Aptamers as a model for functional evaluation of LNA and 2′-amino LNA

The affinity change upon incorporation of LNA and 2′-amino-LNA monomers into an avidin binding DNA aptamer is described. The kinetic profile of selected modified-aptamer was obtained by surface plasmon resonance experiments and compared with the profile of the parent unmodified DNA aptamer. We report significant improvement of avidin binding affinity by the incorporation of single LNA modifications into the aptamer, and successful incorporation of 2′-amino LNA as a novel monomer in aptamers with potential function as carrier unit for additional molecular entities.     

Development of a novel assay for human tyrosyl DNA phosphodiesterase 2

Tyrosyl DNA phosphodiesterase 2 (TDP2), a newly discovered enzyme that cleaves 5′-phosphotyrosyl bonds, is a potential target for chemotherapy. TDP2 possesses both 3′- and 5′-tyrosyl-DNA phosphodiesterase activity, which is generally measured in a gel-based assay using 3′- and 5′-phosphotyrosyl linkage at the 3′ and 5′ ends of an oligonucleotide. To understand the enzymatic mechanism of this novel enzyme, the gel-based assay is useful, but this technique is cumbersome for TDP2 inhibitor screening. For this reason, we have designed a novel assay using p-nitrophenyl-thymidine-5′-phosphate (T5PNP) as a substrate. This assay can be used in continuous colorimetric assays in a 96-well format. We compared the salt and pH effect on product formation with the colorimetric and gel-based assays and showed that they behave similarly. Steady-state kinetic studies showed that the 5′ activity of TDP2 is 1000-fold more efficient than T5PNP. Tyrosyl DNA phosphodiesterase 1 (TDP1) and human AP-endonuclease 1 (APE1) could not hydrolyze T5PNP. Sodium orthovanadate, a known inhibitor of TDP2, inhibits product formation from T5PNP by TDP2 (IC₅₀ = 40 mM). Our results suggest that this novel assay system with this new TDP2 substrate can be used for inhibitor screening in a high-throughput manner.     

Oligomeric Structure Diversity within the GIY-YIG Nuclease Family

The GIY-YIG nuclease domain has been identified in homing endonucleases, DNA repair and recombination enzymes, and restriction endonucleases. The Type II restriction enzyme Eco29kI belongs to the GIY-YIG nuclease superfamily and, like most of other family members, including the homing endonuclease I-TevI, is a monomer. It recognizes the palindromic sequence 5′-CCGC/GG-3′ (“/” marks the cleavage position) and cuts it to generate 3′-staggered ends. The Eco29kI monomer, which contains a single active site, either has to nick sequentially individual DNA strands or has to form dimers or even higher-order oligomers upon DNA binding to make a double-strand break at its target site. Here, we provide experimental evidence that Eco29kI monomers dimerize on a single cognate DNA molecule forming the catalytically active complex. The mechanism described here for Eco29kI differs from that of Cfr42I isoschisomer, which also belongs to the GIY-YIG family but is functional as a tetramer. This novel mechanism may have implications for the function of homing endonucleases and other enzymes of the GIY-YIG family.     

5-Ethynyl-2'-deoxycytidine as a new agent for DNA labeling: detection of proliferating cells

The labeling of newly synthesized DNA in cells to identify cell proliferation is an important experimental technique. The most accurate methods incorporate [³H]thymidine or 5-bromo-2′-deoxyruidine (BrdU) into dividing cells during S phase, which is subsequently detected by autoradiography or immunohistochemistry, directly measuring the newly synthesized DNA. Recently, a novel method was developed to detect DNA synthesis in proliferating cells based on a novel thymidine analog, 5-ethynyl-2′-deoxyuridine (EdU). EdU is incorporated into DNA and subsequently detected with a fluorescent azide via “click” chemistry. This novel technique is highly sensitive and does not require DNA denaturation. However, it was also found that EdU exhibits time-dependent inhibition effects on cell growth. Therefore, here we report a novel deoxycytidine analog, 5-ethynyl-2′-deoxycytidine (EdC), that can be used to detect DNA synthesis in vitro and in vivo at a similar sensitivity level compared with EdU. Furthermore, the EdC-induced cytotoxicity is much less than that of EdU when combined with thymidine. This will be a potential application for the long-term detection of proliferating cells.     

