Search

Base‐filling, i. e., post‐synthetic furnishing of an oligonucleotide scaffold with base moieties or their analogues, is an interesting alternative to the conventional approach of sequential coupling of building blocks (modified or otherwise). Reversible attachment of the base moieties is particularly attractive as it allows the use of dynamic combinatorial chemistry and usually leads to higher fidelity. This concept article summarizes the various backbones and coupling reactions used for base‐filling over the past fifteen years, discusses the impact of base stacking and pairing on efficiency and fidelity and highlights potential and realized applications. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are promising biomarkers especially for early‐stage cancer diagnostics, but the implementation of miRNA‐based diagnostic tests is still hindered by the limitations of current analytical methods. The small size, low concentrations in biofluids and high sequence homology of miRNAs are challenges for assay development. Currently, most of the sensitive detection methods rely on enzymatic amplification steps, which complicate the analysis and can lead to biases in quantitation. Therefore, there is an increasing need to develop enzyme‐free detection methods that are sensitive, specific and user‐friendly. In this study, a simple direct hybridization assay for the DNA analogue of miR‐20a was developed. The assay is based on upconverting nanoparticle labels, which enable ultrasensitive detection, and hairpin structured probes, which provide additional hybridization stability due to base stacking. The limit of detection was 0.73 fM with plasma recoveries between 76 % and 111 %, demonstrating that the assay could be used for direct detection of miRNAs from complex sample matrices without isolation of RNA. Due to the simplicity and the excellent sensitivity for an amplification‐free method, the assay has a great potential for miRNA‐based clinical applications. [ABSTRACT FROM AUTHOR]

Abstract Base‐filling, i. e., post‐synthetic furnishing of an oligonucleotide scaffold with base moieties or their analogues, is an interesting alternative to the conventional approach of sequential coupling of building blocks (modified or otherwise). Reversible attachment of the base moieties is particularly attractive as it allows the use of dynamic combinatorial chemistry and usually leads to higher fidelity. This concept article summarizes the various backbones and coupling reactions used for base‐filling over the past fifteen years, discusses the impact of base stacking and pairing on efficiency and fidelity and highlights potential and realized applications.

Ribonucleic acid (RNA) molecules serve as master regulators of cells by encoding their biological function in the ribonucleotide sequence, particularly their ability to interact with other molecules. To understand how RNA molecules perform their biological tasks and to design new sequences with specific functions, it is of great benefit to be able to computationally predict how RNA folds and interacts in the cellular environment. Our workflow for computational modeling of the 3D structures of RNA and its interactions with other molecules uses a set of methods developed in our laboratory, including MeSSPredRNA for predicting canonical and non-canonical base pairs, PARNASSUS for detecting remote homology based on comparisons of sequences and secondary structures, ModeRNA for comparative modeling, the SimRNA family of programs for modeling RNA 3D structure and its complexes with other molecules, and QRNAS for model refinement. In this study, we present the results of testing this workflow in predicting RNA 3D structures in the CASP15 experiment. The overall high score of the computational models predicted by our group demonstrates the robustness of our workflow and its individual components in terms of predicting RNA 3D structures of acceptable quality that are close to the target structures. However, the variance in prediction quality is still quite high, and the results are still too far from the level of protein 3D structure predictions. This exercise led us to consider several improvements, especially to better predict and enforce stacking interactions and non-canonical base pairs. [ABSTRACT FROM AUTHOR]

Specific recognition of DNA base sequences by proteins is vital for life‐cycles of all organisms. In a large number of crystal structures of protein–DNA complexes, DNA conformation significantly deviates from the canonical B‐DNA structure. A key question is whether such alternate conformations exist prior to protein binding and one is selected for complexation or the structure observed is induced by protein binding. Non‐canonical base pairs, such as Hoogsteen base pairs, are often observed in crystal structures of protein–DNA complexes. We decided to explore whether the occurrence of such non‐canonical base pairs in protein–DNA complexes is induced by the protein or is selected from pre‐existing conformations. Detailed quantum chemical calculations with dispersion‐corrected density functional theory (DFT‐D) indicated that most of the non‐canonical base pairs with DNA bases are stable even in the absence of the interacting amino acids. However, the G:G Hoogsteen base pair, which also appears in the telomere structure, appears to be unstable in the absence of other stabilizing agents, such as positively charged amino acids. Thus, the stability of many of the non‐canonical base pair containing duplexes may be close to the canonical B‐DNA structure and hence energetically accessible in the ground state; suggesting that the selection from pre‐existing conformations may be an important mechanism for observed non‐canonical base pairs in protein–DNA complexes. [ABSTRACT FROM AUTHOR]

