300 research outputs found
Cy3 and Cy5 dyes attached to oligonucleotide terminus stabilize DNA duplexes: Predictive thermodynamic model
AbstractCyanine dyes are important chemical modifications of oligonucleotides exhibiting intensive and stable fluorescence at visible light wavelengths. When Cy3 or Cy5 dye is attached to 5Ⲡend of a DNA duplex, the dye stacks on the terminal base pair and stabilizes the duplex. Using optical melting experiments, we have determined thermodynamic parameters that can predict the effects of the dyes on duplex stability quantitatively (ÎG°, Tm). Both Cy dyes enhance duplex formation by 1.2kcal/mol on average, however, this Gibbs energy contribution is sequence-dependent. If the Cy5 is attached to a pyrimidine nucleotide of pyrimidineâpurine base pair, the stabilization is larger compared to the attachment to a purine nucleotide. This is likely due to increased stacking interactions of the dye to the purine of the complementary strand. Dangling (unpaired) nucleotides at duplex terminus are also known to enhance duplex stability. Stabilization originated from the Cy dyes is significantly larger than the stabilization due to the presence of dangling nucleotides. If both the dangling base and Cy3 are present, their thermodynamic contributions are approximately additive. New thermodynamic parameters improve predictions of duplex folding, which will help design oligonucleotide sequences for biophysical, biological, engineering, and nanotechnology applications
Reconstruction of the JaneÄek mill
Ăkolem diplomovĂŠ prĂĄce bylo vypracovĂĄnĂ stavebnĂ dokumentace pro realizaci stavby a to vÄetnÄ projektu specializace. JednĂĄ se o pĹestavbu objektu stĂĄvajĂcĂho mlĂ˝na a jeho konverzi na objekt penzionu. NavrĹženĂĄ rekonstrukce je ĹeĹĄena se smyslem pro zvĂ˝raznÄnĂ stĂĄvajĂcĂho objektu a vyjĂĄdĹenĂ jeho dominantnosti a funkce v prostĹedĂ kde se nachĂĄzĂ. Z pĹŻvodnĂho stavu zĹŻstala zachovĂĄna pouze ona hlavnĂ budova v historizujĂcĂm stylu a v kontrastu k nĂ byly navrĹženy dva novĂŠ menĹĄĂ objekty se zĂĄzemĂmi. Objekt slouŞà jako penzion pro ubytovĂĄnĂ osob, restaurace a takĂŠ poskytuje prostory pro ĹĄkolenĂ a zasedĂĄnĂ. DĂky protĂŠkajĂcĂmu potĹŻÄku pĹed objektem a umĂstÄnĂ budovy mezi lĂĄzeĹskĂ˝m a mÄstskĂ˝m parkem je toto vyuĹžitĂ objektu vĂce neĹž vyhovujĂcĂ.The task of this diploma thesis was the development of construction documents for the implementation of project, icluding project specialization. This is a rebuilding of the existing mill and it´s conversion to the object. Proposed reconstruction is solved with a sense of highlighting the exist building and expressing his dominance and function in an environment, where the building is located. From the original state was preserved only main building in historical style, and in contrast to this building were proposed two smaller objects with the background. The object is used as a pension for the accommodation for persons, restaurant and also provides rooms for seminars and meetings. Due to a brooklet flowing in front of the building and location of pension between the spa´s and city parks is the use of this building more than suitable.
Material engineering for atopic dermatitis treatment
Atopic dermatitis (AD) is a chronic inflammatory skin disease with a prevalence of 30% for children and to 17% for adults. There is observed an increasing trend of occurring AD over time in the world. Many factors contribute to the development of the disease, such as environmental, genetic and psychological factors. The proper AD treatment should be complexed and consists of skin care with emollients and pharmacological treatment. Most of the topical corticosteroids and other drugs have unpleasant side effects, therefore, developing new therapies is very useful. To minimalize side effects with a simultaneous reduction in the duration, a NPs (nanoparticles) therapy application is highly proposed. On the other hand, hydrogels and their shielding properties with high hydrating level and drug delivery capability are also widely studied. Some works report on the combination of these two solutions with promising results. Material engineering for biomedical applications is a dynamically growing field which offers new drug delivery systems (DDS). In this paper, based on the literature we discuss the new methods of AD treatment using hydrogels and nanotechnology
Design of LNA probes that improve mismatch discrimination
Locked nucleic acids (LNA) show remarkable affinity and specificity against native DNA targets. Effects of LNA modifications on mismatch discrimination were studied as a function of sequence context and identity of the mismatch using ultraviolet (UV) melting experiments. A triplet of LNA residues centered on the mismatch was generally found to have the largest discriminatory power. An exception was observed for GâT mismatches, where discrimination decreased when the guanine nucleotide at the mismatch site or even the flanking nucleotides were modified. Fluorescence experiments using 2-aminopurine suggest that LNA modifications enhance base stacking of perfectly matched base pairs and decrease stabilizing stacking interactions of mismatched base pairs. LNAs do not change the amount of counterions (Na(+)) that are released when duplexes denature. New guidelines are suggested for design of LNA probes, which significantly improve mismatch discrimination in comparison with unmodified DNA probes
Electrical detection of the temperature induced melting transition of a DNA hairpin covalently attached to gold interdigitated microelectrodes
The temperature induced melting transition of a self-complementary DNA strand covalently attached at the 5Ⲡend to the surface of a gold interdigitated microelectrode (GIME) was monitored in a novel, label-free, manner. The structural state of the hairpin was assessed by measuring four different electronic properties of the GIME (capacitance, impedance, dissipation factor and phase angle) as a function of temperature from 25°C to 80°C. Consistent changes in all four electronic properties of the GIME were observed over this temperature range, and attributed to the transition of the attached single-stranded DNA (ssDNA) from an intramolecular, folded hairpin structure to a melted ssDNA. The melting curve of the self-complementary single strand was also measured in solution using differential scanning calorimetry (DSC) and UV absorbance spectroscopy. Temperature dependent electronic measurements on the surface and absorbance versus temperature values measured in solution experiments were analyzed assuming a two-state process. The model analysis provided estimates of the thermodynamic transition parameters of the hairpin on the surface. Two-state analyses of optical melting data and DSC measurements provided evaluations of the thermodynamic transition parameters of the hairpin in solution. Comparison of surface and solution measurements provided quantitative evaluation of the effect of the surface on the thermodynamics of the melting transition of the DNA hairpin
