Search

Cytosine-phosphate-guanine (CpG) DNA is known to increase the potency of vaccines. Here, in vitro and in vivo stimulation of toll-like receptor 9 by CpG DNA incorporated into polypod-like DNA nanostructures was evaluated by measuring the levels of tumor necrosis factor alpha released from macrophage-like RAW 264.7 cells and plasma interleukin (IL)-12p40 in vivo following intravenous injection into mice. Phosphodiester CpG1668 was selected as the CpG DNA, and tripodna and hexapodna, which were CpG1668-containing tripod and hexapod-like DNA nanostructures, respectively, were designed. CpG-tripodna and CpG-hexapodna induced tumor necrosis factor alpha release from RAW 264.7 cells about 10- and ∼30-fold higher than single-stranded CpG1668 (CpG-SS). Moreover, in all cases examined, plasma IL-12p40 concentrations increased after intravenous injection into mice, with peak levels depending on the samples and the doses. The area under the plasma concentration-time curves indicated that the CpG-hexapodna was approximately 20-fold more efficient in inducing IL-12p40 production than CpG-SS. The efficiency of CpG-tripodna and CpG-hexapodna to increase the potency of CpG-SS in vivo was comparable to that observed in cultured RAW 264.7 cells. These results provide experimental evidence that in vitro studies can be used to estimate the in vivo immunostimulatory activity of CpG DNA incorporated into DNA nanostructures. [ABSTRACT FROM AUTHOR]

Internucleoside amide linkages are excellent mimics of phosphodiesters in RNA and may be used to optimize the properties of short interfering RNAs. Herein we report a remarkably straightforward, efficient and step economic synthesis of C3'-homologated uridine and adenosine amino acids starting from nucleosides in six steps (31% overall yield) and eight steps (16% overall yield), respectively. The key enabling step is a one-pot multifunctional group transformation including a stereoselective hydrogenation, termed "Global Hydrogenation". [ABSTRACT FROM AUTHOR]

Cholesterol (Chol) conjugation to the 5′ or 3′ end of antisense oligonucleotide (ASO) enables delivery to the liver, and Chol conjugation at the gap region can also be expected to improve delivery to the liver. In this study, we synthesized ASOs bearing the Chol-conjugated thiono triester and evaluated their activity and hepatic accumulation. We found that Chol conjugations at the gap region improved in vitro activity and hepatic accumulation when compared to unconjugated ASOs. However, Chol conjugation with phosphorothioate linkage did not improve in vivo activity in the liver, suggesting the importance of cleaving the phosphodiester between ASO and Chol. These results offer useful information for tuning the oligonucleotide structure to improve pharmaceutical properties and designing ASOs for multiple ligand conjugations and combinations with end modification. [ABSTRACT FROM AUTHOR]

2-Deoxy-2-fluoromannosyl phosphotriester and phosphodiester derivatives were synthesized and they were demonstrated to be more stable than the 2-OH compounds under acidic conditions. 2-Fluoro substituted analog of α-mannopyranosyl 1-phosphodiester 20 was found to be significantly stable at pH 1, where 2-OH compound 21 was gradually hydrolyzed. In addition, 2-F analogs of α-mannopyranosyl 1-phosphotriesters also was found to be stable under both Brønsted and Lewis acidic conditions. These results strongly suggested that the 2-F substitution is useful for both the synthesis of mannosyl phosphodiester derivatives and improvement of their chemical stability. [ABSTRACT FROM AUTHOR]

Objectives: To assess the changes in phosphodiester (PDE)-levels, detected by 31P magnetic resonance spectroscopy (MRS), over 24-months to determine the potential of PDE as marker for muscle tissue changes in Duchenne Muscular Dystrophy (DMD) patients. Methods: Spatially resolved phosphorous datasets were acquired in the right lower leg of 18 DMD patients (range: 5–15.4 years) and 12 age-matched healthy controls (range: 5–14 years) at three time-points (baseline, 12-months, and 24-months) using a 7T MR-System (Philips Achieva). 3-point Dixon images were acquired at 3T (Philips Ingenia) to determine muscle fat fraction. Analyses were done for six muscles that represent different stages of muscle wasting. Differences between groups and time-points were assessed with non-parametric tests with correction for multiple comparisons. Coefficient of variance (CV) were determined for PDE in four healthy adult volunteers in high and low signal-to-noise ratio (SNR) datasets. Results: PDE-levels were significantly higher (two-fold) in DMD patients compared to controls in all analyzed muscles at almost every time point and did not change over the study period. Fat fraction was significantly elevated in all muscles at all time points compared to healthy controls, and increased significantly over time, except in the tibialis posterior muscle. The mean within subject CV for PDE-levels was 4.3% in datasets with high SNR (>10:1) and 5.7% in datasets with low SNR. Discussion and conclusion: The stable two-fold increase in PDE-levels found in DMD patients in muscles with different levels of muscle wasting over 2-year time, including DMD patients as young as 5.5 years-old, suggests that PDE-levels may increase very rapidly early in the disease process and remain elevated thereafter. The low CV values in high and low SNR datasets show that PDE-levels can be accurately and reproducibly quantified in all conditions. Our data confirms the great potential of PDE as a marker for muscle tissue changes in DMD patients. [ABSTRACT FROM AUTHOR]

The RNA lariat debranching enzyme, Dbr1, is a metallophosphoesterase that cleaves 2′-5′ phosphodiester bonds within intronic lariats. Previous reports have indicated that Dbr1 enzymatic activity is supported by diverse metal ions including Ni2+, Mn2+, Mg2+, Fe2+, and Zn2+. While in initial structures of the Entamoeba histolytica Dbr1 only one of the two catalytic metal-binding sites were observed to be occupied (with a Mn2+ ion), recent structures determined a Zn2+/Fe2+ heterobinucleation. We solved a high-resolution X-ray crystal structure (1.8 Å) of the E. histolytica Dbr1 and determined a Zn2+/Mn2+ occupancy. ICP- AES corroborate this finding, and in vitro debranching assays with fluorescently labeled branched substrates confirm activity. [ABSTRACT FROM AUTHOR]

A versatile conjugatable/bioreduction-responsive protecting group for phosphodiester moieties was designed, synthesized and incorporated into oligonucleotide strands. Subsequently, controlled pore glass-supported oligonucleotides were conjugated to a variety of functional molecules using a copper-catalyzed azide-alkyne cycloaddition reaction. The functionalized protecting groups were deprotected by a nitroreductase/NADH reduction system to give “naked” oligonucleotides. This method allowed the synthesis of oligonucleotide prodrugs bearing the functionalized protecting group at the desired sites and desired residues on oligodeoxyribonucleotide (ODN) backbones. [ABSTRACT FROM AUTHOR]

