3-Nitropyrrole and 5-nitroindole as universal bases in primers for DNA sequencing and PCR.

3-Nitropyrrole and 5-nitroindole have been assessed as universal bases in primers for dideoxy DNA sequencing and in the polymerase chain reaction (PCR). In contrast to a previous report, we have found that the introduction of more than one 3-nitropyrrole residue at dispersed positions into primers significantly reduced their efficiency in PCR and sequencing reactions. Primers containing 5-nitroindole at multiple dispersed positions were similarly affected; for both bases only a small number of substitutions were tolerated. In PCR experiments neither base, when incorporated into primers in codon third positions, was as effective as hypoxanthine, which was incorporated in six codon third positions in a 20mer oligomer. However, primers containing up to four consecutive 5-nitroindole substitutions performed well in both PCR and sequencing reactions. Consecutive 3-nitropyrrole substitutions were tolerated, but less well in comparable reactions.     

INCORPORATION OF 8-HISTAMINYL-DEOXYADENOSINE [8-(2-(4-IMIDAZOLYL)ETHYLAMINO)-2′-DEOXYRIBOADENOSINE] INTO OLIGODEOXYRIBONUCLEOTIDES BY SOLID PHASE PHOSPHORAMIDITE COUPLING

ABSTRACT

The 3′phosphoramidite of 8-histaminyl deoxyadenosine has been prepared and successfully incorporated into a short oligodeoxyribonucleotide. The synthetic methodology leading to this preparation is given and the implications for developing new DNAzymes as well as probing unusual nucleic acid structures are discussed.     

Computational Evaluation of Intermolecular Interactions of a Universal Base 3-Nitropyrrole in Stacked Dimers and DNA Duplexes

Abstract

The stacking interactions between a universal base of 3-nitropyrrole (3NP) and four canonical nucleobases were studied by means of ab initio molecular orbital calculations. The stabilities of the complexes are comparable to those of the stacked dimers of canonical bases reported previously. The detailed analysis of the interaction energies revealed the importance of the dipole-dipole interaction included in the Hartree-Fock terms to determine the geometry dependence of the stacking energies. It was also clarified that the dispersion energies included in the electron-correlation terms were essential to obtain adequate stabilities. The contribution of the nitro group was evaluated by the comparative studies of pyrrole and 3NP. The increased molecular dipole moment and surface are expected to account for the enhancement of the stability of the stacked dimers containing 3NP. The force field parameters required for calculation of the molecular mechanics of 3NP were obtained for 3NP on the basis of these molecular orbital calculations. The energy-minimized structures obtained by the molecular mechanics calculations of 3NP accorded with those obtained by the molecular orbital calculations described above. A DNA duplex structure containing 3NP-A, 3NP-T, or 3NP-C was calculated by use of these force field parameters. In the case of 3NP-A, the computationally calculated structure was in good agreement with that previously determined by use of ¹H-NMR except for the orientation of the nitro group.     

Synthesis and Evaluation of Oligodeoxynucleotides Containing 3′-C-Aminomethyl-and 3′-C-Amethylthymidine

Abstract

3′-C-Aminomethyl- and 3′-C-methylthymidine were synthesized and incorporated into oligodeoxynucleotides. Hybridization and enzyme stability of the modified oligonucleotides containing the 3′-C-branched thymidines are discussed.     

Synthesis of an Asymmetrically Substituted AZA Crown Ether as Metal and Amino Acid Binding Site in DNA Conjugates

Abstract

Crown ether 4 as a receptor core for protonated primary amines such as amino acids has been synthesized and incorporated into oligodeoxynucleotides as dangling ends.     

Peptide nucleic acid-DNA duplexes containing the universal base 3-nitropyrrole

A peptide nucleic acid (PNA) monomer containing the universal base 3-nitropyrrole was synthesized by coupling 1-carboxymethyl-3-nitropyrrole to ethyl N-[2-(tert-butoxycarbonylamino)ethyl]glycinate. The PNA sequence H-TGTACGTXACAACTA-NH2 (X = 3-nitropyrrole and C) and DNA sequence 5'-TGTACGTXACAACTA-3' were synthesized and thermal melting studies with the complementary DNA sequence 5'-TAGTTGTYACGTACA-3' (Y = A,C, G, T) compared. The T(m) data show that 3-nitropyrrole pairs indiscriminately with all four natural nucleobases as a constituent of either DNA or PNA. However, 3-nitropyrrole-containing PNA-DNA (average T(m) value = 51.1 degrees C) is significantly more thermally stable than 3-nitropyrrole-containing DNA-DNA (average T(m) value = 39.6 degrees C). From circular dichroism measurements, it is apparent that 3-nitropyrrole in the PNA strand causes a significant change in duplex structure.     

Synthesis and Hybridization Properties of Oligodeoxynucleotides Containing 3′-Deoxy-3′-C-methyleneuridine

Abstract

3′-Deoxy-3′-C-methyleneuridine nucleoside 1 ¹ has been incorporated into oligodeoxynucleotides. Relative to the unmodified references, oligomers containing nucleoside 1 displayed reduced binding affinities towards complementary DNA and RNA with a tendency towards RNA-selective hybridization.     