Surface-enhanced Raman spectroscopy (SERS), as a label-free, highly sensitive analytical method, has become an important tool for providing substance fingerprints. In this study, silver nanoparticles containing thiosulfate ions and calcium ions (Ag@SCNPs) have been used as an enhanced substrate to eliminate the interference of impurities on DNA signals. Intrinsic structural information on single-strand DNA (ssDNA) was directly obtained through SERS. The improved enhancement system was used to explore the base-stacking rules of ssDNA in a solution environment. For the first time, single-base insertion mutations and deletion mutations, as well as their exact mutation sites, were identified, and Raman spectra with high stability, repeatability, and high signal-to-noise ratio were obtained. The method is simple, fast, and accurate, and the detection process is nondestructive. It has potential to be applied in the fields of medical diagnosis and genetics research. [ABSTRACT FROM AUTHOR]

Complex RNA–RNA interactions are increasingly known to play key roles in numerous biological processes from gene expression control to ribonucleoprotein granule formation. By contrast, the nature of these interactions and characteristics of their interfaces, especially those that involve partially or wholly structured RNAs, remain elusive. Herein, we discuss different modalities of RNA–RNA interactions with an emphasis on those that depend on secondary, tertiary, or quaternary structure. We dissect recently structurally elucidated RNA–RNA complexes including RNA triplexes, riboswitches, ribozymes, and reverse transcription complexes. These analyses highlight a reciprocal relationship that intimately links RNA structure formation with RNA–RNA interactions. The interactions not only shape and sculpt RNA structures but also are enabled and modulated by the structures they create. Understanding this two-way relationship between RNA structure and interactions provides mechanistic insights into the expanding repertoire of noncoding RNA functions, and may inform the design of novel therapeutics that target RNA structures or interactions.

Single nucleotide polymorphisms (SNPs) affect base pair stacking, which is the primary factor for maintaining the stability of DNA. However, the mechanism of how SNPs lead to phenotype variations is still unclear. In this work, we connected SNPs and base pair stacking by a 3-mer base pair stacking free energy matrix. The SNPs with large base pair stacking free energy differences led to phenotype variations. A molecular dynamics (MD) simulation was then applied. Our results showed that base pair stacking played an important role in the transcription factor (TF)-DNA interaction. Changes in DNA structure mainly originate from TF-DNA interactions, and with the increased base pair stacking free energy, the structure of DNA approaches its free type, although its binding affinity was increased by the SNP. In addition, quantitative models using base pair stacking features revealed that base pair stacking can be used to predict TF binding specificity. As such, our work combined knowledge from bioinformatics and structural biology and provided a new understanding of the relationship between SNPs and phenotype variations. The 3-mer base pair stacking free energy matrix is useful in high-throughput screening of SNPs and predicting TF-DNA binding affinity.

Unnatural nucleic acids are promising materials to expand genetic information beyond the natural bases. During replication, substrate nucleotide incorporation should be strictly controlled for optimal base pairing with template strand bases. Base-pairing interactions occur via hydrogen bonding and base stacking, which could be perturbed by the chemical environment. Although unnatural nucleobases and sugar moieties have undergone extensive structural improvement for intended polymerization, the chemical environmental effect on the reaction is less understood. In this study, we investigated how molecular crowding could affect native DNA polymerization along various templates comprising unnatural nucleobases and sugars. Under non-crowding conditions, the preferred incorporation efficiency of pyrimidine deoxynucleotide triphosphates (dNTPs) by the Klenow fragment (KF) was generally high with low fidelity, whereas that of purine dNTPs was the opposite. However, under crowding conditions, the efficiency remained almost unchanged with varying preferences in each case. These results suggest that hydrogen bonding and base-stacking interactions could be perturbed by crowding conditions in the bulk solution and polymerase active center during transient base pairing before polymerization. This study highlights that unintended dNTP incorporation against unnatural nucleosides could be differentiated in cases of intracellular reactions.

Recent years has witnessed increasing development and exploitation of fluorescence techniques which has been spurred by the discovery, design and synthesis of novel fluorescent molecules. In the realm of nucleic acids, both modest and dramatic structural modification of the nucleobase may engender it with fluorescence. This review focuses on the design, synthesis and applications of purine nucleoside analogs possessing fluorescently modified nucleobases, particularly for detection of nucleic acid sequences or for structural studies on nucleic acids. Thus, fluorescent nucleoside/nucleotide analogs that can be incorporated into oligonucleotides and report changes due to base-pairing/stacking or microenvironmental alterations, such as polarity changes in the grooves, are highlighted. Whenever available, the photophysical mechanism giving rise to the environmental responsiveness of the fluorescent nucleoside/tide is presented. [ABSTRACT FROM AUTHOR]

Prominent in the L-shaped three-dimensional structure of tRNAs is the "elbow" where their two orthogonal helical stacks meet. It has a conserved structure arising from the interaction of the terminal loops of the D- and T-stem-loops, and presents to solution a flat face of a tertiary base pair between the D- and T-loops. In addition to the ribosome, which interacts with the elbow in all three of its tRNA binding sites, several cellular RNAs and many proteins are known to recognize the elbow. At least three classes of non-coding RNAs, namely 23S rRNA, ribonuclease P, and the T-box riboswitches, recognize the tRNA elbow employing an identical structural motif consisting of two interdigitated T-loops. In contrast, structural solutions to tRNA-elbow recognition by proteins are varied. Some enzymes responsible for post-transcriptional tRNA modification even disrupt the elbow structure in order to access their substrate nucleotides. The evolutionary origin of the elbow is mysterious, but, because it does not explicitly participate in the flow of genetic information, it has been proposed to be a late innovation. Regardless, it is biologically essential. Even some viruses that hijack the cellular machinery using tRNA decoys have convergently evolved near-perfect mimics of the tRNA elbow. [ABSTRACT FROM AUTHOR]

