Determination of salicin and related compounds in botanical dietary supplements by liquid chromatography with fluorescence detection

The centromeric dodeca-satellite of Drosophila forms unusual DNA structures in which its purine-rich strand (GTACGGGACCGA)n folds into very stable intramolecular hairpins. These intramolecular hairpins contain groups of tandem 5'-GA:GA-3' mismatches that, as judged by gel electrophoresis analysis and UV-melting studies, have a determinant contribution to their stability. Duplexes of the dodeca-satellite purine-rich strand, carrying tandem 5'-GA:GA-3' mismatches, are as stable as equivalent fully Watson-Crick duplexes containing tandem 5'-TA:TA-3' Watson-Crick pairs in place of the non-Watson-Crick G.A pairs. On the other hand, duplexes carrying any of the other three possible tandem combinations of purine.purine mismatches, including G.A pairs on the opposite orientation 5'-AG:AG-3', are very unstable. The high stability of the dodeca-satellite hairplus suggests that the tandem G.A pairs are on the sheared configuration although they are found within the less favourable 5'-G-(G-A)-C-3' sequence context. Other centromeres DNA sequences, including the AAGAG satellite of Drosophila and the mammalian CENP-B box sequence, have the potential of forming intramolecular hairpins stabilised by similar purine.purine interactions.     

Crystal structure of an RNA octamer duplex r(CCCIUGGG)2 incorporating tandem I.U wobbles

The crystal structure of the RNA octamer duplex r(CCCIUGGG)2has been elucidated at 2.5 A resolution. The crystals belong to the space group P21and have unit cell constants a = 33.44 A, b = 43.41 A, c = 49.39 A and beta = 104.7 degrees with three independent duplexes (duplexes 1-3) in the asymmetric unit. The structure was solved by the molecular replacement method and refined to an Rwork/Rfree of 0.185/0.243 using 3765 reflections between 8.0 and 2.5 A. This is the first report of an RNA crystal structure incorporating I.U wobbles and three molecules in the asymmetric unit. Duplex 1 displays a kink of 24 degrees between the mismatch sites, while duplexes 2 and 3 have two kinks each of 19 degrees and 27 degrees, and 24 degrees and 29 degrees, respectively, on either side of the tandem mismatches. At the I.U/U.I mismatch steps, duplex 1 has a twist angle of 33.9 degrees, close to the average for all base pair steps, but duplexes 2 and 3 are underwound, with twist angles of 24.4 degrees and 26.5 degrees, respectively. The tandem I.U wobbles show intrastrand purine-pyrimidine stacking but exhibit interstrand purine-purine stacking with the flanking C.G pairs. The three independent duplexes are stacked non-coaxially in a head-to-tail fashion to form infinite pseudo-continuous helical columns which form intercolumn hydrogen bonding interactions through the 2'-hydroxyl groups where the minor grooves come together.     

Thermodynamics of nonsymmetric tandem mismatches adjacent to G.C base pairs in RNA

The thermodynamic stabilities and structures of a series of RNA duplexes containing nonsymmetric tandem mismatches in the context of , where are tandem mismatches, were studied by UV melting and imino proton NMR. The contribution of one mismatch to the free energy increment for tandem mismatch formation depends on the identity of the other mismatch. Imino proton NMR indicates that this is partly because the structure of a mismatch is dependent on the adjacent mismatch. The results suggest that differences in size, shape, and hydrogen bonding of the adjacent mismatches play important roles in determining loop stability. A model for predicting stabilities of all possible tandem mismatches is proposed based on these and previous results.     

