Synthesis and properties of oligonucleotides containing aminodeoxythymidine units.

Procedures are described for synthesis via solid support methodology of oligonucleotide analogues derived in part from 3'-amino-3'-deoxythymidine or 5'-amino-5'-deoxythymidine. Oligothymidylate decamers terminated with a 3'-amino group or containing a 3'-NHP(O)(O-)O-5' internucleoside link are found to form unusually stable complexes with poly(dA), poly(A), and oligo(dA). For related derivatives of 5'-amino-5'-deoxythymidine enhancement is less or absent, and in the case of multiple substitution destabilization of the heteroduplex may be observed. That the effect of the 3'-amino group is general for oligonucleotide derivatives is indicated by enhanced Tm values for heteroduplex complexes of the mixed-base oligomer, d(TATTCAGTCAT(NH2)), and the methyl phosphonate derivatives, TmTmTmTmTmTmTmTmTmT(NH2) and d(TmAmTmTmCmAmGmTmCmAmT(NH2)).     

Formation of internucleotide 3''-5'' phosphoramidate links by direct coupling of phosphoryl and amino groups.

The stepwise synthesis of oligomers derived from 5'-amino-5'-deoxythymidine 3'-phosphate and 5'-amino-5'-deoxy-3'-O-mono-p-methoxytrityl-thymidine is described. The internucleotide phosphoramidate links were formed by condensation of nucleoside 3'-phosphates with aminonucleosides by means of triphenylphosphine and dipridyl disulfide.     

Acid-mediated cleavage of oligonucleotide P3' --> N5' phosphoramidates triggered by sequence-specific triplex formation

The P-N bond in oligonucleotide P3' --> N5' phosphoramidates (5'-amino-DNA) is known to be chemoselectively cleaved under mild acidic conditions. We prepared homopyrimidine oligonucleotides containing 5'-amino-5'-deoxythymidine (5'-amino-DNA thymine monomer) or its conformationally locked congener, 5'-amino-2',4'-BNA thymine monomer, at midpoint of the sequence. The effect of triplex formation with homopurineohomopyrimidine dsDNA targets on acid-mediated hydrolysis of the P3' --> N5' phosphoramidate linkage was evaluated. Very interestingly, it was found that the triplex formation significantly accelerates the P-N bond cleavage.     

Molecular and crystalline structure of 3'-methylamino-2',3'-dideoxyribosylthymine--a potential inhibitor of HIV]

The structure of 3'-methylamino-2',3'-dideoxyribosylthymine [ddT(3'NHMe)] was determined by X-ray analysis. The space group is P2(1)2(1)2(1). Cell dimensions are: a 5.132(1), b 13.718(1), c 16.947(2) A, V 1193.2 A3, Z 4. The structure was solved by directed methods and refined by the full-matrix least square method to R 4.8%. The molecule of ddT(3'NHMe) has anti-conformation with respect to the glycosidic bond (chi (O4'-C1'-N1-C2) = -106.7 degrees), C3'-endo-C4'-exo puckering of the sugar moiety (P -28.8 degrees, psi m -31.5 degrees) and gauche-gauche conformation about exocyclic C4'-C5' bond (psi(C3'-C4'-C5'-O5') 45.8 degrees). The structure of ddT(3'NHMe) was compared with those of 3'-amino-3'-deoxythymidine, 3'-azido-3'-deoxythymidine and natural thymidine.     

Synthesis of new terminator substrates for DNA-polymerases-- nucleoside-5'-triphosphates with modified carbohydrate residue

Synthesis of 3'-chloro- and 3'-cyanothio-2',3'-dideoxythymidine by the reaction of 2,3'-anhydro-2'-deoxythymidine with ammonium chloride and lithium thiocyanate, respectively, has been developed. In addition, 3'-methanesulphonylamido- and 3'-sulphonylamido-2',3'-dideoxythymidines were synthesized starting from 3'-amino-2',3'-dideoxythymidine. All these compounds along with 2',3'-anhydroriboadenosine,2',3'-anhydrolyxoadenosine, 2',3'-O-isopropylidenecytidine, and 2,3'-anhydro-2'-deoxythymidine were transformed into 5'-triphosphates by treatment with phosphoryl tris-1,2,4-triazolide and then with bis(tri-n-butylammonium)pyrophosphate. All 5'-triphosphates of nucleoside analogues were tested as termination substrates in cell-free systems with various DNA polymerases.     