Synthesis and antiviral evaluation of a mutagenic and non-hydrogen bonding ribonucleoside analogue: 1-beta-D-Ribofuranosyl-3-nitropyrrole

Synthetic small molecules that promote viral mutagenesis represent a promising new class of antiviral therapeutics. Ribavirin is a broad-spectrum antiviral nucleoside whose antiviral mechanism against RNA viruses likely reflects the ability of this compound to introduce mutations into the viral genome. The mutagenicity of ribavirin results from the incorporation of ribavirin triphosphate opposite both cytidine and uridine in viral RNA. In an effort to identify compounds with mutagenicity greater than that of ribavirin, we synthesized 1-beta-D-ribofuranosyl-3-nitropyrrole (3-NPN) and the corresponding triphosphate (3-NPNTP). These compounds constitute RNA analogues of the known DNA nucleoside 1-(2'-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole. The 3-nitropyrrole pseudobase has been shown to maintain the integrity of DNA duplexes when placed opposite any of the four nucleobases without requiring hydrogen bonding. X-ray crystallography revealed that 3-NPN is structurally similar to ribavirin, and both compounds are substrates for adenosine kinase, an enzyme critical for conversion to the corresponding triphosphate in cells. Whereas ribavirin exhibits antiviral activity against poliovirus in cell culture, 3-NPN lacks this activity. Evaluation of 3-NPNTP utilization by poliovirus RNA-dependent RNA polymerase (RdRP) revealed that 3-NPNTP was not accepted universally. Rather, incorporation was only observed opposite A and U in the template and at a rate 100-fold slower than the rate of incorporation of ribavirin triphosphate. This diminished rate of incorporation into viral RNA likely precludes 3-NPN from functioning as an antiviral agent. These results indicate that hydrogen bonding substituents are critical for efficient incorporation of ribonucleotides into RNA by viral RdRPs, thus providing important considerations for the design of improved mutagenic antiviral nucleosides.     

3′-C-Hydroxymethylthymidine: Synthesis and Incorporation into Oligodeoxynucleotide Analogues

Abstract

The stereoselective synthesis of 3′-C-hydroxymethylthymidine (5) in five steps from thymidine has been accomplished and this nucleoside has been incorporated into oligodeoxynucleotides (ODNs) in different ways.     

A general enhancement scheme in heteronuclear multidimensional NMR employing pulsed field gradients

Summary General pulse sequence elements that achieve sensitivity-enhanced coherence transfer from a heteronucleus to protons of arbitrary multiplicity are introduced. The building blocks are derived from the sensitivity-enhancement scheme introduced by Cavanagh et al. ((1991) J. Magn. Reson., 91, 429–436), which was used in conjunction with gradient coherence selection by Kay et al. ((1992) J. Am. Chem. Soc., 114, 10663–10665), as well as from a multiple-pulse sequence effecting a heteronuclear planar coupling Hamiltonian. The building blocks are incorporated into heteronuclear correlation experiments, in conjunction with coherence selection by the formation of a heteronuclear gradient echo. This allows for efficient water suppression without the need for water presaturation. The methods are demonstrated in HSQC-type experiments on a sample of a decapeptide in H₂O. The novel pulse sequence elements can be incorporated into multidimensional experiments.     

7-Deaza-2′-deoxyguanosines Functionalized with 7-(ω-Aminoalk-1-YNYL) Residues

Abstract

The Pd(0)-catalyzed cross coupling reaction of 7-iodo-7-deaza-2′-deoxyguanosine (1, I⁷c⁷G_d) with the phthalimido-protected ω-aminoalkines 2a-c gave the compounds 3a-c. They were converted into the phosphoramidite building block 4a-c as well as the phosphonates 5a-c. Compounds 4a and 4c were incorporated into oligodeoxynucleotides of different sequence and their duplex stabilities were measured and compared with those of the unmodified counterparts.     

Pα-Methyl Deoxynucleoside Triphosphates as Substrates for E. COLI DNA Polymerase I in a Template-Directed Synthesis of DNA

Abstract

Pα-methyl deoxynucleoside triphosphates are used as substrates for E. coli DNA polymerase I in template-directed polymerase reactions. It is shown that the modified compounds are incorporated together with the unmodified deoxynucleoside triphosphates into DNA under both nick-translation and Klenow reaction conditions.     

