Non-standard Base Research Articles

Study of Wear Resistance of a Radial Bearing Covered by a Polymer Coating with an Axial Groove on a Nonstandard Base Surface

This paper is devoted to study of increasing the wear resistance of a radial plain bearing. The operation of a bearing is considered in the hydrodynamic mode by means of application of an antifriction polymer composite coating with an axial groove and the micropolar properties on a nonstandard bearing surface adapted to the friction conditions of a bearing bush. The effect of pressure and temperature in the turbulent friction mode on the rheological properties of the lubricant is taken into account. Based on the equation for the micropolar fluid flow in a “thin layer” as well as on the dependence of the micropolar lubricant on the pressure and temperature and on the continuity equation, a self-similar solution has been found taking into account the axial groove on the surface of a bearing bush and without taking into account the axial groove. As a result, the velocity and pressure fields in the axial groove and on the surface of a polymer antifriction composite coating have been determined as has the load capacity and friction force, which make it possible to increase the load capacity, reduce the friction coefficient (increase wear resistance), and also increase the duration of the hydrodynamic mode. The results of numerical analysis of theoretical models and experimental evaluation of the suggested design are presented to verify and confirm the efficiency of the models obtained.

Study of Wear Resistance of a Radial Bearing Covered by a Polymer Coating with an Axial Groove on a Nonstandard Base Surface

This paper is devoted to study of increasing the wear resistance of a radial plain bearing. The operation of a bearing is considered in the hydrodynamic mode by means of application of an antifriction polymer composite coating with an axial groove and the micropolar properties on a nonstandard bearing surface adapted to the friction conditions of a bearing bush. The effect of pressure and temperature in the turbulent friction mode on the rheological properties of the lubricant is taken into account. Based on the equation for the micropolar fluid flow in a “thin layer” as well as on the dependence of the micropolar lubricant on the pressure and temperature and on the continuity equation, a self-similar solution has been found taking into account the axial groove on the surface of a bearing bush and without taking into account the axial groove. As a result, the velocity and pressure fields in the axial groove and on the surface of a polymer antifriction composite coating have been determined as has the load capacity and friction force, which make it possible to increase the load capacity, reduce the friction coefficient (increase wear resistance), and also increase the duration of the hydrodynamic mode. The results of numerical analysis of theoretical models and experimental evaluation of the suggested design are presented to verify and confirm the efficiency of the models obtained.

A New Perspective on the Maillard Reaction and the Origin of Life

The Maillard reaction, a spontaneous 'one pot' reaction between amino acids and reducing sugars that occurs at low reactant concentrations and low temperatures, is a good candidate for having played a role in the origin of life on the Earth. In view of the probability that RNA and DNA were preceded by an evolutionary forerunner with a more straightforward prebiotic synthesis, it is a testament to the prescience of Oró and colleagues that, in 1975, they drew attention to the Maillard reaction, in particular evidence that melanoidin polymers (the end-product of the reaction) contain '…heterocyclic nitrogen compounds similar to the nitrogenous bases' (Nissenbaum in J Mol Evol 6:253-270, 1975). Indeed, reports of the Maillard reaction product, 2-Acetyl-6-(Hydroxymethyl)-5,6-Dihydro-4H-Pyridinone (AHDP), with a structure reminiscent of the pyrimidine nucleobase uracil, suggest the Maillard reaction might have played a key role in the synthesis of components of a proto-RNA polymer, with AHDP and two structurally related products predicted to be similar to uracil in the latter's ability to form non-standard base pair interactions. It is possible that the primary function of these interactions was to allow molecules such as AHDP to separate out of the prebiotic chemical clutter. If this were the case, catalysis, and coding-made possible by the polymerization of proto-nucleoside monomers into linear sequence strings-would have been evolving properties.

Genotoxic effects of PM10 and PM2.5 bound metals: metal bioaccessibility, free radical generation, and role of iron.

