Mechanism of copper mediated triple helix formation at neutral pH in Drosophila satellite repeats.

The highly repeated Drosophila melanogaster AAGAGAG satellite sequence is present at each chromosome centromere of the fly. We demonstrate here how, under nearly physiological pH conditions, these sequences can form a pyrimidine triple helix containing T.A-T and CCu.G-C base triplets, stabilized by Cu2+ metal ions in amounts mirroring in vivo concentrations. Ultraviolet experiments were used to monitor the triple helix formation at pH 7.2 in presence of Cu2+ ions. Triplex melting is observed at 23 degrees C. Furthermore, a characteristic signature of triple helix formation was obtained by Fourier transform infrared spectroscopy. The stabilization of the C.G-C base triplets at pH 7.2 is shown to occur via interactions of Cu2+ ions on the third strand cytosine N3 atom and on the guanine N7 atom of the polypurine target strand forming CCu.G-C triplets. Under the same neutral pH conditions in absence of Cu2+ ions, the triple helix fails to form. Possible biological implications are discussed.

DNA coiled coils.

We report the coiled-coil structure of DNA, which is generated by the dodecanucleotide d(ATATATATATAT). The structure has been determined by single-crystal x-ray crystallography. The molecules form duplexes with single-stranded overhangs, which associate with neighbor molecules and give rise to infinite double helices in a coiled-coil conformation, with staggered nicks in both strands. The coiled coils have six dodecamer duplexes per turn. Despite the presence of nicks, the structure is very rigid. Statistical disorder is present, which gives rise to continuous scattering along the layer lines. This observation can be interpreted by the classical theory of coiled coils developed for proteins, which we apply here to a DNA structure. A clear splitting of the original layer lines of the DNA double helix is detected. The structure we have found adds a previously unrecognized element to the architectures that can be built from DNA oligonucleotides. Any duplex with complementary single-strand overhangs should be expected to give rise to regular coiled-coil structures.

Comparison between CUUG and UUCG tetraloops: thermodynamic stability and structural features analyzed by UV absorption and vibrational spectroscopy.

CUUG loop is one of the most frequently occurring tetraloops in bacterial 16S rRNA. This tetraloop has a high thermodynamic stability as proved by previous UV absorption and NMR experiments. Here, we present our results concerning the thermodynamic and structural features of the 10mer 5'-r(GCG-CUUG-CGC)-3', forming a highly stable CUUG tetraloop hairpin in aqueous solution, by means of several optical techniques (UV and FT-IR absorption, Raman scattering). UV melting profile of this decamer provides a high melting temperature (60.7 degrees C). A set of Raman spectra recorded at different temperatures allowed us to analyze the order-to-disorder (hairpin-to-random coil) transition. Assignment of vibrational markers led us to confirm the particular nucleoside conformation, and to get information on the base stacking and base pairing in the hairpin structure. Moreover, comparison of the data obtained from two highly stable CUUG and UUCG tetraloops containing the same nucleotides but in a different order permitted an overall discussion of their structural features on the basis of Raman marker evidences.

Solution conformation of an RNA--DNA hybrid duplex containing a pyrimidine RNA strand and a purine DNA strand.

RNA--DNA hybrid duplexes are involved in transcription, replication and reverse transcription of nucleic acids. Information on such duplexes may shed some light on the mechanism of these processes. For this purpose, the influence of base composition on the structure of a polypyrimidine--polypurine RNA--DNA duplex r(cucuccuucucuu). d(GAGAGGAAGAGAA) has been studied using 1H, 31P and 13C NMR experiments, molecular modeling (JUMNA program) and NOE back-calculation methods. The resulting structure of the 13-mer hybrid duplex shows that the RNA strand is in the expected A-type conformation while the DNA strand is in a very flexible conformation. In the DNA strand, the desoxyribose sugars retain the C2'-endo B-type conformation. The duplex helical parameters (such as inclination, twist and displacement of the bases) are close to the A-type conformation. No bending was observed for the global axis curvature. The major groove width is close to the B-form value and the minor groove width is intermediate between standard values for A and B-forms. These results are in favour of the independence of minor groove size (where RNase H interacts) and the base composition of the hybrid duplexes.

Parallel and antiparallel G*G.C base triplets in pur*pur.pyr triple helices formed with (GA) third strands.