Synthesis of 2′-Amino-LNA Purine Nucleosides

The first reported synthesis of 2′-amino-LNA purine nucleosides via a transnucleosidation is accomplished enabling the preparation of oligonucleotides incorporating 2′-amino-LNA with all four natural bases.     

Assay for the determination of urinary 8-hydroxy-2′-deoxyguanosine by high-performance liquid chromatography with electrochemical detection

A high-performance liquid chromatographic procedure with electrochemical detection is described for the determination of urinary 8-hydroxy-2′-deoxyguanosine, a major oxidative DNA lesion induced by radical forming agents. A two-step solid-phase extraction procedure was followed for extracting 8-hydroxy-2′-deoxyguanosine from human urine and the analysis was performed on a RP-18 analytical column under isocratic conditions. The limit of detection of 8-hydroxy-2′-deoxyguanosine in urine was found to be 0.9 nM. The non-invasive assay provides an indirect measurement of oxidative DNA damage.     

An enzyme from the earthworm Eisenia fetida is not only a protease but also a deoxyribonuclease

The earthworm enzyme Eisenia fetida Protease-III-1 (EfP-III-1) is known as a trypsin-like protease which is localized in the alimentary canal of the earthworm. Here, we show that EfP-III-1 also acts as a novel deoxyribonuclease. Unlike most DNases, this earthworm enzyme recognizes 5′-phosphate dsDNA (5′P DNA) and degrades it without sequence specificity, but does not recognize 5′OH DNA. As is the case for most DNases, Mg²⁺ was observed to markedly enhance the DNase activity of EfP-III-1. Whether the earthworm enzyme functioned as a DNase or as a protease depended on the pH values of the enzyme solution. The protein acted as a protease under alkaline conditions whereas it exhibited DNase activity under acid conditions. At pH 7.0, the enzyme could work as either a DNase or a protease. Given the complex living environment of the earthworm, this dual function of EfP-III-1 may play an important role in the alimentary digestion of the earthworm.     

Enzymatic polymerisation involving 2'-amino-LNA nucleotides

The triphosphate of the thymine derivative of 2′-amino-LNA (2′-amino-LNA-TTP) was synthesised and found to be a good substrate for Phusion® HF DNA polymerase, allowing enzymatic synthesis of modified DNA encoded by an unmodified template. To complement this, 2′-amino-LNA-T phosphoramidites were incorporated into DNA oligonucleotides which were used as templates for enzymatic synthesis of unmodified DNA using either KOD, KOD XL or Phusion polymerases. 2′-Amino-LNA-T in the template and 2′-amino-LNA-TTP as a substrate both decreased reaction rate and yield compared to unmodified DNA, especially for sequences with multiple 2′-amino-LNA-T nucleotides.     

Use of domain enzymes from wheat RNA ligase for in vitro preparation of RNA molecules

Wheat RNA ligase can be dissected into three isolated domain enzymes that are responsible for its core ligase, 5′-kinase, and 2′,3′-cyclic phosphate 3′-phosphodiesterase activities, respectively. In the present study, we pursued a practical strategy using the domain enzymes for in vitro step-by-step ligation of RNA molecules. As a part of it, we demonstrated that a novel side reaction on 5′-tri/diphosphate RNAs is dependent on ATP, a 2′-phosphate-3′-hydroxyl end, and the ligase domain. Mass spectroscopy and RNA cleavage analyses strongly suggested that it is an adenylylation on the 5′ terminus. The ligase domain enzyme showed a high productivity for any of the possible 16 combinations of terminal bases and a high selectivity for the 5′-phosphate and 2′-phosphate-3′-hydroxyl ends. Two RNA molecules having 5′-hydroxyl and 2′,3′-cyclic monophosphate groups were ligated almost stoichiometrically after separate conversion of respective terminal phosphate states into reactive ones. As the product has the same terminal state as the starting material, the next rounds of ligation are also possible in principle. Thus, we propose a flexible method for in vitro RNA ligation.     

