Conformational Changes In Nucleic Acids Research Articles

Kinetic FRET Assay to Measure Binding-Induced Conformational Changes of Nucleic Acids.

The interaction of small molecules or proteins with RNA or DNA often involves changes in the nucleic acid (NA) folding and structure. A biophysical characterization of these processes helps us to understand the underlying molecular mechanisms. Here, we propose kinFRET (kinetics Förster resonance energy transfer), a real-time ensemble FRET methodology to measure binding and folding kinetics. With kinFRET, the kinetics of conformational changes of NAs (DNA or RNA) upon analyte binding can be directly followed via a FRET signal using a chip-based biosensor. We demonstrate the utility of this approach with two representative examples. First, we monitored the conformational changes of different formats of an aptamer (MN19) upon interaction with small-molecule analytes. Second, we characterized the binding kinetics of RNA recognition by tandem K homology (KH) domains of the human insulin-like growth factor II mRNA-binding protein 3 (IMP3), which reveals distinct kinetic contributions of the two KH domains. Our data demonstrate that kinFRET is well suited to study the kinetics and conformational changes of NA-analyte interactions.

Thermodynamics of benzoquinone-induced conformational changes in nucleic acids and human serum albumin

Biological macromolecules such as proteins, nucleic acids, carbohydrates and lipids, play a crucial role in biochemical and molecular processes. Thus, the study of the structure-function relationship of biomolecules in presence of ligands is an important aspect of structural biology. The current communication describes the chemico-biological interaction between benzene metabolite para-benzoquinone (BQ) with B-form of nucleic acids (B-DNA) and human serum albumin (HSA). The binding ability of HSA towards bromocresol green (BCG) was significantly suppressed when exposed to increasing concentrations of BQ in the presence of various physiological buffers. Further, the native fluorescence of HSA was drastically reduced and the secondary structures of HSA were significantly compromised with increasing concentrations of BQ. In vitro and in silico studies also revealed that BQ binds to domains I and II of HSA and thus altering the conformation of HSA which may potentially affect plasma osmotic pressure, as well as the binding and transport of numerous endogenous and exogenous molecules. Similarly, BQ interacts directly to the GC region of B-DNA particularly in the minor groove which was also assessed by computational docking studies. Isothermal titration calorimetry data suggest higher binding affinity of BQ towards DNA than HSA. Various spectroscopic observations also suggest that BQ binds to DNA preferably in the minor grooves. Thus, the results revealed that BQ may play a key role in inducing mutagenicity, either by formation of adducts on GC regions or by accelerating oxidative damage to biomacromolecules through chemico-biological interactions.

Drying and temperature induced conformational changes of nucleic acids and stallion sperm chromatin in trehalose preservation formulations

Even though dried sperm is not viable, it can be used for fertilization as long as its chromatin remains intact. In this study, we investigated drying- and temperature-induced conformational changes of nucleic acids and stallion sperm chromatin. Sperm was diluted in preservation formulations with and without sugar/albumin and subjected to convective drying at elevated temperatures on glass substrates. Accumulation of reactive oxygen species was studied during storage at different temperatures, and the sperm chromatin structure assay was used to assess DNA damage. Fourier transform infrared spectroscopy was used to identify dehydration and storage induced conformational changes in isolated DNA and sperm chromatin. Furthermore, hydrogen bonding in the preservation solutions associated with storage stability were investigated. Reactive oxygen species and DNA damage in dried sperm samples were found to accumulate with increasing storage temperature and storage duration. Non-reducing disaccharides (i.e., trehalose, sucrose) and albumin counteracted oxidative stress and preserved sperm chromatin during dried storage, whereas glucose increased DNA damage during storage. When sperm was dried in the presence of trehalose and albumin, no spectral changes were detected during storage at refrigeration temperatures, whereas under accelerated aging conditions, i.e., storage at 37 °C, spectral changes were detected indicating alterations in sperm chromatin structure.

Cu2+-based distance measurements by pulsed EPR provide distance constraints for DNA backbone conformations in solution.

