Chemical ribonucleases: VII. Effect of positively charged RNA-binding domains and hydrophobic fragments of the conjugates based on 1,4-diazabicyclo[2.2.2]octane and imidazol on their ribonuclease activity

The effect of the total positive charge in the RNA-binding domain of chemical ribonucleases that are conjugates of bisquaternary salts of diazabicyclo[2.2.2]octane and imidazol on the cleavage of an HIV-1 RNA fragment was studied. An increase in the positive charge from +2 to +4 was shown to result in a significant growth in the ribonuclease activity. Possible mechanisms of the interactions between structural moieties of chemical ribonucleases and RNA that enable an effective catalysis of the cleavage of phosphodiester bonds are discussed.     

Chemical ribonucleases: VII. Effect of positively charged RNA-binding domains and hydrophobic fragments of the conjugates based on 1,4-diazabicyclo[2.2.2]octane and imidazole on their ribonuclease activity

The effect of the total positive charge in the RNA-binding domain of chemical ribonucleases that are conjugates of bisquaternary salts of 1,4-diazabicylo[2.2.2]octane and imidazole on the cleavage of an HIV-1 RNA fragment was studied. An increase in the positive charge from +2 to +4 was shown to result in a significant growth in the ribonuclease activity. Possible mechanisms of the interactions between structural moieties of chemical ribonucleases and RNA that enable an effective catalysis of the cleavage of phosphodiester bonds are discussed.     

Enhancing sequence-specific cleavage of RNA within a duplex region: incorporation of 1,3-propanediol linkers into oligonucleotide conjugates of serinol-terpyridine.

The syntheses and RNA cleavage efficiencies of a new series of oligonucleotide conjugates of Cu(II)-serinol-terpyridine and 1,3-propanediol are reported. These reagents, termed ribozyme mimics, were designed such that they would yield multiple unpaired RNA residues directly opposite the site of the RNA cleavage catalyst upon ribozyme mimic-RNA duplex formation. This design effect was implemented using the 1,3-propanediol linker 3, which mimics the three-carbon spacing between the 5'- and 3'-hydroxyls of a natural nucleotide. Incorporation of one or more of these 1,3-propanediol linkers at positions directly adjacent to the serinol-terpyridine modification in the ribozyme mimic DNA strand resulted in cleavage at multiple phosphates in a complementary 31-mer RNA target sequence. The linkers effectively created artificial mismatches in the RNA-DNA duplexes, rendering the opposing RNA residues much more susceptible to cleavage via the transesterification/hydrolysis pathway. The RNA cleavage products produced by the various mimics correlated directly with the number and locations of the linkers in their DNA strands, and the most active ribozyme mimic in the series exhibited multiple turnover in the presence of excess 31-mer RNA target.     

Oligoamine-acridine conjugates for promotion of gap-selective DNA hydrolysis by Ce(IV)/EDTA complex

Oligoamines (spermidine, dipropylenetriamine and propylenediamine) were covalently attached to acridine via a hexamethylene linker. These oligoamine–acridine conjugates were efficiently bound to gap sites in substrate DNA, and promoted the DNA hydrolysis by a homogeneous Ce(IV)/ethylenediamine-N,N,N′,N′-tetraacetate (EDTA) complex at these sites. In contrast, the hydrolysis of the double-stranded portion in the DNA was little affected by these conjugates, although they were strongly bound thereto by the intercalation of their acridine moieties. As a result, the gap site was selectively and efficiently hydrolyzed by combining the Ce(IV)/EDTA complex with the oligoamine– acridine conjugate. Either the oligoamine or the acridine was only poorly active for the purpose, substantiating the essential role of cooperation between them. The promotion of gap-selective DNA hydrolysis by the conjugates has been ascribed to electrostatic stabilization of a negatively charged transition state by their positive charges.     

Phosphoinositide-protein interactions of the plasma-membrane Ca2(+)-transport ATPase as revealed by fluorescence energy transfer.

