Nucleic Acids In Solution Research Articles

The free energy landscape of pseudorotation in 3'-5' and 2'-5' linked nucleic acids.

The five-membered furanose ring is a central component of the chemical structure of biological nucleic acids. The conformations of the furanose ring can be analytically described using the concept of pseudorotation, and for RNA and DNA they are dominated by the C2′-endo and C3′-endo conformers. While the free energy difference between these two conformers can be inferred from NMR measurements, a free energy landscape of the complete pseudorotation cycle of nucleic acids in solution has remained elusive. Here, we describe a new free energy calculation method for molecular dynamics (MD) simulations using the two pseudorotation parameters directly as the collective variables. To validate our approach, we calculated the free energy surface of ribose pseudorotation in guanosine and 2′-deoxyguanosine. The calculated free energy landscape reveals not only the relative stability of the different pseudorotation conformers, but also the main transition path between the stable conformations. Applying this method to a standard A-form RNA duplex uncovered the expected minimum at the C3′-endo state. However, at a 2′–5′ linkage, the minimum shifts to the C2′-endo conformation. The free energy of the C3′-endo conformation is 3 kcal/mol higher due to a weaker hydrogen bond and a reduced base stacking interaction. Unrestrained MD simulations suggest that the conversion from C3′-endo to C2′-endo and vice versa is on the nanosecond and microsecond time scale, respectively. These calculations suggest that 2′–5′ linkages may enable folded RNAs to sample a wider spectrum of their pseudorotation conformations.

Calibration and quality assurance procedures at the far UV linear and circular dichroism experimental station DISCO

Circular and Linear dichroism spectroscopy used in biophysics, are both differential absorption techniques, which explore the chirality of complex macromolecular structures such as proteins and nucleic acids in solution. In the past two decades synchrotron radiation facilities throughout the world, have accommodated circular dichroism (SRCD) experiments. These intense VUV light sources have greatly expanded the wavelength range exploitable (down to 120nm) at high constant photon flux improving signal to noise ratio and data acquisition speed. Here we present the calibration procedure for the circular and linear dichroism experiments explored on the SRCD beam line DISCO at the synchrotron SOLEIL as well as the specially designed automated sample rotation chamber.

205 Ultrathin nanopores for structural analysis of small nucleic acids

Pinpointing the mechanisms behind function in biological macromolecules is essential for understanding the emerging and evolving nature of life. Biological macromolecules have evolved over billions of years to function efficiently in the highly heterogeneous, crowded environment of a cell. In particular, non-coding ribonucleic acids (RNAs) and deoxyribonucleic acids (DNAs) are omnipresent in cells, preforming both regulatory and catalytic functions by virtue of their structure. One class of such nucleic acids, ironically termed “junk DNA,”1 is significantly involved in orchestrating gene regulation. The RNA subunits of the ribosome2 are an integral part of the translational machinery for protein synthesis. Many ribozymes and other viral RNAs such as the hammerhead ribozyme and the canonical internal ribosome entry site3 exhibit enzymatic activity. Although the exact mechanisms remain unclear, the uniting theme in these non-coding nucleic acids is that their tertiary (spatial) structure yields specific chemical activities and functions. While existing techniques (e.g. nuclear magnetic resonance, X-ray crystallography) provide detailed structural information, inherent drawbacks such as ensemble averaging errors, crystallization artifacts, low time resolutions, and the need for ample amounts of material limit the availability and relevancy of the obtained structural information. Bioinformatics-based tools aim to bridge the gap in knowledge by proposing a homology-based approach to structural prediction, although viable experimental techniques are required in order to unequivocally support and improve such predictions. In this talk, I will present our group's efforts to fabricate nanoscale pore devices for extracting useful structural information about nucleic acids that display in vivo function. Electron-beam irradiation of various thin freestanding membranes affords nanopores with controlled dimensions and interfacial properties, to a quality level that allows highly sensitive analysis of individual nucleic acids in solution at high-throughput. I will describe the properties of our nanopores, as well as some of our recent explorations that have permitted the analysis of DNA and RNA structures.

