Structural Analysis Of Nucleic Acids Research Articles

Label-Free Detection of DNA Supramolecular Structure Formation by Surface-Enhanced Raman Spectroscopy

The precise self-assembly of DNA molecules can be used to create nanoprecision supramolecular materials. However, the lack of methods to characterize such supramolecular materials limits their development. Surface-enhanced Raman spectroscopy (SERS) is widely used to detect the secondary structure of simple DNA molecules, but its application in the revealing of complex DNA supramolecular information remains challenging. Herein, we proposed a modified SERS-based platform able to provide structural information on DNA supramolecular materials. The silver nanoparticle-enhanced substrate uses acetonitrile as an internal standard and modifier, and calcium ions are used as an aggregating agent to induce the formation of stable "hotspots" of silver nanoparticles, where the base planes in DNA supramolecules are perpendicular to the surface of the substrate, obtaining enhanced Raman signals of base ring in both single-stranded DNA and DNA supramolecules for the first time. The structure of DNA supramolecules was efficiently characterized using this technique, showing the great application potential of this technique in the structural analysis of nucleic acids and their ligands.

Synthesis of ethynylpyrene-modified 3-deaza-2′-deoxyguanosines as environmentally sensitive fluorescent nucleosides: Target DNA-sequence detection via changes in the fluorescence wavelength

Novel pyrene-labeled environmentally sensitive fluorescent (ESF) nucleosides [1p3zG (1a) and 2p3zG (1b)] possessing the 3-deazaguanine skeleton were synthesized. Among them, 1p3zG (1a) exhibited longer fluorescence emission wavelengths compared with the previously reported naphthalene-modified ESF nucleoside 3nzG (48–71 nm longer in polar solvents) and showed remarkable solvent-polarity-sensitive fluorescence properties. Oligodeoxynucleotide (ODN) probes containing 1p3zG (1a) clearly discriminated target DNAs by the change in the emission wavelength and intensity through monitoring the microenvironmental change around the DNA minor groove. Thus, these pyrene-labeled ESF nucleosides can be applied in gene detection and molecular diagnostics, as well as in the structural analysis of nucleic acids.

Modular calibrant sets for the structural analysis of nucleic acids by ion mobility spectrometry mass spectrometry.

This study explored the use of modular nucleic acid (NA) standards to generate calibration curves capable of translating primary ion mobility readouts into corresponding collision cross section (CCS) data. Putative calibrants consisted of single- (ss) and double-stranded (ds) oligo-deoxynucleotides reaching up to ∼40 kDa in size (i.e., 64 bp) and ∼5700 Å(2) in CCS. To ensure self-consistency among reference CCS values, computational data obtained in house were preferred to any experimental or computational data from disparate sources. Such values were obtained by molecular dynamics (MD) simulations and either the exact hard sphere scattering (EHSS) or the projection superposition approximation (PSA) methods, and then plotted against the corresponding experimental values to generate separate calibration curves. Their performance was evaluated on the basis of their correlation coefficients and ability to provide values that matched the CCS of selected test samples mimicking typical unknowns. The results indicated that the predictive power benefited from the exclusion of higher charged species that were more susceptible to the destabilizing effects of Coulombic repulsion. The results revealed discrepancies between EHSS and PSA data that were ascribable to the different approximations used to describe the ion mobility process. Within the boundaries defined by these approximations and the challenges of modeling NA structure in a solvent-free environment, the calibrant sets enabled the experimental determination of CCS with excellent reproducibility (precision) and error (accuracy), which will support the analysis of progressively larger NA samples of biological significance.

Automated band annotation for RNA structure probing experiments with numerous capillary electrophoresis profiles.

Capillary electrophoresis (CE) is a powerful approach for structural analysis of nucleic acids, with recent high-throughput variants enabling three-dimensional RNA modeling and the discovery of new rules for RNA structure design. Among the steps composing CE analysis, the process of finding each band in an electrophoretic trace and mapping it to a position in the nucleic acid sequence has required significant manual inspection and remains the most time-consuming and error-prone step. The few available tools seeking to automate this band annotation have achieved limited accuracy and have not taken advantage of information across dozens of profiles routinely acquired in high-throughput measurements. We present a dynamic-programming-based approach to automate band annotation for high-throughput capillary electrophoresis. The approach is uniquely able to define and optimize a robust target function that takes into account multiple CE profiles (sequencing ladders, different chemical probes, different mutants) collected for the RNA. Over a large benchmark of multi-profile datasets for biological RNAs and designed RNAs from the EteRNA project, the method outperforms prior tools (QuSHAPE and FAST) significantly in terms of accuracy compared with gold-standard manual annotations. The amount of computation required is reasonable at a few seconds per dataset. We also introduce an 'E-score' metric to automatically assess the reliability of the band annotation and show it to be practically useful in flagging uncertainties in band annotation for further inspection. The implementation of the proposed algorithm is included in the HiTRACE software, freely available as an online server and for download at http://hitrace.stanford.edu. sryoon@snu.ac.kr or rhiju@stanford.edu Supplementary data are available at Bioinformatics online.

