Polypyrrole DNA chip on a silicon device: example of hepatitis C virus genotyping.

We describe in this article an oligonucleotide array constructed on a silicon device bearing a matrix of addressable 50-microns microelectrodes. Each electrode was covered by a conducting polymer (polypyrrole) grafted by an oligonucleotide (ODN). The DNA chip was prepared by successive electrochemically addressed copolymerizations of 5' pyrrole-labeled ODN and pyrrole. Following hybridization of the biotinylated amplified sample on the chip bearing a series of probes, detection was carried out by fluorescence microscopy through an R-phycoerythrin label. This technology was successfully applied to the genotyping of hepatitis C virus in blood samples. Results show good sensitivity and a high degree of dimensional resolution.

Solution structure of N-(2-deoxy-D-erythro-pentofuranosyl)urea frameshifts, one intrahelical and the other extrahelical, by nuclear magnetic resonance and molecular dynamics.

The presence of a N-(2-deoxy-D-erythro pentofuranosyl)urea (henceforth referred to as deoxyribosylurea) residue, ring fragmentation product of a thymine, in a frameshift situation in the sequence 5'd(AGGACCACG).d(CGTGGurTCCT) has been studied by 1H and 31P nuclear magnetic resonance and molecular dynamics. At equilibrium, two species are found in slow exchange. We observe that the deoxyribosylurea residue can be either intra- or extrahelical within structures which otherwise do not deviate strongly from that of a B-DNA as observed by NMR. Our study suggests that this is determined by the nature and number of hydrogen bonds which this residue can form as a function of two possible isomers. There are two possible structures for the urea side chain, either cis or trans for the urido bond which significantly changes the hydrogen bonding geometry of the residue. In the intrahelical species, the cis isomer can form two good hydrogen bonds with the bases on the opposite strand in the intrahelical species, A4 and C5, which is not the case for the trans isomer. This results in a kink in the helical axis. For the major extrahelical species, the situation is reversed. The trans isomer is able to form two good hydrogen bonds, with G13 on the same strand and A7 on the opposite strand. For the extrahelical species, the cis isomer can form only one hydrogen bond. In this major structure the NMR data show that the bases which are on either side of the deoxyribosylurea residue in the sequence, G14 and T16, are stacked over each other in a way similar to a normal B-DNA structure. This requires the formation of a loop for the backbone between these two residues. This loop can belong to one of two families, right- or left-handed. In a previous study of an abasic frameshift [Cuniasse et al. (1989) Biochemistry 28, 2018-2026], a left-handed loop was observed, whereas in this study a right-handed loop is found for the first time in solution. The deoxyribosylurea residue lies in the minor groove and can form both an intra- and an interstrand hydrogen bond.

Preparation of a DNA matrix via an electrochemically directed copolymerization of pyrrole and oligonucleotides bearing a pyrrole group.

A new methodology for the preparation of addressed DNA matrices is described. The process includes an electrochemically directed copolymerization of pyrrole and oligonucleotides bearing on their 5' end a pyrrole moiety introduced by phosphoramidite chemistry. The electro-controlled synthesis of the copolymer (poly-pyrrole) gives, in one step, a solid conducting film deposited on the surface of an electrode. The resulting polymer consists of pyrrole chains bearing covalently linked oligonucleotide. The polymer growth is limited to the electrode surface, so that it is possible to prepare a DNA matrix on a multiple electrode device by successive copolymerizations. A support bearing four oligonucleotides was used to detect three ras mutations on a synthetic DNA fragment.

Sterical recognition by T4 polynucleotide kinase of non-nucleosidic moieties 5'-attached to oligonucleotides.