MicroRNA (miRNA), an 18-24-nucleotide noncoding RNA molecule, has become an ideal class of biomarker candidates for clinical diagnosis of cancers. By now, a number of detection methods for miRNAs have been developed on planar arrays and suspension arrays. In this work, we describe a hybridization-triggered fluorescence strategy for label-free and multiplex miRNA detection on graphically encoded silica suspension array. The total RNA is directly applied for analysis with an 8-mer Universal Tag which can be selectively captured by the capture probe via base-stacking effects. Benefiting from base-stacking effects, this novel method exhibits superb discrimination ability toward the 5′ and 3′ end single-nucleotide alteration. Mature miRNAs can be distinguished from their corresponding pre-miRNAs easily. Moreover, the estimated detection limit of 5 amol is comparable to some of the most sensitive methods. All these mentioned characteristics offer exciting possibilities for discovery and clinical applications. [ABSTRACT FROM AUTHOR]

ABSTRACT The structural effects of the commonly occurring modified nucleoside dihydrouridine (D) observed experimentally in model oligonucleotides include a strong destabilization of the C3′-endo sugar conformation of D, the disruption of stacking interactions of neighboring residues with D and a possible destabilization of the C3′-endo sugar pucker of the 5′-neighboring nucleoside. Our simulations with a combination of a set of parameters for modified RNA residues with the recently developed AMBER FF99χ force field having reoptimized glycosidic torsion angle parameters for standard nucleosides was found to reproduce the destabilizing effect of dihydrouridine better than with the AMBER FF99 force field for nucleic acids for which the parameters for the modified residues were originally developed. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 985-991, 2014. [ABSTRACT FROM AUTHOR]

RNA contains different secondary structural motifs like pseudo-helices, hairpin loops, internal loops, etc. in addition to anti-parallel double helices and random coils. The secondary structures are mainly stabilized by base-pairing and stacking interactions between the planar aromatic bases. The hydrogen bonding strength and geometries of base pairs are characterized by six intra-base pair parameters. Similarly, stacking can be represented by six local doublet parameters. These dinucleotide step parameters can describe the quality of stacking between Watson-Crick base pairs very effectively. However, it is quite difficult to understand the stacking pattern for dinucleotides consisting of non canonical base pairs from these parameters. Stacking interaction is a manifestation of the interaction between two aromatic bases or base pairs and thus can be estimated best by the overlap area between the planar aromatic moieties. We have calculated base pair overlap between two consecutive base pairs as the buried van der Waals surface between them. In general, overlap values show normal distribution for the Watson-Crick base pairs in most double helices within a range from 45 to 50 Å irrespective of base sequence. The dinucleotide steps with non-canonical base pairs also are seen to have high overlap value, although their twist and few other parameters are rather unusual. We have analyzed hairpin loops of different length, bulges within double helical structures and pseudo-continuous helices using our algorithm. The overlap area analyses indicate good stacking between few looped out bases especially in GNRA tetraloop, which was difficult to quantitatively characterise from analysis of the base pair or dinucleotide step parameters. This parameter is also seen to be capable to distinguish pseudo-continuous helices from kinked helix junctions. [ABSTRACT FROM AUTHOR]

Abstract: Three-dimensional structures of biomolecules are stabilized by a large number of non-covalent interactions and some of them such as van der Waals, electrostatic and hydrogen bond interactions are well characterized. Delocalized π–electron clouds of aromatic residues are known to be involved in cation–π, CH–π, OH–π and π–π interactions. In proteins, many examples have been found in which the backbone carbonyl oxygen of one residue makes close contact with the aromatic center of aromatic residues. Quantum chemical calculations suggest that such contacts may provide stability to the protein secondary structures. In this study, we have systematically analyzed the experimentally determined high-resolution DNA crystal structures and identified 91 examples in which the aromatic center of one base is in close contact (<3.5Ǻ) with the oxygen atom of preceding (Group-I) or succeeding base (Group-II). Examples from Group-I are overwhelmingly observed and cytosine or thymine is the preferred base contributing oxygen atom in Group-I base pairs. A similar analysis of high-resolution RNA structures surprisingly did not yield many examples of oxygen–aromatic contact of similar type between bases. Ab initio quantum chemical calculations on compounds based on DNA crystal structures and model compounds show that interactions between the bases in base pairs with oxygen–aromatic contacts are energetically favorable. Decomposition of interaction energies indicates that dispersion forces are the major cause for energetically stable interaction in these base pairs. We speculate that oxygen–aromatic contacts in intra-strand base pairs in a DNA structure may have biological significance. [Copyright &y& Elsevier]