Competitive Reporter Monitored Amplification (CMA) - Quantification of Molecular Targets by Real Time Monitoring of Competitive Reporter Hybridization

BackgroundState of the art molecular diagnostic tests are based on the sensitive detection and quantification of nucleic acids. However, currently established diagnostic tests are characterized by elaborate and expensive technical solutions hindering the development of simple, affordable and compact point-of-care molecular tests.Methodology and Principal FindingsThe described competitive reporter monitored amplification allows the simultaneous amplification and quantification of multiple nucleic acid targets by polymerase chain reaction. Target quantification is accomplished by real-time detection of amplified nucleic acids utilizing a capture probe array and specific reporter probes. The reporter probes are fluorescently labeled oligonucleotides that are complementary to the respective capture probes on the array and to the respective sites of the target nucleic acids in solution. Capture probes and amplified target compete for reporter probes. Increasing amplicon concentration leads to decreased fluorescence signal at the respective capture probe position on the array which is measured after each cycle of amplification. In order to observe reporter probe hybridization in real-time without any additional washing steps, we have developed a mechanical fluorescence background displacement technique.Conclusions and SignificanceThe system presented in this paper enables simultaneous detection and quantification of multiple targets. Moreover, the presented fluorescence background displacement technique provides a generic solution for real time monitoring of binding events of fluorescently labelled ligands to surface immobilized probes. With the model assay for the detection of human immunodeficiency virus type 1 and 2 (HIV 1/2), we have been able to observe the amplification kinetics of five targets simultaneously and accommodate two additional hybridization controls with a simple instrument set-up. The ability to accommodate multiple controls and targets into a single assay and to perform the assay on simple and robust instrumentation is a prerequisite for the development of novel molecular point of care tests.

Modelling vibrational coupling in DNA oligomers: a computational strategy combining QM and continuum solvation models

In this paper, a computational strategy, based on DFT calculations at the M06-2X level, combined with the polarizable continuum model, the Hessian matrix reconstruction method and the Partial hessian vibrational approach is applied to evaluate inter- and intra-layer vibrational couplings between hydrogen bonded and stacked DNA base pairs. The present work demonstrates that this computational scheme can effectively predict and interpret the vibrational couplings of nucleic acids in solution. The effect of the environment described in a cluster or in a continuum manner is necessary in order to improve the agreement with the experimental values.

Complex permittivity of representative biological solutions in the 2–67 GHz range

The main purpose of this study is to provide experimental data on the complex permittivity of some biological solutions in the 2-67 GHz range at room and human body temperatures. The permittivity measurements are performed using an open-ended coaxial probe. Permittivity spectra of several representative monomolecular solutions of proteins, amino acids, nucleic acids, and carbohydrates are analyzed and compared. Furthermore, measurements have also been performed for complex biomolecular solutions, including bovine serum albumin (BSA)-DNA-glucose mixture, culture medium, and yeast extract solution. The results demonstrate that for concentrations below 1%, the permittivity spectra of the solutions do not substantially differ from that of distilled water. Measurements carried out for 4% and 20% BSA solutions show that the presence of proteins results in a decrease in permittivity. For highly concentrated RNA solutions (3%), a slight increase in the imaginary part of the permittivity is observed below 10 GHz. Experimental data show that free water permittivity can be used for modeling of the culture medium above 10 GHz. However, at lower frequencies a substantial increase in the imaginary part of the permittivity due to ionic conductivity should be carefully taken into account. A similar increase has also been observed for the yeast extract solution in the lower frequency region of the considered spectrum. Above 10 GHz, the high concentration of proteins and other low-permittivity components of the yeast extract solution results in a decrease in the complex permittivity compared to that of water. Obtained data are of utmost importance for millimeter-wave dosimetry studies.