With the rising incidences of cancer cases, the quest for new metal based anticancer drugs has led to extensive research in cancer biology. Zinc complexes of amino acid residue side chains are well recognized for hydrolysis of phosphodiester bond in DNA at faster rate. In the presented work, a Zn(II) complex of cyclen substituted with two l -tryptophan units, Zn(II)-Cyclen-(Trp) 2 has been synthesized and evaluated for antiproliferative activity. Zn(II)-Cyclen-(Trp) 2 was synthesized in ∼70% yield and its DNA binding potential was evaluated through QM/MM study which suggested good binding (G = −9.426) with B-DNA. The decrease in intensity of the positive and negative bands of CT-DNA at 278 nm and 240 nm, respectively demonstrated an effective unwinding of the DNA helix with loss of helicity. The complex was identified as an antiproliferative agent against U-87 MG cells with 5 fold increase in apoptosis with respect to control (2 h post incubation, IC 50 25 µM). Electrophoresis and comet assay studies exhibited an increase in DNA breakage after treatment with complex while caspase-3/β-actin cleavage established a caspase-3 dependent apoptosis pathway in U-87 MG cells after triggering DNA damage. In vivo tumor specificity of the developed ligand was validated after radiocomplexation with 99m Tc (>98% radiochemical yield and specific activity of 2.56 GBq/µmol). Avid tumor/muscle ratio of >6 was depicted in biodistribution and SPECT imaging studies in U-87 MG xenograft model nude mice. [ABSTRACT FROM AUTHOR]

We have previously described nanocomposites containing conjugates or complexes of native oligodeoxyribonucleotides with poly-L-lysine and TiO nanoparticles. We have shown that these nanocomposites efficiently suppressed influenza A virus reproduction in MDCK cells. Here, we have synthesized previously undescribed nanocomposites that consist of TiO nanoparticles and polylysine conjugates with oligonucleotides that contain phosphoryl guanidine or phosphorothioate internucleotide groups. These nanocomposites have been shown to exhibit antiviral activity in MDCK cells infected with H5N1 influenza A virus. The nanocomposites containing phosphorothioate oligonucleotides inhibited virus replication ~130-fold. More potent inhibition, i.e., ~5000-fold or ~4600-fold, has been demonstrated by nanocomposites that contain phosphoryl guanidine or phosphodiester oligonucleotides, respectively. Free oligonucleotides have been nearly inactive. The antiviral activity of oligonucleotides of all three types, when delivered by Lipofectamine, has been significantly lower compared to the oligonucleotides delivered in the nanocomposites. In the former case, the phosphoryl guanidine oligonucleotide has appeared to be the most efficient; it has inhibited the virus replication by a factor of 400. The results make it possible to consider phosphoryl guanidine oligonucleotides, along with other oligonucleotide derivatives, as potential antiviral agents against H5N1 avian flu virus. [ABSTRACT FROM AUTHOR]

Nucleic acid aptamers are short RNA- or DNA-based affinity reagents typically selected from combinatorial libraries to bind to a specific target such as a protein, a small molecule, whole cells or even animals. Aptamers have utility in the development of diagnostic, imaging and therapeutic applications due to their size, physico-chemical nature and ease of synthesis and modification to suit the application. A variety of oligonucleotide modifications have been used to enhance the stability of aptamers from nuclease degradation in vivo. The non-bridging oxygen atoms of the phosphodiester backbones of RNA and DNA aptamers can be substituted with one or two sulfur atoms, resulting in thioaptamers with phosphorothioate or phosphorodithioate linkages, respectively. Such thioaptamers are known to have increased binding affinity towards their target, as well as enhanced resistance to nuclease degradation. In this review, we discuss the development of phosphorothioate chemistry and thioaptamers, with a brief review of selection methods. [ABSTRACT FROM AUTHOR]

We describe the synthesis and pairing properties of the novel DNA analogue 7′,5′-bicyclo(bc)-DNA. In this analogue, the point of attachment of the connecting phosphodiester group is switched from the 3′ to the 7′ position of the underlying bicyclic sugar unit and is thus in a topological position that is inaccessible in natural DNA. The corresponding phosphoramidite building blocks carrying all natural nucleobases were synthesized and incorporated into oligonucleotides. From Tm experiments of duplexes with complementary DNA and RNA we find that single modifications are generally well tolerated with some variability as to the nature of the nucleobase. Fully modified oligonucleotides show low affinity for RNA and DNA complements. However, they form antiparallel homo-duplexes with similar thermal stability as DNA. CD spectra of the homo-duplexes show distinct changes in the helix conformation compared to natural DNA. A conformational analysis at the ab initio level of the mononucleosides revealed two minimal energy structures which primarily deviate in the conformation of the cyclopentane ring. Molecular dynamics simulation of a 7′,5′-bc-DNA homo-duplex revealed a right-handed structure with a smaller helical rise and a significantly wider minor groove compared to DNA. Interestingly, this duplex is characterized by an atypical, alternating 6′- endo/6′- exo conformational pattern of consecutive nucleotides which seems to be responsible for the poor binding to natural nucleic acids. [ABSTRACT FROM AUTHOR]

Background/Aims: Phosphorus magnetic resonance spectroscopy (31 P-MRS) has previously shown abnormal changes in energy metabolites in the brain of multiple sclerosis (MS) patients. However, the relationship between these energy metabolites - particularly adenosine triphosphate (ATP) - and the disease severity remains unclear. The objective of this study was to determine whether measuring ATP metabolites can help to predict disease severity in MS patients. Methods: 31 P-MRS at 3 tesla was performed in 9 relapsing remitting (RRMS), 9 secondary progressive MS patients (SPMS), and 10 age-matched healthy controls. ATP metabolites (expressed as %) in normally appearing white matter of the centrum semiovale were compared between patients and healthy controls. The relationship between Expanded Disability Status Scale (EDSS) and ATP metabolites was evaluated. Results: RRMS and SPMS patients had higher phosphocreatine (PCr) and lower phosphodiesters than healthy controls. In addition, RRMS patients had higher ß-ATP% than SPMS patients. ß-ATP% was negatively correlated with EDSS in all patients. Conclusion: Our findings suggest a defective PCr metabolism in both patient groups, and a higher state of energy production in RRMS that might reflect a compensatory mechanism in face of the increased needs. The correlation of ß-ATP with EDSS makes it a candidate biomarker for assessing MS disease severity. [ABSTRACT FROM AUTHOR]