Triple helices with G*G.C and A*A.T base triplets with third GA strands either parallel or antiparallel with respect to the homologous duplex strand have been formed in presence of Na (+) or Mg(2+) counterions. Antiparallel triplexes are more stable and can be obtained even in presence of only monovalent Na(+) counterions. A biphasic melting has been observed, reflecting third strand separation around 20 degrees C followed by the duplex -> coil transition around 63 degrees C. Parallel triplexes are far less stable than the antiparallel ones. Their formation requires divalent ions and is observed at low temperature and in high concentration conditions. Different FTIR signatures of G*G.C triplets in parallel and antiparallel triple helices with GA rich third strands have been obtained allowing the identification of such base triplets in triplexes formed by nucleic acids with heterogeneous compositions. Only S-type sugars are found in the antiparallel triplex while some N-type sugar conformation is detected in the parallel triplex.

Parallel intramolecular DNA triple helix with G and T bases in the third strand stabilized by Zn(2+) ions.

We present evidence of formation of an intramolecular parallel triple helix with T*A.T and G*G.C base triplets (where * represents the hydrogen bonding interaction between the third strand and the duplex while. represents the Watson-Crick interactions which stabilize the duplex). The third GT strand, containing seven GpT/TpG steps, targets the polypurine sequence 5'-AGG-AGG-GAG-GAG-3'. The triple helix is obtained by the folding back twice of a 36mer, formed by three dodecamers tethered by hydroxyalkyl linkers (-L-). Due to the design of the oligonucleotide, the third strand orientation is parallel with respect to the polypurine strand. Triple helical formation has been studied in concentration conditions in which native gel electrophoresis experiments showed the absence of intermolecular structures. Circular dichroism (CD) and UV spectroscopy have been used to evidence the triplex structure. A CD spectrum characteristic of triple helical formation as well as biphasic UV and CD melting curves have been obtained in high ionic strength NaCl solutions in the presence of Zn(2+) ions. Specific interactions with Zn(2+) ions in low water activity conditions are necessary to stabilize the parallel triplex.

Headgroup specificity of lecithin cholesterol acyltransferase for monomeric and vesicular phospholipids.

In this study, we investigated how the nature of the phospholipid head group and the macromolecular structure of the phospholipid, either as a monomer or incorporated into a lipid matrix, influence the activity of lecithin cholesterol acyltransferase (LCAT). As substrates we used 1,2-bis-(1-pyrenebutanoyl)-phosphatidylcholine, 1, 2-bis-(1-pyrenebutanoyl)-phosphatidylethanolamine and 1, 2-bis-(1-pyrenebutanoyl)-phosphatidyl-alcohols, either as monomers or incorporated into small unilamellar vesicles consisting of dipalmitoylphosphatidylcholine ether. The rate of hydrolysis of the pyrene-labeled phospholipids was determined both by fluorescence and by high performance liquid chromatography. V(max) and K(m) were calculated for the different substrates. The data show that V(max) is 10- to 30-fold higher for the hydrolysis of monomeric phosphatidylcholine (PC) compared to phosphatidylethanolamine (PE) and the phosphatidylalcohols, while K(m) values are comparable. When the fluorescent substrates were incorporated into dipalmitoylphosphatidylcholine ether vesicles, we observed a 4- to 10-fold increase of V(max) for PE and the phosphatidylalcohols, and no significant change for K(m). V(max) for PC remained the same. Natural LCAT mutants causing Fish-Eye Disease (FED) and analogues of these mutants expressed in Cos-1 cells, had similar activity on monomeric PC and PE. These data suggest that the activity of LCAT is determined both by the molecular structure of the phospholipid and by its macromolecular properties. The LCAT activity on monomeric substrates decreases as: phosphatidylcholine&z. Gt;phosphatidylethanolamine congruent withphosphatidylpropanol congruent withphosphatidylethanol congruent withphosphatidylethyleneglycol. The incorporation of PE and the phosphatidylalcohols into a matrix of dipalmitoylphosphatidylcholine decreases the specificity of the phospholipid head group.

Relationship between structure and biochemical phenotype of lecithin:cholesterol acyltransferase (LCAT) mutants causing fish-eye disease.