Fluorescence energy transfer has been used to study the interaction of various phospholipids with the erythrocyte (Ca2+ + Mg2+)-ATPase. The fluorescence energy transfer between tryptophan residues of the (Ca2+ + Mg2+)-ATPase purified from erythrocytes and pyrene-labelled analogues of phosphatidylcholine (Pyr-PC), phosphatidylinositol (Pyr-PI), phosphatidylinositol 4-phosphate (Pyr-PIP), phosphatidylinositol 4,5-bisphosphate (Pyr-PIP2), phosphatidylglycerol (Pyr-PG) and phosphatidic acid (Pyr-PA) was measured. A positive correlation was found between the number of negative charges on the phospholipids (PIP2 greater than PIP greater than PA greater than PI = PG greater than PC) and the potency of their pyrene-labelled analogues to act as quantum acceptors in fluorescence energy transfer from the tryptophan residues of the (Ca2+ + Mg2+)-ATPase. This is the first time that a physical interaction between PIP/PIP2 and an intrinsic membrane protein has been demonstrated. The dependence of the energy transfer on the number of negative charges of the phospholipids closely resembles the previously demonstrated charge dependence of the enzymatic activity of the (Ca2+ + Mg2+)-ATPase (Missiaen, L., Raeymaekers, L., Wuytack, F., Vrolix, M., Desmet, H. and Casteels, R. (1989) Biochem. J. 263, 687-694). It is concluded that the stimulation of the (Ca2+ + Mg2+)-ATPase activity by negatively charged phospholipids is based on a binding of these lipids to the (Ca2+ + Mg2+)-ATPase and that the negative charges are a major modulatory factor for this interaction.     

Preparation of ribonuclease S domain-swapped dimers conjugated with DNA and PNA: modulating the activity of ribonucleases

Obtaining highly specific and active ribonuclease activities is an important goal with numerous medical and biochemical applications. As a step toward more active and specific ribonucleases, we describe the preparation and the enzymatic and structural properties of RNase S monomers and dimers conjugated to DNA and PNA molecules. Poly(dT)n (2'-oligodeoxyribonucleotides, n = 8, 15) and t8 peptide nucleic acid (PNA) chains have been conjugated to the S-peptide of ribonuclease S. Monomers and dimers of the conjugated enzyme have been obtained and characterized by 1H NMR spectroscopy, showing that DNA or PNA conjugation does not alter the native structure of ribonuclease S. The oligonucleotide-conjugated RNase S monomer and dimer show significant activity against single-stranded RNA and very low/negligible hydrolysis of double-stranded poly(A).poly(U). In contrast, the t8-conjugated RNase S monomer and dimer show substantial activity against both ssRNA and dsRNA. These results highlight the importance of positive charges near but not in the active site in enhancing activity against dsRNA and reveal the promise of PNA-RNase conjugates for modulating RNase activity.     

Oligonucleotides with novel, cationic backbone substituents: aminoethylphosphonates.

Oligonucleotide (2-aminoethyl)phosphonates in which the backbone consisted of isomerically pure, alternating (2-aminoethyl)-phosphonate and phosphodiester linkages have been prepared and characterized. One of these single isomer oligonucleotides (Rp) formed a more stable duplex with DNA or RNA than its corresponding natural counterpart. Hybrid stability was more pH-dependent, but less salt-dependent than a natural duplex. The specificity of hybridization was examined by hybridization of an oligonucleotide containing one (2-aminoethyl)phosphonate to oligonucleotides possessing mismatches in the region opposite to the aminoethyl group. In contrast to oligonucleotides containing (aminomethyl)-phosphonate linkages, oligonucleotide (2-aminoethyl)phosphonates were completely stable to hydrolysis in aqueous solution. These oligonucleotides were resistant to nuclease activity but did not induce RNase H mediated cleavage of a complementary RNA strand. Incubation in a serum-containing medium resulted in minimal degradation over 24 hours. Studies of cell uptake by flow cytometry and confocal microscopy demonstrated temperature dependent uptake and intracellular localization. (2-Aminoethyl)phosphonates represent a novel approach to the introduction of positive charges into the backbone of oligonucleotides.     

Systematic solid-phase synthesis of linear pseudooligolysines containing multiple adjacent CH(2)NH amide bond surrogates: potential agents for gene delivery.