Fungal ribotoxins are highly specific extracellular RNases which cleave a single phosphodiester bond at the ribosomal sarcin-ricin loop, inhibiting protein biosynthesis by interfering with elongation factors. Most ribotoxins show high degree of conservation, with similar sizes and amino acid sequence identities above 85%. Only two exceptions are known: hirsutellin A and anisoplin, produced by the entomopathogenic fungi Hirsutella thompsonii and Metarhizium anisopliae , respectively. Both proteins are similar but smaller than the other known ribotoxins (130 vs 150 amino acids), displaying only about 25% sequence identity with them. They can be considered minimized natural versions of their larger counterparts, best represented by α-sarcin. The conserved α-sarcin active site residue Tyr48 has been replaced by the geometrically equivalent Asp, present in the minimized ribotoxins, to produce and characterize the corresponding mutant. As a control, the inverse anisoplin mutant (D43Y) has been also studied. The results show how the smaller versions of ribotoxins represent an optimum compromise among conformational freedom, stability, specificity, and active-site plasticity which allow these toxic proteins to accommodate the characteristic abilities of ribotoxins into a shorter amino acid sequence and more stable structure of intermediate size between that of other nontoxic fungal RNases and previously known larger ribotoxins. [ABSTRACT FROM AUTHOR]

The rapidly developing field of bionanotechnology requires detailed knowledge of the mechanisms of interaction between inorganic matter and biomolecules. Under conditions different from those in an aqueous solution, however, the chemistry of these systems is elusive and may differ dramatically from their interactions in vitro and in vivo. Here, we report for the first time a photoemission study of a metal silver-DNA interface, formed in vacuo, in comparison with DNA-Ag+ and fluorescent DNA-Ag complexes formed in solution. The high-resolution photoelectron spectra reveal that in vacuo silver atoms interact mainly with oxygen atoms of the phosphodiester bond and deoxyribose in DNA, in contrast to the behavior of silver ions, which interact preferentially with the nitrogen atoms of the bases. This offers new insight into the mechanism of DNA metallization, which is of importance in creating metal-bio interfaces for nanotechnology applications. [ABSTRACT FROM AUTHOR]

Numerous catechol-containing polymers, including biodegradable polymers, are currently heavily discussed for modern biomaterials. However, there is no report combining poly(phosphoester)s (PPEs) with catechols. Adhesive PPEs have been prepared via acyclic diene metathesis polymerization. A novel acetal-protected catechol phosphate monomer was homo- and copolymerized with phosphoester comonomers with molecular weights up to 42000 g/mol. Quantitative release of the catechols was achieved by careful hydrolysis of the acetal groups without backbone degradation. Degradation of the PPEs under basic conditions revealed complete and statistical degradation of the phosphotri- to phosphodiesters. In addition, a phosphodiester monomer with an adhesive P-OH group and no protective group chemistry was used to compare the binding to metal oxides with the multicatechol-PPEs. All PPEs can stabilize magnetite particles (NPs) in polar solvents, for example, methanol, due to the binding of the phosphoester groups in the backbone to the particles. ITC measurements reveal that multicatechol PPEs exhibit a higher binding affinity to magnetite NPs compared to PPEs bearing phosphodi- or phosphotriesters as repeating units. In addition, the catechol-containing PPEs were used to generate organo- and hydrogels by oxidative cross-linking, due to cohesive properties of catechol groups. This unique combination of two natural adhesive motives, catechols and phosphates, will allow the design of novel future gels for tissue engineering applications or novel degradable adhesives. [ABSTRACT FROM AUTHOR]

A new class of phosphanylmethylphosphonate reagents has been developed to enable the controlled synthesis of methylene bisphosphonate mono- and diesters. Condensation of such reagents with an alcohol of choice through azole-mediated phosphoramidite chemistry followed by in situ oxidation provides orthogonally protected methylene bisphosphonate tetraesters. Global deprotection of the tetraester leads to terminal methylene bisphosphonates. Alternatively, selective deprotection at the terminal phosphonate followed by a condensation between the acquired methylene bisphosphonate triester and a second alcohol leads to methylene bisphosphonates diesters. [ABSTRACT FROM AUTHOR]

We give an overview of experimental and computational methods to estimate RNA metabolism rates genome-wide. We then advocate a local definition of RNA metabolism rate at the level of individual phosphodiester bonds. Rates of formation and disappearance of individual bonds are unambiguously defined, in contrast to rates of complete transcripts. We show that over previous approaches, the recently developed transient transcriptome sequencing (TT-seq) protocol allows for estimation of metabolism rates of individual bonds with least positional bias. [ABSTRACT FROM PUBLISHER]

The catalytic activity of some classes of natural RNA, named as ribozymes, has been discovered just in the past decades. In this paper, the cleavage of the RNA phosphodiester backbone has been studied in aqueous solution and in a twister ribozyme from Oryza sativa. The free energy profiles associated with a baseline substrate-assisted mechanism for the reaction in the enzyme and in solution were computed by means of free energy perturbation methods within hybrid QM/MM potentials, describing the chemical system by the M06-2× functional and the environment by means of the AMBER and TIP3P force fields. The results confirm that this is a stepwise mechanism kinetically controlled by the second step that involves the P-O5′ breaking bond concomitant with the proton transfer from the OP1 atom to the leaving O5′ atom. O kinetic isotope effects on the nucleophile and leaving oxygen atoms, in very good agreement with experiments, also support this description. Nevertheless, the free energy profiles in the enzyme and in solution are almost coincident which, despite that the rate-limiting activation free energy is in very good agreement with experimental data of counterpart reactions in solution, rule out this substrate-assisted catalysis mechanism for the twister ribozyme from O. sativa. Catalysis must come from the role of alternative acid-base species not available in aqueous solution, but the rate-limiting transition state must be associated with the P-O5′ bond cleavage. [ABSTRACT FROM AUTHOR]

Xanthomonas translucens pv. translucens (Xtt) is a Gram-negative pathogen of crops from the plant family Poaceae. The lipopolysaccharide (LPS) of Xtt was isolated and chemically characterized. The analyses revealed the presence of rhamnose, xylose, mannose, glucose, galacturonic acid, phosphates, 3-deoxy-D-manno-oct-2-ulopyranosonic acid (Kdo) and fatty acids (10:0, 11:0, 11:0(3-OH) i/a, 11:0(3-OH), 12:0(3-OH) i/a, 12:0(3-OH), 12:0, 13:0(3-OH) i, 13:0(3-OH) a, 13:0(3-OH), 14:0(3-OH) i/a, 14:0(3-OH) and 16:0). The rough type of LPS (lipooligosaccharides; LOS) was isolated and its composition determined utilizing mass spectrometry. The structure of core-lipid A backbone was revealed by nuclear magnetic resonance (NMR) spectroscopy performed on O-deacylated LOS sample, and was shown to be: α-D-Manp-(1→3)-α-D-Manp-(1→3)-β-D-Glcp-(1→4)-α-D-Manp-(1→5)-α-Kdo-(2→6)-β-D-GlcpN-(1→6)-α-D-GlcpN. 4-α-Man and Kdo were further substituted via phosphodiester groups by two galactopyranuronic acids. Xtt LPS elicited a stress response in Nicotiana tabacum suspension cell cultures, namely a transient calcium signal and the generation of H2O2 was observed. Pharmacological studies indicated the involvement of plasma membrane calcium channels, kinases and phospholipase C as key factors in Xtt LPS induced pathogen signaling. [ABSTRACT FROM AUTHOR]

