Hydrogen bonding versus ion pairing in polyelectrolyte multilayers with homopolynucleotides

Homopolynucleotides--poly(adenylic acid), poly(A), and poly(uridylic acid), poly(U)--were assembled, layer-by-layer, into thin films with poly(ethylenimine), PEI. Various combinations and sequences of polynucleotide and PEI were used to highlight contributions of electrostatic versus hydrogen bonding as driving forces for multilayer build-up. Assembly of alternating poly(A) and poly(U) failed to yield growing films, due to excessively strong interactions between these complimentary strands. The surface morphology of multilayers depended on the deposition order and whether films had been annealed by salt. Films assembled from preformed A/U duplexes (having high persistence lengths) were very smooth. Individual adsorption steps, followed by optical waveguide light-mode spectroscopy, showed that only complementary polynucleotides adsorb by H-bonding to the surface of a growing multilayer. In contrast to behavior usually observed for polyelectrolyte multilayer build-up, the films decreased in thickness with increasing salt concentration.     

Elongation factor Tu-induced conformational changes of ribosomes detected by iodination

The effect of elongation factor (EF) Tu, bound to the ribosome with the help of poly(uridylic) acid, Phe-tRNA and guanyl-5-yl methylene diphosphonate, on the conformation and/or chemical environment of ribosomal proteins has been examined using, as a probe, protein iodination. Ribosomes complexed only with poly(uridylic acid) and Phe-tRNA have been used as a control. EF-Tu on the ribosome significantly increases the iodination of proteins S7, S10 and L3 and decreases that of S21 and L18.     

Formaldehyde as a probe of DNA structure. II. Reaction with endocyclic imino groups of DNA bases.

We describe the equilibrium and kinetic aspects of the formaldehyde reaction with the endocyclic imino groups of derivatives of thymine, uracil, and a series of halogenated uracils, as well as poly(uridylic acid) and poly(inosinic acid). The main results are: (i) the equilibrium constants for forming a hydroxymethyl adduct remain quite constant at about 2-2.5 (M-1) for all the compounds studied, independent of their pK; (ii) both forward and reverse rate constants with 5'-TMP are specific base catalyzed in the pH range of about 4-9; (iii) the response of the rate constants to temperature and to several solvent additives are measured; (iv) at neutral pH, for the series of pyrimidine compounds, a linear free energy relation is observed between the logarithm of both the forward and the reverse rate constant and the pK for deprotonation; (v) the unstructured polynucleotides, poly(U) and poly(I) react very similarly to their constituent monomers; (vi) a reaction mechanism is proposed; and (vii) some implications for polynucleotide studies are discussed. In an appendix, a method of spectral analysis is derived to obtain accurate estimates of the quite small equilibrium constants; this should be applicable to all similar two-component systems in which the final product is unobtainable, either by isolation or by saturation. Together with the results of the previous paper on the formaldehyde reaction with exocyclic amino groups (J. D. McGhee and P. H. von Hippel, preceding paper), these results form a reasonably comprehensive account of the basic chemical controls required to use formaldehyde as a quantitative probe of DNA structure.     

Endoribonuclease from bovine adrenal cortex cytosol.

An endoribonuclease which digests a variety of synthetic homoribopolymers and poly(A)-rich mRNA has been identified and purified greater than 500-fold with respect to specific activity from bovine adrenal cortex cytosol. Enzymatic digestion of synthetic poly(riboadenylic acid) was stimulated by Mn-2+ and Mg-2+ and the enzyme exhibited broad pH and salt optima. Poly(cytidylic acid) and poly(uridylic acid), but not poly(guanylic acid), served as substrates for the enzyme preparation; double-stranded RNA, DNA, and DNA-RNA hybrids were not digested by the enzyme. Digestion generated oligonucleotides with 3-hydroxyl and 5'-monophosphoester termini. On isoelectric focusing, the enzymatic activity banded at pH 8.3 plus or minus 0.2. An initial preferential cleavage of the poly(A) tract of poly(A)-rich RNA is suggested by the rapid appearance of a 4-6S digestion product highly enriched for adenylic acid; however, progressive digestion of the RNA occurs with additional incubation.     