The present study was undertaken to examine the possible genotoxicity of ambient particulate matter (PM10 and PM2.5) in Pune city. In both size fractions of PM, Fe was found to be the dominant metal by concentration, contributing 22% and 30% to the total mass of metals in PM10 and PM2.5, respectively. The speciation of soluble Fe in PM10 and PM2.5 was investigated. The average fraction of Fe3+ and Fe2+ concentrations in PM2.5 was 80.6% and 19.3%, respectively, while in PM2.5 this fraction was 71.1% and 29.9%, respectively. The dominance of Fe(III) state in both PM fractions facilitates the generation of hydroxyl radicals (·OH), which can damage deoxyribose nucleic acid (DNA), as was evident from the gel electrophoresis study. The DNA damage by ·OH was supported through the in silico density functional theory (DFT) method. DFT results showed that C8 site of guanine (G)/adenine (A) and C6 site of thymine (T)/cytosine (C) would be energetically more favorable for the attack of hydroxyl radicals, when compared with the C4 and C5 sites. The non-standard Watson-Crick base pairing models of oxidative products of G, A, T and C yield lower-energy conformations than canonical dA:dT and dG:dC base pairing. This study may pave the way to understand the structural consequences of base-mediated oxidative lesions in DNA and its role in human diseases.

Ataluren stimulates ribosomal selection of near-cognate tRNAs to promote nonsense suppression

A premature termination codon (PTC) in the ORF of an mRNA generally leads to production of a truncated polypeptide, accelerated degradation of the mRNA, and depression of overall mRNA expression. Accordingly, nonsense mutations cause some of the most severe forms of inherited disorders. The small-molecule drug ataluren promotes therapeutic nonsense suppression and has been thought to mediate the insertion of near-cognate tRNAs at PTCs. However, direct evidence for this activity has been lacking. Here, we expressed multiple nonsense mutation reporters in human cells and yeast and identified the amino acids inserted when a PTC occupies the ribosomal A site in control, ataluren-treated, and aminoglycoside-treated cells. We find that ataluren's likely target is the ribosome and that it produces full-length protein by promoting insertion of near-cognate tRNAs at the site of the nonsense codon without apparent effects on transcription, mRNA processing, mRNA stability, or protein stability. The resulting readthrough proteins retain function and contain amino acid replacements similar to those derived from endogenous readthrough, namely Gln, Lys, or Tyr at UAA or UAG PTCs and Trp, Arg, or Cys at UGA PTCs. These insertion biases arise primarily from mRNA:tRNA mispairing at codon positions 1 and 3 and reflect, in part, the preferred use of certain nonstandard base pairs, e.g., U-G. Ataluren's retention of similar specificity of near-cognate tRNA insertion as occurs endogenously has important implications for its general use in therapeutic nonsense suppression.

Assessment of the seismic behaviour of braced steel storage racking systems by means of full scale push over tests

Pallet racking systems, made of thin-walled cold formed steel profiles, are commonly used to store valuable goods and products in the logistics industry. In service conditions, longitudinal (down-aisle) stability of the racks is provided by the flexural stiffness of the demountable beam-upright column connections and base joints. Rack designers usually prefer to avoid bracings for a full accessibility of shelves from both aisles. However, under seismic conditions, typical rack connections cannot often provide sufficient flexural performance in terms of stiffness and strength, which deems necessary to introduce spine (longitudinal) bracings in the down-aisle direction. Yet the racks can only be braced at one of their two longitudinal planes to allow pallet loading from the aisle, which results in an asymmetric horizontal bearing configuration. This combined with their perforated upright columns, and non-standard beam-upright and base connections make it even more difficult to estimate their complex global seismic performance. Therefore, full scale experimental investigations are strongly needed in order to understand and quantify the global performance of the braced storage racks, and improve their design for seismic actions.This paper presents the experimental results of the Europe's largest full-scale push-over testing program that has been carried out on racking systems. In particular, experimental global capacity curves of 6 fully-loaded pallet racking specimens with spine bracings, provided by 5 different international rack producers, are presented, discussing the key factors influencing the racks’ response, as well as the failure mechanisms of the different rack typologies. Furthermore, behaviour factor (q) values of each specimen are derived from re-analysis of test results. Vulnerability of braced racks to bracing connection failure is demonstrated, highlighting its causes. Design indications are provided in order to guarantee a globally homogenous ductility under seismic actions.