The molecular properties of the phosphodiester backbone that made it the evolutionary choice for the enzymatic replication of genetic information are not well understood. To address this, and to develop new chemical ligation strategies for assembly of biocompatible modified DNA, we have synthesized oligonucleotides containing several structurally and electronically varied artificial linkages. This has yielded a new highly promising ligation method based on amide backbone formation that is chemically orthogonal to CuAAC "click" ligation. A study of kinetics and fidelity of replication through these artificial linkages by primer extension, PCR, and deep sequencing reveals that a subtle interplay between backbone flexibility, steric factors, and ability to hydrogen bond to the polymerase modulates rapid and accurate information decoding. Even minor phosphorothioate modifications can impair the copying process, yet some radical triazole and amide DNA backbones perform surprisingly well, indicating that the phosphate group is not essential. These findings have implications in the field of synthetic biology. [ABSTRACT FROM AUTHOR]

The purpose of this study was to evaluate the usefulness of in vivo 31P magnetic resonance spectroscopy (MRS) for monitoring changes in growth-related phosphate metabolite concentration and intracellular pH value in rabbit thigh muscle implanted with VX-2 carcinoma. The time-course magnetic resonance imaging (MRI) and in vivo 31P MRS were examined weekly in the course of 10 weeks following the onset of a VX-2 carcinoma implantation. The spectra were quantitatively analyzed to obtain vital information on the time course variation of the phosphorus metabolites and intracellular pH value according to the tumor growth. Elevation in the concentrations of phosphormonoesters (PME), inorganic phosphate (Pi), and phosphodiesters (PDE) was observed over the time course of 3-4 weeks after the implantation of VX2 carcinoma, while the rest of the metabolites, PCr and ATP tended to be constant. The concentration changes of PME, Pi, and PDE were positively correlated with the volumes of tumor necrosis. The intracellular pH values decreased with the time course of tumor growth and the volumes of tumor necrosis. In vivo 31P MRS is capable of non-invasive monitoring of intracellular pH values as well as the concentration changes of phosphate metabolites during tumor growth. [ABSTRACT FROM AUTHOR]

Ribonucleic acids (RNA) frequently associate with proteins in many biological processes to form more or less stable complex structures. The characterization of RNA-protein complex structures and binding interfaces by nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography, or strategies based on chemical crosslinking, however, can be quite challenging. Herein, we have explored the use of an alternative method, native top-down mass spectrometry (MS), for probing of complex stoichiometry and protein binding sites at the single-residue level of RNA. Our data show that the electrostatic interactions between HIV-1 TAR RNA and a peptide comprising the arginine-rich binding region of tat protein are sufficiently strong in the gas phase to survive phosphodiester backbone cleavage of RNA by collisionally activated dissociation (CAD), thus allowing its use for probing tat binding sites in TAR RNA by top-down MS. Moreover, the MS data reveal time-dependent 1:2 and 1:1 stoichiometries of the TAR-tat complexes and suggest structural rearrangements of TAR RNA induced by binding of tat peptide. [ABSTRACT FROM AUTHOR]

Stereoselective synthesis of variably protected α- and β-l-Ara4N glycosyl H-phosphonates as key intermediates in the syntheses of β-l-Ara4N-modified LPS structures and α-l-Ara4N-containing biosynthetic precursors is reported. A facile one-pot approach toward β-l-Ara4N glycosyl H-phosphonates includes anomeric deallylation of protected 4-azido β-l-Ara4N via terminal olefin isomerization followed by ozonolysis and methanolysis of formyl groups to furnish exclusively β-configured lactols that are phosphitylated with retention of configuration. The carbohydrate epitope of β-l-Ara4N-modified Lipid A, βGlcN(1→6)αGlcN(1→P←1)β-l-Ara4N, was stereoselectively synthesized and linked to maleimide-activated bovine serum albumin. [ABSTRACT FROM AUTHOR]

A convenient approach has been proposed to the synthesis of nicotinamide adenine dinucleotide (NAD) mimetics, which comprise morpholino analogues of nucleosides. The approach is based on the use of ADP conjugates containing an amino group, which is tethered to the terminal phosphate through the aliphatic linker by the phosphodiester bond. We have synthesized four series of the NAD mimetics, which differ in the type of the modified nucleoside (2-aminomethylmorpholine (Mor) or 2-aminomethyl-4-carboxymethylmorpholine (MorGly) derivatives), in the linker length, and in the manner of the nucleoside attachment to the ADP derivative. We have studied the efficiency of NAD mimetics in the inhibition of the auto-poly(ADP-ribosyl)ation by the PARP 1 enzyme. The linker length, the mode of the attachment of the morpholino nucleoside analogue, and the nature of the heterocyclic base of the modified nucleoside were shown to inf luence the inhibition efficiency. [ABSTRACT FROM AUTHOR]

We have employed a DNA molecular beacon with a real abasic site, namely a 2-deoxyribose, in a fluorescent high-throughput assay to identify artificial nucleases that cleave at abasic sites. We screened a 1280 compound chemical library and identified a compound that functions as an artificial nuclease. We validated a key structure-activity relationship necessary for abasic site cleavage using available analogs of the identified artificial nuclease. We also addressed the activity of the identified compound with dose titrations in the absence and presence of a source of non-specific DNA. Finally, we characterized the phosphodiester backbone cleavage at the abasic site using denaturing gel electrophoresis. This study provides a useful template for researchers seeking to rapidly identify new artificial nucleases. [ABSTRACT FROM AUTHOR]

Objective: The OAS proteins are characterized by their capacity to synthesize 2′,5′-linked phosphodiester bonds to polymerize ATP into oligomers of adenosine. OAS3, belonging to OASs gene family, synthesizes dimeric 2-5A that binds to RNase L with low affinity and produces 2-5A oligomers shorter than the tri-tetramer 2-5As produced by other family members. Methods: For these studies, we used the open source tools cNLS Mapper, PredictProtein and COMPARTMENTS for the nuclear localization signal prediction, UCSF Chimera for molecular graphics and analyses, The Human Protein Atlas to confirm with the IF the OAS3 cell localization and Ensembl Variation Table to identify the presence of putative single nucleotide polymorphisms in the NLS sequence identification. Results: The analysis of OAS3 protein sequence (NP_006178.2) displayed a putative nuclear localization signal (cNLS Mapper score 8 and PP 100 %), identified by 11 and 5 amino acids ( LQRQL KRPRP V) located in the outer portion ready to interact with the importin α/β. Furthermore, we showed that in all cells lines available in the Human Protein Atlas subcell section, the OAS3 was mainly localized in the cytoplasm and nucleus, but not in the nucleoli. We identify six known variant SNPs mapping in the nuclear import sequence, but only three were associated with a missense variation (rs781335794, rs750458641, rs550465943) and were able to strongly reduce the cNLS score. Conclusions: The catalytically inactive domain of human OAS3 has a potential nuclear import function, susceptible to SNPs, which could determine their roles in the viral infection and IFNs response. [ABSTRACT FROM AUTHOR]