A single replacement of histidine to arginine in EGFR-lytic hybrid peptide demonstrates the improved anticancer activity

Ribonucleotide reductase M1 (RRM1) is the regulatory subunit of the holoenzyme that catalyzes the conversion of ribonucleotides to 2′-deoxyribonucleotides. Its function is indispensible in cell proliferation and DNA repair. It also serves as a biomarker of therapeutic efficacy of the antimetabolite drug gemcitabine (2′,2′-difluoro-2′-deoxycytidine) in various malignancies. However, a mechanistic explanation remains to be determined. This study investigated how the alkylating agent N-ethylmaleimide (NEM) interacts with the inhibitory activity of gemcitabine on its target protein RRM1 in vivo. We found, when cells were treated with gemcitabine in the presence of NEM, a novel 110 kDa band, along with the 90 kDa native RRM1 band, appeared in immunoblots. This 110 kDa band was identified as RRM1 by mass spectrometry (LC–MS/MS) and represented a conformational change resulting from covalent labeling by gemcitabine. It is specific to gemcitabine/NEM, among 11 other chemotherapy drugs tested. It was also detectable in human tumor xenografts in mice treated with gemcitabine. Among mutations of seven residues essential for RRM1 function, C218A, C429A, and E431A abolished the conformational change, while N427A, C787A, and C790A diminished it. C444A was unique since it was able to alter the conformation even in absence of gemcitabine treatment. We conclude that the thiol alkylator NEM can stabilize the gemcitabine-induced conformational change of RRM1, and this stabilized RRM1 conformation has the potential to serve as a specific biomarker of gemcitabine’s therapeutic efficacy.     

A crtA-related gene from Flavobacterium P99-3 encodes a novel carotenoid 2-hydroxylase involved in myxol biosynthesis

A genomic DNA fragment with carotenogenic genes involved in myxol biosynthesis (3′,4′-didehydro-1′,2′-dihydro-β,ψ-carotene-3,1′,2′-triol) was cloned from Flavobacterium P99-3. It contains a gene highly homologous to crtA from purple bacteria encoding there an acyclic carotenoid 2-ketolase. Since no ketolation step is involved in myxol biosynthesis, the function of crtA-OH from Flavobacterium was assigned by complementation in Escherichia coli engineered to synthesize demethylspheroidene and 1′-hydroxy-demethylspheroidene. Upon co-expression of crtA-OH, the formation of 2-hydroxy derivatives of both carotenoids assigns CrtA-OH as a novel carotenoid hydroxylase. The gene was used to re-construct myxol biosynthesis in E. coli successfully. Additionally, 1′,2′-dihydroxytorulene and 1,2,1′-trihydroxy-3,4,3′,4′-tetradehydrolycopene were obtained. Their generation demonstrates that a new class of 2-hydroxy carotenoids can now be pursued by genetic engineering in E. coli.     

DNA-binding and photocleavage studies of [Ru(phen)2(NMIP)]

A novel ligand 2′-(2″-nitro-3″,4″-methylenedioxyphenyl)imidazo[4′,5′-f][1,10]-phenanthroline (NMIP) and its complex [Ru(phen)₂(NMIP)]²⁺ have been synthesized and characterized by mass spectroscopy, ¹H NMR and cyclic voltammetry. Binding of the complex with calf thymus DNA (CT DNA) has been investigated by spectroscopic methods, viscosity and electrophoresis measurements. The experimental results indicate that [Ru(phen)₂(NMIP)]²⁺ binds to DNA via partial intercalative mode and the individual enantiomers of it bind to DNA in different rates. [Ru(phen)₂(NMIP)]²⁺ has also been found to promote cleavage of plasmid pBR 322 DNA from the supercoiled Form I to the open circular Form II upon irradiation.     

1,4-Dioxane-2,5-dione-type monomers derived from l-ascorbic and d-isoascorbic acids. Synthesis and polymerisation

l-Ascorbic and d-isoascorbic acids have been used as the starting materials for the preparation of (3R,4′S)-3-(2′,2′-dimethyl-1′,3′-dioxolan-4′-yl)-1,4-dioxane-2,5-dione (IPTA), (3R and S, 4′S,6R)-3-methyl-6-(2′,2′-dimethyl-1′,3′-dioxolan-4′-yl)-1,4-dioxane-2,5-dione (IPTP) and (3R,4′R)-3-(2′,2′-dimethyl-1′,3′-dioxolan-4′-yl)-1,4-dioxane-2,5-dione (IPEA), three novel 1,4-dioxane-2,5-dione-type monomers. Ring-opening homopolymerisation and copolymerisation of the IPTA monomer, derived from l-ascorbic acid, with d,l-lactide have been performed. The polymers were characterised by elemental microanalysis, as well as IR and ¹H and ¹³C NMR spectroscopies. GPC was used to estimate product molecular weights, and thermal studies (DSC and TGA) revealed that all the polymers were amorphous, being stable up to 250 °C under nitrogen.     