Synthesis and properties of 3'-amino-2',4'-BNA, a bridged nucleic acid with a N3'-->P5' phosphoramidate linkage

The synthesis and properties of a bridged nucleic acid analogue containing a N3'-->P5' phosphoramidate linkage, 3'-amino-2',4'-BNA, is described. A heterodimer containing a 3'-amino-2',4'-BNA thymine monomer, and thymine and methylcytosine monomers of 3'-amino-2',4'-BNA and their 5'-phosphoramidites, were synthesized efficiently. The dimer and monomers were incorporated into oligonucleotides by conventional 3'-->5' assembly, and 5'-->3' reverse assembly phosphoramidite protocols, respectively. Compared to a natural DNA oligonucleotide, modified oligonucleotides containing the 3'-amino-2',4'-BNA residue formed highly stable duplexes and triplexes with single-stranded DNA (ssDNA), single-stranded RNA (ssRNA), and double-stranded DNA (dsDNA) targets, with the average increase in melting temperature (T(m)) against ssDNA, ssRNA and dsDNA being +2.7 to +4.0 degrees C, +5.0 to +7.0 degrees C, and +5.0 to +11.0 degrees C, respectively. These increases are comparable to those observed for 2',4'-BNA-modified oligonucleotides. In addition, an oligonucleotide modified with a single 3'-amino-2',4'-BNA thymine residue showed extraordinarily high resistance to nuclease degradation, much higher than that of 2',4'-BNA and substantially higher even than that of 3'-amino-DNA and phosphorothioate oligonucleotides. The above properties indicate that 3'-amino-2',4'-BNA has significant potential for antisense and antigene applications.     

Reversal of the deoxyhypusine synthesis reaction. Generation of spermidine or homospermidine from deoxyhypusine by deoxyhypusine synthase

Deoxyhypusine synthase catalyzes the first step in hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine) synthesis in a single cellular protein, eIF5A precursor. The synthesis of deoxyhypusine catalyzed by this enzyme involves transfer of the 4-aminobutyl moiety of spermidine to a specific lysine residue in the eIF5A precursor protein to form a deoxyhypusine-containing eIF5A intermediate, eIF5A(Dhp). We recently discovered the efficient reversal of deoxyhypusine synthesis. When eIF5A([3H]Dhp), radiolabeled in the 4-aminobutyl portion of its deoxyhypusine residue, was incubated with human deoxyhypusine synthase, NAD, and 1,3-diaminopropane, [3H]spermidine was formed by a rapid transfer of the radiolabeled 4-aminobutyl side chain of the [3H]deoxyhypusine residue to 1,3-diaminopropane. No reversal was observed with [3H]hypusine protein, suggesting that hydroxylation at the 4-aminobutyl side chain of the deoxyhypusine residue prevents deoxyhypusine synthase-mediated reversal of the modification. Purified human deoxyhypusine synthase also exhibited homospermidine synthesis activity when incubated with spermidine, NAD, and putrescine. Thus it was found that [14C]putrescine can replace eIF5A precursor protein as an acceptor of the 4-aminobutyl moiety of spermidine to form radiolabeled homospermidine. The Km value for putrescine (1.12 mM) as a 4-aminobutyl acceptor, however, is much higher than that for eIF5A precursor (1.5 microM). Using [14C]putrescine as an acceptor, various spermidine analogs were evaluated as donor substrates for human deoxyhypusine synthase. Comparison of spermidine analogs as inhibitors of deoxyhypusine synthesis, as donor substrates for synthesis of deoxyhypusine (or its analog), and for synthesis of homospermidine (or its analog) provides new insights into the intricate specificity of this enzyme and versatility of the deoxyhypusine synthase reaction.     

Fluorescence energy transfer between points in G-actin: the nucleotide-binding site, the metal-binding site and Cys-373 residue

Fluorescence energy transfers were studied in order to investigate the spatial relationships between the nucleotide-binding site, the metal-binding site and the Cys-373 residue in the G-actin molecule. When 1-N6-ethenoadenosine-5'-triphosphate (epsilon-ATP) in the nucleotide-binding site and Co2+ or Ni2+ in the metal-binding site were used as fluorescence donor and acceptor, respectively, the fluorescence intensity of epsilon-ATP was perfectly quenched by Ni2+ or Co2+. This indicated that the nucleotide-binding site is very close to the metal-binding site; the distance should be less than 10 A. When N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS) bound to Cys-373 residue and Co2+ in the metal-binding site were used as a fluorescence donor and an acceptor, respectively, the transfer efficiency was equal to 5 +/- 1%. The corresponding distance was calculated to be 23-32 A, assuming a random orientation factor K2 = 2/3.     