High-throughput single-nucleotide structural mapping by capillary automated footprinting analysis

The use of capillary electrophoresis with fluorescently labeled nucleic acids revolutionized DNA sequencing, effectively fueling the genomic revolution. We present an application of this technology for the high-throughput structural analysis of nucleic acids by chemical and enzymatic mapping (‘footprinting’). We achieve the throughput and data quality necessary for genomic-scale structural analysis by combining fluorophore labeling of nucleic acids with novel quantitation algorithms. We implemented these algorithms in the CAFA (capillary automated footprinting analysis) open-source software that is downloadable gratis from https://simtk.org/home/cafa. The accuracy, throughput and reproducibility of CAFA analysis are demonstrated using hydroxyl radical footprinting of RNA. The versatility of CAFA is illustrated by dimethyl sulfate mapping of RNA secondary structure and DNase I mapping of a protein binding to a specific sequence of DNA. Our experimental and computational approach facilitates the acquisition of high-throughput chemical probing data for solution structural analysis of nucleic acids.

Sequence‐specific and nonspecific mobilities of single‐stranded oligonucleotides observed by changing the borate buffer concentration

The suitability of gel electrophoresis to structural analysis of nucleic acids has been examined, using from borate buffer concentrations (in the range from 4.5 to 450 mM. The gel electrophoretic mobility of single-stranded oligonucleotides was shown to be sequence-dependent at higher concentrations than 27 mM of borate buffer and nondependent at lower one (less than 9 mM). As a result, each dodecamer had a sequence-specific critical concentration of Tris-borate-EDTA (TBE) buffer at which each seems to change its structural state. At the lower concentration than the critical one, all the dodecamers turned to the state of a finite mobility and migrated in a sharp band. This finding is discussed and rationalized by the assumption that the difference in conformational dynamics of oligonucleotides, due to the difference in their sequence, is mainly responsible for the observed difference in their mobility.

Comparison of rRNA Cleavage by Complementary 1,10-Phenanthroline-Cu(II)- and EDTA-Fe(II)-Derivatized Oligonucleotides

The chemical nucleases 1,10-phenanthroline-Cu(II) and EDTA-Fe(II), have proven to be valuable tools for structural analysis of nucleic acids. Both have found applications in footprinting and directed proximity studies of DNA and RNA. Derivatives of each that provide for tethering to nucleic acid or protein are commercially available, allowing their widespread use for structural analysis of macromolecules. Although their applications are somewhat overlapping, differences in their cleavage mechanisms and chemical properties allow them to provide distinct and complementary structural information. The purpose of this study is to compare directly the cleavage patterns of tethered 1,10-phenanthroline-Cu(II) and EDTA-Fe(II) complexes within a similar experimental system. Here, the region surrounding nucleotide 1400 of 16S rRNA from Escherichia coli serves as a substrate for chemical cleavage directed by a derivatized complementary oligonucleotide. This region of rRNA is known to be involved in the decoding of mRNA during translation. The results of this study provide evidence in support of the mechanistic differences previously established for EDTA-Fe(II) and 1,10-phenathroline-Cu(II). The delocalized cleavage envelope produced by EDTA-Fe(II) cleavage suggests the involvement of a diffusible reactive species. On the other hand, rRNA cleavage induced by the tethered 1,10-phenanthroline-Cu(II) complex appears localized to the proximity of the chemical nuclease under normal conditions, although the production of an unknown diffusible species appears to occur during long reaction times.