Structure of a 16-mer RNA duplex r(GCAGACUUAAAUCUGC)2 with wobble C.A+ mismatches

The crystal structure of a 16-mer, the longest known RNA duplex, has been determined at 2.5 A resolution. The hexadecamer r(GCAGACUUAAAUCUGC) contains isolated C.A/A.C mismatches with two hydrogen bonds. The two hydrogen bonds in the mismatches suggests that N1 of A is protonated even though the crystallization was done at neutral pH. Therefore, the C.A mismatch is a C.A+ wobble similar to the G.U wobble. The two C.A+ pairs are isolated by four Watson-Crick pairs and flanked by five Watson-Crick base-pairs on either sides. Kinks/bends of 20 degrees are observed at the wobble sites. The Watson-Crick base-pair A5.U26 on the 5'-side of the first C6.A27(+) wobble has a twist angle of 27 degrees compared to the 3'-side U7.A28 pair of 36 degrees. The twist angles are reversed (37 degrees and 26 degrees) in the second A11(+).C22 wobble because of the approximate dyad in the molecule, the flanking base-pair sequences are A.U pairs. The wobbles expand the major groove to 7.1 A/7.3 A. The duplexes form helical columns and are tightly packed around the 31-screw axis. The minor grooves of adjacent columns in juxtaposition interact through the O2' atoms and the anionic phosphate oxygen atoms.     

Solution structure of a DNA duplex containing the exocyclic lesion 3,N4-etheno-2'-deoxycytidine opposite 2'-deoxyguanosine

Vinyl chloride reacts with cellular DNA producing 3,N4-etheno-2'-deoxycytidine (epsilonC) along with other exocyclic adducts. The solution structure of an oligodeoxynucleotide duplex containing an epsilonC.dG base pair was determined by high-resolution NMR spectroscopy and molecular dynamics simulations. NMR data indicated that the duplex adopts a right-handed helical structure having all residues in anti orientation around the glycosylic torsion angle. The epsilonC adduct has a sugar pucker in the C3'-endo/C4'-exo region while the rest of the residues are in the C2'-endo/C3'-exo range. NOE interactions established Watson-Crick alignments for canonical base pairs of the duplex. The imino proton of the lesion-containing base pair resonated as a sharp signal that was resistant to water exchange, suggesting hydrogen bonding. Restrained molecular dynamics simulations generated three-dimensional models in excellent agreement with the spectroscopic data. The refined structures are slightly bent at the lesion site without major perturbations of the sugar-phosphate backbone. The adduct is displaced and shifted toward the major groove of the helix while its partner on the complementary strand remains stacked. The epsilonC(anti).dG(anti) base pair alignment is sheared and stabilized by the formation of hydrogen bonds. The biological implications of structures of epsilonC-containing DNA duplexes are discussed.     

Solution structure of the tobramycin-RNA aptamer complex

We have solved the solution structure of the aminoglycoside antibiotic tobramycin complexed with a stem-loop RNA aptamer. The 14 base loop of the RNA aptamer 'zippers up' alongside the attached stem through alignment of four mismatches and one Watson-Crick pair on complex formation. The tobramycin inserts into the deep groove centered about the mismatch pairs and is partially encapsulated between its floor and a looped out guanine base that flaps over the bound antibiotic. Several potential intermolecular hydrogen bonds between the charged NH3 groups of tobramycin and acceptor atoms on base pair edges and backbone phosphates anchor the aminoglycoside antibiotic within its sequence/structure specific RNA binding pocket.     

Compensatory neutral mutations and the evolution of RNA

There are many examples of RNA molecules in which the secondary structure has been strongly conserved during evolution, but the base sequence is much less conserved, e.g., transfer RNA, ribosomal RNA, and ribonuclease P. A model of compensatory neutral mutations is used here to describe the evolution of the base sequence in RNA helices. There are two loci (i.e., the two sides of the pair) with four alleles at each locus (corresponding to A, C, G, U). Watson-Crick base pairs (AU, CG, GC, and UA) are each assigned a fitness 1, whilst all other pairs are treated as mismatches and assigned fitness 1-s. A population of N diploid individuals is considered with a mutation rate of u per base. For biologically reasonable parameter values, the frequency of mismatches is always small but the frequency of the four matching pairs can vary over a wide range. Using a diffusion model, the stationary distribution for the frequency x of any of the four matching pairs is calculated. The shape depends on the combination of variables beta = 8Nu2/9s. For small beta, the distribution diverges at the two extremes, x = 0 and x = 1-z, where z is the mean frequency of mismatches. The population typically consists almost entirely of one of the four types of matching pairs, but occasionally makes shifts between the four possible states. The mean rate at which these shifts occur is calculated here. The effect of recombination between the two loci is to decrease the probability density at intermediate x, and to increase the weight at the extremes. The rate of transition between the four states is slowed by recombination (as originally shown by Kimura in a two-allele model with irreversible mutation). A very small recombination rate r approximately u2/s is sufficient to increase the mean time between transitions dramatically. In addition to its application to RNA, this model is also relevant to the 'shifting balance' theory describing the drift of populations between alternative equilibria separated by low fitness valleys. Equilibrium values for the frequencies of the different allele combinations in an infinite population are also calculated. It is shown that for low recombination rates the equilibrium is symmetric, but there is a critical recombination rate above which alternative asymmetric equilibria become stable.     