Solid-phase methodology was used to synthesize a series of fully reduced linear oligolysines (pseudooligolysines, abbreviated herein as PLs) containing up to five adjacent CH2NH peptide bond isosteres. The reduced peptide bonds were introduced by the reductive alkylation reaction between Fmoc-Lys-(Boc)-al and a free alpha-amine moiety on the pseudopeptidyl resin, using sodium cyanoborohydride in an acidified mixture of NMP/CH3OH (1 : 1 v/v). The oligomeric molecules, which can be regarded as polyethylene imine and spermine analogs, possess multiple positive charges under physiological conditions and form tight complexes with plasmid DNA. These characteristics and the increased resistance to hydrolysis by trypsin make these molecules potential candidates for future use as DNA carriers in gene delivery.     

Cooperation of metal-ion fixation and target-site activation for efficient site-selective RNA scission

Iminodiacetate–DNA conjugates and acridine–DNA conjugates were synthesized and combined for site-selective RNA hydrolysis by Lu(III). When these conjugates form a ternary complex with complementary RNA, the Lu(III)–iminodiacetate complex is placed near the target phosphodiester linkage of RNA which is in front of the acridine and is activated by noncovalent interactions. The site-selective hydrolysis by these combinations is several times as fast as that achieved by combining unmodified DNA (without iminodiacetate) and the acridine–DNA conjugate.Electronic Supplementary Material Supplementary material is available for this article at .     

Modifications of major aspects of myocardial ribonucleic acid metabolism as a response to noradrenaline. Action of the hormone on cytoplasmic processing of ribonucleic acid after reserpine treatment.

Treatment of perfused rabbit heart with reserpine causes a decrease of incorporation of labelled precursors into RNA species of subcellular fractions and polyamines. Ornithine decarboxylase, S-adenosylmethionine decarboxylase and cytoplasmic Mn2+-stimulated polyadenylate polymerase activities are not modified. Addition of noradrenaline to reserpine-treated perfused hearts enhances, compared with the control, the incorporation of precursor into RNA in all subcellular fractions other than the nuclear one, restores incorporation of labelled putrescine into polyamines, enhances ornithine decarboxylase and S-adenosylmethionine decarboxylase activities and causes a 12-fold increase in cytoplasmic Mn2+-dependent polyadenylate polymerase activity. After treatment with noradrenaline the increase in radioactivity was found solely in AMP after hydrolysis of microsomal RNA to nucleoside monophosphates.     

The screw-helical voltage gating of ion channels.

In the voltage-gated ion channels of every animal, whether they are selective for K+, Na+ or Ca2+, the voltage sensors are the S4 transmembrane segments carrying four to eight positive charges always separated by two uncharged residues. It is proposed that they move across the membrane in a screw-helical fashion in a series of three or more steps that each transfer a single electronic charge. The unit steps are stabilized by ion pairing between the mobile positive charges and fixed negative charges, of which there are invariably two located near the inner ends of segments S2 and S3 and a third near the outer end of either S2 or S3. Opening of the channel involves three such steps in each domain.     

Negative surface charge near sodium channels of nerve: divalent ions, monovalent ions, and pH.

Evidence is given for a high density of negative surface charge near the sodium channel of myelinated nerve fibres. The voltage dependence of peak sodium permeability is measured in a voltage clamp. The object is to measure voltage shifts in sodium activation as the following external variables are varied: divalent cation concentration and type, monovalent concentration, and pH. With equimolar substitution of divalent ions the order of effectiveness for giving a positive shift is: Ba equals Sr less than Mg less than Ca less than Co approximately equal to Mn less than Ni less than Zn. A tenfold increase of concentration of any of these ions gives a shift of +20 to +25 mV. At low pH, the shift with a tenfold increase in Ca-2+ is much less than at normal pH, and conversely for high pH. Soulutions with no added divalent ions give a shift of minus 18 mV relative to 2 mM Ca-2+. Removal of 7/8 of the cations from the calcium-free solution gives a further shift of minue 35 mV. All shifts are explained quantitatively by assuming that changes in an external surface potential set up by fixed charges near the sodium channel produce the shifts. The model involves a diffuse double layer of counterions at the nerve surface and some binding of H+ions and divalent ions to the fixed charges. Three types of surface groups are postulated: (1) an acid pKa equals 2.88 charge density minus 0.9 nm- minus 2; (i) an acid pKa equals 4.58, charge density minus 0.58 nm- minus 2; (3) a base pKa equals 6.28, charge density +0.33 nm- minus 2. The two acid groups also bind Ca-2+ ions with a dissociation constant K equals 28 M. Reasonable agreement can also be obtained with a lower net surface charge density and stronger binding of divalent ions and H+ ions.     