Abstract: We have employed a laser desorption technique combined with supersonic-jet cooling for producing base pairs of adenine nucleosides, adenosine (Ado) and N6,N6-dimethyladenosine (DMAdo) under low-temperature conditions. The resulting base pairs are then ionized through resonant two-photon ionization (R2PI) and analyzed by time-of-flight mass spectrometry. It is found that dimers of these adenine nucleosides are stable, especially in the case of DMAdo, with respect to those of the corresponding bases, i.e., adenine and N6,N6-dimethyladenine. Structural analysis of the DMAdo dimer is performed based on the IR–UV double resonance measurements and theoretical calculations. The result demonstrates that the dimer possesses a stacked structure being stabilized by the formation of hydrogen-bonding network involving the two sugar groups. The occurrence of the frequency shift and broadening is explained satisfactorily based on the anharmonic coupling of the OH stretching modes with specific bending modes and low-frequency modes of base and sugar moieties. [Copyright &y& Elsevier]

Abstract: We developed a simple method for the detection of platelet-derived growth factors (PDGFs) based on base stacking effect coupled with an unmodified gold nanoparticle (AuNP) indicator. In the absence of a target, an aptamer probe and a capture probe stably co-exist in a solution, as it is difficult to sustain an interaction between both these probes due to the short 8bp duplex. However, when a target protein binds to the aptamer probe, the strong base stacking effect can lead to a favorable and stable interaction between the aptamer and capture probes. Hence, the capture probe dissociates from the AuNP surfaces, inducing AuNP aggregation. Compared with other AuNP-based aptasensors for PDGFs, using this base stacking effect can overcome a structured-aptamer method''s limitation of requiring thiolated-aptamer-modified AuNPs. Under optimal detection conditions, this label-free colorimetric sensor could detect PDGFs down to 6nM with high selectivity in the presence of other interferring proteins. This simple detection approach provides viable methods for a structured-aptamer sensing protocol. [Copyright &y& Elsevier]

The potential energy surfaces of stacked uracil dimer (U/U) and stacked thymine dimer (T/T) have been explored at the counterpoise (CP)-corrected M06-2X/6-31+G(d) level of theory, in the gas phase and in solution (with water and, for U/U, 1,4-dioxane as the solvents) modeled by a continuum solvent using the polarizable continuum model. Potential energy scans were created by rotation of one monomer around its center-of-mass, whereas the other monomer remained still. Both face-to-back (one molecule exactly on top of the other) and face-to-face (one base molecule flipped by 180°) structures were considered. Five or six (dependent on whether CP correction is included or not) stacked uracil dimer minima and six stacked thymine dimer minima were located. A number of transition states on the U/U and T/T potential energy surfaces were likewise identified. The general effect of the continuum solvent is a flattening of the potential energy surface. Comparison of the gas-phase M06-2X/6-31+G(d) U/U interaction energies with estimated CCSD(T)/complete basis set values (where available) show the excellent performance of this functional for stacking energies. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The crystal structure of the PNA (peptide nucleic acid) oligomer H- Lys-HalG- AlaG-HalC- AlaG-HalC- AlaC-Lys-NH2 (PNA1, amino acids with d-configuration are underlined, Ala = alanyl, Hal = homoalanyl) has been determined by ab initio direct methods and refined against 1.0 Å data. The asymmetric unit consists of a tetrameric cage with almost ideal Watson-Crick C-G base pairing of all the guanine and cytosine side-chain substituents. Each PNA strand has a 90°β-turn every second residue, stabilized by three hydrogen bonds between the backbone amides. The first, second, fifth and sixth bases stack on one side of the monomer and pair with the corresponding complementary bases of a second monomer to form a dimer. The two remaining bases on each side of the resulting dimer form Watson-Crick pairs with the complementary bases of a second dimer, leading to a unique cage structure. The extra methylene groups in the homoalanyl residues enable stacking of the bases with an optimal distance between base-planes but also with an appreciable lateral displacement (slide). [ABSTRACT FROM AUTHOR]

Abstract: microRNAs have emerged as the central player in gene expression regulation and have been considered as potent cancer biomarkers for early disease diagnosis. Direct microRNA detection without amplification and labeling is highly desired. Here we present a rapid, sensitive and selective microRNA detection method based on the base stacking hybridization coupling with time-resolved fluorescence technology. Other than planar microarrays, magnetic beads are used as reaction platforms. In this method, one universal tag is used to report all microRNA targets. Its specificity allows for discrimination between microRNAs differing by a single nucleotide, and between precursor and mature microRNAs. This method also provides a high sensitivity down to 20fM. Moreover, the full protocol can be completed in about 3h starting from total RNA. [Copyright &y& Elsevier]

