide contains a non-nucleotide linker, the 1,2,3 propanetriol residue and the corresponding triplex was named 14-mer PT. For the 15-mer oligonucleotide both junctions were alternatively used and the relative triplexes were named 15-mer P and 15-mer PT, respectively. These linkers introduce the appropriate polarity inversion and let the third strand switch from one oligopurine strand of the duplex to the other. Thermal denaturation profiles indicate the initial loss of the third strand followed by the dissociation of the target duplex. Transition enthalpies, entropies and free energies were derived from differential scanning calorimetric measurements. The comparison of Gibbs energies reveals that a more stable triplex is obtained when in the third strand there is the lack of one nucleotide in the junction region and a propanetriol residue as linker was used. The thermodynamic data were discussed in light of molecular mechanics and dynamics calculations.
Oligonucleotides with a 3'-3' inversion of polarity site assured by one lysine residue have been synthesized, characterized and used as third strands in alternate strand triple helix formation. UV melting studies and molecular mechanics calculations have been carried out to investigate the stability and the geometry of these new triplexes.
Differential scanning calorimetric (DSC), circular dichroism (CD) and molecular mechanics studies have been performed on two triple helices of DNA. The target duplex consists of 16 base pairs in alternate sequence of the type 5'-(purine)m(pyrimidine)m-3'. In both the triplexes, the third oligopyrimidine strand crosses the major groove at the purine-pyrimidine junction, with a simultaneous binding of the adjacent purine tracts on alternate strands of the Watson-Crick duplex. The switch is ensured by a non-nucleotide linker, the 1,2,3 propanetriol residue, that joins two 3'-3' phosphodiester ends. The third strands differ from each other for a nucleotide in the junction region. The resulting triple helices were termed 14-mer-PXP and 15-mer-PXP (where P = phosphate and X = 1,2,3-propanetriol residue) according to the number of nucleotides that compose the third strand. DSC data show two independent processes: the first corresponding to the dissociation of the third strand from the target duplex, the second to the dissociation of the double helix in two single strands. The two triple helices show the same stability at pH 6.6. At pH 6.0, the 15-mer-PXP triplex is thermodynamically more stable than the 14-mer-PXP triplex. Thermodynamic data are discussed in relation to structural models. The results are useful when considering the design of oligonucleotides that can bind in an antigene approach to the DNA for therapeutic purposes.
Bovine seminal ribonuclease (BS-RNase) is a dimeric protein with two identical subunits linked by two disulfide bridges, each subunit showing 80% of sequence identity with pancreatic RNase A. BS-RNase exists in two different quaternary conformations in solution: the MxM form, in which each subunit exchanges its alpha-helical N-terminal segment with its partner, and the M=M form with no exchange. By differential scanning microcalorimetry (DSC), the denaturation of the two dimeric forms of BS-RNase was found to be more complex than a simple two-state process. Monomeric derivatives of the dimeric protein follow instead a simple two-state mechanism, but are distinctly less stable than RNase A. The three-state N if I if D denaturation process of the two quaternary isoforms was interpreted by identifying in the dimers a central highly structured core, enclosing the covalently bonded subunit interface, which unfolds only after the periphery (mainly the N-terminal peptide) unfolds. Circular dichroism spectra of the two forms in the far-ultraviolet region show large differences between the secondary structure of the isoforms and that of the native BS-RNase mixture at equilibrium. This has been attributed to the presence in the equilibrium mixture of intermediate forms with displaced and disordered N-terminal alpha-helical segments.
This work analyzed the thermal denaturation process of defatted bovine serum albumin (BSA). DSC measurements were performed on changing the pH, the ionic strength and the sodium dodecyl sulfate (SDS) concentration. These data have been compared with those previously obtained by us and other authors. The purpose of these measurements was to study the correlation between the three-dimensional organization of BSA native protein structure and its thermodynamic stability and to clarify the non-covalent interactions between the globular proteins and amphipathic molecules. These measurements have shown that the thermal denaturation is always irreversible regardless of pH, ionic strength and SDS concentration. The nature of the irreversible process superimposed on the protein unfolding is discussed. The strong stabilizing effect of NaCl on the BSA native structure has been found for the range 0-1.0 M. It is worth noting that the calorimetric curves, confined to the pH region studied, could not be represented by a two-state transition model; they were deconvoluted as the sum of two independent two-state transitions. These transitions were correlated to the domain structure of BSA. Sodium dodecyl sulfate has a net stabilizing effect up to a molar ratio of 10:1 (ligand to protein). In this range of concentrations the presence of SDS cause a biphasic profile of excess heat capacity. A simple thermodynamic model was developed in attempt to reproduce the experimental DSC profiles and collect information regarding the binding equilibrium of SDS.