Evaluation of a new reagent for covalent attachment of polyethylene glycol to proteins.

Methoxypolyethylene glycol of molecular weight 5000 was converted to a reactive succinimidyl carbonate form (SC-PEG). The usefulness of this new polymeric reagent for the covalent attachment of polyethylene glycol to proteins was evaluated. SC-PEG was found to be sufficiently reactive to produce extensively modified proteins under mild conditions within 30 min, showing the highest reactivity around pH 9.3. The commonly used succinimidyl succinate derivative of methoxypolyethylene glycol (SS-PEG) served as a reference standard to which the new reagent was compared. The stability of the polymer-protein linkages, studied on a series of PEG-modified bovine serum albumins, provided the single most important difference between the two activated polymers. Urethane-linked PEG-proteins obtained through the use of SC-PEG showed considerably higher chemical stability than SS-PEG-derived conjugates. The measured rate constants of aminolysis (using N alpha-acetyllysine) and hydrolysis showed that SC-PEG is slightly less reactive yet more selective of the two reagents. Hydrolysis of the active groups on SC-PEG was on average twofold slower than that on SS-PEG. The differences in the rates of aminolysis were even smaller than those in hydrolysis. PEG-trypsin conjugates produced by both activated polymers showed similar properties: they had no proteolytic activity, well-preserved esterolytic activity, and enhanced activity toward p-nitroanilide substrates. Michaelis-Menten constants of the modified enzymes were determined using N alpha-benzyloxycarbonyl-L-arginine p-nitroanilide. These measurements indicated that the attachment of PEG to trypsin caused an increase in both the rate of turnover of the substrate and its affinity toward the modified enzymes. Through a series of experiments involving the appropriate polymeric and low-molecular-weight model compounds, it was demonstrated that these increases in amidolytic activity were unrelated to tyrosyl residues acylation by either one of the activated polymers.     

Vanadyl-induced Fenton-like reaction in RNA. An ESR and spin trapping study.

Vanadyl (VO2+) complexed to RNA reacts with hydrogen peroxide in a Fenton-like manner producing hydroxyl radicals (.OH). The hydroxyl radicals can be spin trapped with 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) forming the DMPO-OH spin adduct. In addition, in the presence of ethanol the formation of the hydroxyethyl radical adduct of DMPO (DMPO-ETOH) confirms the production of hydroxyl radicals by the RNA/VO2+ complex. When the reaction between the RNA/VO2+ complex and H2O2 is carried out in the presence of the spin trap 2-methyl-2-nitrosopropane (MNP), radicals produced in the reaction of .OH with RNA are trapped. Base hydrolysis of the MNP-RNA adducts (pH 12) followed by a reduction in the pH to pH 7 after hydrolysis is complete, yields an MNP adduct with a well-resolved ESR spectrum identical to the ESR spectrum obtained from analogous experiments with poly U. The ESR spectrum consists of a triplet of sextets (aN = 1.48 mT, a beta N = 0.25 mT and a beta H = 0.14 mT), indicating that the unpaired nitroxide electron interacts with the nuclei of a beta-nitrogen and beta-hydrogen. The results suggest that the .OH generated in the RNA/VO2+ reaction with H2O2 add to the C(5) carbon of uracil forming a C(6) carbon centered radical. This radical is subsequently spin trapped by MNP.     

