Affinity labelling in situ of the bL12 protein on E. coli 70S ribosomes by means of a tRNA dialdehyde derivative.

In this report, we have used periodate-oxidized tRNA (tRNAox) as an affinity laleling reagent to demonstrate that: (i) the bL12 protein contacts the CCA-arm of P-site bound tRNA on the Escherichia coli 70S ribosomes; (ii) the stoichiometry of labelling is one molecule of tRNAox bound to one polypeptide chain of endogenous bL12; (iii) cross-linking in situ of bL12 with tRNAox on the ribosomes provokes the loss of activity; (iv) intact tRNA protects bL12 in the 70S ribosomes against cross-linking with tRNAox; (v) both tRNAox and pyridoxal 5'-phosphate (PLP) compete for the same or for proximal cross-linking site(s) on bL12 inside the ribosome; (vi) the stoichiometry of cross-linking of PLP to the recombinant E. coli bL12 protein is one molecule of PLP covalently bound per polypeptide chain; (vii) the amino acid residue of recombinant bL12 cross-linked with PLP is Lys-65; (viii) Lys-65 of E. coli bL12 corresponds to Lys-53 of eL42 which was previously shown to cross-link with P-site bound tRNAox on human 80S ribosomes in situ; (ix) finally, E. coli bL12 and human eL42 proteins display significant primary structure similarities, which argues for evolutionary conservation of these two proteins located at the tRNA-CCA binding site on eubacterial and eukaryal ribosomes.

A STAT3-inhibitory hairpin decoy oligodeoxynucleotide discriminates between STAT1 and STAT3 and induces death in a human colon carcinoma cell line.

BACKGROUND: The Signal Transducer and Activator of Transcription 3 (STAT3) is activated in tumor cells, and STAT3-inhibitors are able to induce the death of those cells. Decoy oligodeoxynucleotides (dODNs), which bind to the DNA Binding Domain (DBD) of STAT3, are efficient inhibitors. However, they also inhibit STAT1, whose activity is essential not only to resistance to pathogens, but also to cell growth inhibition and programmed cell death processes. The aim of this study was to design STAT3-specific dODNs which do not affect STAT1-mediated processes. RESULTS: New dODNs with a hairpin (hpdODNs) were designed. Modifications were introduced, based on the comparison of STAT3- and STAT1-DBD interactions with DNA using 3D structural analyses. The designed hpdODNs were tested for their ability to inhibit STAT3 but not STAT1 by determining: i) cell death in the active STAT3-dependent SW480 colon carcinoma cell line, ii) absence of inhibition of interferon (IFN) γ-dependent cell death, iii) expression of STAT1 targets, and iv) nuclear location of STAT3 and STAT1. One hpdODN was found to efficiently induce the death of SW480 cells without interfering with IFNγ-activated STAT1. This hpdODN was found in a complex with STAT3 but not with STAT1 using an original in-cell pull-down assay; this hpdODN also did not inhibit IFNγ-induced STAT1 phosphorylation, nor did it inhibit the expression of the STAT1-target IRF1. Furthermore, it prevented the nuclear transfer of STAT3 but not that of IFNγ-activated STAT1. CONCLUSIONS: Comparative analyses at the atomic level revealed slight differences in STAT3 and STAT1 DBDs' interaction with their DNA target. These were sufficient to design a new discriminating hpdODN that inhibits STAT3 and not STAT1, thereby inducing tumor cell death without interfering with STAT1-dependent processes. Preferential interaction with STAT3 depends on oligodeoxynucleotide sequence modifications but might also result from DNA shape changes, known to modulate protein/DNA interactions. The finding of a STAT3-specific hpdODN establishes the first rational basis for designing STAT3 DBD-specific inhibitors.

Solution structure of a truncated anti-MUC1 DNA aptamer determined by mesoscale modeling and NMR.

Mucin 1 is a well-established target for the early diagnosis of epithelial cancers. The nucleotides of the S1.3/S2.2 DNA aptamer involved in binding to variable number tandem repeat mucin 1 peptides have been identified using footprinting experiments. The majority of these binding nucleotides are located in the 25-nucleotide variable region of the total aptamer. Imino proton and 2D NMR spectra of truncated and total aptamers in supercooled water reveal common hydrogen-bonding networks and point to a similar secondary structure for this 25-mer sequence alone or embedded within the total aptamer. NMR titration experiments confirm that the TTT triloop structure is the primary binding site and show that the initial structure of the truncated aptamers is conserved upon interaction with variable number tandem repeat peptides. The thermal dependence of the NMR chemical shift data shows that the base-paired nucleotides melt cooperatively at 47 ± 4°C. The structure of the 25-mer oligonucleotide was determined using a new combined mesoscale molecular modeling, molecular dynamics and NMR spectroscopy investigation. It contains three Watson-Crick pairs, three consecutive mispairs and four Watson-Crick pairs capped by a TTT triloop motif. The 3D model structures (PDB 2L5K) and biopolymer chain elasticity molecular models are consistent with both NMR and long unconstrained molecular dynamics (10 ns) in explicit water, respectively. Database Structural data are available in the Protein Data Bank and BioMagResBank databases under the accession numbers 2L5K and 17129, respectively.