The review considers different experimental and theoretical approaches to the investigation of RNA folding and identification of nucleotides that critically affect the folding of molecules, such as tRNA, and several classes of ribozymes. For instance, it has been shown that nucleotides of the D- and T-loop regions are the last to be involved in the tRNA structure, or, rather, they are not included in the tRNA folding nucleus. A specially developed SHAPE method was used to show that the long-recognized hierarchical folding model does not hold true for tRNA folding. In the second part of the review, algorithms and programs used for the prediction of RNA secondary structures, as well as for modeling RNA folding, are considered. [ABSTRACT FROM AUTHOR]

To elucidate the physical origin of the preference of nucleic acid bases for stacking over hydrogen bonding in water, Monte Carlo simulations were performed starting from Watson-Crick structures of the adenine-thymine, adenine-uracil and guanine-cytosine base pairs, as well as from the Hoogsteen adenine-thymine base pair, in clusters comprising 400 and 800 water molecules. The simulations employed a newly implemented Metropolis Monte Carlo algorithm based on the extended cluster approach. All simulations reached stacked structures, confirming that such structures are preferred over the hydrogen-bonded Watson-Crick and Hoogsteen base pairs. The Monte Carlo simulations show the complete transition from hydrogen-bonded base pairs to stacked structures in the Monte Carlo framework. Analysis of the average energies shows that the preference of stacked over hydrogen-bonded structures is due to the increased water-base interaction in these structures. This is corroborated by the increased number of water-base hydrogen bonds in the stacked structures. [ABSTRACT FROM AUTHOR]

The adenine-thymine (AT), adenine-uracil (AU) and guanine-cytosine (GC) base associates in clusters containing 400 water molecules were studied using a newly implemented Metropolis Monte Carlo algorithm based on the extended cluster approach. Starting from the hydrogen-bonded Watson-Crick geometries, all three base pairs are transformed into more favorable stacked configurations during the simulation. The obtained results show, for the first time, the transition from planar base pairs to stacked base associates in the Monte Carlo framework. Analysis of the interaction energies shows that, in the water cluster, the stacked dimers are energetically preferable compared to the corresponding Watson-Crick base pairs. This is due to the larger base-water interaction in the stacked structures. The water-water interaction is one of the main factors promoting the formation of stacked dimers, and the obtained data confirm the crucial role of the water-water interactions in base stacking. [ABSTRACT FROM AUTHOR]

Atomistic simulations of nucleic acids are prohibitively expensive and, consequently, reduced models of these compounds are of great interest in the field. In this work, we propose a physics-based coarse-grained model of nucleic-acid bases in which each base is represented by several (3–5) interaction centers. van der Waals interactions are modeled by Lennard-Jones spheres with a 12–6 potential energy function. The charge distribution is modeled by a set of electric dipole moments located at the centers of the Lennard-Jones spheres. The method for computing the Lennard-Jones parameters, electric dipole moments (their magnitude and orientation) and positions of the interaction centers is described. Several models with different numbers of interaction centers were tested. The model with three-center cytosine, four-center guanine, four-center thymine, and five-center adenine satisfactorily reproduces the canonical Watson–Crick hydrogen bonding and stacking interaction energies of the all-atom AMBER model. The computation time with the coarse-grained model is reduced seven times compared with that of the all-atom model. © 2009 Wiley Periodicals, Inc. J Comput Chem, 2010 [ABSTRACT FROM AUTHOR]

The double helix of DNA, when composed of dinucleotide purine-pyrimidine repeats, can adopt a left-handed helical structure called Z-DNA. For reasons not entirely understood, such dinucleotide repeats in genomic sequences have been associated with genomic instability leading to cancer. Adoption of the left-handed conformation results in the formation of conformational junctions: A B-to-Z junction is formed at the boundaries of the helix, whereas a Z-to-Z junction is commonly formed in sequences where the dinucleotide repeat is interrupted by single base insertions or deletions that bring neighboring helices out of phase. B-Z junctions are shown to result in exposed nucleotides vulnerable to chemical or enzymatic modification. Here we describe the three-dimensional structure of a Z-Z junction stabilized by Zα, the Z-DNA binding domain of the RNA editing enzyme ADAR1. We show that the junction structure consists of a single base pair and leads to partial or full disruption of the helical stacking. The junction region allows intercalating agents to insert themselves into the left-handed helix, which is otherwise resistant to intercalation. However, unlike a B-Z junction, in this structure the bases are not fully extruded, and the stacking between the two left-handed helices is not continuous. [ABSTRACT FROM AUTHOR]

Abstract: RNA structures are very dynamic and the dynamic motions result in a heterogeneous conformational ensemble. It is crucial to illustrate the role of conformational dynamics in RNA function. A variety of spectroscopic methods have been used to investigate the dynamic aspects of RNA structures. Recently, ultrafast time-resolved spectroscopy, a well-established technique, has been introduced as a new tool in this field. With femtosecond time-resolution, one can resolve the heterogeneous nature of RNA conformational ensemble quantitatively, detect and characterize minor unusual conformations, and capture folding events that may occur on a fast timescale. Here, we review the technical aspects of using an ultrafast fluorescence up-conversion technique to probe the heterogeneous base stacking patterns in RNA motifs and dynamic base motions that allow sampling of multiple states. [Copyright &y& Elsevier]

