Conformational Transitions Of Nucleic Acids Research Articles

Probing the mechanical properties, conformational changes, and interactions of nucleic acids with magnetic tweezers.

Nucleic acids are central to the storage and transmission of genetic information. Mechanical properties, along with their sequence, both enable and fundamentally constrain the biological functions of DNA and RNA. For small deformations from the equilibrium conformations, nucleic acids are well described by an isotropic elastic rod model. However, external forces and torsional strains can induce conformational changes, giving rise to a complex force-torque phase diagram. This review focuses on magnetic tweezers as a powerful tool to precisely determine both the elastic parameters and conformational transitions of nucleic acids under external forces and torques at the single-molecule level. We review several variations of magnetic tweezers, in particular conventional magnetic tweezers, freely orbiting magnetic tweezers and magnetic torque tweezers, and discuss their characteristic capabilities. We then describe the elastic rod model for DNA and RNA and discuss conformational changes induced by mechanical stress. The focus lies on the responses to torque and twist, which are crucial in the mechanics and interactions of nucleic acids and can directly be measured using magnetic tweezers. We conclude by highlighting several recent studies of nucleic acid-protein and nucleic acid-small-molecule interactions as further applications of magnetic tweezers and give an outlook of some exciting developments to come.

Empirical Corrections to the Amber RNA Force Field with Target Metadynamics.

The computational study of conformational transitions in nucleic acids still faces many challenges. For example, in the case of single stranded RNA tetranucleotides, agreement between simulations and experiments is not satisfactory due to inaccuracies in the force fields commonly used in molecular dynamics simulations. We here use experimental data collected from high-resolution X-ray structures to attempt an improvement of the latest version of the AMBER force field. A modified metadynamics algorithm is used to calculate correcting potentials designed to enforce experimental distributions of backbone torsion angles. Replica-exchange simulations of tetranucleotides including these correcting potentials show significantly better agreement with independent solution experiments for the oligonucleotides containing pyrimidine bases. Although the proposed corrections do not seem to be portable to generic RNA systems, the simulations revealed the importance of the α and ζ backbone angles for the modulation of the RNA conformational ensemble. The correction protocol presented here suggests a systematic procedure for force-field refinement.

RNA Conformational Ensembles: Narrowing the GAP between Experiments and Simulations with Metadynamics

The computational study of conformational transitions in nucleic acids still faces many challenges. For example, in the case of single stranded RNA tetranucleotides, agreement between simulations and experiments is not satisfactory due to inaccuracies in the force fields commonly used in molecular dynamics. Improvement of force fields is however hindered by the difficulties of decoupling those errors from the statistical errors caused by insufficient sampling. We here tackle both problems simultaneously by introducing a novel enhancing sampling method and using experimental data to improve RNA force fields.In this novel method, concurrent well-tempered metadynamics are integrated in a Hamiltonian replica-exchange scheme. The ladder of replicas is built with different strength of the bias potential exploiting the tunability of well-tempered metadynamics so as to scale barriers on individual collective variables [1]. At the same time, the metadynamics algorithm is modified so as to allow enforcing a target distribution of backbone and sugar-base torsion angles taken from experimental structures, using a procedure related to two recently introduced techniques. Replica-exchange simulations of several RNA tetranucleotides with experimental corrections show significantly better agreement with NMR experimental data and suggest a systematic procedure for force field refinement.[1] Gil-Ley, A.; Bussi, G. J. Chem. Theory Comput. 2015, 11, 1077-1085.

Conformational Transitions of Nucleic Acids under External Forces: Computer Simulations and a Stochastic Theory for their Kinetics

I will present molecular dynamics simulations of several examples of conformational transitions that nucleic acids and their complexes undergo upon the application of external forces and/or torques: (1) DNA supercoil relaxation by topoisomerases, (2) the condensation of DNA by dendrimers and, (3) RNA unfolding. Then I will showcase the use of the formalism of stochastic path integrals to deduce the kinetics of these transitions, from simulation trajectories or experimental single molecule recordings of the transition, under other conditions that those that are actually simulated or recorded.

5-Fluoro pyrimidines: labels to probe DNA and RNA secondary structures by 1D 19 F NMR spectroscopy

19F NMR spectroscopy has proved to be a valuable tool to monitor functionally important conformational transitions of nucleic acids. Here, we present a systematic investigation on the application of 5-fluoro pyrimidines to probe DNA and RNA secondary structures. Oligonucleotides with the propensity to adapt secondary structure equilibria were chosen as model systems and analyzed by 1D 19F and 1H NMR spectroscopy. A comparison with the unmodified analogs revealed that the equilibrium characteristics of the bistable DNA and RNA oligonucleotides were hardly affected upon fluorine substitution at C5 of pyrimidines. This observation was in accordance with UV spectroscopic melting experiments which demonstrated that single 5-fluoro substitutions in double helices lead to comparable thermodynamic stabilities. Thus, 5-fluoro pyrimidine labeling of DNA and RNA can be reliably applied for NMR based nucleic acid secondary structure evaluation. Furthermore, we developed a facile synthetic route towards 5-fluoro cytidine phosphoramidites that enables their convenient site-specific incorporation into oligonucleotides by solid-phase synthesis.