To construct the model of metallohydrolase, two inclusion complexes [MLCl 2 (β-CD)] ( 1 , M = Zn(II); 2 , M = Cu(II); L = N,N′ -bis(2-pyridylmethyl)amantadine; β-CD = β-cyclodextrin) were synthesized by mixing β-CDs with the pre-synthesized complexes G1 , [ZnLCl 2 ] and G2 , [CuLCl 2 ]. Structures of G1 , G2 , 1 and 2 were characterized by X-ray crystallography, respectively. In solution, two chloride anions of G1 and G2 underwent ligand exchange with solvent molecules according to ESI-MS analysis. The chemical equilibrium constants were determined by potentiometric pH titration. The kinetics of bis(4-nitrophenyl) phosphate (BNPP) hydrolysis catalyzed by G1 , G2 , 1 and 2 were examined at pHs ranging from 7.50 to 10.50 at 308 ± 0.1 K. The pH profile of rate constant of BNPP hydrolysis catalyzed by 1 exhibited an exponential increase with the second-order rate constant of 2.68 × 10 − 3 M − 1 s − 1 assigned to the di-hydroxo species, which was approximately an order of magnitude higher than those of reported mono-Zn(II)-hydroxo species. The high reactivity was presumably hydroxyl-rich microenvironment provided by β-CDs, which might effect in stabilizing either the labile zinc-hydroxo species or the catalytic transition state. [ABSTRACT FROM AUTHOR]

A multi-nucleating azacrown conjugate ( 5a ) consisting of two 3,5-bis(1,5,9-triazacyclododecan-3-yloxymethyl)benzyl groups attached to 1 and 7 sites of cyclen was prepared and tested as an artificial ribonuclease. The conjugate in the presence of five equivalents of zinc nitrate expectedly showed uridine selectivity comparable to that 1,3,5-tris(1,5,9-triazacyclododecan-3-yl)benzene ( 2 ), a compound known to bind to two adjacent uridine residues and cleave the intervening phosphodiester bond. 5a was, however, unable to discriminate between two and three adjacent uridine residues, but cleaved oligonucleotides containing a UpU and UpUpU site at a comparable rate, even when present at sub-saturating concentrations. [ABSTRACT FROM AUTHOR]

A series of dinuclear metal(ii)-acetato complexes: [Ni2(μ-LClO)(μ2-OAc)2](PF6)·3H2O (1), [Ni2(μ-LClO)(μ2-OAc)2](ClO4)·CH3COCH3 (2), [Cu2(μ-LClO)(μ2-OAc)(ClO4)](ClO4) (3), [Cu2(μ-LClO)(OAc)2](PF6)·H2O (4), [Zn2(μ-LClO)(μ2-OAc)2](PF6) (5) and [Mn2(LCl-O)(μ2-OAc)2](ClO4)·H2O (6), where LClO− = 2,6-bis[bis(2-pyridylmethyl)aminomethyl]-4-chlorophenolate, were synthesized. The complexes were structurally characterized by spectroscopic techniques and single crystal X-ray crystallography. Six-coordinate geometries with doubly bridged acetato ligands were found in Ni(ii), Zn(ii) and Mn(ii) complexes 1, 2, 5 and 6, whereas with Cu(ii) complexes a five-coordinate species was obtained with 4, and mixed five- and six-coordinate geometries with a doubly bridged dimetal core were observed in 3. The magnetic properties of complexes 1–4 and 6 were studied at variable temperatures and revealed weak to very weak antiferromagnetic interactions in 1, 2, 4 and 6 (J = −0.55 to −9.4 cm−1) and ferromagnetic coupling in 3 (J = 15.4 cm−1). These results are consistent with DFT calculations performed at the B3LYP/def2-TZVP(-f) level of theory. Under physiological conditions, the interaction of the dinculear complexes 1–5 with supercoiled plasmid ds-DNA did not show any pronounced nuclease activity, but Ni(ii) complexes 1 and 2 revealed a strong ability to unwind the supercoiled conformation of ds-DNA. The mechanistic studies performed on the interaction of the Ni(ii) complexes with DNA demonstrated the important impact of the nickel(ii) ion in the unwinding process. In combination with the DNA study, the phosphatase activity of complexes 1, 3, and 5 was examined by the phosphodiester hydrolysis of bis(2,4-dinitrophenol)phosphate (BDNPP) in the pH range of 5.5–10.5 at 25 °C. The Michaelis–Menten kinetics performed at pH 7 and 10.7 showed that catalytic efficiencies kcat/KM (kcat = catalytic rate constant, KM = substrate binding constant) decrease in the order: Ni(ii), 1 ＞ Zn(ii), 5 ＞ Cu(ii), 3. A similar trend was also observed with the turnover numbers at pH = 7. The results are discussed in relation to the coordination geometry and nature of the metal center as well as the steric environment imposed by the compartmental phenoxido ligand. [ABSTRACT FROM AUTHOR]

A 2,7-disubstituted phosphodiester-linked phenanthrene trimer forms tubular structures in aqueous media. Chromophores are arranged in H-aggregates. Incorporation of small quantities of pyrene results in the development of light-harvesting nanotubes in which phenanthrenes act as antenna chromophores and pyrenes as energy acceptors. Energy collection is most efficient after excitation at the phenanthrene H-band. Fluorescence quantum yields up to 23 % are reached in pyrene doped, supramolecular nanotubes. [ABSTRACT FROM AUTHOR]

The chemical synthesis of phosphorothioate/phosphodiester analogues of 2-methoxy-lysophosphatidylethanolamine has been described. For the preparation of phosphorothioate derivatives oxathiaphospholane approach has been employed. The phosphodiester compounds were prepared by OXONE® oxidation of corresponding phosphorothioates. Each lysophospholipid analogue was synthesized as a series of four compounds, bearing different fatty acid residues both saturated (14:0, 16:0, 18:0) and unsaturated (18:1). The methylation of glycerol 2-hydroxyl function was applied in order to increase the stability of prepared analogues by preventing 1→2 acyl migration. The cytotoxicity of newly synthesized 2-methoxy-lysophosphatidylethanolamine derivatives was evaluated with resazurin-based method in prostate cancer PC3 cell line. The highest reduction of cell viability was noted for LPE analogues containing myristoyl acyl chain. [ABSTRACT FROM AUTHOR]