Electron paramagnetic resonance (EPR) has become an important tool to probe conformational changes in nucleic acids. An array of EPR labels for nucleic acids are available, but they often come at the cost of long tethers, are dependent on the presence of a particular nucleotide or can be placed only at the termini. Site directed incorporation of Cu2+-chelated to a ligand, 2,2′dipicolylamine (DPA) is potentially an attractive strategy for site-specific, nucleotide independent Cu2+-labelling in DNA. To fully understand the potential of this label, we undertook a systematic and detailed analysis of the Cu2+-DPA motif using EPR and molecular dynamics (MD) simulations. We used continuous wave EPR experiments to characterize Cu2+ binding to DPA as well as optimize Cu2+ loading conditions. We performed double electron-electron resonance (DEER) experiments at two frequencies to elucidate orientational selectivity effects. Furthermore, comparison of DEER and MD simulated distance distributions reveal a remarkable agreement in the most probable distances. The results illustrate the efficacy of the Cu2+-DPA in reporting on DNA backbone conformations for sufficiently long base pair separations. This labelling strategy can serve as an important tool for probing conformational changes in DNA upon interaction with other macromolecules.

A semipermeable enzymatic nanoreactor as an efficient modulator for reversible pH regulation.

Here we propose a new concept for the fabrication of a semipermeable enzymatic nanoreactor as an efficient modulator to reversibly switch the pH of an aqueous environment. We used amino-functionalized, expanded mesoporous silica nanoparticles (EMSN) as a model nanocarrier to load enzymes. In order to protect enzymes from the interference of a complicated environment, polyelectrolyte multilayers (PEMs) were coated on the surface of the EMSN through layer by layer (LbL) assembly. These PEMs can serve as semipermeable membranes, allowing small molecules to diffuse in and out freely while trapping the enzymes in the nanoreactors. Compared with traditional electrochemical stimulation or optical control methods, our enzymatic regulation platform is easy to operate without complicated instruments. In addition, this system can cover a wide range of pH values and conveniently regulate pH values by simply controlling the concentrations of catalysts or reactants. Meanwhile, this strategy could be generalized to other enzymes or nanocarriers to achieve reversible pH regulation for different purposes. The switched pH values can be implemented for the modulation of the conformational changes of nucleic acids and activation of the charge conversion in drug delivery applications.

Advances in the determination of nucleic acid conformational ensembles.

Conformational changes in nucleic acids play a key role in the way genetic information is stored, transferred, and processed in living cells. Here, we describe new approaches that employ a broad range of experimental data, including NMR-derived chemical shifts and residual dipolar couplings, small-angle X-ray scattering, and computational approaches such as molecular dynamics simulations to determine ensembles of DNA and RNA at atomic resolution. We review the complementary information that can be obtained from diverse sets of data and the various methods that have been developed to combine these data with computational methods to construct ensembles and assess their uncertainty. We conclude by surveying RNA and DNA ensembles determined using these methods, highlighting the unique physical and functional insights obtained so far.

In vivo and in situ monitoring of the nitric oxide stimulus response of single cancer cells by Raman spectroscopy

Raman spectroscopy is capable of studying time-resolved information of selected biomolecular distributions inside individual cells without labeling. In this study, Raman spectroscopy was for the first time utilized to in vivo and in situ monitor the cellular response to nitric oxide (NO) in single oral squamous cell carcinoma (OSCC) cells over a period of 24 h. Sodium nitroprusside (SNP) was chosen as a NO donor to be incubated with the OSCC cell line (TCA8113) for certain time intervals. In vivo and in situ Raman analysis revealed that the degradation and conformational changes of nucleic acids, lipids and proteins could be directly observed by changes in the characteristic Raman bands. In comparison with conventional flow cytometric analysis, Raman spectroscopy not only detected more subtle NO-induced chemical changes of cells, where the SNP concentration could be even less than 1 mM, but also provided a full view of the whole chemical components of single cells. Raman spectroscopy therefore is an important candidate for label-free, nondestructive and in situ monitoring of cellular changes in response to chemotherapeutic agents, which could potentially be used in rapid screening of novel drugs.

2-Aminopurine Single-Molecule Fluorescence

Base flipping is a local conformational change that results in the rotation of a nucleotide out of its helical structure. This is frequently observed in numerous nucleic acid structural rearrangements, such as in DNA-protein interactions and in different RNA enzymes. 2-Aminopurine (2AP) is a fluorescent nucleotide analog extensively used to probe local conformational changes in nucleic acids in bulk experiments, but no single molecule approaches have been developed to study these mechanisms using 2AP. One challenge towards this goal is that 2AP fluorescence is very sensitive to the interaction with neighboring nucleotides, and its emission spectrum partially overlaps with that of tryptophan. We have developed new click-chemistry-based surface immobilization approach that enables us to monitor, in real time base flipping in DNA. We have characterized the fluorescence properties of single 2AP labelled DNAs. Our results show that nucleotides are very dynamic in a single- or double-stranded DNA and that bases can flip out of the helix, suggesting that the base flipping occurs frequently and in a much slower time scale that previously believe. This new assay can also be used to study other local conformational changes in nucleic acids at the single-molecule level by using 2AP as the base flipping probe.