Degradation of phosphatidylnucleosides induced by phospholipases

Hydrolysis of phosphatidylnucleosides, 5'-(rac-1-hexadecyl-2-palmitoyl-sn-glycero-3-phosphoryl)-3'-azido- 3'-deoxythymidine and -2',3'-didehydro-3'-deoxythymidine, effected by phospholipases (PL) A2, C, and D was studied to reveal the metabolism of these derivatives. It was shown that PLA2 deacetylates the glycerol residue at position 2, PLC is inactive, and PLD hydrolyzes the phosphatidylnucleosides to give free nucleosides.     

Enzymatic synthesis of duplex circular phiX174 DNA containing phosphoramidate bonds in the (-) strand.

Duplex circular phiX174 DNA (RF I) containing some phosphoramidate links in the backbone chain of the (-) strand was synthesized by reaction of 5'-amino-5'-deoxythymidine 5'-triphosphate, dCTP, dGTP, and 3H-dATP with DNA polymerase I and DNA ligase (T4) on a (+) strand phiX174 amber 3 DNA template. The yield of duplex DNA was higher when dTTP was included along with the amino analog in the initial reaction system or was added late in the synthesis. RF I DNA was observed as a rapidly sedimenting species in an alkaline sucrose gradient, and the presence of phosphoramidate linkages was demonstrated by the unusual lability of the duplex DNA in a weakly acidic solution.     

Crisscross enzymatic reaction between the two molecules in the active dimeric P69 form of the 2'-5' oligodenylate synthetase

2'-5' oligoadenylate (2-5 (A)) synthetases are major components of the antiviral pathways induced by interferons. In the presence of double-stranded RNA, they polymerize ATP to form 2-5 (A) oligomers that, in turn, activate the latent ribonuclease RNase L, causing mRNA degradation. These enzymes, unlike other nucleotidyl transferases, catalyze 2'-5', not 3'-5', phosphodiester bond formation between substrates bound to the acceptor and donor sites. Moreover, unlike other members of this extended family, the P69 isozyme of 2-5 (A) synthetase functions as a homodimer. Here, we report that the need for P69 dimerization is because of a crisscross enzyme reaction joining two substrate molecules bound to two opposite subunits. Consequently, although homodimers of mutants in the previously identified acceptor site, the donor site, or the catalytic site were inactive, selective heterodimers of the mutants were active because of subunit complementation. The catalytic site had to be present in the same subunit that contained the acceptor site, whereas the donor site had to be provided by the other subunit. These results allowed us to design a mutant protein that acted as a dominant-negative inhibitor of wt P69 but not of another isozyme of 2-5 (A) synthetase.     

Fluorescence resonance energy transfer studies on the proximity between lysine-107 and cysteine-239 in rabbit muscle aldolase

Spatial relationships between Lys-107, which binds the C-6 phosphate group of the substrate, and fast-reacting Cys-239, located outside the active site of rabbit muscle aldolase, were studied by means of resonance energy transfer. The Lys-107 residue was covalently linked to pyridoxal phosphate (fluorescence donor) and the Cys-239 residue was modified by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (fluorescence acceptor). The energy transfer between donor and acceptor has been demonstrated. The steady-state and the lifetime measurements indicate that in solution the distance between Lys-107 and Cys-239 in the aldolase molecule is 12.4 A assuming chi 2 = 2/3.     

Identification of pheromones in mouse urine by head-space solid phase microextraction followed by gas chromatography-mass spectrometry

Transmission of HIV from mother to infant can be effectively prevented by zidovudine (3'-azido-3'-deoxythymidine; AZT) alone or in combination with other anti-retroviral drugs; however, significant evidence for genotoxicity, including transplacental carcinogenicity in mice, has been reported for AZT. A method, based upon solid phase extraction (SPE) in the 96-well format, gradient liquid chromatography (LC), and electrospray mass spectrometry (MS), was developed and validated to measure serum concentrations in maternal C57BL/6N and fetal B6C3F1 mice of the nucleoside analogs AZT, lamivudine ((-)2',3'-dideoxy-3'-thiacytidine; 3TC), and several metabolites selected based on importance in detoxification and bioactivation reactions. After intravenous (i.v.) and oral dosing with either 400 mg/kg AZT or 200 mg/kg 3TC, pharmacokinetics were determined for AZT, AZT-5'-glucuronide, 3'-amino-3'-deoxythymidine (AMT), AZT-5'-phosphate, 3TC, and 3TC-5'-phosphate in serum of adult female mice. Pharmacokinetics were also determined in spleen for AZT-5'-phosphate and 3TC-5'-phosphate following i.v. dosing. In addition, a preliminary assessment was made of placental transfer of AZT and 3TC and the presence of metabolites in the fetal compartment. The method described provides a means to evaluate thoroughly metabolism and disposition of anti-retroviral nucleoside analogs in maternal and fetal mice for comprehensive studies of genotoxicity.     