In order to test the hypothesis that fish-eye disease (FED) is due to a deficient activation of lecithin:cholesterol acyltransferase (LCAT) by its co-factor apolipoprotein (apo) A-I, we overexpressed the natural mutants T123I, N131D, N391S, and other engineered mutants in Cos-1 cells. Esterase activity was measured on a monomeric phospholipid enelogue, phospholipase A(2) activity was measured on reconstituted high density lipoprotein (HDL), and acyltransferase activity was measured both on rHDL and on low density lipoprotein (LDL). The natural FED mutants have decreased phospholipase A(2) activity on rHDL, which accounts for the decreased acyltransferase activity previously reported. All mutants engineered at positions 131 and 391 had decreased esterase activity on a monomeric substrate and decreased acyltransferase activity on LDL. In contrast, mutations at position 123 preserved these activities and specifically decreased phospholipase A(2) and acyltransferase activites on rHDL. Mutations of hydrophilic residues in amphipathic helices alpha 3;-4 and alpha His to an alanine did not affect the mutants' activity on rHDL. Based upon the 3D model built for human LCAT, we designed a new mutant F382A, which had a biochemical phenotype similar to the natural T123I FED mutant. These data suggest that residues T123 and F382, located N-terminal of helices alpha 3-4 and alpha His, contribute specifically to the interaction of LCAT with HDL and possibly with its co-factor apoA-I. Residues N131 and N391 seem critical for the optimal orientation of the two amphipathic helices necessary for the recognition of a lipoprotein substrate by the enzyme.

Dimer initiation sequence of HIV-1Lai genomic RNA: NMR solution structure of the extended duplex.

The genome of all retrovirus consists of two copies of genomic RNA which are noncovalently linked near their 5' end. A sequence localized immediately upstream from the splice donor site inside the HIV-1 psi-RNA region was identified as the domain responsible for the dimerization initiation. It was shown that a kissing complex and a stable dimer are both involved in the HIV-1Lai RNA dimerization process in vitro. The NCp7 protein activates the dimerization by converting a transient loop-loop complex into a more stable dimer. The structure of this transitory loop-loop complex was recently elucidated by Mujeeb et al. In work presented here, we use NMR spectroscopy to determine the stable extended dimer structure formed from a 23 nucleotides RNA fragment, part of the 35 nucleotides SL1 sequence. By heating to 90 degrees C, then slowly cooling this sequence, we were able to show that an extended dimer is formed. We present evidence for the three dimensional structure of this dimer. NMR data yields evidence for a zipper like motif A8A9.A16 existence. This motif enables the surrounding bases to be positioned more closely and permit the G7 and C17 bases to be paired. This is different to other related sequences where only the kissing complex is observed, we suggest that the zipper like motif AA.A could be an important stabilization factor of the extended duplex.

Trans-complementation of the second step of pre-mRNA splicing by exogenous 5' exons.

During splicing of nuclear pre-mRNAs, the first step liberates the 5' exon (exon 1) and yields a lariat intron-3'exon (intron-exon 2) intermediate. The second step results in exon ligation. Previous results indicated that severe truncations of the 5' exon of the actin pre-mRNA result in a block to the second splicing step in vitro in yeast extracts, leading to an accumulation of intron-exon 2 lariat intermediates. We show that exogenous exon 1 RNA oligonucleotides can chase these stalled intermediates into lariat intron and spliced exons. This reaction requires some of the cis elements and trans-acting factors that are required for a normal second step. There is no strong sequence requirement for the exon 1 added in trans, but oligonucleotides with complementarity to the U5 snRNA conserved loop perform the chase more efficiently. Using a dominant negative mutant of the DEAH-box ATPase Prp16p and ATP depletion, we show that the stalled intermediate is blocked after the Prp16p-dependent step. These results show that exogenous RNAs with various sequences but containing no splicing signals can be incorporated into spliceosomes and undergo RNA recombination and exon shuffling during the second step of pre-mRNA splicing.

Unusual nucleotide conformations in GNRA and UNCG type tetraloop hairpins: evidence from Raman markers assignments.

High resolution NMR data on UNCG and GNRA tetraloops (where N is any of the four nucleotides and R is a purine) have shown that they contain ribonucleosides with unusual 2'-endo/anti and 3'-endo/syn conformations, in addition to the 3'-endo/anti ones which are regularly encountered in RNA chains. In the current study, Raman spectroscopy has been used to probe these nucleoside conformations and follow the order (hairpin) to disorder (random chain) structural transitions in aqueous phase in the 5-80 degreesC temperature range. Spectral evolution of GCAA and GAAA tetraloops, as formed in very short hairpins with only three G.C base pairs in their stems (T m >60 degreesC), are reported and compared with those previously published on UUCG and UACG tetraloops, for which the syn orientation of the terminal guanine as well as the 2'-endo/anti conformation of the third rC residue have been confirmed by means of vibrational marker bands. Raman data obtained as a function of temperature show that the first uracil in the UUCG tetraloop is stacked and the two middle residues (rU and rC) are in the 2'-endo/anti conformation, in agreement with the previously published NMR results. As far as the new data concerning the GNRA type tetraloops are concerned, they lead us to conclude that: (i) in both cases (GCAA and GAAA tetraloops) the adenine bases are stacked; (ii) the second rC residue in the GCAA tetraloop has a 3'-endo/anti conformation; (iii) the sugar pucker associated with the third rA residue in both tetraloops possibly undergoes a 3'-endo/2'-endo interconversion as predicted by NMR results; (iv) the stem adopts a regular A-form structure; (v) all other nucleosides of these two GNRA tetraloops possess the usual 3'-endo/anti conformation.