Photocontrol of Gene Expressions Systems Using Light-Induced Conformational Changes of Nucleic Acids

To understand non-trivial biological functions, it is crucial to develop minimal synthetic models that capture their basic features. Here, we demonstrate a sequence-independent, reversible control of transcription and gene expression using a photosensitive nucleic acid binder (pNAB). By introducing a pNAB whose affinity for nucleic acids is tuned by light, in vitro RNA production, EGFP translation, and GFP expression were successfully inhibited in the dark and recovered after a short illumination at 365 nm. Our results indicate that the accessibility of the protein machinery to one or several nucleic acid binding sites can be efficiently regulated by changing the conformational/condensation state of the nucleic acid (DNA conformation or mRNA aggregation), thus regulating gene activity in an efficient, reversible, and sequence-independent manner. The possibility offered by our approach to use light to trigger various gene expression systems in a system-independent way opens interesting perspectives to study gene expression dynamics as well as to develop photocontrolled biotechnological procedures.Ref: A. Estevez-Torres, C. Crozatier, A. Diguet, T. Hara, H. Saito, K. Yoshikawa, D. Baigl, Proc. Natl. Acad. Sci. USA 2009, 106, 12219-12223.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Measuring nanometer distances in nucleic acids using a sequence-independent nitroxide probe

This protocol describes the procedures for measuring nanometer distances in nucleic acids using a nitroxide probe that can be attached to any nucleotide within a given sequence. Two nitroxides are attached to phosphorothioates that are chemically substituted at specific sites of DNA or RNA. Inter-nitroxide distances are measured using a four-pulse double electron-electron resonance technique, and the measured distances are correlated to the parent structures using a Web-accessible computer program. Four to five days are needed for sample labeling, purification and distance measurement. The procedures described herein provide a method for probing global structures and studying conformational changes of nucleic acids and protein/nucleic acid complexes.

Novel tethered particle motion analysis of CI protein-mediated DNA looping in theregulation of bacteriophage lambda

The tethered particle motion (TPM) technique has attracted great interest because of itssimplicity and the wealth of information that it can provide on protein-induced conformationalchanges in nucleic acids. Here we present an approach to TPM methodology and analysisthat increases the efficiency of data acquisition and facilitates interpretation of TPMassays. In particular, the statistical analysis that we propose allows fast data processing,minimal data selection and visual display of the distribution of molecular behaviour. Themethodology proved useful in verifying CI protein-mediated DNA looping in bacteriophageλ and in differentiating between two different types of loops, stable and dynamic, whoserelative occurrence seems to be a function of the distance between the operators as well astheir relative angular orientation. Furthermore, the statistical analysis indicates that CIbinding per se slightly shortens the DNA.

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.

Reactivity of Nucleic Acids with Ozone: An FT-IR Microspectroscopy Study

Chemical changes were observed by Fourier transform microspectroscopy (FT-IR-M) in DNA and RNA samples treated with increasing amounts of ozone. The nucleobase reactivity and conformational changes in nucleic acids were followed through the trend of certain spectroscopic parameters calculated from the spectra. The P1 and P2 parameters (calculated from the ratios of the optical densities of the bands at 1692 and 1654 and at 1728 and 1654 cm−1, respectively), involving base absorptions, are sensitive to the chemical action of ozone on covalent bonds of purinic and pirimidinic bases. The P3 parameter (calculated from the ratios of the optical densities of the bands at 1230 and 1090 cm−1) is sensitive to conformational changes in the phosphate-ribose backbone of nucleic acids. We found that, as the amount of ozone increases, the P1, P2, and P3 parameters increase for both the DNA and RNA sample spectra, but the curve trend and the concentration range that gave significant parameter changes were different for DNA and RNA. DNA spectra showed a sigmoidal increase in P1 and P2 parameters ( C1/2 = 1.6 mg/mL, the concentration of ozone at which the parameters observed changed by 50%), a plateau between 2 and 4.5 mg/mL of ozone, and a slow linear increase for ozone concentrations >5 mg/mL. Instead, a C1/2 = 2.5 mg/mL was evaluated from the P3 vs. ozone concentration plot. The plots of P1, P2, and P3 parameters calculated from the RNA sample spectra vs. ozone concentration showed a saturation-shape curve that reached its maximum value between 0 and 1.9 mg/mL. On the basis of these results, several hypotheses are suggested to explain the reaction mechanism of DNA and RNA nucleobases and the conformational changes in nucleic acids.