The ability of T4 polynucleotide kinase (PNK) to phosphorylate non-nucleosidic moieties 5'-attached to oligodeoxynucleotides (ODNs) has been investigated. Non-nucleosidic phosphoramidite units were prepared from ethane-1,2-diol and propane-1,3-diol backbones. Some of them corresponded to pure enantiomers. They were used to obtain the corresponding 5'-end modified oligothymidylates X(pdT)10. The free primary hydroxyl of the non-nucleosidic moieties (X) of these oligomers was phosphorylated by PNK. We report the stereoselective phosphorylation of the L form of the 5'-end attached non-nucleosidic chiral fragments; the non-chiral moieties were completely phosphorylated. Dimers of glycerol analogue and thymidine 3'-phosphate were not recognized by PNK and the shortest modified ODN able to be phosphorylated was a trinucleotide X(pdT)3. A modified X(pdT)10, bearing a cyclic abasic site (X) at its 5'-end, was prepared by chemical synthesis from 1,2-dideoxyribose phosphoramidite and was phosphorylated with a 90% yield.

Detection of HIV1 DNA in biological samples by an homogeneous assay: fluorescence measurement of double-stranded RNA synthesized from amplified DNA.

A nonisotopic homogeneous detection of nucleic acid sequences after amplification is described. We show that a DNA fragment bearing T7 RNA polymerase promoters on each extremity is able to be transcribed in two complementary RNAs, leading to a high yield direct synthesis of double-stranded RNA (dsRNA). Thus, this dsRNA can be easily detected and quantified in solution by fluorescence in the presence of propidium iodide. This reaction, used as a postamplification step, has been associated with a nested polymerase chain reaction (PCR); the second PCR round allowing the incorporation of the T7 promoters. This leads to a very efficient homogeneous assay. The fluorescence signal is proportional to the concentration of PCR product and is highly specific. This method can be easily carried out with currently available reagents and with unsophisticated instrumentation. This homogeneous procedure has been evaluated for the detection of HIV1 in blood samples; the sensitivity and the specificity appear to be equivalent to that of the radioactive method.

Fast solid support detection of human papillomavirus in in vitro amplified DNA using a DNP-anti DNP monoclonal antibody couple.

In some anogenital lesions the detection of certain types of human papilloma virus, especially oncogenic types, is of interest. In a first step during a prospective study, we compared two methods for the detection of human papillomavirus (HPV) DNA in clinical samples: Southern blotting followed by hybridization with a cloned radioactive genomic probe and a classical polymerase chain reaction (PCR) followed by hybridization with a 32P-labelled oligonucleotide probe. 118 biopsies and swabs were examined for HPV 6/11, 16, 18 and 33, 67 positive reactions were found by both methods, 5 positives only by PCR and 2 positives only by Southern blot for unidentified HPV. Patients with anogenital condylomas, dysplasias and carcinomas or asymptomatic patients were studied. Most high grade (II and III) dysplasias were associated with HPV 16 and HPV 18. Condylomata lesions and low grade dysplasia (grade I) were associated mostly with HPV 6/11, mixed type of HPV, less frequently with HPV 16 or HPV 18. As a second step a nested PCR coupled to solid support detection method was used as described by Sauvaigo et al. (1990) Nucleic Acids Res. 18, 3175-3183) to study a panel of 30 previously qualified different HPV DNA extracts. In this procedure the second round of PCR amplification involves biotinylated and dinitrophenylated labelled primers allowing the capture of PCR amplified HPV DNA sequences on streptavidin coated tubes and its revelation. We describe an improvement of HPV DNA detection by means of single-step immunoenzymatic revelation involving anti-DNP monoclonal antibodies conjugated to horseradish peroxidase enzyme. A perfect correlation with the previous results was obtained. This solid support method allows a faster and easier HPV typing compared to methods using membrane transfer.

The crystal structure of N4-methylcytosine.guanosine base-pairs in the synthetic hexanucleotide d(CGCGm4CG).