The crystal structure of the RNA/DNA hybrid r(GAAGAGAAGC). d(GCTTCTCTTC) shows significant differences to that found in solution

The crystal structure of the RNA/DNA hybrid r(GAAGAGAAGC). d(GCTTCTCTTC) has been solved and refined at 2.5 A resolution. The refinement procedure converged at R = 0.181 for all reflections in the range 20.0-2.5 A. In the crystal, the RNA/DNA hybrid duplex has an A' conformation with all but one of the nucleotide sugar moieties adopting a C3'- endo (N) conformation. Both strands in the double helix adopt a global conformation close to the A-form and the width of the minor groove is typical of that found in the crystal structures of other A-form duplexes. However, differences are observed between the RNA and DNA strands that make up the hybrid at the local level. In the central portion of the duplex, the RNA strand has backbone alpha, beta and gamma torsion angles that alternate between the normal gauche -/ trans / gauche + conformation and an unusual trans / trans / trans conformation. Coupled with this so-called 'alpha/gamma flipping' of the backbone torsion angles, the distance between adjacent phosphorous atoms on the RNA strand systematically varies. Neither of these phenomena are observed on the DNA strand. The structure of the RNA/DNA hybrid presented here differs significantly from that found in solution for this and other sequences. Possible reasons for these differences and their implications for the current model of RNase H activity are discussed.     

The high resolution crystal structure of the DNA decamer d(AGGCATGCCT)

The crystal structure of the DNA decamer d(AGGCATGCCT) has been determined to a resolution of 1.3 A and R factor of 13.9%. The structure has a unique conformation with each of the decamer single strands forming base-pairing interactions with two symmetry-related strands. The central eight bases of the decamer form an A-DNA octamer duplex with one symmetry-related strand whilst the terminal 5'-A and T-3' bases are flipped out and away from the octamer helix axis to form base-pairing interactions with a second symmetry-related strand. These A.T base-pairs lie perpendicular to the crystallographic c axis and pack within the unit cell in conjunction with a symmetry-related A.T base-pair displaced by 3.4 A degrees along the c axis. A novel base triplet interaction of the type A*(G.C) is present in the structure with interaction from the major groove side of the terminal 5'-A base to the minor groove of the central A-DNA octamer. This structure reports the first example of cobalt hexammine binding to a right-handed DNA duplex. The crystallographic asymmetric unit contains two cobalt hexammine ligands with one site in the major groove coordinating via hydrogen bonds to the 5'-AGG bases, and the second site located between DNA molecules and interacting with the oxygen atoms of phosphate groups.     