RNA folding with hard and soft constraints.

BackgroundA large class of RNA secondary structure prediction programs uses an elaborate energy model grounded in extensive thermodynamic measurements and exact dynamic programming algorithms. External experimental evidence can be in principle be incorporated by means of hard constraints that restrict the search space or by means of soft constraints that distort the energy model. In particular recent advances in coupling chemical and enzymatic probing with sequencing techniques but also comparative approaches provide an increasing amount of experimental data to be combined with secondary structure prediction.ResultsResponding to the increasing needs for a versatile and user-friendly inclusion of external evidence into diverse flavors of RNA secondary structure prediction tools we implemented a generic layer of constraint handling into the ViennaRNA Package. It makes explicit use of the conceptual separation of the “folding grammar” defining the search space and the actual energy evaluation, which allows constraints to be interleaved in a natural way between recursion steps and evaluation of the standard energy function.ConclusionsThe extension of the ViennaRNA Package provides a generic way to include diverse types of constraints into RNA folding algorithms. The computational overhead incurred is negligible in practice. A wide variety of application scenarios can be accommodated by the new framework, including the incorporation of structure probing data, non-standard base pairs and chemical modifications, as well as structure-dependent ligand binding.Electronic supplementary materialThe online version of this article (doi:10.1186/s13015-016-0070-z) contains supplementary material, which is available to authorized users.

DNA Duplex Stability of the Thio-Iso-Guanine•Methyl-Iso-Cytosine Base Pair

We report the synthesis, incorporation into oligonucleotides, and base-pairing properties of the 2-thio-variant of iso-guanine. Iso-guanine is the purine component of a nonstandard base pair with 5-methyl-iso-cytosine. The 2-thio-iso-guanine•5-methyl-iso-cytosine base pair is found to have similar stability to an adenine•thymine pair.

Assessment of chemical content of base oil blends used as alternative diesel fuel for environmental safety

Increases in crude oil prices have led the transportation sector drivers to seek alternative cheaper fuels for diesel engines. A blend of virgin and/or non-standard base oils with diesel fuel called Number 10 lube (NTL) has been widely used for such purposes in public buses or trucks all over Turkey. This study was conducted as a first step to assess the occurrence, spatial distribution, and potential sources of Ag, As, Ba, Bi, Cd, Cl, Co, Cr, Fe, Li, Mn, Mo, Ni, Pb, Rb, Se, Sr, Tl, V, and Zn in this generic oil to make further evaluation of environmental and public health. A microwave-assisted combustion procedure was applied for the determination of metals and metalloids in NTL samples by inductively coupled plasma-mass spectrometry while a field test method was used for Cl. The range of total metallic/metalloid elements and chlorine concentration was between 0.04 and 189μg/g, and from below detection to 825μg/g, respectively. Unexpectedly, samples varied in types and levels of constituents between the western and eastern parts of the country. The enrichment of Zn, Mo, and Cl in NTL samples suggests that some sort of waste oils, lubricating oils, chlorinated solvents, or transformer oils were mixed with base oils. The metal emission rates derived from the annual consumption of NTLs were far beyond the estimations for diesel vehicles and industrial sources. Therefore, the problem, which leads to financial, environmental, and health concerns in Turkey, could probably be experienced in other countries and, thus, should be managed properly.