Single-molecule derivation of salt dependent base-pair free energies in DNA
Accurate knowledge of the thermodynamic properties of nucleic acids is
crucial to predicting their structure and stability. To date most measurements
of base-pair free energies in DNA are obtained in thermal denaturation
experiments, which depend on several assumptions. Here we report measurements
of the DNA base-pair free energies based on a simplified system, the mechanical
unzipping of single DNA molecules. By combining experimental data with a
physical model and an optimization algorithm for analysis, we measure the 10
unique nearest-neighbor base-pair free energies with 0.1 kcal mol-1 precision
over two orders of magnitude of monovalent salt concentration. We find an
improved set of standard energy values compared with Unified Oligonucleotide
energies and a unique set of 10 base-pair-specific salt-correction values. The
latter are found to be strongest for AA/TT and weakest for CC/GG. Our new
energy values and salt corrections improve predictions of DNA unzipping forces
and are fully compatible with melting temperatures for oligos. The method
should make it possible to obtain free energies, enthalpies and entropies in
conditions not accessible by bulk methodologies.Comment: Main text: 27 pages, 4 figures, 2 tables. Supporting Information: 51
pages, 19 figures, 4 table
Temperature and electrolyte optimization of the Îą-hemolysin latch sensing zone for detection of base modification in double-stranded DNA
The latch region of the wild-type protein pore Îą-hemolysin (Îą-HL) constitutes a sensing zone for individual abasic sites (and furan analogs) in double-stranded DNA (dsDNA). The presence of an abasic site or furan within a DNA duplex, electrophoretically captured in the Îą-HL vestibule and positioned at the latch region, can be detected based on the current blockage prior to duplex unzipping. We investigated variations in blockage current as a function of temperature (12â35°C) and KCl concentration (0.15â1.0 M) to understand the origin of the current signature and to optimize conditions for identifying the base modification. In 1 M KCl solution, substitution of a furan for a cytosine base in the latch region results in an âź8 kJ molâ1 decrease in the activation energy for ion transport through the protein pore. This corresponds to a readily measured âź2 pA increase in current at room temperature. Optimal resolution for detecting the presence of a furan in the latch region is achieved at lower KCl concentrations, where the noise in the measured blockage current is significantly lower. The noise associated with the blockage current also depends on the stability of the duplex (as measured from the melting temperature), where a greater noise in the measured blockage current is observed for less stable duplexes
Non-specific binding of Na and Mg to RNA determined by force spectroscopy methods
RNA duplex stability depends strongly on ionic conditions, and inside cells
RNAs are exposed to both monovalent and multivalent ions. Despite recent
advances, we do not have general methods to quantitatively account for the
effects of monovalent and multivalent ions on RNA stability, and the
thermodynamic parameters for secondary structure prediction have only been
derived at 1M [Na]. Here, by mechanically unfolding and folding a 20 bp RNA
hairpin using optical tweezers, we study the RNA thermodynamics and kinetics at
different monovalent and mixed monovalent/Mg salt conditions. We measure
the unfolding and folding rupture forces and apply Kramers theory to extract
accurate information about the hairpin free energy landscape under tension at a
wide range of ionic conditions. We obtain non-specific corrections for the free
energy of formation of the RNA hairpin and measure how the distance of the
transition state to the folded state changes with force and ionic strength. We
experimentally validate the Tightly Bound Ion model and obtain values for the
persistence length of ssRNA. Finally, we test the approximate rule by which the
non-specific binding affinity of divalent cations at a given concentration is
equivalent to that of monovalent cations taken at 100 fold that concentration
for small molecular constructs.Comment: main paper (32 pages, 11 figures, 1 table) + supplementary
information (15 pages
TmPrime: fast, flexible oligonucleotide design software for gene synthesis
Herein we present TmPrime, a computer program to design oligonucleotide sets for gene assembly by both ligase chain reaction (LCR) and polymerase chain reaction (PCR). TmPrime offers much flexibility with no constraints on the gene and oligonucleotide lengths. The program divides the long input DNA sequence based on the input desired melting temperature, and dynamically optimizes the length of oligonucleotides to achieve homologous melting temperatures. The output reports the melting temperatures, oligonucleotide sequences and potential formation of secondary structures. Our program also provides functions on sequence pooling to separate long genes into smaller pieces for multi-pool assembly and codon optimization for expression. The software has been successfully used in the design and synthesis of green fluorescent protein fragment (GFPuv) (760 bp), human protein kinase B-2 (PKB2) (1446 bp) and the promoter of human calcium-binding protein A4 (S100A4) (752 bp) using real-time PCR assembly with LCGreen I, which offers a novel approach to compare the efficiency of gene synthesis. The purity of assembled products is successfully estimated with the use of melting curve analysis, which would potentially eliminate the necessity for agarose gel electrophoresis. This program is freely available at http://prime.ibn.a-star.edu.sg
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