Design of Functional Nucleic Acid Systems for Biomolecular Analysis

Abstract With the aim of realizing an in situ nucleic acid analysis, we have developed functional nucleic acid probes that can autonomously detect and analyze nucleic acid sequences of interest. Fluorophore-labeled artificial oligonucleotide probes with target-dependent self-ligation or self-digestion functionalities can be used to sense nucleic acids in solution, on solid supports, or even in cells. In addition, we have successfully established a new sensing system using nonmodified nucleic acids as a probe. These probes and sensors are all composed of nonmodified natural-type nucleic acids, and thus can be transcribed directly from dsDNA, giving a high potency for in situ or in-cell application.

Microfabrication of biomedical lab-on-chip devices. A review

Lab-on-chip systems are a class of miniaturized analytical devices that integrate fluidics, electronics and various sensorics. They are capable of analysing biochemical liquid samples, like solutions of metabolites, macromolecules, proteins, nucleic acids, or cells and viruses. Supple- mentary to their measuring capabilities, the lab-on-chip devices facilitate fluidic transportation, sorting, mixing, or separation of liquid samples. A type of lab-on-chip devices, named biochip, is devoted specifically to genomic, proteomic and pharmaceutical tests. The significance of such miniaturized devices lies in their potentiality of automating laboratory procedures, which highly reduces the time of biomedical tests and laboratory work. This review summarizes numerous fabrication methods and procedures for producing lab-on-chip devices and also envisages future evolution.

Effects of Chinese herbal medicine Bushen Gubiao Recipe on toll-like receptor 4 and CD4+CD25+foxp3+ regulatory T cells in mice with recurrent respiratory tract infections

To evaluate the effects of Bushen Gubiao Recipe (BGR), a compound traditional Chinese herbal medicine, on toll-like receptor 4 (TLR4) signaling pathway and CD4(+)CD25(+)foxp3(+)regulatory T cells (CD4(+)CD25(+)foxp3(+)Tregs) in mice with recurrent respiratory tract infections (RRTIs). A mouse model of kidney-yang deficiency which simulated physical characteristics of RRTIs was established by intraperitoneal injection of hydrocortisone for 14 d. The model mice were divided into 4 groups, model group, high-dose BGR group, low-dose BGR group, and nucleic acid and casein oral solution group. They were administered respectively with distilled water, high-dose BGR (50 g/kg body weight), low-dose BGR (25 g/kg body weight) and nucleic acid and casein oral solution. Besides, a normal control group was set up and gastrogavage with distilled water. The effect of intervention was evaluated 4 weeks later by estimating the changes in behaviors of mice. Expressions of TLR4 and nuclear factor-kappa B (NF-κB) p65 in lung tissue were detected by immunohistochemical SABC method, the expression of TLR4 mRNA in lung tissue was detected by fluorescence quantitative-polymerase chain reaction (FQ-PCR), and the level of blood CD4(+)CD25(+)foxp3(+) Tregs was determined by flow cytometry. A kidney-yang deficiency mouse model with RRTIs was successfully established by intraperitoneal injection of hydrocortisone. BGR could improve the abnormal behavioral condition of the mice and enhance the expressions of TLR4 and NF-κB p65 in the lung tissue. Expression of TLR4 mRNA in high-dose BGR group was higher than that in model group (P<0.05), while the difference was not statistically significant between high-dose BGR group and low-dose BGR group (P>0.05). Levels of CD4(+)CD25(+)foxp3(+)Tregs in high-dose BGR group and nucleic acid and casein oral solution group were lower than that in model group (P<0.05), while the difference was not statistically significant between high-dose BGR group and low-dose BGR group (P>0.05). BGR can improve the behavior of the kidney-yang deficiency mice, and improve the innate immune function by up-regulating TLR/NF-κB signaling pathway. BGR can adjust the immune imbalance of T-helper cell (Th) 1/Th2 through reducing the activity of CD4(+)CD25(+)foxp3(+)Tregs and enhancing the immune response of Th1 type.