12] Systems for the NMR study of modified nucleoside-dependent, metal-ion induced conformational changes in nucleic acids

This chapter discusses systems for the nuclear magnetic resonance (NMR) study of modified nucleoside-dependent, metal-ion induced conformational changes in nucleic acids. RNAs have more than 90 nucleoside modifications and function through a large variety of interactions with other macromolecules. The structure–function interrelationships of modified nucleosides and their interactions with the ribose 2'OH and metal ions are largely unknown. Modified nucleosides and stable isotope-labeled nucleosides are chemically synthesized and introduced into RNA by solid phase manual synthesis or by automated chemical synthesis. The latter requires production of the protected phosphoramidites. Modified nucleoside-containing stem-loop domains and smaller sequences of RNAs and their DNA analogs are produced, and their physical, chemical, and biological properties are studied. Modified nucleosides have evolved to alter the RNA chemistry and structure and the potential to accomplish this is as varied as are the chemistries and structures of the modifications. There are three methods of incorporation of modified nucleosides and stable isotope-enriched nucleosides into nucleic acids. From the in vivo incorporation of modified nucleosides and stable isotope-enriched nucleosides, a fully mature, completely modified, and isotopically enriched RNA can be isolated from the cells grown in culture and purified by chromatography.

Implications and concepts of polyamine‐nucleic acid interactions

Modeling, x-ray diffraction, and solution studies have contributed to the understanding of interactions between polyamines and nucleic acids. Polyamines stabilize a variety of unusual DNA structures and conformations in vitro, including both the left-handed Z and the right-handed A DNA. In addition, polyamines condense DNA and may be important in bending specific sequences. Investigations into the mechanisms of these effects provide support for both specific and nonspecific interactions between polyamines and DNA. Although exact relationships between the binding of polyamines and conformational changes in nucleic acids are still being clarified, polyamines remain important candidates for regulators of DNA conformation in vivo.

Ligand-Induced Conformational Changes in Ribonucleic Acids

Publisher Summary This chapter discusses the ligand-induced conformational changes in ribonucleic acids (RNAs). This chapter also discusses the newer results on induced structural changes in transfer ribonucleic acids (tRNA), focusing on codon-induced changes, and then emphasize a new aspect— namely, the evidence for allosteric conformational changes in nucleic acids. Transfer RNAs are recognized by two different classes of proteins: those that bind only to their cognate tRNAs (like aminoacyl synthetases) and the elongation factor EF-Tu, that form complexes with all aminoacylated tRNAs. Transfer RNA (tRNA) may exist as both aminoacyl- or peptidyl-tRNA and several isoacceptor tRNAs may exist for only one amino acid. Besides a complicated tertiary structure and many modified nucleosides, there is a third way to ease the correlation of structure, with the many tRNA functions. Like proteins, tRNA may undergo induced structural transitions and there may be one defined tRNA conformation for every biochemical process. The chapter also discusses induced allosteric conformational changes in tRNA.

Conformational changes of nucleic acids and poly (d(A-T)-d(A-T)) caused by photoaddition of furocoumarins.

DNA's of various AT content, poly[d(A-T)-d(A-T)], and double-stranded RNA were irradiated with UV light at 365 nm in the presence of linear (xanthotoxin) or angular (angelicin) furocoumarins. The covalent photobinding is strongly dependent on the spatial arrangement of furocoumarin molecules at the polymer conformation. CD measurements demonstrate that the bifunctional photochemical binding of xanthotoxin with double-stranded DNA's and poly[d(A-T)-d(A-T)] is accompanied by conformational changes which involve probably decreasing helical twisting of the double helix. This effect is greatly enhanced with increasing AT content. The formation of A-like structures is very unlikely since the B leads to A transition induced by ethanol addition was found to be strongly suppressed in xanthotoxin photoreacted DNA. The B-type helix appears to be the most sensitive conformation with minor restriction to produce photochemically induced cross-links.

Infrared spectroscopic study of desoxyribonucleic acids, their derivates and analogs. 8. Determination of the out-of-plane NH deformation vibrations

The NH out-of-plane deformation and the NH2 wagging vibrations of base-pairing models of DNA and related model substances in the solid state are assigned by deuteration shifts, cooling shifts, and shifts caused by the complementary base-pairing. The sensitivity of the frequencies of these vibrations to variations of the hydrogen bond strength may be useful to follow conformational changes of nucleic acids.

Conformational changes of nucleic acids in methanol-water solutions at low temperature

Conformational changes of native DNA in aqueous methanolic solution at low temperature are described. The enthalpy difference between two “native” forms is on the order of 6.5 kcal/mole of nucleotide.