The DNA sequence specificity of the cancer chemotherapeutic agent, bleomycin, was determined with high precision in purified plasmid DNA using an improved technique. This improved technique involved the labelling of the 5′- and 3′-ends of DNA with different fluorescent tags, followed by simultaneous cleavage by bleomycin and capillary electrophoresis with laser-induced fluorescence. This permitted the determination of bleomycin cleavage specificity with high accuracy since end-label bias was greatly reduced. Bleomycin produces single- and double-strand breaks, abasic sites and other base damage in DNA. This high-precision method was utilised to elucidate, for the first time, the DNA sequence specificity of bleomycin-induced DNA damage at abasic sites. This was accomplished using endonuclease IV that cleaves DNA at abasic sites after bleomycin damage. It was found that bleomycin-induced abasic sites formed at 5′-GC and 5′-GT sites while bleomycin-induced phosphodiester strand breaks formed mainly at 5′-GT dinucleotides. Since bleomycin-induced abasic sites are produced in the absence of molecular oxygen, this difference in DNA sequence specificity could be important in hypoxic tumour cells. [ABSTRACT FROM AUTHOR]

Polymorphonuclear leukocytes (PMNLs, neutrophils) play a major role in the initiation and resolution of the inflammatory response, and neutrophil apoptosis is a critical step in resolving inflammation. We examined the effects of oligodeoxynucleotide (ODN) species with different numbers of phosphodiester and phosphorothioate bonds on leukotriene synthesis in PMNLs and on neutrophil apoptosis. Our modifications were based on the well-known ODN2216 molecule (Krug et al., 2001). Treatment of cultured human neutrophils with ODN2216 accelerated apoptosis except in the case of a species with only phosphodiester bonds. The ODNs with poly( g ) (phosphorothioate) sequences at both ends and a phosphodiester inner core had maximal effects on leukotriene synthesis in neutrophils and inhibited formation of 5-lipoxygenase metabolites. Addition of phosphodiester and phosphorothioate ODNs to PMNLs produced distinct effects on superoxide and nitric oxide formation: phosphorothioate-containing ODNs concomitantly stimulated production of nitric oxide and superoxide, which may rapidly combine to generate peroxynitrite. Altogether, our results describe strong activation of neutrophil's cellular responses by phosphorothioate ODN2216. We propose that phosphorothioate modification of ODNs represents a potential mechanism of PMNL activation. [ABSTRACT FROM AUTHOR]

T4 polynucleotide kinase is widely used for 5'-phosphorylation of DNA and RNA oligonucleotide termini, but no natural protein enzyme is capable of 3'-phosphorylation. Here, we report the in vitro selection of deoxyribozymes (DNA enzymes) capable of DNA oligonucleotide 3'-phosphorylation, using a 5'-triphosphorylated RNA transcript (pppRNA) as the phosphoryl donor. The basis of selection was the capture, during each selection round, of the 3'-phosphorylated DNA substrate terminus by 2-methylimidazole activation of the 3'-phosphate (forming 3'-MeImp) and subsequent splint ligation with a 5'-amino DNA oligonucleotide. Competing and precedented DNA-catalyzed reactions were DNA phosphodiester hydrolysis or deglycosylation, each also leading to a 3'-phosphate but at a different nucleotide position within the DNA substrate. One oligonucleotide 3'-kinase deoxyribozyme, obtained from an N40 random pool and named 3'Kin1, can 3'-phosphorylate nearly any DNA oligonucleotide substrate for which the 3'-terminus has the sequence motif 5'-NKR-3', where N denotes any oligonucleotide sequence, K = T or G, and R = A or G. These results establish the viabilty of in vitro selection for identifying DNA enzymes that 3'-phosphorylate DNA oligonucleotides. [ABSTRACT FROM AUTHOR]

Cyclic dinucleotides are able to trigger the innate immune system by activating STING. It was found that the binding affinity of asymmetric 2'3'-cGAMP to symmetric dimer of STING is 3 orders of magnitude higher than that of the symmetric 3'3'-cyclic dinucleotides. Such a phenomenon has not been understood yet. Here we show that the subtle changes in phosphodiester linkage of CDNs lead to their distinct structural properties which correspond to the varied binding affinities. 2'-5' and/or 3'-5' linked CDNs adopt specific while different types of ribose puckers and backbone conformations. That ribose conformations and base types have different propensities for anti or syn glycosidic conformations further affects the overall flexibility of CDNs. The counterbalance between backbone ring tension and electrostatic repulsion, both affected by the ring size, also contributes to the different flexibility of CDNs. Our calculations reveal that the free energy cost for 2'3'-cGAMP to adopt the STING-bound structure is smaller than that for 3'3'-cGAMP and cyclic-di-GMP. These findings may serve as a reference for design of CDN-analogues as vaccine adjuvants. Moreover, the cyclization pattern of CDNs closely related to their physiological roles suggests the importance of understanding structural properties in the study of protein-ligand interactions. [ABSTRACT FROM AUTHOR]

Replicative DNA polymerases (DNAPs) require divalent metal cations for phosphodiester bond formation in the polymerase site and for hydrolytic editing in the exonuclease site. Me2+ ions are intimate architectural components of each active site, where they are coordinated by a conserved set of amino acids and functional groups of the reaction substrates. Therefore Me2+ ions can influence the noncovalent transitions that occur during each nucleotide addition cycle. Using a nanopore, transitions in individual &#109229 DNAP complexes are resolved with single-nucleotide spatial precision and sub-millisecond temporal resolution. We studied Mg2+ and Mn2+, which support catalysis, and Ca2+, which supports deoxynucleoside triphosphate (dNTP) binding but not catalysis. We examined their effects on translocation, dNTP binding, and primer strand transfer between the polymerase and exonuclease sites. All three metals cause a concentration-dependent shift in the translocation equilibrium, predominantly by decreasing the forward translocation rate. Me2+ also promotes an increase in the backward translocation rate that is dependent upon the primer terminal 3'-OH group. Me2+ modulates the translocation rates but not their response to force, suggesting that Me2+ does not affect the distance to the transition state of translocation. Absent Me2+, the primer strand transfer pathway between the polymerase and exonuclease sites displays additional kinetic states not observed at>1mM Me2+. Complementary dNTP binding is affected by Me2+ identity, with Ca2+ affording the highest affinity, followed by Mn2+, and then Mg2+. Both Ca2+ and Mn2+ substantially decrease thedNTPdissociation rate relative toMg2+, while Ca2+ also increases the dNTP association rate. [ABSTRACT FROM AUTHOR]