Multivariate curve resolution: a powerful tool for the analysis of conformational transitions in nucleic acids.

A successful application is reported of the multivariate curve resolution alternating least-squares method (MCR-ALS) for the analysis of nucleic acid melting and salt-induced transitions. Under conditions where several structures co-exist in a conformational equilibrium, MCR-ALS analysis of the UV and circular dichroism (CD) spectra at different temperatures, ionic strength and oligonucleotide concentration allows for the resolution of concentration profiles and pure spectra of the different species. The methodology is illustrated by the case of the cyclic oligonucleotide d<pTGCTCGCT>. The melting transition of this molecule at different oligonucleotide concentrations was studied at 0, 2 and 10 mM MgCl2 by UV and CD spectroscopy. In addition, salt titration experiments were carried out at 21.0 and 54.0 degrees C. The MCR-ALS analysis indicates that three different conformations of this molecule co-exist in solution. In agreement with previous NMR studies, these conformations were assigned to a monomeric dumbbell-like structure, a dimeric four-stranded conformation and a disordered (random coil) structure. The MCR-ALS methodology allows for a detailed analysis of how this equilibrium is affected by temperature, salt and oligonucleotide concentration.

The decisive role of the water structure in changes of conformation of nucleic acids.

This review summarizes data on the structure and properties of water under normal conditions, at high salt concentration and under high pressure. We correlate the observed conformational transitions in nucleic acids with changes in water structure and activity, and suggest a mechanism of conformational transitions of nucleic acid involving these changes. We conclude that the Z-DNA form is induced only at low water activity caused by high salt concentrations and/or high pressure.

The role of water structure in conformational changes of nucleic acids in ambient and high-pressure conditions.

This review describes and summarizes data on the structure and properties of water under normal conditions, at high salt concentration and under high pressure. We correlate the observed conformational changes in nucleic acids with changes in water structure and activity, and suggest a mechanism of conformational transitions of nucleic acids which accounts for changes in the water structure. From the biophysical, biochemical and crystallographic data we conclude that the Z-DNA form can be induced only at low water activity produced by high salt concentrations or high pressure, and accompanied by the stabilizing conjugative effect of the cytidine O4' electrons of the CG base pairs.

Observing the helical geometry of double-stranded DNA in solution by fluorescence resonance energy transfer.

The efficiency of fluorescence resonance energy transfer (FRET) between fluorescein and rhodamine covalently attached to both 5' termini of a series of double-stranded DNA species (ranging from 8 to 20 bp) was measured. FRET efficiency varied with a dependence compatible with dye-to-dye distances (R) calculated on the basis of double-stranded B-DNA structure; the helical geometry of double-stranded DNA in solution is clearly evident. The experimental data were consistent with a 1/[1 + (R/R0)6] dependence of FRET efficiency characteristic for the Förster dipole-dipole mechanism. The thermal dissociation of the strands of the duplex DNA species can be followed by using FRET, and from these data we have been able to obtain enthalpies of duplex formation in good agreement with earlier measurements using alternative techniques. FRET measurements at very different salt concentrations can be accurately compared. We conclude that FRET is a reliable and valuable method for studying structure and conformational transitions in nucleic acids.

4] Use of denaturing gradient gel electrophoresis to study conformational transitions in nucleic acids

Publisher Summary This chapter presents the study of conformational transitions in nucleic acids using denaturing gradient gel electrophoresis (DGGE). The term melting is used to denote, for double-stranded DNA (dsDNA), the temperature- or solvent-induced transition from a double-stranded, helical conformation to a single-stranded, random coil conformation. Melting is influenced by (among other things) solvent, DNA concentration, base composition, and sequence. DGGE is an experimental method in which solvent is varied to exploit the sensitivity of melting to changes in sequence. DNA fragments that differ by a single base or even by a single methyl group can be separated. The chapter also reviews the relevant principles of DNA melting, illustrate the use of computer programs that greatly aid in the design and interpretation of experiments, provide protocols for procedures, and describe the versatility of DGGE as a tool for detecting various covalent modifications to double-stranded nucleic acids. It emphasize work on dsDNA, the principles apply to dsRNA or RNA–DNA hybrids. Denaturing gradient gel electrophoresis is technically simple: DNA fragments are electrophoresed through a polyacrylamide gel that contains a concentration gradient of chemical denaturant. To obtain conditions where dsDNA is close to melting, the gel is immersed in a heated bath of electrophoresis buffer. Denaturant concentration and bath temperature are selected so that DNA fragments will partially melt during electrophoresis. Sequence-dependent conformational changes are detected as variations in migration distance arising from the effect of partial melting on electrophoretic mobility: partially melted molecules are severely retarded in the gel.

