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.

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.

Characterization of base-pair opening in deoxynucleotide duplexes using catalyzed exchange of the imino proton.

Using nuclear magnetic resonance line broadening, longitudinal relaxation and magnetization transfer from water, we have measured the imino proton exchange times in the duplex form of the 10-mer d-CGCGATCGCG and in seven other deoxy-duplexes, as a function of the concentration of exchange catalysts, principally ammonia. All exchange times are catalyst dependent. Base-pair lifetimes are obtained by extrapolation to infinite concentration of ammonia. Lifetimes of internal base-pairs are in the range of milliseconds at 35 degrees C and ten times more at 0 degrees C. Lifetimes of neighboring pairs are different, hence base-pairs open one at a time. Lifetimes of d(G.C) are about three times longer than those of d(A.T). The nature of neighbors usually has little effect, but lifetime anomalies that may be related to sequence and/or structure have been observed. In contrast, there is no anomaly in the A.T base-pair lifetimes of d-CGCGA[TA]5TCGCG, a model duplex of poly[d(A-T)].poly[d(A-T)]. The d(A.T) lifetimes are comparable to those of r(A.U) that we reported previously. End effects on base-pair lifetimes are limited to two base-pairs. The low efficiency of exchange catalysts is ascribed to the small dissociation constant of the deoxy base-pairs, and helps to explain why exchange catalysis had been overlooked in the past. This resulted in a hundredfold overestimation of base-pair lifetimes. Cytosine amino proteins have been studied in the duplex of d-CGm5CGCG. Exchange from the closed base-pair is indicated. Hence, the use of an amino exchange rate to evaluate the base-pair dissociation constant would result in erroneous, overestimated values. Catalyzed imino proton exchange is at this time the safest and most powerful, if not the only probe of base-pair kinetics. We propose that the single base-pair opening event characterized here may be the only mode of base-pair disruption, at temperatures well below the melting transition.

Structure of the d(CGCCCGCGGGCG) dodecamer: a kinked A-DNA molecule showing some B-DNA features.

We have determined the structure of the dodecamer duplex d(CGCCCGCGGGCG)2. A careful use of the molecular replacement programme AMoRe has been essential in order to solve the structure. This dodecamer shows a unique conformation, quite different from all the previously studied oligonucleotide duplexes: the central octamer has an A conformation, but with a sharp 65 degrees kink in the centre; the terminal base-steps have a B-like conformation; the major groove is completely closed in the centre, a hollow molecule is thus found. The results obtained confirm the high degree of variability of DNA structure. A new type of kink and an intermediate A/B double-helical conformation have been found. Such intermediate conformation differs from those described in DNA polymerase complexes.

Recombination-like structure of d(CCGCGG).

We have solved the single crystal X-ray structure of the synthetic DNA hexamer d(CCGCGG). The central alternating tetramer forms a Z-DNA duplex. The initial cytosine of each strand of the duplex swings out and forms a Watson-Crick base-pair with the terminal guanine of a symmetry-related molecule. Thus, two symmetry-related DNA molecules form a twin with intermolecular base-pairs at both ends. Such a twin is additionally stabilized by a sodium ion located on a dyad axis between two DNA duplexes. The total structure has recombination-like features. It also provides a model for B/Z junctions. The crystal used in this study belongs to space group C222(1) with a = 34.33 A, b = 44.04 A and c = 38.27 A. The structure was solved by molecular replacement using partial models, and refined by molecular dynamics simulated annealing and positional treatment. The refinement has been concluded with an R-factor of 18.5% for 2377 reflections with F > or = 2 sigma (F) in the resolution region 8.0 to 1.92 A. The asymmetric unit contains two strands of d(CCGCGG) and 38 water molecules.

Molecular structure of a complete turn of A-DNA.

We have determined the crystal structure of the dodecamer d(CCCCCGCGGGGG), showing for the first time a complete turn of A-DNA. It has average structural parameters similar to those determined in fibres. Nevertheless it shows a considerable local variation in structure which is in part associated with the presence of a bound spermine molecule. We conclude that the local DNA conformation does not only depend on the base sequence, but may be strongly modified upon interaction with other molecules. In particular, the CpG sequence, which is found in hypersensitive regions of the genome, appears to be able to easily change its conformation under external influences.

Structure of the complex between the HIV-1 nucleocapsid protein NCp7 and the single-stranded pentanucleotide d(ACGCC).