The structure of d(CGCGm4CG) were m4C = N4-methylcytosine has been determined by crystallographic methods. The crystals are multifaced prisms, with orthorhombic space group P2(1)2(1)2(1) and unit cell dimensions of a = 17.98, b = 30.77 and c = 44.75A. The asymmetric unit consists of one duplex of hexanucleotide and 49 waters. The R-factor is 0.189 for 1495 reflections with F > or = sigma(F) to a resolution limit of 1.8A. The double helix has a Z-DNA type structure which appears to be intermediate in structure to the two previously characterised structure types for Z-DNA hexamers. The two m4C.G base-pairs adopt structures that are very similar to those of the equivalent base-pairs in the structure of the native sequence d(CGCGCG) except for the presence of the methyl groups which are trans to the N3 atoms of their parent nucleotides and protrude into the solvent region. The introduction of the modified base-pairs into the d(CGCGCG) duplex appears to have a minimal effect on the overall base-pair morphology of the Z-DNA duplex.

End attachment of phenol-oligonucleotide conjugates to diazotized cellulose.

The synthesis of a novel phosphoramidite reagent with a hexanediol backbone is described. This reagent has been used to incorporate a phenol moiety on an oligonucleotide (ODN) directly in the course of its automated synthesis. Multiple phenol attachments can be achieved by repetitive coupling cycles. A simple and rapid immobilization method is described where phenol-modified ODNs are covalently attached to diazotized cellulose. The binding capacity of the membrane can be modulated, depending on the ODN concentration used, to ca. 180 pmol/cm2. There is at least 80% end attachment of the ODN through the phenol group. In addition, the phenol residue can be used as a carrier for the radiolabeling with 125I. The non-nucleosidic hexanediol derivative incorporated at the 5'-end of the ODN is recognized as a substrate by the T4 polynucleotide kinase and the terminal hydroxyl group is successfully phosphorylated allowing its 32P labeling.

Standardized nested polymerase chain reaction-based assay for detection of human immunodeficiency virus type 1 DNA in whole blood lysates.

The routine detection of human immunodeficiency virus type 1 (HIV-1) proviral DNA in clinical samples requires a standardized, simple, and sensitive test. To identify the HIV-1 proviral DNA in blood, we used a solid-phase assay based on the affinity capture and the gamma counting of the amplified product after a nested polymerase chain reaction (AMPLICIS test). In order to simplify the general process, whole-blood lysates rather than peripheral blood mononuclear cell lysates were used for the amplifications. The solid-phase capture and counting of the final amplified products allowed us to define precise interpretive criteria to determine the positivity level of the test. Three new primer sets located in the gag and pol structural genes and in the tat regulatory gene of HIV-1 were studied. The results obtained in 54 seropositive and 120 seronegative individuals demonstrated the ability of the AMPLICIS test to be used for HIV-1 provirus detection: 53 of 54 of the seropositive specimens were found to be positive with at least two primer sets. We also assessed the usefulness of this test for the estimation of the HIV-1 DNA load by the end point dilution method with serial dilutions of blood lysates from 26 HIV-1-seropositive patients.

Solution conformation of an oligonucleotide containing a urea deoxyribose residue in front of a thymine.

Urea residues are produced by ionizing radiation on thymine residues in DNA. We have studied an oligodeoxynucleotide containing a thymine opposite the urea residue, by one and two dimensional NMR spectroscopy. The urea deoxyribose exists as two isomers with respect to the orientation about the peptide bond. For the trans isomer we find that the thymine and urea site are positioned within the helix and are probably hydrogen bonded. The oligonucleotide adopts a globally B form structure although conformational changes are observed around the mismatch site. A minor species is observed, in which the urea deoxyribose and the opposite base adopt an extrahelical position and this corresponds to the isomer cis for the peptide bond.

Chemical synthesis of a biologically active natural tRNA with its minor bases.

The complete chemical synthesis of an E. coli tRNA(Ala) with its specific minor nucleosides, dihydrouridine, ribothymidine and pseudouridine, is reported. The method makes use of protected 2'-O-tertiobutyldimethylsilyl-ribonucleoside-3'-O-(2-cyanoethyl-N- ethyl-N- methyl)phosphoramidites. The exocyclic amino functions of the bases were protected by the phenoxyacetyl group for purines and acetyl for cytosine. The assembling has been performed on a silica support with coupling yield better than 98% within 2 min of condensation. Triethylamine tris-hydrofluoride allowed a clean and complete deprotection of the tBDMS groups. The synthetic tRNA(Ala) has been transcribed into cDNA by reverse transcriptase and sequenced. With E. coli alanyl-tRNA synthetase the alanyl acceptance activity and kcat/Km were 672 pmol/A260 and 6 x 10(4)M-1s-1, respectively.