The structural basis for specificity in human ABO(H) blood group biosynthesis

The human ABO(H) blood group antigens are produced by specific glycosyltransferase enzymes. An N-acetylgalactosaminyltransferase (GTA) uses a UDP-GalNAc donor to convert the H-antigen acceptor to the A antigen, whereas a galactosyltransferase (GTB) uses a UDP-galactose donor to convert the H-antigen acceptor to the B antigen. GTA and GTB differ only in the identity of four critical amino acid residues. Crystal structures at 1.8-1.32 A resolution of the GTA and GTB enzymes both free and in complex with disaccharide H-antigen acceptor and UDP reveal the basis for donor and acceptor specificity and show that only two of the critical amino acid residues are positioned to contact donor or acceptor substrates. Given the need for stringent stereo- and regioselectivity in this biosynthesis, these structures further demonstrate that the ability of the two enzymes to distinguish between the A and B donors is largely determined by a single amino acid residue.     

Identification of a mung bean arabinofuranosyltransferase that transfers arabinofuranosyl residues onto (1, 5)-linked alpha-L-arabino-oligosaccharides

Arabinofuranosyltransferase activity was identified in Golgi membranes obtained from mung bean (Vigna radiata) hypocotyls. The enzyme transfers the arabinofuranosyl (Araf) residue from UDP-beta-L-arabinofuranose to exogenous (1, 5)-linked alpha-L-arabino-oligosaccharides labeled at their reducing ends with 2-aminobenzamide. The transferred residue was shown, using 1H-nuclear magnetic resonance spectroscopy and alpha-L-arabinofuranosidase treatment, to be alpha-L-Araf and to be linked to O-5 of the nonreducing terminal Araf residue of the acceptor oligosaccharide. The enzyme was nonprocessive because only a single Araf residue was added to the acceptor molecule. Arabino-oligosaccharides with a degree of polymerization between 3 and 8 were acceptor substrates. The 2-aminobenzamide-labeled arabino-tetra- and pentasaccharides were the most effective acceptor substrates analyzed. The enzyme has a pH optimum between 6.5 and 7.0 and its activity is stimulated by Mn2+ and Co2+ ions. The apparent Km and Vmax values of the arabinofuranosyltransferase for UDP-arabinofuranose are 243 microm and 243 pmol min(-1) mg protein(-1), respectively. The highest enzyme activity was detected in the elongating regions of mung bean hypocotyls. The data show that UDP-arabinofuranose is the donor molecule for the generation of arabino-oligosaccharides composed of Araf residues.     

Synaptic complex formation of two retrovirus DNA attachment sites by integrase: a fluorescence energy transfer study

The integration of retroviral DNA by the viral integrase (IN) into the host genome occurs via assembled preintegration complexes (PIC). We investigated this assembly process using purified IN and viral DNA oligodeoxynucleotide (ODN) substrates (93 bp in length) that were labeled with donor (Cy3) and acceptor fluorophores (Cy5). The fluorophores were attached to the 5' 2 bp overhangs of the terminal attachment (att) sites recognized by IN. Addition of IN to the assay mixture containing the fluorophore-labeled ODN resulted in synaptic complex formation at 14 degrees C with significant fluorescence resonance energy transfer (FRET) occurring between the fluorophores in close juxtaposition (from approximately 15 to 100 A). Subsequent integration assays at 37 degrees C with the same ODN (32P-labeled) demonstrated a direct association of a significant FRET signal with concerted insertion of the two ODNs into the circular DNA target, here termed full-site integration. FRET measurements (deltaF) show that IN binds to a particular set of 3' OH recessed substrates (type I) generating synaptic complexes capable of full-site integration that, as shown previously, exhibit IN mediated protection from DNaseI digestion up to approximately 20 bp from the ODN att ends. In contrast, IN also formed complexes with nonspecific DNA ends and loss-of-function att end substrates (type II) that had significantly lower deltaF values and were not capable of full-site integration, and lacked the DNaseI protection properties. The type II category may exemplify what is commonly understood as "nonspecific" binding by IN to DNA ends. Two IN mutants that exhibited little or no integration activity gave rise to the lower deltaF signals. Our FRET analysis provided the first direct physical evidence that IN forms synaptic complexes with two DNA att sites in vitro, yielding a complex that exhibits properties comparable to that of the PIC.     