The nucleocapsid protein NCp7 of HIV-1 Mal contains two successive Zn knuckles of the CX2CX4HX4C type and plays a major role in virion morphogenesis, genomic RNA packaging and viral infectivity, mainly through single-stranded nucleic acid binding. We report here the study by 1H 2D NMR of the complex formed between the (12-53)NCp7, encompassing the two Zn knuckles, and d(ACGCC), a deoxynucleotide sequence analog corresponding to the shortest NCp7 binding site. Ten structures of the (12-53)NCp7/d(ACGCC) complex have been obtained from 607 NOE-derived distance constraints, 28 of which are intermolecular, and from molecular dynamics studies. The oligonucleotide is almost perpendicular to the sequence linking the two Zn knuckles. The Trp37 indole ring is inserted between the C2 and G3 bases and stacked on the latter. The complex is stabilized by hydrophobic interactions and hydrogen bonds, and accounts for the observed loss of virus infectivity induced by mutations in the Zn knuckle domain. Thus, the interaction between d(ACGCC) and the inactive mutant Cys23 (12-53)NCp7 was found by NMR to be completely different from that observed with the wild-type peptide. A mechanism of action for NCp7 in virus morphogenesis and replication is proposed from these results, which could facilitate the design of possible antiviral agents acting by a new mechanism.

Exploring the folding pathways of annexin I, a multidomain protein. I. non-native structures stabilize the partially folded state of the isolated domain 2 of annexin I.

Proteins of the annexin family constitute very attractive models because of their four approximately 70 residue domains, D1 to D4, exhibiting an identical topology comprising five helix segments with only a limited sequence homology of approximately 30%. We focus on the isolated D2 domain, which is only partially folded. A detailed analysis of this equilibrium partially folded state in aqueous solution and micellar solution using 15N-1H multidimensional NMR is presented. Comparison of the residual structure of the entire domain with that of shorter fragments indicates the presence of long-range transient hydrophobic interactions that slightly stabilize the secondary structure elements. The unfolded domain tends to behave as a four-helix, rather than as a five-helix domain. The ensemble of residual structures comprises: (i) a set of native structures consisting of three regions with large helix populations, in rather sharp correspondence with A, B and E helices, and a small helix population in the second part of the C helix; (ii) a set of non-native local structures corresponding to turn-like structures stabilized by several side-chain to side-chain interactions and helix-disruptive side-chains to backbone interactions. Remarkably, residues involved in these local non-native interactions are also involved, in the native structure, in structurally important non-local interactions. During the folding process of annexin I, the local non-native interactions have to switch to native long-range interactions. This structural switch reveals the existence of a sequence-encoded regulation of the folding pathways and kinetics, and emphasizes the key role of the non-native local structures in this regulation.

A conformational equilibrium in a protein fragment caused by two consecutive capping boxes: 1H-, 13C-NMR, and mutational analysis.

The conformational properties of an 18 residues peptide spanning the entire sequence, L1KTPA5QFDAD10ELRAA15MKG, of the first helix (A-helix) of domain 2 of annexin I, were thoroughly investigated. This fragment exhibits several singular features, and in particular, two successive potential capping boxes, T3xxQ6 and D8xxE11. The former corresponds to the native hydrogen bond network stabilizing the alpha helix N-terminus in the protein; the latter is a non-native capping box able to break the helix at residue D8, and is observed in the domain 2 partially folded state. Using 2D-NMR techniques, we showed that two main populations of conformers coexist in aqueous solution. The first corresponds to a single helix extending from T3 to K17. The second corresponds to a broken helix at residue Ds. Four mutants, T3A, F7A, D8A, and E11A, were designed to further analyze the role of key amino acids in the equilibrium between the two ensembles of conformers. The sensitivity of NMR parameters to account for the variations in the populations of conformers was evaluated for each peptide. Our data show the delta13Calpha chemical shift to be the most relevant parameter. We used it to estimate the population ratio in the various peptides between the two main ensembles of conformers, the full helix and the broken helix. For the WT, E11A, and F7A peptides, these ratios are respectively 35/65, 60/40, 60/40. Our results were compared to the data obtained from helix/coil transition algorithms.

Sequence-dependence of the conformational changes induced by the 5-methyl cytosine in synthetic RNA oligomers.

The RNA hexamer containing a 5-methyl cytosine (m5C) r(CGUAm5CG) was studied by 1H and 31P NMR at 500 MHz and 121 MHz, respectively. In contrast to r(CGm5CGCG) which exhibits an atypic duplex structure [(1987) J. Am. Chem. Soc. 109, 2539-2541], r(CGUAm5CG) adopts a classical A-type conformation. This result demonstrates that the influence of the m5C on the conformation of RNA hexamers is sequence-dependent.