Conformation of guanine-8-oxoadenine base pairs in the crystal structure of d(CGCGAATT(O8A)GCG).

The structure of the synthetic deoxydodecamer d(CGCGAATT(O8A)GCG)2 (O8A = 8-oxoadenine) has been determined by single-crystal X-ray diffraction techniques. The oligonucleotide crystallizes in the orthorhombic space group P2(1)2(1)2(1) with cell dimensions of a = 25.48 A, b = 41.84 A, and c = 64.91 A. The refinement has converged with an R-factor of 0.151 for 1119 reflections in the resolution range 8.0-2.25 A. Sixty-seven solvent molecules were located during the course of the refinement. The B-DNA helix consists of ten Watson-Crick base pairs and two guanine-8-oxoadenine (G.O8A) base pairs. In order to achieve hydrogen-bonding complementarity between the two bases, an unusual G(anti).O8A-(syn) wobble conformation is adopted. It is proposed that the G.O8A mispairs are held together by a network of four interbase hydrogen bonds which are the result of the formation of two reverse three-center hydrogen-bonding systems. These involve one carbonyl oxygen lone pair interacting with two hydrogen atoms. In a departure from previous observations of the characteristics of purine-purine anti-syn base pairs, lambda 1 and lambda 2, the angles between the glycosidic bonds and the C1'-C1' vector, are symmetric. A reassessment of the other purine-purine mispairs suggests that similar three-center hydrogen bonds may occur and make a contribution to stabilizing other base pairings.

[Total chemical synthesis of natural transfer RNA]. / Synthèse chimique totale d'un ARN de transfert naturel.

New improvements in the chemical synthesis of oligoribonucleotides are reported and they are applied to the first total chemical synthesis of a natural RNA. This E. coli K12 alanine tRNA contains in its sequence dihydrouridine, ribothymidine and pseudo-uridine. The synthetic tRNA was fully sequenced and showed a 42% aminoacyl acceptance activity. When tRNA was used as a template, reverse transcriptase directed the incorporation of adenine opposite dihydrouridine, ribothymidine and pseudouridine.

Incorporation by chemical synthesis and characterization of deoxyribosylformylamine into DNA.

2-deoxyribosylformylamine is a major oxidative DNA damage type which occurs upon the action of ionizing radiation on DNA. The protected 2-deoxyribosylformylamine phosphoramidite was synthesized and used in conjunction with previously reported alkali labile base protected phosphoramidites ('PAC phosphoramidites') for the preparation of oligodeoxyribonucleotides containing this lesion. Final deprotection of the oligonucleotides was performed under mild alkaline conditions to preserve the integrity of the fragile defect. The presence of formylamino deoxyribosyl residue was confirmed by FAB mass spectrometry sequencing. Oligonucleotides bearing deoxyribosyl formylamine were used as templates for studying in vitro replication. They direct the insertion of guanine or induce a deletion opposite the lesion.

The solution structure of a DNA hairpin containing a loop of three thymidines determined by nuclear magnetic resonance and molecular mechanics.

We have determined by two-dimensional nuclear magnetic resonance studies and molecular mechanics calculations the three-dimensional solution structure of a 21 residue oligonucleotide capable of forming a hairpin structure with a loop of three thymidine residues. This structure is in equilibrium with a duplex form. At 33 degrees C, low ionic strength and in the presence of MgCl2 the hairpin form dominates in solution. Six Watson-Crick base pairs are formed topped by the loop structure. The residues 1-3 and 18-21 are not complementary and form dangling ends. Distance constraints have been derived from nuclear Overhauser enhancement measurements. These, together with molecular mechanics calculations, have been used to determine the structure. We do not observe stacking of thymidine residues either over the 3' or the 5' end of the stem.