Conformational change in poly-L-lysine on reaction with polyacids

When solutions of poly-L -lysine and poly(acrylic acid) in salt solutions at neutral pH are mixed, complexes are formed. Optical rotatory dispersion studies of these complexes away from the equivalence point reveals the formation of α-helices. The α-helix content of the polylysine never rises above about 50%. On addition of 25% by volume of dioxane, the turbidity of the complex solutions disappears, and there is some enhancement of the α-helix content. Poly(phosphoric acid) behaves essentially in the same way as poly(acrylic acid). Poly(uridylic acid) forms complexes with polylysine but with no detectable formation of α-helix. The same is true of ribosomal RNA and native and denatured DNA. A number of other polyacid polybase systems involving basic proteins have been examined, but again no α-helix formation is induced. The results are related to the case of biological nucleoproitein complexes, and the conformational specificity of these systems is discussed. The effect of scattering on measured optical rotation has been investigated and is shown to be unimportant up to quite high levels.     

Selective and effective cleavage of RNAs by vinyladenine-vinylamine copolymer.

Poly(uridylic acid) (poly[U]) and poly(adenylic acid) (poly[A]) are efficiently cleaved, via phosphodiester linkage hydrolysis, at pH 8, 50 degrees C by use of poly(vinyladenine-co-vinylamine) as catalyst. The catalytic activity of the copolymer surpasses the value of poly(vinylamine), indicating a significant role of the adenine residue in the copolymer for the effective catalysis.     

Optically detected magnetic resonance of tryptophan residues in complexes formed between a bacterial single-stranded DNA binding protein and heavy atom modified poly(uridylic acid)

Optically detected magnetic resonance (ODMR) methods were employed to study three single-stranded DNA binding (SSB) proteins encoded by plasmids of enteric bacteria: pIP71a, R64, and F. Equilibrium binding isotherms obtained by fluorescence titrations reveal that the complexes of the plasmid SSB proteins with heavy atom modified polynucleotides are readily disrupted by salt. Since all the plasmid SSB proteins show limited solubility at low ionic strength (pIP71a greater than R64 greater than F), we were able to bind only the pIP71a protein to mercurated poly(uridylic acid) [poly(5-HgU)] and brominated poly(uridylic acid) [poly(5-BrU)]. ODMR results reveal the existence of at least one heavy atom perturbed, red-shifted, stacked Trp residue in these complexes. Amplitude-modulated phosphorescence microwave double resonance spectra display selectively the phosphorescence associated with Hg-perturbed Trp residue(s) in the pIP71a SSB protein-poly(5-HgU) complex, which has a broad, red-shifted 0,0-band. Our results suggest that Trp-135 in Escherichia coli SSB, which is absent in the plasmid-encoded SSB proteins, is located in a polar environment and is not involved in stacking interactions with the nucleotide bases. Phosphorescence spectra and lifetime measurements of the pIP71a SSB protein-poly (5-BrU) complex show that at least one Trp residue in the complex does not undergo stacking. This sets a higher limit of two stacking interactions of Trp residues with nucleotide bases in complexes of pIP71a SSB with single-stranded polynucleotides.     

The determination of secondary structure in the poly(C) tract of encephalomyocarditis virus RNA with sodium bisulphite.

The degree of secondary structure in the poly(C) tract of encephalomyocarditis virus (EMCV) RNA has been investigated using sodium bisulphite, which brings about the hydrolysis of non-base-paired cytidylic acid to uridylic acid in RNA. The percentage conversion of C to U in the poly(C) region of native EMCV RNA was similar to that found in a synthetic polynucleotide lacking secondary structure [poly(C)]. When poly(I) was annealed to either native or denatured EMCV RNA, it protected the poly(C) tract from the action of bisulphite. It is concluded that the poly(C) tract of EMCV RNA in solution is very largely single-stranded.     

The interaction of 2,6,8-triaminopurine with poly(uridylic acid).

Equilibrium dialysis measurements have shown that poly(uridylic acid) binds 2,6,8-triaminopurine in a strongly cooperative manner to form a stable 2 : 1 complex at pH 7.8, 0.15 M Na+. The thermal dissociation of the complex has been characterized by ultraviolet absorbance versus temperature profiles. From the variation of Tm with the concentration of uncomplexed triaminopurine at this temperature, the partial molar enthalpy and entropy of formation of the complex have been calculated as --87 (+/- 2) kJ/mol of triaminopurine and --236 (+/- 7) J/mol of triaminopurine per K, respectively. In terms of Tm, the complex is approximately 4 degrees C more stable than the corresponding 2 : 1 complex of poly(U) with 2-aminoadenine. This stabilization is attributed to the existence of an additional hydrogen-bonding interaction, in the poly(U)-triaminopurine complex, between the 8-amino group of 2,6,8-triaminopurine and O(2) of the uracil moiety which is base paired with it in Hoogsteen fashion.     