Structural features of the UCCG and UGCG tetraloops in very short hairpins as evidenced by optical spectroscopy.

Structures of the UCCG and UGCG tetraloops formed in octamer ribonucleotidic hairpin sequences, i.e., 5'-r[GC(UCCG)GC]-3' and 5'-r[GC(UGCG)GC]-3', have been studied in aqueous solution by methods of optical spectroscopy. UV absorption melting profiles of these short hairpins, containing only two closing GC base pairs in the stem, are consistent with a monophasic, completely reversible order-to-disorder transition and clearly confirm their unusual structural stability (with Tm congruent with 50 degrees C). To establish structural characteristics of these tetraloops, Raman and FTIR spectroscopies have been used and vibrational conformation markers arising from the phosphate backbone and various nucleosides have been analyzed. They have been assigned on the basis of known unambiguous vibrational markers established for DNA and RNA chains. Surprisingly, they are easily transferable to short oligonucleotidic sequences. Intensities and wavenumbers of these conformation markers have been monitored in the 0-70 degrees C temperature range, i.e., in going from an ordered to a disordered structure. The main structural features of the UCCG and UGCG tetraloops are similar to those previously found in the UUCG and UACG tetraloops by means of NMR and vibrational spectroscopies, except those of the second nucleosides of the tetraloops (rC and rG, respectively) which adopt a 3'-endo/anti rather than a 2'-endo/anti conformation.

Common structural features of UUCG and UACG tetraloops in very short hairpins determined by UV absorption, Raman, IR and NMR spectroscopies.

Thermodynamic and structural properties of two UNCG tetraloops in very short hairpin octamers, 5'-r(GCUUCGGC)-3' and 5'-r(GCUACGGC)-3', have been studied by means of various physical techniques. Melting profiles of both octamers, obtained from UV absorption spectra taken as a function of temperature, are consistent with a monophasic, progressive and completely reversible order-to-disorder transition and confirm their unusual structural stability (Tm > 51 degrees C). The 1H, 13C and 31P NMR chemical shifts and coupling constants of the UACG loop nucleotides are comparable with those reported previously for UUCG loops, i.e. 2'-endo/anti conformation of the second and third nucleotide of the loop as well as the syn orientation of the ultimate guanine base and the A-type double helical conformation of the hairpin stem. Simulation of quantitative NOESY volumes shows that the UACG octamer adopts a very rigid compact structure which is well represented by an average order parameter of 0.9. Three base-pairs and four additional strong hydrogen bonds are undoubtedly responsible for such limited flexibility. Raman and infrared spectra as a function of temperature reflect the order-to-disorder transition, as well. Vibrational conformational markers in low temperature spectra of both octamers indicate the hairpin structure as the major conformer in aqueous phase. These spectra further support the structural features of most of the nucleotides involved in the tetraloops and clearly demonstrate the structural similarities of the phosphodiester backbone in both hairpins. Consequently, on the basis of all present results, one can deduce that the conformational features of the UUCG and UACG tetraloops seem to be inherent to the UNCG type tetraloops, regardless of either the nature of the tetraloop second base or the stem length.

Structural characterization of an intramolecular RNA triple helix by NMR spectroscopy.

A chemically synthesized 29-base RNA oligomer, designed to fold to form an intramolecular triple helix at acid pH, has been studied by NMR spectroscopy. The molecule consisted of seven U.A.U or C+.G.C base triples joined by two pyrimidine tetra-loops. The fold was such that the third strand was Hoogsteen base-paired in the major groove of a Watson-Crick paired double helix. The nature and size of the molecule required the use of an assignment strategy using two- and three-dimensional homonuclear methods, complemented by a natural abundance 13C correlation experiment. The assignment of the majority of the exchangeable and non-exchangeable resonances is presented. The data suggest a C3'-endo sugar puckering for all the nucleotides involved in base triples. A preliminary structural model consistent with the NMR data is presented.