Keto-fluorothiopyranosyl nucleosides: a convenient synthesis of 2- and 4-keto-3-fluoro-5-thioxylopyranosyl thymine analogs

A novel series of fluorinated keto-β-d-5-thioxylopyranonucleosides bearing thymine as the heterocyclic base have been designed and synthesized. Deprotection of 3-deoxy-3-fluoro-5-S-acetyl-5-thio-d-xylofuranose (1) and selective acetalation gave the desired isopropylidene 5-thioxylopyranose precursor 3. Acetylation and isopropylidene removal followed by benzoylation led to 3-deoxy-3-fluoro-1,2-di-Ο-benzoyl-4-O-acetyl-5′-thio-d-xylopyranose (6). This was condensed with silylated thymine and selectively deacetylated to afford 1-(2′-Ο-benzoyl-3′-deoxy-3′-fluoro-5′-thio-β-d-xylopyranosyl)thymine (8). Oxidation of the free hydroxyl group in the 4′-position of the sugar led to the formation of the target 4′-keto compound together with the concomitant displacement of the benzoyl group by an acetyl affording, 1-(2′-O-acetyl-3′-deoxy-3′-fluoro-β-d-xylopyranosyl-4′-ulose)thymine (9). Benzoylation of 3 and removal of the isopropylidene group followed by acetylation, furnished 3-deoxy-3-fluoro-1,2-di-Ο-acetyl-4-O-benzoyl-5′-thio-d-xylopyranose (12). Condensation of thiosugar 12 with silylated thymine followed by selective deacetylation led to the 1-(4′-Ο-benzoyl-3′-fluoro-5′-thio-β-d-xylopyranosyl)thymine (14). Oxidation of the free hydroxyl group in the 2′-position and concomitant displacement of the benzoyl group by an acetyl gave target 1-(4′-O-acetyl-3′-deoxy-3′-fluoro-β-d-xylopyranosyl-2′-ulose)thymine (15).     

ALCAPs induce mitochondrial apoptosis and activate DNA damage response by generating ROS and inhibiting topoisomerase I enzyme activity in K562 leukemia cell line

Endemic Alkanna cappadocica was used to isolate novel antitumor molecules from Turkish landscapes in our previous studies. In this study, deoxyalkannin (ALCAP1), β,β-dimethylacrylalkannin (ALCAP2), acetylalkannin (ALCAP3), and alkannin (ALCAP4) as well as the novel isolated compounds 5-methoxydeoxyalkannin (ALCAP5), 8-methoxydeoxyalkannin (ALCAP6), 5-methoxyacetylalkannin (ALCAP7), 5-methoxy-β,β-dimethylacrylalkannin (ALCAP8) were characterized. The topoisomerase I (topo I) inhibitory activity of ALCAPs was investigated using in vitro plasmid relaxation assay and found that ALCAP2, 3, 4 and 7 were potent inhibitors at 2–6 μM concentrations. Further, DNA damage response to ALCAP treatments was also studied by measuring the H2AX^(S139) and ATM^(S1981) phosphorylations. ALCAP2, 7 and 8 induced the DNA damage and apoptosis, consistently resulted in PARP cleavage at nanomolar concentrations in K562 leukemia cells. Moreover, when the free radical (ROS) generating capacity of the compounds was studied by 2′,7′-dichlorofluorescein-diacetate assay using flow cytometry, we found that a known antioxidant N-acetyl-cysteine almost completely abrogated the H2AX^(S139) phosphorylations and the caspase 3 cleavage and activation. Thus, γH2AX^(S139) foci formation remained higher than the control, and an increase in CHK2^(T68) phosphorylation was observed by ALCAP2 and 7 treatments suggested that, these compounds can be potential therapeutics against tumor cell growth because of their unique DNA damaging abilities additional to enzyme inhibition similar to those of doxorubicin.     