Catalytic DNAs, also known as deoxyribozymes, are of practical value for the synthesis of structurally or topologically complex RNAs, but little is known about the molecular details of DNA catalysis. We have investigated a deoxyribozyme that catalyzes the formation of a specific intramolecular 2′,5′-phosphodiester bond to produce lariat RNA, which is an important biological intermediate in eukaryotic mRNA splicing. The results of combinatorial mutation interference analysis (CoMA) allowed us to shrink the catalytic core to 70 % of its original length and revealed that the essential part of the deoxyribozyme sequence contained more than 50 % guanosines. Nucleotide analogue interference mapping (dNAIM) and dimethyl sulfate interference (DMSi) experiments provided atomic details of individual guanosine functional groups. Additional spectroscopic experiments and structural probing data identified conformational changes upon metal-ion binding and catalysis. Overall, this comprehensive analysis of the DNA-catalyzed reaction has provided specific insights into the synthesis of 2′,5′-branched RNA, and suggested the general features of deoxyribozymes that catalyze nucleic acid ligation reactions. [ABSTRACT FROM AUTHOR]

Density functional theory calculations have been used to investigate the reaction mechanisms of phosphodiester hydrolysis and transesterification catalyzed by a dinuclear zinc complex of the 2-(N-isopropyl-N-((2-pyridyl)methyl)aminomethyl)-6-(N-(carboxylmethyl)-N-((2-pyridyl)methyl)amino-methyl)-4-methylphenol (IPCPMP) ligand, mimicking the active site of zinc phosphotriesterase. The substrates bis(2,4)-dinitrophenyl phosphate (BDNPP) and 2-hydroxypropyl-p-nitrophenyl phosphate (HPNP) were employed as analogues of DNA and RNA, respectively. A number of different mechanistic proposals were considered, with the active catalyst harboring either one or two hydroxide ions. It is concluded that for both reactions the catalyst has only one hydroxide bound, as this option yields lower overall energy barriers. For BDNPP hydrolysis, it is suggested that the hydroxide acts as the nucleophile in the reaction, attacking the phosphorus center of the substrate. For HPNP transesterification, on the other hand, the hydroxide is proposed to act as a Brønsted base, deprotonating the alcohol moiety of the substrate, which in turn performs the nucleophilic attack. The calculated overall barriers are in good agreement with measured rates. Both reactions are found to proceed by essentially concerted associative mechanisms, and it is demonstrated that two consecutive catalytic cycles need to be considered in order to determine the rate-determining free energy barrier. [ABSTRACT FROM AUTHOR]

Purpose: The identification of the phosphodiester (PDE) 31P MR signals in the healthy human breast at ultra-high field. Methods: In vivo 31P MRS measurements at 7 T of the PDE signals in the breast were performed investigating the chemical shifts, the transverse- and the longitudinal relaxation times. Chemical shifts and transverse relaxation times were compared with non-ambiguous PDE signals from the liver. results: The chemical shifts of the PDE signals are shifted -0.5 ppm with respect to glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE), and the transverse and longitudinal relaxation times for these signals are a factor 3 to 4 shorter than expected for aqueous GPC and GPE. conclusion: The available experimental evidence suggests that GPC and GPE are not the main source of the PDE signals measured in fibroglandular breast tissue at 7 T. These signals may predominantly originate from mobile phospholipids. [ABSTRACT FROM AUTHOR]

A methodology employing high performance liquid chromatography coupled with matrix-assisted laser desorption and ionization time-of-flight mass spectrometry has been utilized to determine the quality of phospholipid classes. Home-made phosphoester chemically bonded stationary phase containing diol, phosphate and octadecyl groups (Diol-P-C18) has been employed in the separation of polar lipids from milk. Each phospholipid fraction was collected manually and identified by MALDI-TOF MS. [ABSTRACT FROM AUTHOR]

Understanding the structural rules that govern specific, high-affinity binding characteristic of aptamer–protein interactions is important in view of the increasing use of aptamers across many applications. From the modest number of 16 aptamer–protein structures currently available, trends are emerging. The flexible phosphodiester backbone allows folding into precise three-dimensional structures using known nucleic acid motifs as scaffolds that orient specific functional groups for target recognition. Still, completely novel motifs essential for structure and function are found in modified aptamers with diversity-enhancing side chains. Aptamers and antibodies, two classes of macromolecules used as affinity reagents with entirely different backbones and composition, recognize protein epitopes of similar size and with comparably high shape complementarity. [ABSTRACT FROM AUTHOR]

Raman difference spectroscopy is used to monitor the conformational changes associated with dNMP incorporation in single crystals of a RB69 DNA polymerase (RB69pol) ternary complex. An inactive crystal of L415G RB69pol exo- with a primer/template duplex and a cognate dATP was produced in the presence of Ca2+. The Raman spectrum of the crystallized complex was obtained using a Raman microscope. Phosphodiester bond formation is triggered by soaking in Mg2+. The Raman difference spectra [ie, (ternary complex + Mg2+)-(ternary complex with no Mg2+)] show that conformational changes are complete prior to the acquisition of the first Raman spectrum following magnesium incorporation into the crystal. Polyacrylamide gel electrophoresis experiments carried in parallel show that dNMP incorporation occurs rapidly in the crystal. The Raman data indicate that significant changes in protein amide features, tyrosine ring modes, and nucleic acid base backbone modes have occurred. Overall, they are dominated by the dichroic effect with the molecular groups changing orientation with respect to the laser beam following nucleotide incorporation. The amide I and amide III α-helix features are unusually narrow and are assigned to movement of the P and N helices. These results demonstrate the ability of Raman spectroscopy to monitor conformational changes in proteins via dichroism. The Raman spectra and the gel electrophoresis support the hypothesis that the major spectroscopic changes are because of structural changes in the ternary complex crystal following correct dNMP incorporation. Copyright © 2015 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Intrastrand cross-links (IaCL) connecting two purine nucleobases in DNA pose a challenge to high-fidelity replication in the cell. Various repair pathways or polymerase bypass can cope with these lesions. The influence of the phosphodiester linkage between two neighboring 2'-deoxyguanosine (dG) residues attached through the O6 atoms by an alkylene linker on bypass with human DNA polymerase η (hPol η) was explored in vitro. Steady-state kinetics and mass spectrometric analysis of products from nucleotide incorporation revealed that although hPol η is capable of bypassing the 3'-dG in a mostly error-free fashion, significant misinsertion was observed for the 5'-dG of the IaCL containing a butylene or heptylene linker. The lack of the phosphodiester linkage triggered an important increase in frameshift adduct formation across the 5'-dG by hPol η, in comparison to the 5'-dG of IaCL DNA containing the phosphodiester group. [ABSTRACT FROM AUTHOR]

High affinity RNA-protein interactions are critical to cellular function, but directly identifying the determinants of binding within these complexes is often difficult. Here, we introduce a stable isotope mass labeling technique to assign specific interacting nucleotides in an oligonucleotide-protein complex by photo-cross-linking. The method relies on generating site-specific oxygen-18-labeled phosphodiester linkages in oligonucleotides, such that covalent peptide-oligonucleotide cross-link sites arising from ultraviolet irradiation can be assigned to specific sequence positions in both RNA and protein simultaneously by mass spectrometry. Using Lin28A and a let-7 pre-element RNA, we demonstrate that mass labeling permits unambiguous identification of the cross-linked sequence positions in the RNA-protein complex. [ABSTRACT FROM AUTHOR]