Nucleic acid folding determined by mesoscale modeling and NMR spectroscopy: solution structure of d(GCGAAAGC).

Determination of DNA solution structure is a difficult task even with the high-sensitivity method used here based on simulated annealing with 35 restraints/residue (Cryoprobe 750 MHz NMR). The conformations of both the phosphodiester linkages and the dinucleotide segment encompassing the sharp turn in single-stranded DNA are often underdetermined. To obtain higher quality structures of a DNA GNRA loop, 5'-d(GCGAAAGC)-3', we have used a mesoscopic molecular modeling approach, called Biopolymer Chain Elasticity (BCE), to provide reference conformations. By construction, these models are the least deformed hairpin loop conformation derived from canonical B-DNA at the nucleotide level. We have further explored this molecular conformation at the torsion angle level with AMBER molecular mechanics using different possible (epsilon,zeta) constraints to interpret the 31P NMR data. This combined approach yields a more accurate molecular conformation, compatible with all the NMR data, than each method taken separately, NMR/DYANA or BCE/AMBER. In agreement with the principle of minimal deformation of the backbone, the hairpin motif is stabilized by maximal base-stacking interactions on both the 5'- and 3'-sides and by a sheared G.A mismatch base pair between the first and last loop nucleotides. The sharp turn is located between the third and fourth loop nucleotides, and only two torsion angles beta6 and gamma6 deviate strongly with respect to canonical B-DNA structure. Two other torsion angle pairs epsilon3,zeta3 and epsilon5,zeta5 exhibit the newly recognized stable conformation BIIzeta+ (-70 degrees, 140 degrees). This combined approach has proven to be useful for the interpretation of an unusual 31P chemical shift in the 5'-d(GCGAAAGC)-3' hairpin.

Dinucleotide TpT and its 2'-O-Me analogue possess different backbone conformations and flexibilities but similar stacked geometries.

UV irradiation at 254 nm of 2'-O,5-dimethyluridylyl(3'-5')-2'-O,5-dimethyluridine (1a) and of natural thymidylyl(3'-5')thymidine (1b) generates the same photoproducts (CPD and (6-4)PP; responsible for cell death and skin cancer). The ratios of quantum yields of photoproducts obtained from 1a (determined herein) to that from 1b are in a proportion close to the approximately threefold increase of stacked dinucleotides for 1a compared with those of 1b (from previous circular dichroism results). 1a and 1b however are endowed with different predominant sugar conformations, C3'-endo (1a) and C2'-endo (1b). The present investigation of the stacked conformation of these molecules, by unrestrained state-of-the-art molecular simulation in explicit solvent and salt, resolves this apparent paradox and suggests the following main conclusions. Stacked dinucleotides 1a and 1b adopt the main characteristic features of a single-stranded A and B form, respectively, where the relative positions of the backbone and the bases are very different. Unexpectedly, the geometry of the stacking of two thymine bases, within each dinucleotide, is very similar and is in excellent agreement with photochemical and circular dichroism results. Analyses of molecular dynamics trajectories with conformational adiabatic mapping show that 1a and 1b explore two different regions of conformational space and possess very different flexibilities. Therefore, even though their base stacking is very similar, these molecules possess different geometrical, mechanical, and dynamical properties that may account for the discrepancy observed between increased stacking and increased photoproduct formations. The computed average stacked conformations of 1a and 1b are well-defined and could serve as starting models to investigate photochemical reactions with quantum dynamics simulations.

In vitro selection of halo-thermophilic RNA reveals two families of resistant RNA.

