Residue Interactions Affecting The Deprotonation of Internal Guanine Moieties In Oligodeoxyribonucleotides, Calculated By FMO Methods

The effect of vicinal molecular groups on the intrinsic acidity of a central guanine residue in short single-stranded DNA models, and the potentials exerted by the backbone and the nucleobases on the leaving proton were determined by the Fragment Molecular Orbital (FMO) method, in terms of quantum descriptors (QD) and pair interaction interfragment decomposition analysis (PIEDA). The acidity of the central guanine moiety decreased with increasing oligonucleotide length, in response to changes by less than1 eV in the ionization potential, global softness, electrophilicity index and electronegativity descriptors. The differences in these descriptors were majorly interpreted in terms of the electrostatic influence of the negative charges residing on the backbone of the molecule. Additionally, this electric-field effect was determined explicitly for the displacement of the test hydronium ion to a distance of 250 Å from its original position, resulting in good agreement with calculations of the variation in Gibbs free energies, obtained from physical experiments conducted on the identical oligonucleotide sequences. The reported results are useful for biophysical applications of deoxyriboligonucleotides containing guanine residues in order to induce local negative charges at specific positions in the DNA chain.

Alkaline Agarose Gel Electrophoresis

Alkaline agarose gels are run at high pH, which causes each thymine and guanine residue to lose a proton and thus prevents the formation of hydrogen bonds with their adenine and cytosine partners. The denatured DNA is maintained in a single-stranded state and migrates through an alkaline agarose gel as a function of its size. Other denaturants such as formamide and urea do not work well because they cause the agarose to become rubbery.

Stimuli-Responsive Nucleotide–Amino Acid Hybrid Supramolecular Hydrogels

The ability to assemble chemically different gelator molecules into complex supramolecular hydrogels provides excellent opportunities to construct functional soft materials. Herein, we demonstrate the formation of hybrid nucleotide–amino acid supramolecular hydrogels. These are generated by the silver ion (Ag+)-triggered formation of silver–guanosine monophosphate (GMP) dimers, which undergo self-assembly through non-covalent interactions to produce nanofilaments. This process results in a concomitant pH reduction due to the abstraction of a proton from the guanine residue, which triggers the in situ gelation of a pH-sensitive amino acid, N-fluorenylmethyloxycarbonyl tyrosine (FY), to form nucleotide–amino acid hybrid hydrogels. Alterations in the supramolecular structures due to changes in the assembly process are observed, with the molar ratio of Ag:GMP:FY affecting the assembly kinetics, and the resulting supramolecular organisation and mechanical properties of the hydrogels. Higher Ag:GMP stoichiometries result in almost instantaneous gelation with non-orthogonal assembly of the gelators, while at lower molar ratios, orthogonal assembly is observed. Significantly, by increasing the pH as an external stimulus, nanofilaments comprising FY can be selectively disassembled from the hybrid hydrogels. Our results demonstrate a simple approach for the construction of multicomponent stimuli-responsive supramolecular hydrogels with adaptable network and mechanical properties.

Evaluation of Antibiotic Residue in Cow Milk and Its Impact on Danio Rerio

Ceftriaxone (CEFT), a widely used wide-spectrum beta -lactam cephalosporin antibiotic, is used to treat bovine mastitis, which is caused by a variety of bacteria. When this antibiotic is used injudiciously, it leaves a residue that persists after pasteurization. Antibiotic residue contamination occurs when antibiotic residue exceeds its Maximum Residue Limits (MRLs).This has a negative impact on both public health and the environment. The aim of a recent study was to determine the concentration of ceftriaxone residue (CEFTR) in raw and pasteurized mastitis cow milk, as well as its role in developmental toxicity and genotoxicity in zebra fish model. CEFTR concentrations in raw and pasteurized milk were several times higher than CEFT's MRL. CEFTR showed a decrease in body length and yolk sac region of zebra fish larvae 7-amino cephalosporanic acid (7-ACA), C3 and C7 are the cephalosporin constituents that produced by the breakdown of CEFT that may present in CEFTR, have an impact on the zebra fish embryo in this stage of development. Comet Assay or single cell gel electrophoresis (SCGE) also exhibited highest percentage of tail DNA, and tail moment that is the ultimate indicator of DNA damage by breaking DNA strands and incorporating guanine residue in the genome that ultimately damages DNA. As a result, the CEFTR is extremely concerning for public health and the environment. The toxic effects of the CEFTR in zebra fish model have not been studied yet. This is the first comprehensive study.

Analysis of biomolecules in cochineal dyed archaeological textiles by surface-enhanced Raman spectroscopy

SERS spectroscopy is successfully employed in this work to reveal different components integrating the cochineal colorant employed for dying archaeological textile samples from the Arica Region in North Chile. This analysis was done by in-situ experiments that does not imply the material (colorant and biomolecules) extraction. The spectroscopic analysis of the archaeological textiles by SERS reveals the presence of bands attributed to carminic acid and nucleobases, mainly adenine and guanine. The identification of these biomolecules was also verified in raw cochineal extract and in cochineal dyed replica wool fibers fabricated by us following ancient receipts. The effect of Al on the complexation of carminic acid and other biomolecules was also tested in order to understand the changes induced by the metal interaction on the colorant structure. This study revealed that Al can also complex biomolecules existing in the cochineal extract. In particular, guanine residue seems to interact strongly with the metal, since SERS bands of this residue are enhanced. Furthermore, a theoretical analysis on the interaction of carminic acid and a silver surface was also performed in order to better understand the interaction mechanism between carminic acid and a metal surface that leads to the final SERS spectrum. The results of the present work will be very useful in the identification of different molecules and metal complexes that may be forming part of the cochineal colorant found in archaeological materials.