Influence of network structure on the degradation of photo-cross-linked PLA-b-PEG-b-PLA hydrogels

Triblock copolymers of functionalized poly(lactic acid)-b-poly(ethylene glycol)-b-poly(lactic acid) (PLA-b-PEG-b-PLA) have been widely investigated as precursors for fabricating resorbable polymeric drug delivery vehicles and tissue engineering scaffolds. Previous studies show degradation and erosion behavior of PLA-b-PEG-b-PLA hydrogels to rely on macromer chemistry as well as structural characteristics of the cross-linked networks. In this research, the degradation kinetics of diacrylated PLA-b-PEG-b-PLA copolymers as soluble macromers and cross-linked gels are directly compared as a function of macromer concentration, buffer pH, and ionic strength. The pseudo first-order rate constants for degradation of soluble macromers increase with water concentration and show a minimum at intermediate pH values, but are insensitive to ionic strength. The degradation rate constants for covalently cross-linked gels display a greater sensitivity to local water concentration and a minimum at lower pH values than corresponding soluble macromers. In addition, ionic strength significantly affects the rate of gel degradation due to the direct correlation between the degree of network ionization and gel water content.     

Base composition of duck 10S RNA and its poly(A) segment

The base composition of the poly(A) segment of duck 10S RNA was determined to be 92% AMP and 8% GMP. The GMP was probably the result of contamination of poly(A) with other segments of the RNA. A comparison of the theoretical and determined base compositions of the whole 10S RNA molecule suggested that it contains, besides a coding sequence, two noncoding sequences only one of which is poly(A).Abbreviations AMP adenylic acid - GMP guanylic acid - CMP cytidylic acid - UMP uridylic acid - SDS sodium dodecyl sulfate - EDTA ethylene diamine tetraacetic acid - TCA trichloroacetic acid     

Boronic acids for affinity chromatography: spectral methods for determinations of ionization and diol-binding constants

Arylboronic acids attached to solid matrices have proved useful for the diol-specific chromatography of biomolecules and affinity purification of enzymes by exchangeable-ligand chromatography. The latter use has been limited by the intrinsic ionization constant (pKa approximately 9) of the most common commercial products. The synthesis of several arylboronic acids with ionization constants near neutrality are described, and the application of a new general, spectral-difference method for determining acid ionization constants and formation constants with fructose is developed. In particular 4-(N-methyl) carboxamido-benzeneboronic acid was found to have a pKa of 7.86 and a formation constant with D-fructose of 8600. It was stable toward acid or base hydrolysis. We suggest that 4-carboxybenzeneboronic acid might be useful for preparing matrices for enzyme affinity chromatography.     

Regeneration of enzyme activity after western blot: activation of RNase L by 2-5A on filter--importance for its detection.

A rapid and convenient new procedure for detecting RNase L activity following Western blot by renaturation of the enzyme on the nitrocellulose sheets is described. This method allows the simultaneous analysis of enzymatic activity (e.g., cleavage of poly(uridylic acid)-3'-[32P]pCp) and RNase L binding to radioactivE probes (e.g., 2-5A-3'-[32P]pCp) in the same sample. Unlike previously published methods, this procedure eliminates interference from proteases or other RNases during the analysis of RNase L activity. The detection of RNase(s) L is also affected by the presence of endogenous 2-5A, 2-5A derivatives, or other possible "inhibitors" in cell extracts; this Western blot assay allows of RNase(s) L to be detected independently of intracellular 2-5A or analogs. Differences between the procedures used so far and this Western blot technique can indeed be demonstrated. It is shown with this Western blot assay that although RNase L has been described as a protein of 185-200 kDa under nondenaturating conditions, its 80-kDa (and 40-kDa) component is able to bind 2-5A and to cleave poly(uridylic acid) in a 2-5A-dependent way, independently of other subunit(s) or cofactor(s).     