Conformations within soluble oligomers and insoluble aggregates revealed by resonance energy transfer

A fluorescently labeled 20‐residue polyglutamic acid (polyE) peptide 20 amino acid length polyglutamic acid (E₂₀) was used to study structural changes which occur in E₂₀ as it co‐aggregates with other unlabeled polyE peptides. Resonance energy transfer (RET) was performed using an o‐aminobenzamide donor at the N‐terminus and 3‐nitrotyrosine acceptor at the C‐terminus of E₂₀. PolyE aggregates were not defined as amyloid, as they were nonfibrillar and did not bind congo red. Circular dichroism measurements indicate that polyE aggregation involves a transition from α‐helical monomers to aggregated β‐sheets. Soluble oligomers are also produced along with aggregates in the reaction, as determined through size exclusion chromatography. Time‐resolved and steady‐state RET measurements reveal four dominant E₂₀ conformations: (1) a partially collapsed conformation (24 Å donor–acceptor distance) in monomers, (2) an extended conformation in soluble oligomers (>29 Å donor–acceptor distance), (3) a minor partially collapsed conformation (22 Å donor‐acceptor distance) in aggregates, and (4) a major highly collapsed conformation (13 Å donor–acceptor distance) in aggregates. These findings demonstrate the use of RET as a means of determining angstrom‐level structural details of soluble oligomer and aggregated states of proteins. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 299–317, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Synthesis and intermembrane transfer of pyrene-labelled liponucleotides: ceramide phosphothymidines.

Phospholipid conjugates of 3'-azido-3'-deoxythymidine (AZT) show activity against human immunodeficiency virus (HIV) in vitro. Here we report on the synthesis and characterization of two pyrene containing conjugates: 2-N-(4-(pyren-1-yl)butanoyl)ceramide 5'-phosphothymidine (Pbs-Cer-P-T) (XII) and 2-N-(10-(pyren-1-yl)decanoyl)ceramide 5'-phosphothymidine (Pds-Cer-P-T) (XIII). These fluorescent labelled conjugates served as model compounds to study incorporation of sphingoliponucleotides into membranes. The complex compounds were prepared by condensation of 3'-acetylthymidine and labelled ceramides using the phosphite triester coupling procedure. UV absorption, fluorimetry as well as 1H-, 31P-, 13C-NMR analyses were used for structure confirmation of the synthesized substances. When incorporated into small unilamellar 1-palmitoyl-2-oleoyl-glycerophosphatidyl-choline (POPC) vesicles and incubated with unlabelled acceptor POPC vesicles, the compounds (XII) and (XIII) exhibited spontaneous transfer. Kinetic data suggest that transfer from donor to acceptor vesicles occurred via the intervening aqueous phase. The non-specific lipid transfer protein from bovine liver stimulated the transfer of Pds-Cer-P-T between phospholipid vesicles in a concentration dependent manner.     

Antisense oligodeoxynucleotides targeting internal exon sequences efficiently regulate TNF-alpha expression.

ABSTRACT Exon sequences upstream of splice sites play a critical role in mRNA processing, which is dependent on spliceosome interactions with these sites. Using antisense oligodeoxynucleotides (ODN), we targeted these and other sequences of the proinflammatory tumor necrosis factor-alpha (TNF-alpha) gene because it is multiply spliced and has been difficult to regulate with ODN in the past. ODN targeting exon sequences upstream of the donor splice sites of internal exons 2 (ORF4) and 3 (ORF6) significantly reduced TNF-alpha levels in stimulated U937 cells by 62%+/-7% and 51%+/-9%, respectively, in a dose-dependent manner but did not affect interleukin-6 (IL-6) levels. In contrast, ODN targeting the exon sequences downstream of the acceptor splice sites of exons 1, 2, and 3 failed to reduce TNF-alpha levels significantly under the same conditions. End-phosphorothioated ORF4 (ORF4-PE) significantly reduced TNF-alpha mRNA levels by greater than 80% (p < 0.001) and protein levels by 60% (p < 0.001) in U937 cells. ORF4-PE reduced newly synthesized TNF-alpha protein levels by >80% in lipopolysaccharide (LPS)-stimulated human macrophages, by greater than 60% in phorbol myristate acetate/phyto-hemagglutinin (PMA/PHA)-stimulated human peripheral blood mononuclear cells (PBMC), and by approximately 50% in LPS-stimulated murine monocytes. These results suggest that exon sequences flanking donor splice sites are highly susceptible target domains for antisense inhibition of TNF-alpha gene expression.