Analysis of Biomolecules in Cochineal Dyed Archaeological Textiles by Surface-Enhanced Raman Spectroscopy.

SERS spectroscopy is successfully employed in this work to reveal different components integrating the cochineal colorant employed for dying archaeological textile samples from the Arica region in North Chile. This analysis was done by in-situ experiments that does not imply the material (colorant and biomolecules) extraction. The spectroscopic analysis of the archaeological textiles by SERS reveals the presence of bands attributed to carminic acid and nucleobases, mainly adenine and guanine. The identification of these biomolecules was also verified in raw cochineal extract and in cochineal dyed replica wool fibers fabricated by us following ancient receipts. The effect of Al on the complexation of carminic acid and other biomolecules was also tested in order to understand the changes induced by the metal interaction on the colorant structure. This study revealed that Al can also complex biomolecules existing in the cochineal extract. In particular, guanine residue seems to interact strongly with the metal, since SERS bands of this residue are enhanced. Furthermore, a theoretical analysis on the interaction of carminic acid and a silver surface was also performed in order to better understand the interaction mechanism between carminic acid and a metal surface that leads to the final SERS spectrum. The results of the present work will be very useful in the identification of different molecules and metal complexes that may be forming part of the cochineal colorant found in archaeological materials.

Dynamical ensemble of the active state and transition state mimic for the RNA-cleaving 8–17 DNAzyme in solution

We perform molecular dynamics simulations, based on recent crystallographic data, on the 8–17 DNAzyme at four states along the reaction pathway to determine the dynamical ensemble for the active state and transition state mimic in solution. A striking finding is the diverse roles played by Na+ and Pb2+ ions in the electrostatically strained active site that impact all four fundamental catalytic strategies, and share commonality with some features recently inferred for naturally occurring hammerhead and pistol ribozymes. The active site Pb2+ ion helps to stabilize in-line nucleophilic attack, provides direct electrostatic transition state stabilization, and facilitates leaving group departure. A conserved guanine residue is positioned to act as the general base, and is assisted by a bridging Na+ ion that tunes the pKa and facilitates in-line fitness. The present work provides insight into how DNA molecules are able to solve the RNA-cleavage problem, and establishes functional relationships between the mechanism of these engineered DNA enzymes with their naturally evolved RNA counterparts. This adds valuable information to our growing body of knowledge on general mechanisms of phosphoryl transfer reactions catalyzed by RNA, proteins and DNA.

RNA ligands activate the Machupo virus polymerase and guide promoter usage

Segmented negative-sense (SNS) RNA viruses initiate infection by delivering into cells a suite of genomic RNA segments, each sheathed by the viral nucleocapsid protein and bound by the RNA-dependent RNA-polymerase (RdRP). For the orthomyxovirus influenza and the bunyavirus La Crosse, the 5′ end of the genomic RNA binds as a hook-like structure proximal to the active site of the RdRP. Using an in vitro assay for the RNA-dependent RNA-polymerase (RdRP) of the arenavirus Machupo (MACV), we demonstrate that the 5′ genomic and antigenomic RNAs of both small and large genome segments stimulate activity in a promoter-specific manner. Functional probing of the activating RNAs identifies intramolecular base-pairing between positions +1 and +7 and a pseudotemplated 5′ terminal guanine residue as key for activation. Binding of structured 5′ RNAs is a conserved feature of all SNS RNA virus polymerases, implying that promoter-specific RdRP activation extends beyond the arenaviruses. The 5′ RNAs and the RNA binding pocket itself represent targets for therapeutic intervention.

Towards Understanding of Polymorphism of the G-rich Region of Human Papillomavirus Type 52

The potential to affect gene expression via G-quadruplex stabilization has been extended to all domains of life, including viruses. Here, we investigate the polymorphism and structures of G-quadruplexes of the human papillomavirus type 52 with UV, CD and NMR spectroscopy and gel electrophoresis. We show that oligonucleotide with five G-tracts folds into several structures and that naturally occurring single nucleotide polymorphisms (SNPs) have profound effects on the structural polymorphism in the context of G-quadruplex forming propensity, conformational heterogeneity and folding stability. With help of SNP analysis, we were able to select one of the predominant forms, formed by G-rich sequence d(G3TAG3CAG4ACACAG3T). This oligonucleotide termed HPV52(1–4) adopts a three G-quartet snap back (3 + 1) type scaffold with four syn guanine residues, two edgewise loops spanning the same groove, a no-residue V loop and a propeller type loop. The first guanine residue is incorporated in the central G-quartet and all four-guanine residues from G4 stretch are included in the three quartet G-quadruplex core. Modification studies identified several structural elements that are important for stabilization of the described G-quadruplex fold. Our results expand set of G-rich targets in viral genomes and address the fundamental questions regarding folding of G-rich sequences.