Watson-Crick base-pairing properties of bicyclo-DNA

A series of sequences of the DNA analog bicyclo-DNA, 6-12 nucleotides in length and containing all four natural nucleobases, were prepared and their Watson-Crick pairing properties with complementary RNA and DNA, as well as in its own series, were analyzed by UV-melting curves and CD-spectroscopy. The results can be summarized as follows: bicyclo-DNA forms stable Watson-Crick duplexes with complementary RNA and DNA, the duplexes with RNA generally being more stable than those with DNA. Pyrimidine-rich bicyclo-DNA sequences form duplexes of equal or slightly increased stability with DNA or RNA, whereas purine-rich sequences show decreased affinity to complementary DNA and RNA when compared with wild-type (DNA-DNA, DNA-RNA) duplexes. In its own system, bicyclo-DNA prefers antiparallel strand alignment and strongly discriminates for base mismatches. Duplexes are always inferior in stability compared with the natural ones. A detailed analysis of the thermodynamic properties was performed with the sequence 5'-GGATGGGAG-3'x 5'-CTCCCATCC-3' in both backbone systems. Comparison of the pairing enthalpy and entropy terms shows an enthalpic advantage for DNA association (delta deltaH = -18 kcal x (mol)-1)) and an entropic advantage for bicyclo-DNA association (delta deltaS = 49 cal x K(-1) x mol(-1), leading to a delta deltaG 25 degrees C of -3.4 kcal x mol(-1) in favor of the natural duplex. The salt dependence of Tm for this sequence is more pronounced in the case of bicyclo-DNA due to increased counter ion screening from the solvent. Furthermore bicyclo-DNA sequences are more stable towards snake venom phosphodiesterase by a factor of 10-20, and show increased stability in fetal calf serum by a factor of 8 compared with DNA.     

Specificity and fidelity of strand joining by Chlorella virus DNA ligase

Chlorella virus PBCV-1 DNA ligase seals nicked duplex DNA substrates consisting of a 5'-phosphate-terminated strand and a 3'-hydroxyl-terminated strand annealed to a bridging template strand, but cannot ligate a nicked duplex composed of two DNAs annealed on an RNA template. Whereas PBCV-1 ligase efficiently joins a 3'-OH RNA to a 5'-phosphate DNA, it is unable to join a 3'-OH DNA to a 5'-phosphate RNA. The ligase discriminates at the substrate binding step between nicked duplexes containing 5'-phosphate DNA versus 5'-phosphate RNA strands. PBCV-1 ligase readily seals a nicked duplex DNA containing a single ribonucleotide substitution at the reactive 5'-phosphate end. These results suggest a requirement for a B-form helical conformation of the polynucleotide on the 5'-phosphate side of the nick. Single base mismatches at the nick exert disparate effects on DNA ligation efficiency. PBCV-1 ligase tolerates mismatches involving the 5'-phosphate nucleotide, with the exception of 5'-A:G and 5'-G:A mispairs, which reduce ligase activity by two orders of magnitude. Inhibitory configurations at the 3'-OH nucleotide include 3'-G:A, 3'-G:T, 3'-T:T, 3'-A:G, 3'-G:G, 3'-A:C and 3'-C:C. Our findings indicate that Chlorella virus DNA ligase has the potential to affect genome integrity by embedding ribonucleotides in viral DNA and by sealing nicked molecules with mispaired ends, thereby generating missense mutations.     

RNA hydration: a detailed look

The crystal structure of the RNA duplex [r(CCCCGGGG)]2 has been refined to 1.46 A resolution with room temperature synchrotron diffraction data. This represents the highest resolution reported to date for an all-RNA oligonucleotide and is well beyond the best resolution ever achieved with an A-form DNA duplex. The analysis of the ordered hydration around the octamer duplex reveals conserved regular arrangements of water molecules in both grooves. In the major groove, all located first shell water molecules can be fitted into a pattern that is repeated through all eight base pairs, involves half the phosphate oxygens, and joins the two strands. In the minor groove, roughly across its narrowest dimension, tandem water molecules link the 2'-hydroxyl groups of adjacent nucleotides in base-pair steps in a similarly regular fashion. The structure provides evidence for an important role of the 2'-hydroxyl groups in the thermodynamic stabilization of RNA, beyond their known functions of locking the sugar pucker and mediating 3' --> 5' intrastrand O2'...O4' hydrogen bonds. The ribose 2'-hydroxyls lay the foundation for the enthalpic stability of the RNA relative to the DNA duplex, both as a scaffold for the water network in the minor groove and through their extensive individual hydration.     

