Summary: The determination of annealing temperature is a critical step in PCR design. This parameter is typically derived from the melting temperature of the PCR primers, so for successful PCR work it is important to determine the melting temperature of primer accurately. We introduced several enhancements in the widely used primer design program Primer3. The improvements include a formula for calculating melting temperature and a salt correction formula. Also, the new version can take into account the effects of divalent cations, which are included in most PCR buffers. Another modification enables using lowercase masked template sequences for primer design.

Availability: Features described in this article have been implemented into the development code of Primer3 and will be available in future versions (version 1.1 and newer) of Primer3. Also, a modified version is compiled under the name of mPrimer3 which is distributed independently. The web-based version of mPrimer3 is available at http://bioinfo.ebc.ee/mprimer3/ and the binary code is freely downloadable from the URL http://bioinfo.ebc.ee/download/.

Contact: [email protected]

/pdf/enhancements-and-modifications-of-primer-design-program-3734t4bych.pdf

Enhancements and modifications of primer design program Primer3

DNA is increasingly being used as the engineering material of choice for the construction of nanoscale circuits, structures, and motors. Many of these enzyme-free constructions function by DNA strand displacement reactions. The kinetics of strand displacement can be modulated by toeholds, short single-stranded segments of DNA that colocalize reactant DNA molecules. Recently, the toehold exchange process was introduced as a method for designing fast and reversible strand displacement reactions. Here, we characterize the kinetics of DNA toehold exchange and model it as a three-step process. This model is simple and quantitatively predicts the kinetics of 85 different strand displacement reactions from the DNA sequences. Furthermore, we use toehold exchange to construct a simple catalytic reaction. This work improves the understanding of the kinetics of nucleic acid reactions and will be useful in the rational design of dynamic DNA and RNA circuits and nanodevices.

http://nablab.rice.edu/publications/JACS2009.pdf

Control of DNA Strand Displacement Kinetics Using Toehold Exchange

The DINAMelt web server simulates the melting of one or two single-stranded nucleic acids in solution. The goal is to predict not just a melting temperature for a hybridized pair of nucleic acids, but entire equilibrium melting profiles as a function of temperature. The two molecules are not required to be complementary, nor must the two strand concentrations be equal. Competition among different molecular species is automatically taken into account. Calculations consider not only the heterodimer, but also the two possible homodimers, as well as the folding of each single-stranded molecule. For each of these five molecular species, free energies are computed by summing Boltzmann factors over every possible hybridized or folded state. For temperatures within a user-specified range, calculations predict species mole fractions together with the free energy, enthalpy, entropy and heat capacity of the ensemble. Ultraviolet (UV) absorbance at 260 nm is simulated using published extinction coefficients and computed base pair probabilities. All results are available as text files and plots are provided for species concentrations, heat capacity and UV absorbance versus temperature. This server is connected to an active research program and should evolve as new theory and software are developed. The server URL is http://www.bioinfo.rpi.edu/applications/hybrid/.

/pdf/dinamelt-web-server-for-nucleic-acid-melting-prediction-45n2jzglwg.pdf

DINAMelt web server for nucleic acid melting prediction

Biofilms are widespread in nature and constitute an important strategy implemented by microorganisms to survive in sometimes harsh environmental conditions. They can be beneficial or have a negative impact particularly when formed in industrial settings or on medical devices. As such, research into the formation and elimination of biofilms is important for many disciplines. Several new methodologies have been recently developed for, or adapted to, biofilm studies that have contributed to deeper knowledge on biofilm physiology, structure and composition. In this review, traditional and cutting-edge methods to study biofilm biomass, viability, structure, composition and physiology are addressed. Moreover, as there is a lack of consensus among the diversity of techniques used to grow and study biofilms. This review intends to remedy this, by giving a critical perspective, highlighting the advantages and limitations of several methods. Accordingly, this review aims at helping scientists in finding the most appropriate and up-to-date methods to study their biofilms.

https://tandfonline.com/doi/pdf/10.1080/1040841X.2016.1208146

Critical review on biofilm methods

MicroRNAs (miRNAs) have emerged as important post-transcriptional regulators of gene expression in many developmental and cellular processes. Moreover, there is now ample evidence that perturbations in the levels of individual or entire families of miRNAs are strongly associated with the pathogenesis of a wide range of human diseases. Indeed, disease-associated miRNAs represent a new class of targets for the development of miRNA-based therapeutic modalities, which may yield patient benefits unobtainable by other therapeutic approaches. The recent explosion in miRNA research has accelerated the development of several computational and experimental approaches for probing miRNA functions in cell culture and in vivo. In this review, we focus on the use of antisense oligonucleotides (antimiRs) in miRNA inhibition for loss-of-function studies. We provide an overview of the currently employed antisense chemistries and their utility in designing antimiR oligonucleotides. Furthermore, we describe the most commonly used in vivo delivery strategies and discuss different approaches for assessment of miRNA inhibition and potential off-target effects. Finally, we summarize recent progress in antimiR mediated pharmacological inhibition of disease-associated miRNAs, which shows great promise in the development of novel miRNA-based therapeutics.

/pdf/inhibition-of-microrna-function-by-antimir-oligonucleotides-y4tz49pdm8.pdf

Inhibition of microRNA function by antimiR oligonucleotides

Melting temperatures, Tm, were systematically studied for a set of 92 DNA duplex oligomers in a variety of sodium ion concentrations ranging from 69 mM to 1.02 M. The relationship between Tm and ln [Na+] was nonlinear over this range of sodium ion concentrations, and the observed melting temperatures were poorly predicted by existing algorithms. A new empirical relationship was derived from UV melting data that employs a quadratic function, which better models the melting temperatures of DNA duplex oligomers as sodium ion concentration is varied. Statistical analysis shows that this improved salt correction is significantly more accurate than previously suggested algorithms and predicts salt-corrected melting temperatures with an average error of only 1.6 °C when tested against an independent validation set of Tm measurements obtained from the literature. Differential scanning calorimetry studies demonstrate that this Tm salt correction is insensitive to DNA concentration. The Tm salt correction function ...

Effects of sodium ions on DNA duplex oligomers: improved predictions of melting temperatures.

Accurate predictions of DNA stability in physiological and enzyme buffers are important for the design of many biological and biochemical assays. We therefore investigated the effects of magnesium, potassium, sodium, Tris ions, and deoxynucleoside triphosphates on melting profiles of duplex DNA oligomers and collected large melting data sets. An empirical correction function was developed that predicts melting temperatures, transition enthalpies, entropies, and free energies in buffers containing magnesium and monovalent cations. The new correction function significantly improves the accuracy of predictions and accounts for ion concentration, G-C base pair content, and length of the oligonucleotides. The competitive effects of potassium and magnesium ions were characterized. If the concentration ratio of [Mg2+]0.5/[Mon+] is less than 0.22 M−1/2, monovalent ions (K+, Na+) are dominant. Effects of magnesium ions dominate and determine duplex stability at higher ratios. Typical reaction conditions for PCR an...

Predicting Stability of DNA Duplexes in Solutions Containing Magnesium and Monovalent Cations

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 G–T 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

Bernardo G. Moreira

Papers

Effects of sodium ions on DNA duplex oligomers: improved predictions of melting temperatures.

Predicting Stability of DNA Duplexes in Solutions Containing Magnesium and Monovalent Cations

Design of LNA probes that improve mismatch discrimination

Effects of fluorescent dyes, quenchers, and dangling ends on DNA duplex stability.

Cy3 and Cy5 dyes attached to oligonucleotide terminus stabilize DNA duplexes: predictive thermodynamic model.