Leishmania amazonensis: characterization of an ecto-3'-nucleotidase activity and its possible role in virulence

Ecto-3′-nucleotidase/nuclease (3′NT/NU) is a membrane-bound enzyme that plays a key role in the nutrition of Leishmania sp. protozoan parasites. This enzyme generates nucleosides via hydrolyzes of 3′mononucleotides and nucleic acids, which enter the cell by specific transporters. In this work, we identify and characterize Leishmania amazonensis ecto-3′-nucleotidase activity (La3′-nucleotidase), report ammonium tetrathiomolybdate (TTM) as a novel La3′-nucleotidase inhibitor and approach the possible involvement of ecto-3′-nucleotidase in cellular adhesion. La3′-nucleotidase presented characteristics similar to those reported for the class I single-strand nuclease family; a molecular weight of approximately 40 kDa and optimum activity in an alkaline pH range were observed. Although it is conserved among the genus, La3′-nucleotidase displays different kinetic properties; it can be inhibited by vanadate, molybdate and Cu²⁺ ions. Interestingly, ecto-3′-nucleotidase activity is 60-fold higher than that of ecto-5′-nucleotidase in L. amazonensis. Additionally, ecto-3′-nucleotidase activity is two-fold higher in virulent L. amazonensis cells than in avirulent ones. Notably, macrophage–parasite attachment/invasion was increased by 400% in the presence of adenosine 3′-monophosphate (3′AMP); however, this effect was reverted by TTM treatment. We believe that La3′-nucleotidase may play a significant role in the generation of adenosine, which may contribute to mammalian host immune response impairment and establishment of infection.     

Crystal structures of Streptococcus mutans 2'-deoxycytidylate deaminase and its complex with substrate analog and allosteric regulator dCTP x Mg2+

2′-Deoxycytidylate deaminase [or deoxycytidine-5′-monophosphate (dCMP) deaminase, dCD] catalyzes the deamination of dCMP to deoxyuridine-5′-monophosphate to provide the main nucleotide substrate for thymidylate synthase, which is important in DNA synthesis. The activity of this homohexameric enzyme is allosterically regulated by deoxycytidine-5′-triphosphate (dCTP) as an activator and by deoxythymidine-5′-triphosphate as an inhibitor. In this article, we report the crystal structures of dCMP deaminase from Streptococcus mutans and its complex with dCTP and an intermediate analog at resolutions of 3.0 and 1.66 Å. The protein forms a hexamer composed of subunits adopting a three-layer α/β/α sandwich fold. The positive allosteric regulator dCTP mainly binds at the interface between two monomers in a molar ratio of 1:1 and rearranges the neighboring interaction networks. Structural comparisons and sequence alignments revealed that dCMP deaminase from Streptococcus mutans belongs to the cytidine deaminase superfamily, wherein the proteins exhibit a similar catalytic mechanism. In addition to the two conserved motifs involved in the binding of Zn^2 +, a new conserved motif, (G⁴³YNG⁴⁶), related to the binding of dCTP was also identified. N-terminal Arg4, a key residue located between two monomers, binds strongly to the γ phosphate group of dCTP. The regulation signal was transmitted by Arg4 from the allosteric site to the active site via modifications in the interactions at the interface where the substrate-binding pocket was involved and the relocations of Arg26, His65, Tyr120, and Arg121 to envelope the active site in order to stabilize substrate binding in the complex. Based on the enzyme-regulator complex structure observed in this study, we propose an allosteric mechanism for dCD regulation.     

pH- and DNA-induced dual molecular light switches based on a novel ruthenium(II) complex

A novel Ru(II) complex, [Ru(bpy)₂(btppz)]Cl₂, where bpy = 2,2′-bipyridine and btppz = benzo[h]tripyrido[3,2-a:2′,3′-c:2″,3″-j]phenazine, has been synthesized and characterized. The pH effects on UV-visible (UV-vis) absorption and emission spectra of the complex have been studied and ground- and excited-state ionization constants of the complex have been derived. The calf thymus DNA (ct-DNA) binding properties of the complex were investigated with UV-vis absorption and luminescence spectrophotometric titrations, steady-state emission quenching by [Fe(CN)₆]⁴⁻, DNA competitive binding with ethidium bromide, DNA melting experiments, reverse salt titrations and viscosity measurements. The complex was demonstrated to act as dual molecular switches: pH-induced “on-off” emission switch with an on-off intensity ratio of ∼54 which is favorably compared with those reported for structurally analogous Ru(II) complexes, and a DNA molecular light switch with a luminescence enhancement factor of 22 as it intercalatively bound to the DNA.