Kinetics for hybridization of peptide nucleic acids (PNA) with DNA and RNA studied with the BIAcore technique

The binding of a mixed-sequence pentadecamer PNA (peptide nucleic acid) containing all four nucleobases to the fully complementary as well as various singly mismatched RNA and DNA oligonucleotides has been systematically investigated using thermal denaturation and BIAcore surface-interaction techniques. The rate constants for association (k(a)) and dissociation (k(d)) of the duplex formation as well as the thermal stability (melting temperature, T(m)) of the duplexes have been determined. Upon binding to PNA tethered via a biotin-linker to streptavidin at the dextran/gold surface, DNA and RNA sequences containing single mismatches at various positions in the center resulted in increased dissociation and decreased association rate constants. T(m) values for PNA x RNA duplexes are on average 4 degrees C higher than for PNA x DNA duplexes and follow quantitatively the same variation with mismatches as do the PNA x DNA duplexes. Also a faster k(a) and a slower k(d) are found for PNA x RNA duplexes compared to the PNA x DNA duplexes. An overall fair correlation between T(m), k(a), and k(d) is found for a series of PNA x DNA and PNA x RNA duplexes although the determination of k(a) seemed to be prone to artifacts of the method and was not considered capable of providing absolute values representing the association rate constant in bulk solution.     

Effects of the introduction of L-nucleotides into DNA. Solution structure of the heterochiral duplex d(G-C-G-(L)T-G-C-G).d(C-G-C-A-C-G-C) studied by NMR spectroscopy

The effect of the substitution of a L-nucleoside for a D-nucleoside in the duplex d(G-C-G-T-G-C-G).d(C-G-C-A-C-G-C) was studied by UV and NMR spectroscopy. These unnatural oligonucleotides have potential for antisense DNA technology [Damha, M. J., Giannaris, P. A., & Marfey, P. (1994) Biochemistry (preceding paper in this issue)]. The thermal stability of such duplexes is lower than that of the natural one and is dependent on the nucleotide type and/or sequence. Interestingly, inversion of the chirality of thymidine but not adenosine coincides with a large stabilizing enthalpy change. The structure of the heterochiral duplex d(G1-C2-G3-(L)T4-G5-C6-G7).d(C8-G9-C10-A11-C12-G13- C14), where (L)T denotes the mirror image of the natural thymidine, has been determined by NMR spectroscopy. The sugar conformation was determined using the sum of coupling constants and the distances using a model free relaxation matrix approach. The torsion angles of the backbone follow from 3JHH, 3JHP, and 4JHP coupling constants. The structure of the duplex was calculated by metric matrix distance geometry followed by simulated annealing. The structure is close to that of B-DNA. The base pair formed by (L)T and A is of the Watson-Crick type. All sugars adopt an S-type pucker. The incorporation of the L-sugar in the duplex is accomplished by changes in the backbone torsion angles around the phosphates and the glycosidic torsion angle of (L)T. The modification induces changes in the natural strand as well. The structure exhibits an unusual interaction between the aromatic rings of the (L)T4.A11 and G3.C12 base pairs, which provides a plausible explanation of the unusual thermodynamic properties of the duplex.     

Interaction of the phage T4 Dam DNA-[N6-adenine] methyltransferase with oligonucleotides containing native or modified (defective) recognition sites

The DNA-[N 6-adenine]-methyltransferase (Dam MTase) of phage T4 catalyzes methyl group transfer from S-adenosyl-l-methionine (AdoMet) to the N6-position of adenine in the palindromic sequence, GATC. We have used a gel shift assay to monitor complex formation between T4 Dam and various synthetic duplex oligonucleotides, either native or modified/defective. The results are summarized as follows. (i) T4 Dam bound with approximately 100-fold higher affinity to a 20mer specific (GATC-containing) duplex containing the canonical palindromic methylation sequence, GATC, than to a non-specific duplex containing another palindrome, GTAC. (ii) Compared with the unmethylated duplex, the hemimethylated 20mer specific duplex had a slightly increased ( approximately 2-fold) ability to form complexes with T4 Dam. (iii) No stable complex was formed with a synthetic 12mer specific (GATC-containing) duplex, although T4 Dam can methylate it. This indicates that there is no relation between formation of a catalytically competent 12mer-Dam complex and one stable to gel electrophoresis. (iv) Formation of a stable complex did not require that both strands be contiguous or completely complementary. Absence of a single internucleotide phosphate strongly reduced complex formation only when missing between the T and C residues. This suggests that if T4 Dam makes critical contact(s) with a backbone phosphate(s), then the one between T and C is the only likely candidate. Having only one half of the recognition site intact on one strand was sufficient for stable complex formation provided that the 5'G.C base-pairs be present at both ends of the palindromic, GATC. Since absence of either a G or C abolished T4 Dam binding, we conclude that both strands are recognized by T4 Dam.     