Abstract: A simple and efficient dual preconcentration method of on-column liquid–liquid–liquid microextraction (LLLME) coupled with base stacking was developed for capillary zone electrophoresis (CZE) in this paper. Four N-methyl carbamates were used as target compounds to evaluate the enrichment means. The carbamates in sample solutions (donor phase) were extracted into a dodecanol phase immobilized on a porous hollow fiber, hydrolyzed and back extracted into 0.20μL running buffer (acceptor phase) of 30mmol/L methylamine hydrochloride (pH 11.6) containing 0.5mmol/L tetradecyltrimethylammonium bromide inside the hollow fiber, stacked further with 0.5mol/L NaOH injected at −10kV for 60s, and separated by CZE. Analytical parameters affecting the LLLME, base stacking and CZE were investigated, including sample solution volume, pH and temperature, extraction time, stirring rate, buffer component, buffer pH, NaOH concentration, stacking time, etc. The enrichment factors of the carbamates were higher than 1100. The relative standard deviation (RSD) of peak height and limits of detection (LODs) were 4.5–5.5% (n =6) and 2–4ng/mL (S/N =3) for standard solutions, respectively. The proposed method was applied to the analysis of vegetable and fruit samples with the RSD less than 6.0% (n =3) and LODs of 6–10ng/g (S/N =3). The calibration solutions were prepared by diluting the stock solutions with blank sample solutions, and the calibration concentrations ranged from 0.012 to 1.0μg/mL (r >0.9951). The analytical results demonstrated that the LLLME coupled with base stacking was a simple, convenient and reliable on-column sample pretreatment method for the analysis of anionic analytes in CZE. [Copyright &y& Elsevier]

Complex RNA–RNA interactions are increasingly known to play key roles in numerous biological processes from gene expression control to ribonucleoprotein granule formation. By contrast, the nature of these interactions and characteristics of their interfaces, especially those that involve partially or wholly structured RNAs, remain elusive. Herein, we discuss different modalities of RNA–RNA interactions with an emphasis on those that depend on secondary, tertiary, or quaternary structure. We dissect recently structurally elucidated RNA–RNA complexes including RNA triplexes, riboswitches, ribozymes, and reverse transcription complexes. These analyses highlight a reciprocal relationship that intimately links RNA structure formation with RNA–RNA interactions. The interactions not only shape and sculpt RNA structures but also are enabled and modulated by the structures they create. Understanding this two-way relationship between RNA structure and interactions provides mechanistic insights into the expanding repertoire of noncoding RNA functions, and may inform the design of novel therapeutics that target RNA structures or interactions. [ABSTRACT FROM AUTHOR]

The planar H-bonded adenine-thymine (A • T) base pair and four stacked hetero associates (A/T) of these bases in a cluster containing 200 water molecules were studied by the new semi-empirical PM6 method. The formation of the planar A • T base pair in the water cluster was found to be energetically unfavourable, primarily due to the destabilising contribution of the base-water interactions. Under complete geometry optimisation, the planar A • T base pair converted into a nonplanar propeller-twisted and buckled associate. In contrast, the formation of all stacked dimers studied was found to be favourable, with formation energies ranging from -11 to -20 kcal/mol. The preference for the formation of these stacks results from the favourable change in the water structure and distortion of the paired and isolated bases during the base association reaction. [ABSTRACT FROM AUTHOR]

Abstract: The hydrogen atoms transfer process in a generic polynucleotide of DNA has been studied with a theoretical approach. The role of time-dependent interaction among these processes on different base pairs has been analysed. An Hamiltonian with a time-dependent coupling among the nearest neighbor base pairs has been utilised. This general model evidences a complete hydrogen atoms transfer in a base pair with at least one equal nearest neighbor base pair along the chains. [Copyright &y& Elsevier]

The crystal structure of a self-complementary RNA duplex r(GGCGBrUGCGCU)2 with terminal G·U and internal tandem G·U base pairs has been determined at 2.1 Å resolution. The crystals belong to the tetragonal space group P43, with unit-cell parameters a = b = 37.69, c = 96.28 Å and two duplexes in the asymmetric unit. The two strands of each duplex are related by a pseudodyad axis. The structure was refined to final Rwork and Rfree values of 20.9 and 25.3%, respectively. The duplexes stack in an end-to-end manner, forming infinite columns along the c axis. This is the first structural study of an RNA duplex containing G·U pairs at the termini. The stacking overlaps of the terminal G·U base pairs with their adjacent Watson–Crick base pairs are larger than those of Watson–Crick base pairs of the 5′-YR-3′/3′-RY-5′ type. The terminal G·U base pairs of neighbouring duplexes are also stacked with each other. An alternating underwound–overwound pattern of the twist angles is seen at each step along the duplex. This observation is typical for internal tandem G·U pairs, while the terminal G·U base pairs exhibit high twist angles with the adjacent Watson–Crick pairs. The 3′-­side of U of the internal G·U base pair, which is unstacked, appears to be stabilized by π-cation interaction with an Mg2+ ion. [ABSTRACT FROM AUTHOR]