Gamma-radiolysis of poly(A) in aqueous solution: efficiency of strand break formation by primary water radicals

gamma-Radiation-induced single-strand break formation (ssb) in polyadenylic acid (poly(A] has been determined in Ar and N2O-saturated aqueous solution in the presence of different concentrations of t-butanol. Strand breaks were monitored by a low-angle laser light-scattering technique. The efficiencies for strand breakage caused by solvated electrons, hydrogen atoms and OH radicals have been found to be 0.25, 0.20 and 7.8 per cent, respectively. The efficiency of OH radicals depends only slightly on pH (pH 5.0, 7.5 and 9.0) and is independent of the presence of salt (0.01 mol dm-3 NaC1O4) and of the irradiation temperature (20 degrees C and 70 degrees C). The efficiency of OH for ssb formation obtained in this work with poly(A) is much smaller than that of poly(dA). This is explained by the different molecular conformations of the sugar moiety of poly(A) (3'-endo) and poly(dA) (2'-endo). With increasing t-butanol concentration more strand breaks are formed than expected from simple homogeneous competition kinetics of poly(A) and t-butanol for OH radicals. This result is considered to be due to nonhomogeneous reaction kinetics in the above-mentioned competition. The rate constants for the reaction of OH and H with poly(A) have been determined.     

Triple helical polynucleotidic structures: an FTIR study of the C+ .G. Ctriplet.

Triple helixes containing one homopurine poly dG or poly rG strand and two homopyrimidine poly dC or poly rC strands have been prepared and studied by FTIR spectroscopy in H2O and D2O solutions. The spectra are discussed by comparison with those of the corresponding third strands (auto associated or not) and of double stranded poly dG.poly dC and poly rG.poly rC in the same concentration range and salt conditions. The triplex formation is characterized by the study of the base-base interactions reflected by changes in the spectral domain involving the in-plane double bond vibrations of the bases. Modifications of the initial duplex conformation (A family form for poly rG.poly rC, B family form for poly dG.poly dC) when the triplex is formed have been investigated. Two spectral domains (950-800 and 1450-1350 cm-1) containing absorption bands markers of the N and S type sugar geometries have been extensively studied. The spectra of the triplexes prepared starting with a double helix containing only riboses (poly rC+.poly rG.poly rC and poly dC+.poly rG.poly rC) as well as that of poly rC+.poly dG.poly dC present exclusively markers of the North type geometry of the sugars. On the contrary in the case of the poly dC+.poly dG.poly dC triplex both N and S type sugars are shown to coexist. The FTIR spectra allow us to propose that in this case the sugars of the purine (poly dG) strand adopt the S type geometry.     

Poly(adenylic acids) containing the antibiotic tubercidin -- base pairing and hydrolysis by nuclease S1.

Poly(adenylic acids) containing the antibiotic tubercidin (7-deazaadenosine) form double strands with poly(uridylic acid) by Watson-Crick base pairing. The stability of these complexes is enhanced by an increasing adenosine content of the polymers. Whereas poly(tubercidylic acid) can bind only one poly(U) chain, the copolymers of adenylic and tubercidylic acid bind a second strand of poly(U). The melting temperatures imply a triple strand formation in a similar geometry as found for poly(A).2poly(U). The diminished hypochromicity of those complexes suggests semi-Hoogsteen base pairs, caused by the lack of N-7 in the antibiotic. As found for poly(A).poly(U), the double-stranded poly(Tu).poly(U) is not hydrolyzed by nuclease S1. In contrast to the four regular homopolyribonucleotides the single-stranded poly(Tu) is cleaved very rapidly. This may be due to a great flexibility of the polynucleotide chain. Moreover TuMP does not inhibit the enzymic digestion. Both phenomena imply a mechanism for the antibiotic action of tubercidin on the polymer level.     

Length of the polyadenylated sequence in cytoplasmic ribonucleic acid isolated from fetal calf myoblasts differentiating in vitro

Poly(adenylic acid) [poly(A)] containing cytoplasmic ribonucleic acid (RNA) in differentiating fetal calf myoblasts cultivated in vitro was examined by hybridization with radioactive poly(uridylic acid). The size distribution of the poly(A)-containing RNA after sucrose-gradient centrifugation was similar in cells before and after differentiation. There was no apparent correlation between the length of the poly(A) segment and the change in stability of messenger RNA which occurs on differentiation, nor with the polysomal or nonpolysomal localization of the RNA in the cytoplasm. The average length of the poly(A) segments in cytoplasmic RNA in the steady state was found to be dependent on the size of the RNA: the longer the RNA, the longer the average length of the poly(A) sequence. In contrast, in pulse-labeled RNa, the length of poly(A) is similar in all size classes of RNa.     