Molecular recognition in the FMN-RNA aptamer complex

We report on a combined NMR-molecular dynamics calculation approach that has solved the solution structure of the complex of flavin mononucleotide (FMN) bound to the conserved internal loop segment of a 35 nucleotide RNA aptamer identified through in vitro selection. The FMN-RNA aptamer complex exhibits exceptionally well-resolved NMR spectra that have been assigned following application of two, three and four-dimensional heteronuclear NMR techniques on samples containing uniformly 13C, 15N-labeled RNA aptamer in the complex. The assignments were aided by a new through-bond NMR technique for assignment of guanine imino and adenine amino protons in RNA loop segments. The conserved internal loop zippers up through the formation of base-pair mismatches and a base-triple on complex formation with the isoalloxazine ring of FMN intercalating into the helix between a G.G mismatch and a G.U.A base-triple. The recognition specificity is associated with hydrogen bonding of the uracil like edge of the isoalloxazine ring of FMN to the Hoogsteen edge of an adenine at the intercalation site. There is significant overlap between the intercalated isoalloxazine ring and its adjacent base-triple platform in the complex. The remaining conserved residues in the internal loop participate in two G.A mismatches in the complex. The zippered-up internal loop and flanking stem regions form a continuous helix with a regular sugar-phosphate backbone except at a non-conserved adenine, which loops out of the helix to facilitate base-triple formation. Our solution structure of the FMN-RNA aptamer complex is to our knowledge the first structure of an RNA aptamer complex and outlines folding principles that are common to other RNA internal and hairpin loops, and molecular recognition principles common to model self-replication systems in chemical biology.     

Variability in the stability of DNA-peptide nucleic acid (PNA) single-base mismatched duplexes: real-time hybridization during affinity electrophoresis in PNA-containing gels

The stability of all single-base mismatched pairs between a peptide nucleic acid 11-mer and its complementary DNA has been quantified in terms of their melting temperature and compared with the limited amount of data published to date. The strength of the interaction was determined by an automated affinity-electrophoretic approach permitting the visualization, in real time, of hybridization between a physically immobilized peptide nucleic acid and a complementary DNA migrating in an electric field. The dissociation constants are in the range of 10(-7) M (for mismatches) to 10(-10) M (for fully complementary DNA), which are in excellent agreement with solution studies. These and other thermodynamic constants can be accurately, rapidly, and reproducibly measured in this system at concentrations approaching dissociation conditions by using fluorescently labeled DNA in conjunction with commercial DNA sequencers. The stability of single-base mismatched peptide nucleic acid-DNA duplexes depends both on the position as well as on the chemical nature of the mismatch. The stability is at a minimum when the mutation is positioned 4 bases from either terminus (a loss of 20 degreesC or more in the melting temperature) but regains substantial stability when the mismatch is at the center of the duplex. The most stable mismatched pairs are G:T and T:T whereas destabilization is maximal for A:A and G:G. These observations are of significance in the design of probes for detecting mutations by hybridization.     