Conversion Strategy Using an Expanded Genetic Alphabet to Assay Nucleic Acids

Methods to detect DNA and RNA (collectively xNA) are easily plagued by noise, false positives, and false negatives, especially with increasing levels of multiplexing in complex assay mixtures. Here, we describe assay architectures that mitigate these problems by converting standard xNA analyte sequences into sequences that incorporate nonstandard nucleotides (Z and P). Z and P are extra DNA building blocks that form tight nonstandard base pairs without cross-binding to natural oligonucleotides containing G, A, C, and T (GACT). The resulting improvements are assessed in an assay that inverts the standard Luminex xTAG architecture, placing a biotin on a primer (rather than on a triphosphate). This primer is extended on the target to create a standard GACT extension product that is captured by a CTGA oligonucleotide attached to a Luminex bead. By using conversion, a polymerase incorporates dZTP opposite template dG in the absence of dCTP. This creates a Z-containing extension product that is captured by a bead-bound oligonucleotide containing P, which binds selectively to Z. The assay with conversion produces higher signals than the assay without conversion, possibly because the Z/P pair is stronger than the C/G pair. These architectures improve the ability of the Luminex instruments to detect xNA analytes, producing higher signals without the possibility of competition from any natural oligonucleotides, even in complex biological samples.

Incorporation of non-natural nucleotides into template-switching oligonucleotides reduces background and improves cDNA synthesis from very small RNA samples

BackgroundThe template switching PCR (TS-PCR) method of cDNA synthesis represents one of the most straightforward approaches to generating full length cDNA for sequencing efforts. However, when applied to very small RNA samples, such as those obtained from tens or hundreds of cells, this approach leads to high background and low cDNA yield due to concatamerization of the TS oligo.ResultsIn this study, we describe the application of nucleotide isomers that form non-standard base pairs in the template switching oligo to prevent background cDNA synthesis. When such bases are added to the 5' end of the template switching (TS) oligo, they inhibit MMLV-RT from extending the cDNA beyond the TS oligo, thus increasing cDNA yield by reducing formation of concatamers of the TS oligo that are the source of significant background.ConclusionsOur results demonstrate that this novel approach for cDNA synthesis has valuable utility for application of ultra-high throughput technologies, such as whole transcriptome sequencing using 454 technology, to very small biological samples comprised of tens of cells as might be obtained via approaches like laser microdissection.

Examination of the Effect of the Annealing Cation on Higher Order Structures Containing Guanine or Isoguanine Repeats

Isoguanine (2-oxo-6-amino-guanine), a natural but non-standard base, exhibits unique self-association properties compared to its isomer, guanine, and results in formation of different higher order DNA structures. In this work, the higher order structures formed by oligonucleotides containing guanine repeats or isoguanine repeats after annealing in solutions containing various cations are evaluated by electrospray ionization mass spectrometry (ESI-MS) and circular dichroism (CD) spectroscopy. The guanine-containing strand (G9) consistently formed quadruplexes upon annealing, whereas the isoguanine strand (Ig9) formed both pentaplexes and quadruplexes depending on the annealing cation. Quadruplex formation with G9 showed some dependence on the identity of the cation present during annealing with high relative quadruplex formation detected with six of ten cations. Analogous annealing experiments with Ig9 resulted in complex formation with all ten cations, and the majority of the resulting complexes were pentaplexes. CD results indicated most of the original complexes survived the desalting process necessary for ESI-MS analysis. In addition, several complexes, especially the pentaplexes, were found to be capable of cation exchange with ammonium ions. Ab initio calculations were conducted for isoguanine tetrads and pentads coordinated with all ten cations to predict the most energetically stable structures of the complexes in the gas phase. The observed preference of forming quadruplexes versus pentaplexes as a function of the coordinated cation can be interpreted by the calculated reaction energies of both the tetrads and pentads in combination with the distortion energies of tetrads.

Conserved unpaired adenine residues are important for ordered structures of 5S ribosomal RNA. An infrared study of the secondary and tertiary structure of Thermus thermophilus 5S rRNA.