Structure and in vitro replication of DNA templates containing 7,8-dihydro-8-oxoadenine.

Adenine residues in DNA are oxidized under the action of ionizing radiation at the C-8 position to give 7,8-dihydro-8-oxoadenine. The formation of this lesion can be considered a cause of mutations and carcinogenesis. Oligodeoxyribonucleotides 39 and 47 bases long containing a single 7,8-dihydro-8-oxoadenine (8-hydroxyadenine) residue were synthesized by using nucleoside phosphoramidites. They were used as templates to study the copies obtained in vitro by the Klenow fragment and the thermostable Taq DNA polymerase. 7,8-Dihydro-8-oxoadenine does not block the replication and thymine is incorporated opposite the damage. The modifications of the DNA duplex conformation provoked by 7,8-dihydro-8-oxoadenine are minor. 1H-NMR spectroscopy shows that the duplex is in a B form, the sugar in a normal position in the helix and the modified base in the anti position. NMR confirms that 7,8-dihydro-8-oxoadenine exists predominantly in the keto form.

Lack of HIV tat-gene amplification in blood monocytes compared to T lymphocytes.

T-lymphocytes (T-Ly) and monocytes/macrophages are thought to be the main in vivo targets for HIV 1. We previously demonstrated, using the polymerase chain reaction (PCR), that HIV provirus could be detected in 20 out of 21 T-Ly samples and 13 out of 21 monocyte samples from HIV 1-seropositive individuals, with at least gag, env or LTR primers. In the present study, we wanted to find out whether the HIV 1 tat gene could be detected in 14 of these circulating monocyte and T-Ly samples. The tat primers were chosen in order to amplify the overall second exon of this regulatory gene. This new set of primers could not detect HIV provirus in monocytes but it did in T-Ly, among cells previously shown to be positive with one of the other 3 primer pairs. Further molecular studies should help characterize these probable monocytotropic variants and elucidate their contribution to HIV pathogenesis.

Mammalian cell processing of a unique uracil residue in simian virus 40 DNA.

The processing of a unique uracil in DNA has been studied in mammalian cells. A synthetic oligodeoxyribonucleotide carrying a potential Bgl II restriction site, where one base has been substituted with a uracil, was inserted in the early intron of SV40 genome. Various heteroduplexes were constructed in such a manner that the restitution of an active Bgl II restriction site corresponds in each case to the specific substitution of the uracil by one of the four bases normally present in the DNA. DNA cuts by this restriction enzyme in one or several constructed heteroduplexes immediately determine the type of base pair substitution produced at the site of the U residue. When the uracil is inserted opposite a purine it is fully repaired; when facing a guanine it is replaced by a cytosine and opposite an adenine it is replaced by a thymine. These results indicate the error-free repair of uracil when it appears in the cell with the usual mechanisms such as cytosine deamination or incorporation of dUTP in place of dTTP during replication. When the uracil is inserted opposite a pyrimidine no error free repair at all is detected for U:C or U:T mismatches. It appears, moreover, that in approximately 18% of the cases U:T mismatch leads to a C:G base pairing. In the majority of the U:pyrimidine mismatches, mutations occur in the vicinity of the uracil, including base substitutions and frameshifts by addition of one or several bases.

Fast solid support detection of PCR amplified viral DNA sequences using radioiodinated or hapten labelled primers.

Oligonucleotides with novel modifications have been synthesized and incorporated into enzymatically amplified DNA sequences. They allow the fast detection of viral DNA sequences after two rounds of amplification. The hybrids formed are immobilized by affinity on coated tubes and detected by direct beta (32P) or gamma (125I) counting or by colorimetric revelation. The effect of a dilution step between the two amplifications is studied to obtain optimal sensitivity and specificity. This test is used to detect Human Papillomavirus types 16 and 18 in cells and biopsies and for the specific colorimetric detection of HIV1 in extracted DNA.