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ABSTRACT: We studied the kinetic and thermodynamic effects of locked nucleic acid (LNA) modifications on parallel and antiparallel DNA duplexes. The LNA modifications were introduced at cytosine bases of the pyrimidine strand. Kinetic parameters evaluated from melting and annealing curves showed that the association and dissociation rate constants for the formation of the LNA-modified parallel duplex at 25.0 °C were 3 orders of magnitude larger and 6 orders of magnitude smaller, respectively, than that of the unmodified parallel duplex. The activation energy evaluated from the temperature-dependent rate constants was largely altered by the LNA modifications, suggesting that the LNA modifications affected a prenucleation event in the folding process. Moreover, thermodynamic parameters showed that the extent of stabilization by the LNA modification for parallel duplexes (3.6 kcal mol(-1) per one modification) was much more significant than that of antiparallel duplexes (1.6 kcal mol(-1)). This large stabilization was due to the decrease in ΔH° that was more favorable than the decrease in TΔS°. These quantitative parameters demonstrated that LNA modification specifically stabilized the noncanonical parallel duplex. On the basis of these observations, we succeeded to stabilize the parallel duplex by LNA modification at the physiological pH. These results can be useful in the rational design of functional molecules such as more effective antisense and antigene strands, more sensitive strands for detection of target DNA and RNA strands, and molecular switches responding to solution pH.
Biochemistry 08/2011; 50(34):7414-25. · 3.42 Impact Factor
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ABSTRACT: We use a variety of biophysical techniques to determine thermodynamic profiles, including hydration, for the unfolding of DNA stem-loop motifs (hairpin, a three-way junction and a pseudoknot) and their interaction with netropsin and random cationic copolymers. The unfolding thermodynamic data show that their helix-coil transition takes place according to their melting domains or sequences of their stems. All hairpins adopted the B-like conformation and their loop(s) contribute with an immobilization of structural water. The thermodynamic data of netropsin binding to the (5')-AAATT-(3')/TTTAA site of each hairpin show affinities of ~10(6-7)M(-1), 1:1 stoichiometries, exothermic enthalpies of -7 to -12 kcal mol(-1) (-22 kcal mol(-1) for the secondary site of the three-way junction), and water releases. Their interaction with random cationic copolymers yielded higher affinities of ~10(6)M(-1) with the more hydrophobic hairpins. This information should improve our current picture of how sequence and loops control the stability and melting behavior of nucleic acid molecules.
Biophysical chemistry 06/2011; 159(1):162-71. · 2.28 Impact Factor
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Journal of nucleic acids 01/2011; 2011:547245.
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01/2007: pages 191 - 226; , ISBN: 9780470152928
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ABSTRACT: Perfluorocarbon-exposed sonicated dextrose albumin (PESDA) microbubbles bind the antisense to the c-myc protooncogene (anti-c-myc) which prevents neointimal hyperplasia following vascular endothelial injury. The microbubbles also adhere to sites of damaged vascular endothelium and thus may be a method of systemically targeting delivery of anti-c-myc.
Laser scanning microscopy was performed on the aorta of 10 mice (five which were complement depleted) that received intravenous FITC-PESDA following aortic endothelial injury. C-myc expression was quantified following selective intracoronary injury in nine pigs that received intravenous (IV) anti-c-myc bound to PESDA. Finally, neointimal formation was measured following intracoronary stent deployment in 30 pigs that received either IV anti-c-myc alone or the same dose bound to PESDA.
Fluorescent microscopy confirmed selective PESDA microbubble adherence to aortic endothelium in all mice with aortic injury. This binding was nearly abolished when serum complement was depleted prior to injury. C-myc expression at the site of coronary endothelial injury was significantly lower in pigs treated with systemic anti-c-myc bound to PESDA. There was a 33% reduction in % stenosis and a 28% reduction in intimal area at 45 days post-stent deployment in pigs that received IV antisense plus PESDA. The stent margins also had reduced neointimal formation.
Systemic administration of anti-c-myc bound to PESDA microbubbles may be a good method for preventing coronary neointimal formation within and around implanted stents.
Cardiovascular Revascularization Medicine 7(1):25-33.
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ABSTRACT: DNA intramolecular hairpins are appropriate models for the thermodynamic description of the pseudo-intramolecular melting behavior of native DNA. To improve our understanding of the stability and melting behavior of DNA secondary structures and of the physical properties of nucleic acids, we have carried out a thermodynamic investigation of all possible bulges and mismatches in the hairpin molecule: d(GCNGCT5GCGC) and d(GCNGCT5GCMGC), where N represents a bulged base and N-M represents a W-C or mismatched base pair. We used circular dichroism spectroscopy to determine the overall conformation of each hairpin and UV melting and differential scanning calorimetry techniques to characterize their unfolding thermodynam-ics. The majority of hairpins melted in two-state monophasic transitions with transition temperatures independent of strand concentration. Relative to the host hairpin with 4 dG-dC base pairs in the stem, all hairpins with a bulge or a mismatch are less stable, while the hairpins with an extra canonical base pair are more stable. In both cases, the effects are enthalpy driven, indicating a loss or gain in base-pair stacking interactions, respectively. We also obtained linear enthalpy-entropy compensations with slopes (or compensating temperatures) of 317 K (hairpins with lesions) and 395 K (hairpins with fully paired stems) which are indicative of processes that are driven by solute-solvent interactions. These compensating temperatures are in excellent agreement with the results of similar analysis of data sets of other laboratories. Therefore, the relative change in enthalpy contribution for a given hairpin is partially compensated by a change in their overall hydration. These results suggest that the inclusion of bulges and mismatches immobilizes additional structural water while the addition of a W-C base pair immobilizes electrostricted water.