The 1.8 A crystal structure of a statically disordered 17 base-pair RNA duplex: principles of RNA crystal packing and its effect on nucleic acid structure

The crystal structure of a 17 base RNA oligomer, r(CACCGGAUG GUUCGGUG), has been solved to a resolution of 1.8 A through a combination of molecular replacement, multiple isomorphous replacement phasing, and analysis of observed intensity distributions. The oligomer, which forms a stem-loop in solution, crystallized as a pseudo-infinite duplex in spacegroup P321. The asymmetric unit of the crystal contains four superimposed orientations of the duplex that are out of register, such that backbones superimpose, but base identity differs. This static disorder was initially discovered by brominating a single residue per strand in the sequence, and observing four peaks per strand in difference maps phased with a native molecular replacement solution. The presence of four superimposed duplex "motifs" related by non-crystallographic hypersymmetry was detected by computing /2 and Wilson ratios for the observed intensities. The observed ratios matched those produced from calculated intensities of a 4-fold statically disordered model. Multi-conformer simulated annealing refinement against a maximum-likelihood target incorporating experimental phase information was used to refine the 4-fold disordered model to an Rfree and R of 29.35% and 25.5%, respectively. The resulting structure reveals four distinct conformations of the duplex, with an average pairwise backbone rmsd of 2.35 A. The structural differences between the four conformations, which can be attributed to differences in packing environment, highlight the possible influence of crystal packing forces on nucleic acid X-ray structures. Analysis of inter-helical packing between symmetry-related molecules reveals an RNA "zipper" that mediates direct phosphate oxygen-2' hydroxyl interactions between close-packed phosphate-sugar backbones. This may be a general mode for RNA tertiary interaction that does not depend on metal ions or primary sequence.     

Thermodynamic parameters for an expanded nearest-neighbor model for formation of RNA duplexes with Watson-Crick base pairs

Improved thermodynamic parameters for prediction of RNA duplex formation are derived from optical melting studies of 90 oligoribonucleotide duplexes containing only Watson-Crick base pairs. To test end or base composition effects, new sets of duplexes are included that have identical nearest neighbors, but different base compositions and therefore different ends. Duplexes with terminal GC pairs are more stable than duplexes with the same nearest neighbors but terminal AU pairs. Penalizing terminal AU base pairs by 0.45 kcal/mol relative to terminal GC base pairs significantly improves predictions of DeltaG degrees37 from a nearest-neighbor model. A physical model is suggested in which the differential treatment of AU and GC ends accounts for the dependence of the total number of Watson-Crick hydrogen bonds on the base composition of a duplex. On average, the new parameters predict DeltaG degrees37, DeltaH degrees, DeltaS degrees, and TM within 3.2%, 6.0%, 6.8%, and 1.3 degreesC, respectively. These predictions are within the limit of the model, based on experimental results for duplexes predicted to have identical thermodynamic parameters.     

The folding and quaternary structure of trimeric 2-keto-3-deoxy-6-phosphogluconic aldolase at 3.5-A resolution

An X-ray crystallographic structure determination has been carried out on 2-keto-3-deoxy-6-phosphogluconic (KDPG) aldolase at 3.5-A resolution using the multiple isomorphous replacement method with three heavy atom derivatives along with anomalous dispersion contributions from two of the derivatives. Crystals grown from ammonium sulfate-phosphate buffered (pH 3.5) solutions were: cubic, a= 103.40 (4) A, space group P213. KDPG aldolase consists of trimeric heterologous assemblages utilizing crystallographic threefold symmetry. The overall profile of the oligomeric structure viewed down the threefold axis resembles that of a ship propeller while the subunits are approximate irregular oblate ellipsoids (25 X 45 X 45 A). The folding of most of the polypeptide chain was traced unambiguously. Secondary structural features consist of nine helical regions (75 residues, 35%) and a pair of two parallel chains. The subunit contains a long empty channel which is about 9 X 9 X 30 A with one of the pair of parallel chains forming part of the wall. Three mercury binding sites are located in this channel. These might correspond to the two readily accessible and one of the two buried cysteine residues of each subunit. The channel terminates with another cavity of about 8 X 10 X 25 A near the surface of the oligomeric structure. The regions of the subunits near the threefold axis are characterized by a high degree of secondary structural organization and these make close intersubunit contacts. Quarternary interactions are due mainly to side-chain interactions of helices.