Telomeres are specialized protein-DNA complexes that compose the ends of eukaryotic chromosomes. Telomeres protect chromosome termini from degradation and recombination and act together with telomerase to ensure complete genome replication. We have determined the crystal structure of the two-subunit Oxytricha nova telomere end binding protein (OnTEBP) complexed with single strand telomeric DNA at 2.8 A resolution. The structure reveals four oligonucleotide/oligosaccharide-binding folds, three of which form a deep cleft that binds the ssDNA, and a fourth that forms an unusual protein-protein interaction between the alpha and beta subunits. This structure provides a molecular description of how the two subunits of OnTEBP recognize and bind ssDNA to form a sequence-specific, telomeric nucleoprotein complex that caps the very 3' ends of chromosomes.

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RNA is known to fold into a variety of structural elements, many of which have sufficient sequence complexity to make the thermodynamic study of each possible variant impractical. We previously reported a method for isolating stable and unstable RNA sequences from combinatorial libraries using temperature gradient gel electrophoresis (TGGE). This method was used herein to analyze a six-nucleotide RNA hairpin loop library. Three rounds of in vitro selection were performed using TGGE, and unusually stable RNAs were identified by cloning and sequencing. Known stable tetraloops were found, including sequences belonging to the UNCG motif closed by a CG base pair, and the CUUG motif closed by a GC base pair. In addition, unknown tetraloops were found that were nearly as stable as cUNCGg, including sequences related through substitution of the U with a C (Y), the C with an A (M), or both. These substitutions allow hydrogen bonding and stacking interactions in the UNCG loop to be maintained. Thermodynamic analysis of YNMG and variant loops confirmed optimal stability with Y at position 1 and M at position 3. Similarity in structure and stability among YNMG loops was further supported by deoxyribose substitution, CD, and NMR experiments. A conserved tertiary interaction in 16S rRNA exists between a YAMG loop at position 343 and two adenines in the loop at position 159 (Escherichia coli numbering). NMR and functional group substitution experiments suggest that YNAG loops in particular have enhanced flexibility, which allows the tertiary interaction to be maintained with diverse loop sequences at position 159. Taken together, these results support the existence of an extended family of UNCG-like tetraloops with the motif cYNMGg that are thermodynamically stable and structurally similar and can engage in tertiary interactions in large RNA molecules.

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Prediction of nucleic acid structure from sequence requires thermodynamic parameters for a variety of motifs, many of which are complex and consist of a large number of possible sequence combinations. Here we report an experimental approach for identifying the stable and unstable members of an RNA combinatorial library. Short model RNA hairpins consisting of 13 base pairs (bp) flanked by primer binding sites are constructed and separated according to their relative thermodynamic stabilities using temperature gradient gel electrophoresis (TGGE). Partially denaturing TGGE is carried out with potassium chloride, sodium chloride, or magnesium chloride salts in the gel. The TMs of model hairpins can be tuned by adjusting the concentration of urea in the gel while maintaining the correct order of stabilities for the hairpins. Mixtures of RNAs differing by a single Watson-Crick base pair are resolved according to their relative thermodynamic stabilities, as are mixtures of GC or AU base pair transversions differing in DeltaG degrees37 by only 0.3-0.5 kcal/mol. In addition, a simple combinatorial library with one position of randomization opposite a guanosine is prepared and separated into its four members by parallel and perpendicular TGGE. The order of thermodynamic stabilities for the library determined by TGGE is shown to be the same when assayed by UV-melting experiments. Analysis of the thermodynamics of folding of combinatorial libraries is general and may be applied to a wide variety of complex nucleic acid secondary and tertiary motifs in order to identify the stable and unstable members.

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The synthesis, characterization, and emission properties of a series of Pt(diphosphine)(dithiolate) complexes are reported. Diphosphine ligands include l,2-bis(diphenylphosphino)ethane(dppe), l,2-bis(diphenylphosphino)ethylene (dppv), l,2-bis(diphenylphosphino)benzene (dppb), l,2-bis(dicyclohexylphosphino)ethane (chpe), bis(diphenylphosphino)methane (dppm), l,2-bis(dimethoxyphosphino)ethane (pompom), triphenylphosphine (PPh3), dimethylphenylphosphine (PMe2Ph), tricyclohexylphosphine (PCy3), triphenyl phosphite (P(OPh)3), and triisopropyl phosphite (P( 0-/-Pr)3). Dithiolate ligands include maleonitriledithiolate (mnt) and l-(ethoxycarbonyl)-l-cyanoethylene-2,2-dithiolate (ecda). The complexes are readily synthesized by the addition of the diphosphine ligand to Pt-(COD) (dithiolate). On the basis of characterizations using NMR and infrared spectroscopies, all of the complexes are assigned square planar coordination geometries with varying degrees of distortion determined by ligand steric and electronic effects. The assignment of square planar coordination is confirmed by single crystal structure