Effect of the Nature of the 3β-Substitution in Manoyl Oxides on the Thermotropic Behavior of DPPC Lipid Bilayer and on DPPC Liposomes

Functionalized manoyl oxide derivatives have been proved over the years to evoke several biological responses. Among them, 3β-hydroxy-manoyl oxide (1) and 3β-acetoxy-manoyl oxide (2) have been shown to exhibit in vitro antimicrobial and cytotoxic activity, while N-imidazole-3 β-thiocarbonyl ester of manoyl oxide (3) was found to exhibit potent cytotoxic effect. Their partitioning into phospholipid bilayers may lead to membrane structure modifications that are crucial in liposome development as they may influence their maintenance and integrity. DSC was used to study the modifications induced in DPPC bilayers by incorporating increasing concentrations of the three manoyl oxide derivatives. All derivatives were found to strongly affect the bilayer structural organization in terms of a decrease of the cooperativity, the fluidization and partially destabilization of the gel phase and the induction of a lateral phase separation in clustering domains. Derivatives 1 and 3 were incorporated into DPPC liposomes and their physicochemical stability was monitored at 4°C. The stability of liposomes was strongly influenced by the presence of 1 and 3 at any molar ratio studied. DPPC/1 liposomes were found to retain its stability for 48 h at low concentration of 10% mol, while at higher concentrations up to 30% mol they collapsed into aggregated material. In all cases DPPC/3 liposomes were found unstable and sticky aggregated structures precipitated from the bulk suspension.     

Development of a New DNA Sequencing Method: 3′-Ester Cleavage Catalyzed by Taq DNA Polymerase

Abstract

A new 3′-esterified dTTP is incorporated into DNA by Taq DNA polymerase but does not act as a chain terminator. The esterase activity of the polymerase seems to be template dependent and occurs only if the next correct nucleotide is present.     

Reinvestigation of 4-Thiothymidine-5′-triphosphate Synthesis

Abstract

The 4-Thiothymidine-5′-triphosphate 1 (S⁴TTP) was known to be a substrate for polymerase, however a commercial sample of this compound failed to be incorporated into DNA. Mass spectrometry combined to alkaline phosphatase digestion and ³¹P-NMR showed that this sample was in fact 5′-chloro-5′-deoxy-4-thiothymidine-3′-triphosphate 2 . The desired S⁴TTP was synthesized by two alternate routes, was fully characterized and was shown to be incorporated in a DNA polymerase assay.     

Endcaps for Stabilizing Short DNA Duplexes

Abstract

The syntheses of endcaps for covalently linking the 3′ and 5′ hydroxyl groups of blunt end double-stranded DNA are described. Endcap diols were converted into DMTr protected phosphoramidites and incorporated between nucleotides 4 and 5 of a self-complementary octamer. The stabilizing effect of the endcaps on duplex DNA was determined by T_m experiments on the self-complementary octamer.     

α-cycloPNA: 1-Aminocylopentane-1-carboxylic Acid-Derived Peptide Nucleic Acid

Abstract

All four diastereoisomers of 3-thymine-1-(^tbutoxycarbonyl)aminocyclopentane- 1-carboxylic acid have been synthesised from (S)-dimethyl malate and thymine monomer 12 has been incorporated into an α-cycloPNA oligomer.     

Conformationally Locked Nucleosides. Synthesis of Oligodeoxynucleotides Containing 3′-Amino-3′-deoxy-3′-N,5′(R)-C-ethylenethymidine

Abstract

3′-Amino-3′-deoxy-5′-O-(4,4′-dimethoxytrityl)-3′-N,5′(R)-C-ethylenethymidine (6) was synthesized starting from 3′-azido-3′-deoxythymidine. Condensation of 6 with 5′-O-(H-phosphonyl)thymidine and 5′-O-(p-nitrophenoxycarbonyl)thymidine derivatives gave dinucleotide and dinucleoside derivatives, respectively, which were incorporated into oligodeoxynucleotides (ODNs). Tm data of the modified ODNs are also presented.     

Incorporation of the Lipopolysaccharide and Polysaccharide from Aeromonas Salmonicida Into Liposomes

Abstract

Incorporation of the lipopolysaccharide (LPS) and polysaccharide (PS) from Aeromonas salmonicida into liposomes of varying lipid composition and lamellarity as a function of the LPS and PS concentration was investigated. Positively-charged multilamellar vesicles (MLV) composed of phosphatidylcholine (PC): cholesterol (CH): stearylamine (SA) (6:3:1, mole: mole: mole) incorporated the LPS more readily than negatively-charged liposomes composed of PC: CH: phosphatidylglycerol (PG) in the same molar ratios. Regardless of surface charge, more LPS was incorporated into MLV than into vesicles prepared by relatively mild sonication (SV) or large unilamellar vesicles prepared via extrusion through 200 nm pore size filters (LUVET₂₀₀). In contrast, SV and LUVET₂₀₀ incorporated more PS than did MLV. The total amount of liposomally-incorporated LPS or PS among the three vesicle types was proportional to the concentration of antigen in the hydrating solutions.     

Synthesis of a new Photo-Cross-Linking Nucleoside Analogue Containing an Aryl(Trifluoromethyl)Diazirine Group: Application for Eco RII and Mva I Restriction-Modification Enzymes

Abstract

A new photo-cross-linking dU analog, 5-[4-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenyl]-2′-deoxyuridine, was synthesized and incorporated into the recognition site of Eco RII and Mva I restriction-modification enzymes. The resulting base-modified 14-mer substrate was tested for cross-linking to these enzymes. Cross-linking is effected by irradiation of the enzyme-substrate complexs at 366 nm.