Applicability of commonly used atom-atom type potential energy functions in structural analysis of nucleic acids. The role of electrostatic interactions

Applicability of five popular force fields (AMBER, CFF91, CVFF CHARMM and GROMOS) to the structural analysis of nucleic acids without explicit solvent has been examined. Structures of nine deoxyoligonucleotides in the A- and B-forms were optimized by energy minimization and compared with well-refined crystallographic data. Several models for electrostatic interactions were tested including constant and distance dependent dielectric functions in combination with varied effective charges of the phosphate groups. Similarity tests of the theoretical and experimental structures were based on a distance analysis in a conformational parameter space of the DNA molecules and on a proposed scoring scheme.

Structural analysis of nucleic acids by precise denaturing gradient gel electrophoresis: I. Methodology.

A method to convert the conventional denaturing gradient gel electrophoresis into a highly reproducible experimental system was developed. It was based on the following experimental findings; (i) dyes, which are small molecules, do not exhibit mobility changes attributed to their conformational change while nucleic acids do; and (ii) most of the mobility shifts caused by experimental fluctuations could be cancelled by normalizing the mobility of a sample with respect to the corresponding one of a dye. The method involves co-migration of internal reference dyes with samples (nucleic acids), and computer-aided data processing, allowing us to obtain the relative mobility of nucleic acids with respect to a dye throughout the denaturing gradient. The overall pattern of the relative mobilities thus obtained, named the normalized mobility profile (NMP), corresponded well to conformational changes of a macromolecule induced by denaturing effects. This method provides us with objective data without using internal macromolecular references, which not only guarantees the precision but also extends the range of application of the denaturing gradient method.

Structural analysis of nucleic acids by precise denaturing gradient gel electrophoresis: II. Applications to the analysis of subtle and drastic mobility changes of oligo- and polynucleotides.

Precise denaturing gradient gel electrophoresis was effectively applied to various kinds of oligo- and polynucleotides. The analyses on oligonucleotides revealed that every oligonucleotide has its own characteristic normalized mobility profile (NMP), which can be used to identify, characterize and classify the molecules. The precise system also enabled us to obtain unequivocally the mobility transitions corresponding to the melting of hairpin structures of oligonucleotides, single-stranded (ss) DNAs, and RNAs. Another application to co-migration and separate migration experiments demonstrated that there were significant binding interactions between two species of ss molecules of similar mobility, even when they have little complementarity with each other. When the precise temperature gradient gel electrophoresis was applied to double-stranded DNAs, it could be confirmed with high reliability that the mobility transitions observed correspond to cooperative meltings and strand dissociations. Through these experiments, mu m, a parameter defined as a mobility transition point, was shown to be effective to deal with those phenomena quantitatively.

Application of the z-COSY technique with a modified pulse sequence to measurement of coupling constants in macromolecules

Coupling constants can provide useful supplementary information in the structural analysis of nucleic acids and proteins. It is often difficult, however, to determine their values as the large linewidths tend to obscure the multiplet patterns. For this reason it is desirable to interpret reduced rather than full multiplet effects in COSY spectra (I, 2). To this end several alternative methods have been developed. These include /3COSY (2), bilinear COSY (3), E.COSY (4), and z-COSY (5). The z-COSY and E.COSY techniques both yield spectra with reduced multiplet patterns and pure absorption lineshapes for all peaks in the spectrum. While full suppression of the undesired multiplet components can be achieved with E.COSY, the extent of suppression in z-COSY depends on the choice of flip angle for the two mixing pulses. The effects of strong coupling, however, are not as severe for z-COSY as it uses only small pulses (6). In this paper we demonstrate the application of z-COSY to macromolecules using the DNA hexamer 5’d(GCATGC)2 as an example and discuss the effects resulting from incomplete suppression of the undesired multiplet components of the cross peaks. Further, we show that in order to obtain a spectrum of sufficient quality for the measurement of coupling constant, the z-COSY technique should be applied in a modified manner with respect to the suppression of zero-quantum coherences. The basic pulse sequence for z-COSY is 90”-t,+?-7,-&t,. The phase cycling must be designed in such a manner that coherences of the order p = t1 are retained during t, and only populations and zero-quantum coherences are selected during the delay 7,. In the case of macromolecules, the application of the normal NOESY phase-cycle selecting coherences of the order p = 0 f 4n (n = integer) during the delay T, is sufficient. To obtain a z-COSY spectrum with pure absorption lineshapes it is necessary to suppress the zero-quantum transitions (ZQT) appropriately. Because of the intrinsic similarity of the pulse sequences, the methods used for NOESY experiment (7-9) can also be applied to z-COSY. In the original description of the z-COSY experiment (5), zero-quantum suppression was achieved by a random variation of the delay between the two mixing pulses. Unfortunately, this introduces noise into the spectrum due to the effective smearing of the zero-quantum peaks along the w1 axis and the generation oft, noise arising from the random variation in the relaxation of the diagonal peaks