An improved set of infrared calibration spectra for the determination of G X C and A X U base pairs leads to 32 +/- 3 G X C (+ G X U) and 4 +/- 1 A X U base pairs for Thermus thermophilus 5S RNA in the presence and absence of Mg2+. These results give further support for the consensus secondary structure of 5S RNA recently proposed by several groups. T. thermophilus 5S RNA shows, in the presence of Mg2+, a distinct two-step thermal melting of its ordered structure. Based on new data about the stacking dependence of infrared intensities of unpaired ribonucleotides the spectral changes of the low-temperature transition should be explained by melting of stacked arrangements of unpaired bases and/or non-standard base pairs. Striking is the reduction in A stacking, which is not related to the melting of A X U base pairs, indicating the importance of the mostly conserved unpaired adenines for the Mg2+ stabilized higher-order structures especially within internal loops of 5S RNA.

Non-standard base pairing and stacked structures in methyl xanthine clusters

We present resonant two-photon ionization and IR-UV double resonance spectra of methylated xanthine derivatives including 7-methylxanthine dimer and theobromine dimer seeded in a supersonic jet by laser desorption. For 7-methylxanthine, theophylline and theobromine monomer we assign the lowest energy tautomer based on comparison with IR-UV double resonance spectra and calculated IR frequencies. For the 7-methylxanthine dimer, we observe hydrogen bonding on the N3H position suggesting 3 possible combinations, one that is reverse Watson-Crick type and two that are reverse Hoogsteen type. For the theobromine dimer, we observe a stacked structure. For trimethylxanthine dimers we infer a stacked structure as well.

Influence of Secondary Structure on Electronic Energy Relaxation in Adenine Homopolymers

Electronic energy relaxation in the adenine homopolymers poly(A) and poly(dA), as well as in the adenine mononucleotide, was studied by the femtosecond transient absorption technique and by steady-state absorption and emission spectroscopies in aqueous solution. The excited-state lifetime of the adenine mononucleotide was determined to be 370 ± 40 fs at room temperature. Strikingly, the singlet excited states formed in adenine homopolymers decay on time scales ranging from femtoseconds to nanoseconds, and three decay components are required to adequately describe their transient signals. Increasing the temperature decreases the amplitude of the two slowest decay components, indicating that the long-lived excited states are formed in base-stacked regions of the polymers. At room temperature and neutral pH, the two slowest decay components have significantly larger amplitudes in poly(dA) than in poly(A). No evidence is obtained for excited-state quenching of the adenine chromophore by the nonstandard base p...

Nucleic acid analysis using an expanded genetic alphabet to quench fluorescence.

Organic chemistry has made possible the synthesis of molecules that expand on Nature's genetic alphabet. Using the previously described nonstandard DNA base pair constructed from isoguanine and 5-methylisocytosine, we report a highly specific and sensitive method that allows for the fast and specific quantitation of genetic sequences in a closed tube format. During PCR amplification, enzymatic site-specific incorporation of a quencher covalently linked to isoguanine allows for the simultaneous detection and identification of multiple targets. The specificity of method is then established by analysis of thermal denaturation or melting of the amplicons. The appropriate functions of all reactions are further verified by incorporation of an independent target into the reaction mixture. We report that the method is sensitive down to the single copy level, and specificity is demonstrated by multiplexed end-point genotypic analysis of four targets simultaneously using four separate fluorescent reporters. The method is general enough for quantitative and qualitative analysis of both RNA and DNA using previously developed primer sets. Though the method described employs the commonly used PCR, the enzymatic incorporation of reporter groups into DNA site-specifically should find broad utility throughout molecular biology.

PCR amplification of DNA containing non-standard base pairs by variants of reverse transcriptase from Human Immunodeficiency Virus-1.