The structure of the most studied DNA fragment changes under the influence of ions: a new packing of d(CGCGAATTCGCG).

The title oligonucleotide and many related dodecamers have been extensively studied alone and as DNA-drug complexes. In practically all cases they were found to crystallize in the same space group, stabilized by interactions among the terminal guanine bases. Here we report new packing interactions (R3) in the presence of Ca2+. The oligonucleotides interact by placing their terminal guanines in the narrow groove of a neighbor molecule, an interaction which had never been found in dodecamers.

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.

Investigating the conformational coupling between the transmembrane and cytoplasmic domains of a single-spanning membrane protein. A 1H-NMR study.

PMP1 is a 38-residue single-spanning membrane protein whose C-terminal cytoplasmic domain, Y25-F38, is highly positively charged. The conformational coupling between the transmembrane span and the cytoplasmic domain of PMP1 was investigated from 1H-nuclear magnetic resonance data of two synthetic fragments: F9-F38, i.e. 80% of the whole sequence, and Y25-F38, the isolated cytoplasmic domain. Highly disordered in aqueous solution, the Y25-F38 peptide adopts a well-defined conformation in the presence of dodecylphosphocholine micelles. Compared with the long PMP1 fragment, this structure exhibits both native and non-native elements. Our results make it possible to assess the influence of a hydrophobic anchor on the intrinsic conformational propensity of a cytoplasmic domain.

500 MHz 1H-NMR study of the interaction of daunomycin with B and Z helices of d(CGm5CGCG).

The interaction of daunomycin with B and Z helices of a self-complementary DNA fragment d(CGm5CGCG) in solution was studied by 1H-NMR spectroscopy at 500 MHz. The results show that the B-Z transition kinetics is not affected by addition of daunomycin. Daunomycin binds exclusively to the B form of d(CGm5CGCG). Z exchanges with B while the latter also exchanges with the B duplex-daunomycin complexes.

Simple chemical synthesis and hybridization properties of non-radioactive DNA probes.

A simple chemical method for the synthesis of non-radioactive DNA probes is described: triazolyl-containing sequences were built by incorporation of 4-triazolylpyrimidin-2-ones instead of cytidines during oligodeoxyribonucleotide synthesis. The activating triazolyl groups were then displaced by a diamine which was further derivatized by a label, such as biotin. Synthesized DNA probes were oligonucleotides complementary to a cloned human antithrombin III DNA sequence. These probes, containing the same label at different positions of the sequence, were hybridized to their target DNA immobilized on nitrocellulose. Their hybridization specificity and stability were studied. Hybrid detection was performed either colorimetrically by the streptavidin-alkaline phosphatase-based system or by autoradiography after 5'-32P labeling of the probes: 15 fmol (0.05 microgram) of complementary sequence could be visualized in the two cases.

1H- and 2H-NMR studies of a fragment of PMP1, a regulatory subunit associated with the yeast plasma membrane H(+)-ATPase. Conformational properties and lipid-peptide interactions.

PMP1 is a 38-residue polypeptide associated with the yeast plasma membrane H(+)-ATPase, found to regulate the enzyme activity. To investigate the molecular basis of the PMP1 biological function, the conformational properties of a synthetic PMP1 fragment, A18-F38, comprising the predicted C-terminal cytoplasmic domain and a part of the transmembrane anchor have been studied by 1H- and 2H-NMR spectroscopies. High resolution 1H-NMR experiments showed that, in deuterated DPC micelles, the A18-G34 segment adopts a well defined helix conformation. Our data suggest that the whole PMP1 molecule forms a unique helix whose axis might be slightly tilted with respect to the bilayer normal. Protonated DPC, DMPC and DMPS were incorporated in deuterated micelles containing the PMP1 fragment for studying lipid-peptide interactions. Unusually strong and selective intermolecular NOEs between lipid chain and peptide side chain protons, especially those of the unique Trp residue, were observed. Solid state 2H-NMR experiments performed on pure deuterated POPC and mixed deuterated POPC:POPS (5:1) bilayers revealed that the PMP1 fragment specifically interacts with negatively charged PS lipids.

Chemical and enzymatic oxidative coupling of 5-hydroxy-N,N-dimethyltryptamine with amines.

As part of a program aiming to obtain a covalent labeling of serotoninergic receptors we have studied the oxidative coupling of serotonin derivatives with amino compounds. The oxidation of bufotenine (2) by MnO2 and human ceruloplasmin followed by the Michael type addition with dansylcadaverine and dansyllysine gave a fluorescent adduct identified as fused oxazole structure 4.

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.