Conformational transitions of nucleic acids studied by IR and Raman spectroscopies

In this short review we first summarize briefly the comparative results obtained in our laboratory by IR and Raman spectroscopies on the right-handed B and the left-handed Z geometries of polynucleotides. The marker IR bands and Raman lines of these conformations are used to present new results concerning the polymorphism of several oligonucleotidic sequences, studied in crystal, solutions and hydrated films by Raman, IR, microRaman and, for the first time, microIR spectroscopies. Finally we give an example of how vibrational spectroscopy can help in the study of interactions between DNAs and drugs. This experimental work has been performed in parallel with a normal coordinate analysis of the DNA marker peaks and this review includes some of the results concerning these calculations and in particular those recently obtained involved with sugar pucker and phosphate backbone markers.

Temperature‐Gradient gel electrophoresis of nucleic acids: Analysis of conformational transitions, sequence variations, and protein‐nucleic acid interactions

Temperature-gradient gel electrophoresis (TGGE) is applied to analyze conformational transitions and sequence variations of nucleic acids and protein-nucleic acid interactions. A linear and highly reproducible temperature-gradient is established perpendicular or parallel to the direction of the electrophoresis. The instrument consists of an electrically insulated metal plate, which is heated at one edge and cooled at the other edge by two thermostating baths and is used as an ancillary device for commercial horizontal gel electrophoresis instruments. Biopolymers are separated in TGGE according to size, shape and thermal stability of their conformational transitions. If the temperature-gradient is established perpendicular to the electrophoresis, monomolecular conformational transitions of nucleic acids show up as continuous transition curves; strand-separation leads to discontinuous transitions. In the studies on viroid RNA it was shown that natural circular viroid RNA undergoes one highly cooperative transition detected by TGGE as a drastic retardation in mobility. Oligomeric replication intermediates of viroids exhibit coexisting structures which could not be detected by any other technique. Double-stranded satellite RNA from cucumber mosaic virus is a mixture of sequence variants, all of which have the identical length of 335 nucleotides. In TGGE six different strains were resolved. Sequence variants of viroids were analyzed by hybridizing viroid RNA to (-)strand viroid RNA transcripts from viroid cDNA clones. Sequence variations lead to mismatches in the double strands and thereby to a shift of the transition curve to lower temperature. Mutations in plasmids, particularly in cloned inserts, were detected by mixing plasmids of two different clones, linearizing, denaturing, renaturing, and searching for shifts in the transition curves, which are generated by mismatch-formation during the renaturation of (+)- and (-)strands from different clones. Examples are given for different viroid clones and HIV-clones from one and the same patient. In another example, clones with point mutations from site-directed mutagenesis are analyzed and selected by TGGE. TGGE is also applied to study the effect of amino acid exchanges in the Tet repressor from E. coli on the thermal stability of the repressor and on the mode of binding of the repressor to the operator DNA. The results are discussed under the aspect that TGGE may be applied as routine analytical laboratory procedure.

Influence of lipid membranes on the conformational transitions of nucleic acids.

The conformational transitions of nucleic acids which were enclosed in reverse phase evaporation vesicles (REV) were studied by thermal denaturation with optical recording. Cloned fragments of double-stranded DNA containing 179 base pairs and 187 base pairs, respectively, and polyA.polyU were enclosed in REV with a yield up to every vesicle containing 50 nucleic acid molecules. With the 179 base pairs DNA enclosed in the vesicle from egg lecithin two well resolved helix-coil transitions could be measured; one is very similar in the midpoint-temperature Tm and halfwidth delta T1/2 to the transition of the free nucleic acid, and the other transition occurs stabilized at a 3.5 degrees C higher Tm-value and with a broader delta T1/2, 2.7 degrees C instead of 0.6 degree C. Both transitions are from nucleic acids inside the vesicles. Varying the surface charge of the lipid membrane by adding the negatively charged phosphatidylserine or phosphatidylglycerol, an optimum in the yield of enclosure and a maximum in the increase in Tm (4.5 degrees C) and delta T1/2 (5.5 degrees C instead of 1.0 degrees C) was obtained at 20% phosphatidylserine or phosphatidylglycerol. In vesicles from pure negatively charged lipids no second population of nucleic acids was observed. Qualitatively, similar effects were observed with polyA.polyU. Stabilization and broadening of the second transition is higher for nucleic acids inside vesicles from lipids with unsaturated fatty acids, as dioleoyl-phosphatidylcholine, than with saturated fatty acids, dipalmitoyl-phosphatidylcholine. Stabilization and broadening decrease with increasing ionic strength, whereas the relative contributions of both transitions to the total hypochromicity remain unchanged; the second transition coincides with the first at 90 mM Na+. From the experimental results it was concluded that the interaction of nucleic acids and lipid membranes is mainly of electrostatic nature. The nucleic acids exist inside the vesicles in two populations, one behaving like nucleic acid free in solution and one influenced by the contact with the membrane. All results are in accordance with a model in which the interaction between the nucleic acid and the membrane is in competition with the dipole-dipole interaction inside the membrane surface.