Terahertz spectroscopy of biological molecules

We study experimentally the absorption and refraction spectra for a number of amino acids, proteins and nucleic acids in the frequency range 0.1–3.5 THz. The characteristic absorption lines are revealed. The spectroscopy of water solutions of proteins and nucleic acids is carried out under conditions of total internal refraction. This method can be used for studies of the functional properties of biomolecules and the dynamics of their interaction.

Conformational properties of nucleic acids in solution

Principles which govern the conformational properties of nucleic acid structures in aqueous solution are derived from extensive nuclear magnetic resonance studies of nucleic acid components. These principles are then utilized to project the solution conformation of tRNA.

Dissolved oxygen alteration of the spectrophotometric analysis and quantification of nucleic acid solutions

Nucleic acids are routinely and readily analysed using the A(260)/A(280) ratio, although this method is known to be prone to erroneous results owing to contaminants in solution that absorb at similar wavelengths. The aim of the present review, while highlighting the problems and alternatives of using UV spectrophotometry for nucleic acid measurements, is to bring forth an observational result from our recent studies, namely that DO (dissolved oxygen) and nitrogen can alter the A(260) of aqueous DNA solutions. Our finding is of importance because DO is highly variable between protocols and storage conditions of DNA preparations. The physicochemical nature of the oxygen-DNA interactions is briefly discussed.

Current-Controlled Nanospray Ionization Mass Spectrometry

The hypothesis that direct determination of electrospray current would provide a viable method for maintaining spray stability to enable optimal nanospray analysis was tested by building a feedback apparatus capable of reading the current and readjusting the emitter voltage in real time. The apparatus consists of a current-sensing circuit that reads the voltage drop across a resistor located between the high-voltage power supply and the nanospray emitter. A low voltage proportional to the observed current is generated and sent to a data acquisition card. The information is used by a proportional-derivative-integral (PID) algorithm to calculate the magnitude of a low-voltage signal that is used to control the power supply output. Any variation of current across the sensing resistor is thus counteracted by an opposite-direction variation of the high voltage applied to the nanospray emitter. In this way, the apparatus adjusts the emitter voltage to achieve a preset value of current, which it strives to maintain over time in spite of any possible variation of the parameters influencing the spray regime. Preliminary results have shown that the feedback apparatus is capable of establishing and maintaining stable spray for samples that are usually considered challenging in traditional voltage-controlled analysis, such as those consisting of nucleic acid solutions with high salt loads. For these types of samples, the total ion count recorded in current-controlled mode was significantly more stable than that observed in voltage-controlled mode. At the same time, overall signal intensities and signal-to-noise ratios were also significantly improved. Setting the target nanospray current to a predefined value and letting the apparatus reach the target without operator intervention enabled the acquisition of viable data from solutions containing up to 2.5 M ammonium acetate, which are ordinarily difficult by traditional manual tuning. A deeper understanding of the current-voltage relationships for samples of very different compositions is expected to enable one not only to predict the target current that should be used for a certain analysis, but also to devise algorithms to change such target as a function of predictable variations of sample properties and analytical conditions. This will allow for optimal performance to be maintained during on-line gradient chromatography in which the nature of the sprayed solution may vary very widely during the course of the analysis.

CdTe Nanoparticles Display Tropism to Core Histones and Histone‐Rich Cell Organelles