We report the crystal structures of a translesion DNA polymerase, Dpo4, complexed with a matched or mismatched incoming nucleotide and with a pyrophosphate product after misincorporation. These structures suggest two mechanisms by which Dpo4 may reject a wrong incoming nucleotide with its preformed and open active site. First, a mismatched replicating base pair leads to poor base stacking and alignment of the metal ions and as a consequence, inhibits incorporation. By replacing Mg2+ with Mn2+, which has a relaxed coordination requirement and tolerates misalignment, the catalytic efficiency of misincorporation increases dramatically. Mn2+ also enhances translesion synthesis by Dpo4. Subtle conformational changes that lead to the proper metal ion coordination may, therefore, be a key step in catalysis. Second, the slow release of pyrophosphate may increase the fidelity of Dpo4 by stalling mispaired primer extension and promoting pyrophosphorolysis that reverses the polymerization reaction. Indeed, Dpo4 has robust pyrophosphorolysis activity and degrades the primer strand in the presence of pyrophosphate. The correct incoming nucleotide allows DNA synthesis to overcome pyrophosphorolysis, but an incorrect incoming nucleotide does not. [ABSTRACT FROM AUTHOR]

The free energy change of the stacking process of DNA dimers has been investigated by potential of mean force (PMF) calculations. Two reaction coordinates were considered. One is the distance R between the glycosidic nitrogen atoms of the bases. The other is the pseudo dihedral angle X(N–Cl′–Cl′–N). All 16 possible DNA dimers composed of the adenine, cytosine, guanine, or thymine bases in 5′ and 3′ positions were considered. From the free energy profiles, we observed good stacking for all DNA dimers and sequence-dependent stacking stability. This sequence dependence of the stacking free energy is in good agreement with the experimental results. We also observed that the PMF is the lowest at R = 4.0~4.4 Å and X = 20~40° for all the DNA dimers except for the dGpdA dimer. These values are close to those of the canonical B-DNA (4.4 Å and 29°). [ABSTRACT FROM AUTHOR]

In order to elucidate the stacking-unstacking process of DNA dimers, we have performed molecular dynamics simulations based on replica-exchange umbrella sampling (REUS), which is one of powerful conformational sampling techniques. We studied four DNA dimers composed of the adenine and thymine bases in both the 5′ and the 3′ positions (dApdA, dApdT, dTpdA, and dTpdT). We examined the time series of the distance between the glycosidic nitrogen atoms, root-mean-square deviations from A-DNA and B-DNA, various backbone and glycosidic torsion angles, and the pseudorotation phase angles as functions of the simulation time step. All these time series imply that the present simulation has indeed sampled a very wide conformational space. The results for the backbone and glycosidic torsion angles and pseudorotation phase angles imply that B-DNA structures are the dominant motif of the stacked dimers, while a small population of A-DNA also exists in the stacked states. [ABSTRACT FROM AUTHOR]

Local base stacking and conformational mobility play a major role in the structure and function of nucleic acids. We have recently shown that the low-energy CD spectrum of 2-aminopurine (2-AP), i.e., the CD spectral region above 300 nm, can be used to monitor conformational changes in polynucleotides at or near mono- and dinucleotide 2-AP residues that replace adenine residues in DNA and RNA. Here, we extend this technique to pyrrolo-cytosine (PC), a fluorescent analogue of cytosine. The low-energy CD spectrum of a PC dinucleotide in dsDNA exhibits an exciton couplet with two bands of opposite sign centered at 350 nm. This signal is characteristic of base stacking between adjacent PC residues in a right helical conformation. Isolated PC nucleotide residues inserted into polynucleotide chains also display chirality that reflects the asymmetric environment of their sequence context. Thus, we show that the low-energy CD spectra of C(PC)A and A(PC)C sequences in dsDNA have opposite signs. It appears that the measurement of the low-energy CD spectra of PC residues will usefully complement 2-AP measurements by serving to characterize the local conformations and dynamics of nucleic acids near C residues and G-C base pairs. [ABSTRACT FROM AUTHOR]