Monospecific Antibody against 5-Methyl-Cytidine for the Structural Analysis of Nucleic Acids

Anti 5-methyl-cytidine antibodies might be useful agents for the detection and localization of 5-methyl-cytidine of nucleic acids, but only if the antibodies recognize this nucleoside with sufficient specificity. A conjugate containing 18 moles of 5-methyl-cytidine per mole of BSA was prepared and antibodies directed against this nucleoside hapten were produced by immunization of rabbits (as determined by gel diffusion in agar containing excessive amounts of the carrier). A slight crossreaction of cytidine-BSA was eliminated by adsorption on the cross-reacting antigen. Further purification of the antibodies was effected by chromatography on DEAE-Sephadex A-50 and a method for the rapid quantitation of the antibodies showed that 12.7% of the IgG protein are monospecific against 5-methyl-cytidine-BSA. Hydrolysis of antibodies with insolubilized papain produced monovalent Fab fragments which were identified by SDS-Disk-electrophoresis. A two stage method for cross linking the immunoproteins to ferritin by glutaraldehyde was used. The isolation of immunoferritin conjugates by Bio-Gel A 1.5 m column chromatography is described. The identification of the effluents was made by glycerin density gradient ultracentrifugation. The results were visualized by electron microscopy after the treatment of immunoferritin conjugates with (methylated and unmethylated) denaturated DNA, fractionation on the glycerine density gradient, and the spreading by a modification of drop technique.

Nucleotide-specific antibodies as potential blocking agents in the structural analysis of nucleic acids

Anti-nucleotide antibodies might be useful blocking agents in directing the nuclease catalyzed hydrolysis of RNA for sequence analysis, but only if the antibodies recognized a nucleotide or sequence with sufficient specificity and affinity to compete with a nuclease at only specific sites in an RNA. As a test, antibodies directed against mononucleotide haptens were induced with 5′-nucleotide (oxidized with periodate) coupled to bovine serum albumin. Nucleotide-specific antibodies, isolated by affinity chromatography on nucleotide-agarose columns, were at least 95 % pure 7-S γ-globulin. The antibodies precipitated RNA, homologous nucleotide-rabbit albumin conjugates, and to a lesser extent, heterologous conjugates. Antibodies further purified by passage through an affinity column of heterologous nucleotides showed specificity for the single homologous nucleotide. Equilibrium dialysis measurements gave dissociation constants for the antibody-homologous nucleotide complex of about 10 −5 M, while binding of a heterologous nucleotide was insufficient to be measured. The 5′-phosphate group, the base, and the ribose derivative in the conjugate were all elements of structure recognized by the antibodies. Hydrolysis of antibodies with insolubilized papain produced monovalent Fab fragments with dissociation constants essentially as determined for intact antibodies. Antibodies were found to compete with pancreatic ribonuclease for dinucleoside phosphate substrates in a manner consistent with the binding results. Anti-nucleotide Fab fragments inhibited the nuclease hydrolysis of f2 RNA, but no directed cleavage to give altered RNA fragmentation patterns was observed.

The nucleases from Acrocylindrium sp. 3. General properties and specificity of endonuclease.

The general properties and substrate specificity of the endonuclease from Acrocylindrium sp. were examined. 1. The optimum pH of the endonuclease is 8.0. 2. The endonuclease requires divalent cations such as Mn2+, Co2+, Mg2+, Pb2+, Ca2+, etc. for its activity. 3. The endonuclease is relatively stable towards heat treatment (100°C, 5 min, at pH 7.5). 4. The order of relative rate of hydrolysis of substrates is native DNA>heat-denatured DNA>RNA>poly C>poly U>poly A>poly I. 5. Oligonucleotides thus produced are terminated by 5'-phosphoryl groups and showed an average chain length of 4.4. 6. The enzyme hydrolyzes double-stranded polyribonucleotides such as poly I: poly C or poly A: poly U more rapidly than single-stranded polyribonucleotides. 7. The usefulness of this enzyme as a reagent for the structural analysis of nucleic acids, together with the exonuclease from Acrocylindrium sp., is discussed.