As the next step towards generating a synthetic biology from artificial genetic information systems, we have examined variants of HIV reverse transcriptase (RT) for their ability to synthesize duplex DNA incorporating the non-standard base pair between 2,4-diaminopyrimidine (pyDAD), a pyrimidine presenting a hydrogen bond 'donor-acceptor-donor' pattern to the complementary base, and xanthine (puADA), a purine presenting a hydrogen bond 'acceptor-donor-acceptor' pattern. This base pair fits the Watson-Crick geometry, but is joined by a pattern of hydrogen bond donor and acceptor groups different from those joining the GC and AT pairs. A variant of HIV-RT where Tyr 188 is replaced by Leu, has emerged from experiments where HIV was challenged to grow in the presence of drugs targeted against the RT, such as L-697639, TIBO and nevirapine. These drugs bind at a site near, but not in, the active site. This variant accepts the pyDAD-puADA base pair significantly better than wild type HIV-RT, and we used this as a starting point. A second mutation, E478Q, was introduced into the Y188L variant, in the event that the residual nuclease activity observed is due to the RT, and not a contaminant. The doubly mutated RT incorporated the non-standard pair with sufficient fidelity that the variant could be used to amplify oligonucleotides containing pyDAD and puADA through several rounds of a polymerase chain reaction (PCR) without losing the non-standard base pair. This is the first time where DNA containing non-standard base pairs with alternative hydrogen bonding patterns has been amplified by a full PCR. This work also illustrates a research strategy that combines in clinico pre-evolution of proteins followed by rational design to obtain an enzyme that meets a particular technological specification.

Recognition of a non-standard base pair by thermostable DNA polymerases.

Examination of several commercially available thermostable DNA polymerases identifies 9 degrees N DNA polymerase as single enzyme that could incorporate two components of an expanded genetic alphabet, 2,4-diaminopyrimidine and xanthosine as deoxynucleoside triphosphate opposite their cognate base in a DNA template.

Non-Watson-Crick base associations in DNA and RNA revealed by single crystal x-ray diffraction methods: Mismatches, modified bases, and nonduplex DNA

Single crystal x-ray diffraction methods have been used to characterize numerous oligonucleotide structures, providing valuable information on the fine structure of DNA, oligonucleotide hydration, interactions with small molecule ligands and proteins. There has been a particular focus on nonstandard base associations and a number of groups have sought to characterize different non-Watson-Crick base pairs to further the understanding of their influence on the structure of duplex DNA and RNA, and to investigate which structural features might be utilized by enzymes in recognition and repair of these errors in DNA. Bases that have been chemically damaged by mutagenic or carcinogenic agents have distinctive modified hydrogen-bonding patterns and these have been investigated. The structure determination of a series of nonduplex DNA structures including examples of a triplex, quadruplexes, and a novel DNA loop have recently been published. In this article we survey the structures of a series of non-Watson-Crick base associations in duplex DNA and RNA. We show how nonstandard base pairs, base triads, and tetrads play an important role in stabilizing nonduplex structures. © 1997 John Wiley & Sons, Inc. Biopoly 44: 91–103, 1997

HMG box proteins interact with multiple tandemly repeated (GCC)n (GGC)m DNA sequences.

A number of tandemly repeated DNA sequences have the ability to form hairpin structures by forming non-standard base pairs. When (GCC)15 and (GGC)15 strands are annealed together, the expected duplex is the only product. However, when (GCC)15 is annealed with (GCC)10, depending on the relative concentrations, up to five complexes can be detected in native gels. Three of these species are susceptible to limited digestion by Exo VII, suggesting they are duplexes containing single stranded tails. The remaining two bands are resistant to the enzyme, and have low mobility on native gels, consistent with branched structures. The latter complexes bind HMG box proteins, members of a highly abundant class of non-histone proteins of the nucleus. These proteins, modeled in this study by the second box fragment from rat HMG1, HMGb, interact strongly with branched or chemically modified DNA, relative to normal duplexes. The expansion of triplet repeats in genomic DNA is associated with tumor formation as well with a variety of heritable neurologiocal disorders. It is our thesis that the stability of branched intermediate structures that arise in replication of these sequences and promote expansion can be influenced directly by the presence of two highly abundant proteins in the cell nucleus: the HMG box proteins, HMG1/2, and the histone H1, which associates with HMG1/2.