Evidence for regulation of protein synthesis at the elongation step by CDK1/cyclin B phosphorylation

Eukaryotic elongation factor 1 (eEF-1) contains the guanine nucleotide exchange factor eEF-1B that loads the G protein eEF-1A with GTP after each cycle of elongation during protein synthesis. Two features of eEF-1B have not yet been elucidated: (i) the presence of the unique valyl-tRNA synthetase; (ii) the significance of target sites for the cell cycle protein kinase CDK1/cyclin B. The roles of these two features were addressed by elongation measurements in vitro using cell-free extracts. A poly(GUA) template RNA was generated to support both poly(valine) and poly(serine) synthesis and poly(phenylalanine) synthesis was driven by a poly(uridylic acid) template. Elongation rates were in the order phenylalanine > valine > serine. Addition of CDK1/cyclin B decreased the elongation rate for valine whereas the rate for serine and phenylalanine elongation was increased. This effect was correlated with phosphorylation of the eEF-1delta and eEF-1gamma subunits of eEF-1B. Our results demonstrate specific regulation of elongation by CDK1/cyclin B phosphorylation.     

Model building of DNA/RNA triple helix containing L-deoxyadenosine.

A three-dimensional model of DNA/RNA triple helix that contains a poly(L-deoxyadenosine) (L-dA) chain is proposed based on computer-assisted model building and energy calculations. The model building was performed by a new method that systematically searches possible conformations of nucleotide units in the helical chains. Two possible orientations of sugar-phosphate chains, in which two homopyrimidine strands are parallel or antiparallel with each other, were considered in the systematic search. Several possible base-pairing models, in which there are one Watson-Crick base pair and one other base pair, were also considered. Many possible models selected by the systematic search were further refined through molecular mechanics calculation incorporating a helical boundary condition. The preferred model, which was selected on the basis of potential energy, was the one with Watson-Crick and Hoogsteen base pairs and with its two polypyrimidine chains in the antiparallel orientation. The model can explain the experimental observation that poly(L-dA) forms a stable triple helix with poly(uridylic acid) (U) but not with poly(deoxythymidylic acid) (dT).     

Single-stranded RNA molecular weight and shape determination by differential pressure capillary viscometry, sedimentation velocity, and gel electrophoresis.

Determination of the size of a population of nucleic acids can be achieved by several distinct methods. Most of these methods are cumbersome and require complicated equipment or techniques. We demonstrate here the use of a differential pressure capillary viscometer for the rapid and simple determination of RNA molecular weight. This highly sensitive viscometer allowed single viscosity determinations on dilute solutions of RNA, providing a direct measure of the intrinsic viscosity without the need to extrapolate from several concentrations. The molecular weights and conformations of the linear single-stranded RNA homopolymer poly(inosinic acid) (poly(I] and the single-stranded RNA (ssRNA) copolymer poly(cytidylic acid:uridylic acid, 12:1) (poly(C12,U], were determined. The ssRNAs were synthesized in a range of sizes (100 to 100,000 bases). They were widely polydisperse. The Mandelkern-Flory equation (1952, J. Chem. Phys. 20, 212-214), which requires both the intrinsic viscosity and sedimentation coefficient of a macromolecule, was used to calculate the molecular weights. The molecular weights determined by agarose gel electrophoresis were compared to those determined by intrinsic viscosity plus sedimentation coefficient. The correlation between the molecular weights determined by these two methods was good, at R2 greater than or equal to 0.92. The conformations of the RNAs were determined by application of the Mark-Houwink equation. The Mark-Houwink exponents for poly(I) and poly(C12,U) intrinsic viscosities were 0.90 and 0.84, respectively. When compared to other nucleic acid polymers, for which the conformations have been established by several methods, poly(I) and poly(C12,U) are rigid, extended random coils, in a low-salt buffer (15 mM).(ABSTRACT TRUNCATED AT 250 WORDS)