Binding of HIV-1 TAR mRNA to a peptide nucleic acid oligomer and its conjugates with metal-ion-binding multidentate ligands

A peptide nucleic acid (PNA) oligomer and a series of PNA conjugates featuring covalently attached pendant 1,4,7,10-tetraazacyclododecane (cyclen) or bis((pyridin-2-yl)methyl)amine (DPA) moieties have been synthesized that are complementary to regions of the HIV-1 TAR messenger RNA stem-loop. Thermal denaturation studies, in conjunction win with native gel shift assays, suggest that the PNAs “invade” TAR to produce a mixture of two 1:1 PNA–TAR adducts, tentatively assigned as an “open-duplex” structure, in which the TAR stem-loop dissociates and the PNA hybridizes with its RNA complement via Watson–Crick base-pairing, and a triplex-type structure, in which the initially displaced RNA segment is bound to the PNA:RNA duplex through Hoogsteen base-pairing. Thermal denaturation experiments with the TAR sequence and single-stranded RNA and DNA oligonucleotides, both in the presence and in the absence of Zn²⁺ ions, show that the introduction of cyclen or DPA ligand arms into the PNA oligomer leads to a small but reproducible increase in the T _m values. This is attributed to hydrogen-bonding and/or electrostatic interactions between protonated forms of cyclen/DPA and the cognate RNA or DNA oligonucleotide targets. Contrary to expectations, the addition of Zn²⁺ ions did not further enhance duplex formation through binding of Zn(II)–cyclen or Zn(II)–DPA moieties to the complementary RNA or DNA. Native gel shift assays further confirmed the stability increase of the metal-free cyclen- and DPA-modified PNA hybrids as compared with a control PNA sequence. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Studies on the mechanism of inhibition of bacterial ribonuclease P by aminoglycoside derivatives

Ribonuclease P (RNase P) is a Mg²⁺-dependent endoribonuclease responsible for the 5′-maturation of transfer RNAs. It is a ribonucleoprotein complex containing an essential RNA and a varying number of protein subunits depending on the source: at least one, four and nine in Bacteria, Archaea and Eukarya, respectively. Since bacterial RNase P is required for viability and differs in structure/subunit composition from its eukaryal counterpart, it is a potential antibacterial target. To elucidate the basis for our previous finding that the hexa-arginine derivative of neomycin B is 500-fold more potent than neomycin B in inhibiting bacterial RNase P, we synthesized hexa-guanidinium and -lysyl conjugates of neomycin B and compared their inhibitory potential. Our studies indicate that side-chain length, flexibility and composition cumulatively account for the inhibitory potency of the aminoglycoside-arginine conjugates (AACs). We also demonstrate that AACs interfere with RNase P function by displacing Mg²⁺ ions. Moreover, our finding that an AAC can discriminate between a bacterial and archaeal (an experimental surrogate for eukaryal) RNase P holoenzyme lends promise to the design of aminoglycoside conjugates as selective inhibitors of bacterial RNase P, especially once the structural differences in RNase P from the three domains of life have been established.     

An examination of the electrostatic interactions between the N-terminal tail of the Brome Mosaic Virus coat protein and encapsidated RNAs

The coat protein of positive-stranded RNA viruses often contains a positively charged tail that extends toward the center of the capsid and interacts with the viral genome. Electrostatic interaction between the tail and the RNA has been postulated as a major force in virus assembly and stabilization. The goal of this work is to examine the correlation between electrostatic interaction and amount of RNA packaged in the tripartite Brome Mosaic Virus (BMV). Nanoindentation experiment using atomic force microscopy showed that the stiffness of BMV virions with different RNAs varied by a range that is 10-fold higher than that would be predicted by electrostatics. BMV mutants with decreased positive charges encapsidated lower amounts of RNA while mutants with increased positive charges packaged additional RNAs up to ～900 nt. However, the extra RNAs included truncated BMV RNAs, an additional copy of RNA4, potential cellular RNAs, or a combination of the three, indicating that change in the charge of the capsid could result in several different outcomes in RNA encapsidation. In addition, mutant with specific arginines changed to lysines in the capsid also exhibited defects in the specific encapsidation of BMV RNA4. The experimental results indicate that electrostatics is a major component in RNA encapsidation but was unable to account for all of the observed effects on RNA encapsidation. Thermodynamic modeling incorporating the electrostatics was able to predict the approximate length of the RNA to be encapsidated for the majority of mutant virions, but not for a mutant with extreme clustered positive charges. Cryo-electron microscopy of virions that encapsidated an additional copy of RNA4 revealed that, despite the increase in RNA encapsidated, the capsid structure was minimally changed. These results experimentally demonstrated the impact of electrostatics and additional restraints in the encapsidation of BMV RNAs, which could be applicable to other viruses.