The "RNA world" hypothesis proposes that early in the evolution of life, RNA was responsible both for the storage and transfer of genetic information and for the catalysis of biochemical reactions. One of the problems of the hypothesis is that RNA is known to be temperature sensitive. Nevertheless, different types of sequences with a thermostable phenotype may exist. In order to test this possibility, we applied an in vitro evolution method (SELEX) to isolate RNA molecules that are resistant at high temperatures (80 degrees C for 65 h) and high salt concentrations (2 M NaCl). The sequences of the resulting cloned halo-thermophilic RNAs can be grouped in two families (I and II) possessing very different thermal and chemical stabilities and very different secondary structures. The selected RNA molecules illustrate two different possibilities leading to thermal resistance which may be related to primitive conditions. We propose that members of family I constitute a good means of storing sequence information while members of family II are less efficient but replicate faster in early steps of the SELEX. These selected RNA behaviors may be related to primitive conditions and could allow to define limits for survival, and demonstrate that what is at stake for RNA molecules, as for living organisms, is survival and reproduction.

Spectroscopic and structural impact of a stem base-pair change in DNA hairpins: GTTC-ACA-GAAC versus GTAC-ACA-GTAC.

Successive investigations over the last decade have revealed and confirmed a stable loop closure in a family of d-[GTAC-5Pur6N7N-GTAC] hairpins, where 5Pur6N7N is a AAA, GAG and AXC loop (X being any nucleotide). The trinucleotide loop is characterized by a well defined 5Pur-7N mispairing mode, and by upfield chemical shifts for three sugar protons of the apical nucleotide 6N. The GTTC-ACA-GAAC DNA hairpin, of interest for its likely involvement in Vibrio cholerae genome mutations, has now been investigated. The GTAC-ACA-GTAC DNA hairpin has also been studied because it is intermediate between the other structures, as it contains the loop of the hairpin under consideration and the stem of the above family. The two hairpins with the ACA loop are stable. They show the same mispairing mode and similar upfield shifts as the previous family, but GTTC-ACA-GAAC seems to be slightly less compact than any other. GTTC-ACA-GAAC is remarkable in that it exhibits a B(II) character for the phosphate-ester conformation at 8Gp9A, together with a swing of the upper hairpin into the major groove that, in particular, brings 6CH1' roughly as close to 7AH2 as to 6CH6. These unexpected structural features are qualitatively deduced from (1)H and (31)P NMR spectra, and confirmed by Raman spectroscopy. This comparative study shows that not only the loop sequence but also the stem sequence may control hairpin structures.

Biopolymer Chain Elasticity: A novel concept and a least deformation energy principle predicts backbone and overall folding of DNA TTT hairpins in agreement with NMR distances.

A new molecular modelling methodology is presented and shown to apply to all published solution structures of DNA hairpins with TTT in the loop. It is based on the theory of elasticity of thin rods and on the assumption that single-stranded B-DNA behaves as a continuous, unshearable, unstretchable and flexible thin rod. It requires four construction steps: (i) computation of the tri-dimensional trajectory of the elastic line, (ii) global deformation of single-stranded helical DNA onto the elastic line, (iii) optimisation of the nucleoside rotations about the elastic line, (iv) energy minimisation to restore backbone bond lengths and bond angles. This theoretical approach called 'Biopolymer Chain Elasticity' (BCE) is capable of reproducing the tri-dimensional course of the sugar-phosphate chain and, using NMR-derived distances, of reproducing models close to published solution structures. This is shown by computing three different types of distance criteria. The natural description provided by the elastic line and by the new parameter, Omega, which corresponds to the rotation angles of nucleosides about the elastic line, offers a considerable simplification of molecular modelling of hairpin loops. They can be varied independently from each other, since the global shape of the hairpin loop is preserved in all cases.

DNA tri- and tetra-loops and RNA tetra-loops hairpins fold as elastic biopolymer chains in agreement with PDB coordinates.

The biopolymer chain elasticity (BCE) approach and the new molecular modelling methodology presented previously are used to predict the tri- dimensional backbones of DNA and RNA hairpin loops. The structures of eight remarkably stable DNA or RNA hairpin molecules closed by a mispair, recently determined in solution by NMR and deposited in the PDB, are shown to verify the predicted trajectories by an analysis automated for large numbers of PDB conformations. They encompass: one DNA tetraloop, -GTTA-; three DNA triloops, -AAA- or -GCA-; and four RNA tetraloops, -UUCG-. Folding generates no distortions and bond lengths and bond angles of main atoms of the sugar-phosphate backbone are well restored upon energy refinement. Three different methods (superpositions, distance of main chain atoms to the elastic line and RMSd) are used to show a very good agreement between the trajectories of sugar-phosphate backbones and between entire molecules of theoretical models and of PDB conformations. The geometry of end conditions imposed by the stem is sufficient to dictate the different characteristic DNA or RNA folding shapes. The reduced angular space, consisting of the new parameter, angle Omega, together with the chi angle offers a simple, coherent and quantitative description of hairpin loops.