The disclosure of the mechanisms of nanoparticle interaction with specific intracellular targets represents one of the key tasks in nanobiology. Unmodified luminescent semiconductor nanoparticles, or quantum dots (QDs), are capable of a strikingly rapid accumulation in the nuclei and nucleoli of living human cells, driven by processes of yet unknown nature. Here, it is hypothesized that such a strong tropism of QDs could be mediated by charge-related properties of the macromolecules presented in the nuclear compartments. As the complex microenvironment encountered by the QDs in the nuclei and nucleoli of live cells is primarily presented by proteins and other biopolymers, such as DNA and RNA, the model of human phagocytic cell line THP1, nuclear lysates, purified protein, and nucleic acid solutions is utilized to investigate the interactions of the QDs with these most abundant classes of intranuclear macromolecules. Using a combination of advanced technological approaches, including live cell confocal microscopy, fluorescent lifetime imaging (FLIM), spectroscopic methods, and zeta potential measurements, it is demonstrated that unmodified CdTe QDs preferentially bind to the positively charged core histone proteins as opposed to the DNA or RNA, resulting in a dramatic shift off the absorption band, and a red shift and decrease in the pholuminescence (PL) intensity of the QDs. FLIM imaging of the QDs demonstrates an increased formation of QD/protein aggregates in the presence of core histones, with a resulting significant reduction in the PL lifetime. FLIM technology for the first time reveals that the localization of negatively charged QDs to their ultimate nuclear and nucleolar destinations dramatically affects the QDs' photoluminescence lifetimes, and offers thereby a sensitive readout for physical interactions between QDs and their intracellular macromolecular targets. These findings strongly suggest that charge-mediated QD/histone interactions could provide the basis for QD nuclear localization downstream of intracellular transport mechanisms.

FIT probes: Peptide nucleic acid probes with a fluorescent base surrogate enable real-time DNA quantification and single nucleotide polymorphism discovery

The ability to accurately quantify specific nucleic acid molecules in complex biomolecule solutions in real time is important in diagnostic and basic research. Here we describe a DNA–PNA (peptide nucleic acid) hybridization assay that allows sensitive quantification of specific nucleic acids in solution and concomitant detection of select single base mutations in resulting DNA–PNA duplexes. The technique employs so-called FIT (forced intercalation) probes in which one base is replaced by a thiazole orange (TO) dye molecule. If a DNA molecule that is complementary to the FIT–PNA molecule (except at the site of the dye) hybridizes to the probe, the TO dye exhibits intense fluorescence because stacking in the duplexes enforces a coplanar arrangement even in the excited state. However, a base mismatch at either position immediately adjacent to the TO dye dramatically decreases fluorescence, presumably because the TO dye has room to undergo torsional motions that lead to rapid depletion of the excited state. Of note, we found that the use of d-ornithine rather than aminoethylglycine as the PNA backbone increases the intensity of fluorescence emitted by matched probe–target duplexes while specificity of fluorescence signaling under nonstringent conditions is also increased. The usefulness of the ornithine-containing FIT probes was demonstrated in the real-time PCR analysis providing a linear measurement range over at least seven orders of magnitude. The analysis of two important single nucleotide polymorphisms (SNPs) in the CFTR gene confirmed the ability of FIT probes to facilitate unambiguous SNP calls for genomic DNA by quantitative PCR.

Quantitation of DNA and RNA

INTRODUCTIONThere are several ways to quantitate solutions of nucleic acids. If the solution is pure, one can use a spectrophotometer to measure the amount of ultraviolet radiation absorbed by the bases. DNA can also be quantified by measuring the UV-induced emission of fluorescence from intercalated ethidium bromide. This method is useful if there is not enough DNA to quantify with a spectrophotometer, or if the DNA solution is contaminated. Strategies for accurately quantifying nucleic acids using these approaches are discussed here.

Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions.

The gel electrophoresis mobility shift assay (EMSA) is used to detect protein complexes with nucleic acids. It is the core technology underlying a wide range of qualitative and quantitative analyses for the characterization of interacting systems. In the classical assay, solutions of protein and nucleic acid are combined and the resulting mixtures are subjected to electrophoresis under native conditions through polyacrylamide or agarose gel. After electrophoresis, the distribution of species containing nucleic acid is determined, usually by autoradiography of 32P-labeled nucleic acid. In general, protein-nucleic acid complexes migrate more slowly than the corresponding free nucleic acid. In this protocol, we identify the most important factors that determine the stabilities and electrophoretic mobilities of complexes under assay conditions. A representative protocol is provided and commonly used variants are discussed. Expected outcomes are briefly described. References to extensions of the method and a troubleshooting guide are provided.