Nucleolytic ribozymes catalyze site-specific cleavage of their phosphodiester backbones. A minimal version of the twister ribozyme is reported that lacks the phylogenetically conserved stem P1 while retaining wild-type activity. Atomic mutagenesis revealed that nitrogen atoms N1 and N3 of the adenine-6 at the cleavage site are indispensable for cleavage. By NMR spectroscopy, a pKa value of 5.1 was determined for a 13C2-labeled adenine at this position in the twister ribozyme, which is significantly shifted compared to the pKa of the same adenine in the substrate alone. This finding pinpoints at a potential role for adenine-6 in the catalytic mechanism besides the previously identified invariant guanine-48 and a Mg2+ ion, both of which are directly coordinated to the nonbridging oxygen atoms of the scissile phosphate; for the latter, additional evidence stems from the observation that Mn2+ or Cd2+ accelerated cleavage of phosphorothioate substrates. The relevance of this metal ion binding site is further emphasized by a new 2.6 A X-ray structure of a 2'-OCH3-U5 modified twister ribozyme. [ABSTRACT FROM AUTHOR]

Cleavage of five different nucleoside diphosphosugars has been studied in the presence of Cu and Zn complexes. The results show that metal ion catalysts promote the cleavage via intramolecular transesterification whenever a neighbouring HO group can adopt a cis-orientation with respect to the phosphate. The HO group attacks the phosphate and two monophosphate products are formed. If such a nucleophile is not available, Cu complexes are able to promote a nucleophilic attack of an external nucleophile, e.g. a water molecule or metal ion coordinated HO ligand, on phosphate. With the Zn complex, this was not observed. Graphical Abstract: [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

To date, little is known about how neurodegeneration and neuroinflammation propagate in Huntington's disease (HD). Unfortunately, no treatment is available to cure or reverse the progressive decline of function caused by the disease, thus considering HD a fatal disease. Mutation gene carriers typically remain asymptomatic for many years although alterations in the basal ganglia and cortex occur early on in mutant HD gene-carriers. Positron Emission Tomography (PET) is a functional imaging technique of nuclear medicine which enables in vivo visualization of numerous biological molecules expressed in several human tissues. Brain PET is most powerful to study in vivo neuronal and glial cells function as well as cerebral blood flow in a plethora of neurodegenerative disorders including Parkinson's disease, Alzheimer's and HD. In absence of HD-specific biomarkers for monitoring disease progression, previous PET studies in HD were merely focused on the study of dopaminergic terminals, cerebral blood flow and glucose metabolism in manifest and premanifest HD-gene carriers. More recently, research interest has been exploring novel PET targets in HD including the state of phosphodiesterse expression and the role of activated microglia. Hence, a better understanding of the HD pathogenesis mechanisms may lead to the development of targeted therapies. PET imaging follow-up studies with novel selective PET radiotracers such as 11C-IMA-107 and 11C-PBR28 may provide insight on disease progression and identify prognostic biomarkers, elucidate the underlying HD pathology and assess novel pharmaceutical agents and over time. [ABSTRACT FROM AUTHOR]

Ribonuclease (RNase) is hydrolytic enzyme that catalyzes the cleavage of phosphodiester bonds in RNA. RNases play an important role in the metabolism of cellular RNAs, such as mRNA and rRNA or tRNA maturation. Besides their cellular roles, RNases possess biological activity, cell stimulating properties, cytotoxicity and genotoxicity. Cytotoxic effect of particular microbial RNases was comparable to that of animal derived counterparts. In this respect, microbial RNases have a therapeutic potential as anti-tumor drugs. The significant development of DNA vaccines and the progress of gene therapy trials increased the need for RNases in downstream processes. In addition, RNases are used in different fields, such as food industry for single cell protein preparations, and in some molecular biological studies for the synthesis of specific nucleotides, identifying RNA metabolism and the relationship between protein structure and function. In some cases, the use of bovine or other animal-derived RNases have increased the difficulties due to the safety and regulatory issues. Microbial RNases have promising potential mainly for pharmaceutical purposes as well as downstream processing. Therefore, an effort has been given to determination of optimum fermentation conditions to maximize RNase production from different bacterial and fungal producers. Also immobilization or strain development experiments have been carried out. [ABSTRACT FROM AUTHOR]

Indoor environmental pollutants are a threat to human health. In the current study, we analysed 25 per- and polyfluoroalkyl substances (PFASs) in seven different size fraction of house dust including the two relevant for exposure via ingestion and inhalation. The highest PFAS concentration is found in the inhalable particulate fraction which is explained by the increased surface area as the particulate's sizes decrease. The estimated daily intake (EDI) of the individual PFAS and exposure pathways were calculated for children and adults. In addition, the total EDI for PFOA and its precursors was estimated. The polyfluoroalkyl phosphoric acid diesters (diPAP), followed by PFOA and PFHxA fluortelomer, showed the highest concentrations of PFAS analysed. The cumulative EDI of PFAS for children was 3.0 ng/kg bw per day, a worst-case scenario, which is 17 times higher than the calculated EDI for adults. For children, ingestion of dust was found to result in 800 times higher PFOA exposure than via inhalation. The contribution from PFOA precursors corresponded to only 1% of the EDI from dust indicating PFOA as the main source of exposure. The EDI's of PFOA and PFOS from dust were lower than the calculated EDI's from food ingestion reported by the Swedish Food Agency. Our data indicate that the EDI for the sum of four PFASs: PFOA, PFNA, PFHxS and PFOS from dust intake alone is close to the established tolerable weakly intake of 4.4 ng/kg bw in children, set by European Food Safety Authority (EFSA) in 2020. The combined EDI levels PFOA and PFOS from both dust and food exceeded the EFSA TWI for both children and adults. This study demonstrates that dust is a relevant exposure pathway for PFAS intake and that analysis of relevant particle size fractions is important for evaluation of dust as an exposure pathway. [Display omitted] • Inhalation of house dust constitutes a minor route of exposure to PFAS compared to ingestion of dust. • The fluortelomer phosphate esters constitute the highest concentrations of PFASs analysed in dust. • The exposure to PFOA from the precursors to PFOA is low compared to the direct exposure to PFOA. • The sum of PFOA, PFNA, PFHxS and PFOS from house dust exposure in children is close to the TWI value presented by EFSA, 2020. [ABSTRACT FROM AUTHOR]

The article presents the discussion on the synthesis of oligonucleotides. Topics include used for native phosphodiester either diastereomer of phosphorothioate and phosphorodithioate linkages; switching efficacy, ribonuclease H activity, enzymatic stability, toxicity, protein corona formation, and biodistribution; and improvement for liquid-phase technologies and the sustainable production of oligonucleotides.