RNA exhibits a higher structural diversity than DNA and is an important molecule in the biology of life. It shows a number of secondary structures such as duplexes, hairpin loops, bulges, internal loops, etc. However, in natural RNA, bases are limited to the four predominant structures U, C, A, and G and so the number of compounds that can be used for investigation of parameters of base stacking, base pairing, and hydrogen bond is limited. We synthesized different fluoromodifications of RNA building blocks: 1′-deoxy-1′-phenyl-β-d-ribofuranose (B), 1′-deoxy-1′-(4-fluorophenyl)-β-d-ribofuranose (4 FB), 1′-deoxy-1′-(2,4-difluorophenyl)-β-d-ribofuranose (2,4 DFB), 1′-deoxy-1′-(2,4,5-trifluorophenyl)-β-d-ribofuranose (2,4,5 TFB), 1′-deoxy-1′-(2,4,6-trifluorophenyl)-β-d-ribofuranose, 1′-deoxy-1′-(pentafluorophenyl)-β-d-ribofuranose (PFB), 1′-deoxy-1′-(benzimidazol-1-yl)-β-d-ribofuranose (BI), 1′-deoxy-1′-(4-fluoro-1H-benzimidazol-1-yl)-β-d-ribofuranose (4 FBI), 1′-deoxy-1′-(6-fluoro-1H-benzimidazol-1-yl)-β-d-ribofuranose (6 FBI), 1′-deoxy-1′-(4,6-difluoro-1H-benzimidazol-1-yl)-β-d-ribofuranose (4,6 DFBI), 1′-deoxy-1′-(4-trifluoromethyl-1H-benzimidazol-1-yl)-β-d-ribofuranose (4 TFM), 1′-deoxy-1′-(5-trifluoromethyl-1H-benzimidazol-1-yl)-β-d-ribofuranose (5 TFM), and 1′-deoxy-1′-(6-trifluoromethyl-1H-benzimidazol-1-yl)-β-d-ribofuranose (6 TFM). These amidites were incorporated and tested in a defined A, U-rich RNA sequence (12-mer, 5′-CUU UUC XUU CUU-3′ paired with 3′-GAA AAG YAA GAA-5′). Only one position was modified, marked as X and Y, respectively. UV melting profiles of those oligonucleotides were measured. [ABSTRACT FROM AUTHOR]

Abstract: We evaluated a novel strategy for high-sensitivity DNA fragment analysis in a conventional glass double-T microfluidic chip. The microchip allows for a DNA on-channel concentration based on base stacking (BS) with a microchip capillary gel electrophoretic (MCGE) separation step in a poly(vinylpyrrolidone) (PVP) sieving matrix. Depending if low conductivity caused a neutralization reaction between the hydroxide ions and the run buffer component Tris+, the stacking of DNA fragments were processed in the microchip. Compared to a conventional MCGE separation with a normal electrokinetic injection, the peak heights of 50–2650-base pair (bp) DNA fragments on the MCGE-BS separation were increased 3.9–8.0-fold. When we applied the MCGE-BS method to the analysis of a clinical sample of bovine theileria after PCR reaction, the peak height intensity of the amplified 816-bp DNA fragment from the 18S rRNA of T. buffeli was enhanced 7.0-fold compared to that of the normal injection method. [Copyright &y& Elsevier]

Stability of duplex DNA with respect to separation of complementary strands is crucial for DNA executing its major functions in the cell and it also plays a central role in major biotechnology applications of DNA: DNA sequencing, polymerase chain reaction, and DNA microarrays. Two types of interaction are well known to contribute to DNA stability: stacking between adjacent base-pairs and pairing between complementary bases. However, their contribution into the duplex stability is yet to be determined. Now we fill this fundamental gap in our knowledge of the DNA double helix. We have prepared a series of 32, 300 bp-long DNA fragments with solitary nicks in the same position differing only in base-pairs flanking the nick. Electrophoretic mobility of these fragments in the gel has been studied. Assuming the equilibrium between stacked and unstacked conformations at the nick site, all 32 stacking free energy parameters have been obtained. Only ten of them are essential and they govern the stacking interactions between adjacent base-pairs in intact DNA double helix. A full set of DNA stacking parameters has been determined for the first time. From these data and from a well-known dependence of DNA melting temperature on G·C content, the contribution of base-pairing into duplex stability has been estimated. The obtained energy parameters of the DNA double helix are of paramount importance for understanding sequence-dependent DNA flexibility and for numerous biotechnology applications. [Copyright &y& Elsevier]

Circular dichroism is commonly used to investigate the conformations of nucleic acids. However, many biochemical processes implicate conformational changes of particular nucleotide residues within DNA or RNA that cannot be studied by this method, because the CD of these residues is buried in the total signal of the polynucleotide. Here, we report a method to study local conformations of DNA or RNA that is based on the use of the CD of 2-aminopurine (AP) residues as a probe. AP is readily incorporated into DNA in place of adenine and does not significantly after DNA structure. Unlike adenine, AP is fluorescent and this property has been used for many years to investigate local nucleic acid structure. We show here that the CD spectrum of AP dinucleotide, (AP)2, exhibits a positive CD band at 326 nm, a spectral region in which nucleic acids (and proteins) do not absorb. Our results show that the bases of (AP)2 are stacked in a right-handed helical conformation. A low-energy CD band is also observed when this nucleotide dimer is incorporated into double-stranded DNA. Control experiments show that this signal comes from the stacking of adjacent AP residues. We have used this CD signal to provide information about the conformation of the AP dinucleotide at a defined position within single- and double-stranded nucleic acids. [ABSTRACT FROM AUTHOR]