Parameterization of Peptide 13C Carbonyl Chemical Shielding Anisotropy in Molecular Dynamics Simulations

NMR chemical shielding anisotropy (CSA) relaxation is an important tool in the study of dynamical processes in proteins and nucleic acids in solution. Herein, we investigate how dynamical variations in local geometry affect the chemical shielding anisotropy relaxation of the carbonyl carbon nucleus, using the following protocol: 1) Using density functional theory, the carbonyl (13)C' CSA is computed for 103 conformations of the model peptide group N-methylacetamide (NMA). 2) The variations in computed (13)C' CSA parameters are fitted against quadratic hypersurfaces containing cross terms between the variables. 3) The predictive quality of the CSA hypersurfaces is validated by comparing the predicted and de novo calculated (13)C' CSAs for 20 molecular dynamics snapshots. 4) The CSA fluctuations and their autocorrelation and cross correlation functions due to bond-length and bond-angle distortions are predicted for a chemistry Harvard molecular mechanics (CHARMM) molecular dynamics trajectory of Ca(2+)-saturated calmodulin and GB3 from the hypersurfaces, as well as for a molecular dynamics (MD) simulation of an NMA trimer using a quantum mechanically correct forcefield. We find that the fluctuations can be represented by a 0.93 scaling factor of the CSA tensor for both R(1) and R(2) relaxations for residues in helix, coil, and sheet alike. This result is important, as it establishes that (13)C' relaxation is a valid tool for measurement of interesting dynamical events in proteins.

PH‐Tunable Endosomolytic Oligomers for Enhanced Nucleic Acid Delivery

AbstractOligomeric sulfonamides (OSAs) are explored as a tool for the effective endosomal release of polyplexes or delivery of nucleic acid. The OSAs tested in this study show varying proton‐buffering regions and pH‐dependent solubility transitions within the endosomal pH range, and are influenced by the pKa value and hydrophobicity of a given sulfonamide group. In addition, OSA presents negligible toxicity. The oligomers are added to the nucleic acid solution for polyplex formation with positively charged polymeric nucleic acid carriers. The resulting nanoscale, positively charged, and OSA‐incorporated poly(L‐lysine) (PLL)/DNA complexes (OSA‐polyplexes) show a 4–55‐fold increase in in vitro gene expression compared to PLL/DNA (control), depending upon the cell line and the nature of the used OSA. In cellular uptake and intracellular trafficking studies using pH‐sensitive or pH‐insensitive dye‐labeled DNAs, there is no significant difference in the amount of DNA uptake using OSA polyplexes and PLL/DNA. However, OSA–polyplexes induce a broader intracellular distribution of the DNA than PLL/DNA complexes do. These results, coupled with the enhanced DNA transfection using OSA–polyplexes, indicate a mechanism by which OSA induces endosomal release of polyplexes and/or nucleic acids. The findings suggest that OSA could enhance polymer‐based nucleic acid delivery. Furthermore, such materials offer significant potential for effective cytosolic delivery of chemical, biological, and diagnostic therapeutics.

Comment on “Solving the riddle of the bright mismatches: Labeling and effective binding in oligonucleotide arrays”

In a recent paper [Phys. Rev. E 68, 011906 (2003)], Naef and Magnasco suggested that the "bright" mismatches observed in Affymetrix microarray experiments are caused by the fluorescent molecules used to label RNA target sequences, which would impede target-probe hybridization. Their conclusion is based on the observation of "unexpected" asymmetries in the affinities obtained by fitting microarray data from publicly available experiments. We point out here that the observed asymmetry is due to the inequivalence of RNA and DNA, and that the reported affinities are consistent with stacking free energies obtained from melting experiments of unlabeled nucleic acids in solution. The conclusion of Naef and Magnasco is therefore based on an unjustified assumption.