Spectroscopic studies of chimeric DNA-RNA and RNA 29-base intramolecular triple helices.

Fourier transform infrared (FTIR), UV absorption and exchangeable proton NMR spectroscopies have been used to study the formation and stability of two intramolecular pH-dependent triple helices composed by a chimeric 29mer DNA-RNA (DNA double strand and RNA third strand) or by the analogous 29mer RNA. In both cases decrease of pH induces formation of a triple helical structure containing either rU*dA.dT and rC+*dG.dC or rU*rA.rU and rC+*rG.rC triplets. FTIR spectroscopy shows that exclusively N-type sugars are present in the triple helix formed by the 29mer RNA while both N- and S-type sugars are detected in the case of the chimeric 29mer DNA-RNA triple helix. Triple helix formation with the third strand RNA and the duplex as DNA appears to be associated with the conversion of the duplex part from a B-form secondary structure to one which contains partly A-form sugars. Thermal denaturation experiments followed by UV spectroscopy show that a major stabilization occurs upon formation of the triple helices. Monophasic melting curves indicate a simultaneous disruption of the Hoogsteen and Watson-Crick hydrogen bonds in the intramolecular triplexes when the temperature is increased. This is in agreement with imino proton NMR spectra recorded as a function of temperature. Comparison with experiments concerning intermolecular triplexes of identical base and sugar composition shows the important role played by the two tetrameric loops in the stabilization of the intramolecular triple helices studied.

Spectroscopic evidence for an intramolecular RNA triple helix.

A 29-base RNA oligomer has been chemically synthesized and shown to form an intramolecular triple helix in solution at acidic pH. Assignment of the majority of the exchangeable proton NMR resonances demonstrated the Watson-Crick and Hoogsteen base pairings consistent with folding to form pyrimidine-purine-pyrimidine base triplets. FTIR spectroscopy provided independent evidence of base triplet formation, and indicated a predominately C3'-endo sugar pucker. UV absorption as a function of temperature suggested monophasic melting behaviour, which was confirmed by NMR of the imino protons.

Improved brain delivery of AZT using a glycosyl phosphotriester prodrug.

The concentration of AZT in mice plasma and brain was measured using HPLC after an ingestion of 20 mg/kg of AZT or the molar equivalent of hexadecyl 2-(alpha-D-mannopyranosidyl)ethyl 3'-azido-3'-deoxy-5'-thymidinyl phosphate 3. The results demonstrated the promising qualities of the prodrug 3 which gave AZT-5'-phosphate as the main metabolite: the total concentration of AZT derivatives detected in brain presented a peak of 156 nmol/g (5 nmol/g for AZT) at 1 h; the half-life was about 24 h (1 h for AZT) with an AUC of 4366 nmol h/g as compared to 4 nmol h/g for AZT. The lipophilic properties of 3 were confirmed by its in vitro transport of inside synaptosomes. The derivative 2-(alpha-D-mannopyranosidyl)ethyl 3'-azido-3'-deoxy-5'-thymidinyl phosphate (2) provided also a good delivery of AZT to the central nervous system, with values intermediate between those of AZT and 3.

Lipophilic glycosyl phosphotriester derivatives of AZT: synthesis, NMR transmembrane transport study, and antiviral activity.

Phosphate derivatives of AZT esterified with a carbohydrate (D-glucose, D-mannose, and ethyl D-mannopyranoside) and a hexadecyl chain were prepared from glucose 6-phosphate and D-mannose precursors. The 31P NMR study of the mannosyl phosphotriester series in the presence of large unilamellar vesicles demonstrated either an interaction with the external lipid layer or a transmembrane transport into the intravesicular interface. The antiviral activity, measured by the inhibition of cytopathogenicity on different infected cells and of reverse transcriptase activity in the supernatant of cultures, appeared to be comparable to that of AZT, in the micromolar range.

Glucosyl phosphotriesters of nucleosides: exchange mechanism of transmembrane transport and application to 5-fluoro-deoxyuridine.

The structure and membrane interactions of lipophilic glucosyl phosphotriester derivatives of thymidine and 5-fluoro-deoxy thymidine are investigated by NMR spectroscopy. The self-association of these molecules, found in different solvents, presents a diastereoisomeric effect which is also observed in the transmembrane transport inside large unilamellar vesicles. The influence of the hydrophobic chain and the nature of the nucleoside in the water-membrane exchange process is discussed.