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Biomedical subjects

H Lönnberg

Publications and source records attributed to H Lönnberg.

At least 19 recordsLinked to original sources
PubMed2001

The effect of secondary structure on cleavage of the phosphodiester bonds of RNA.

This review discusses the effects the secondary structure of an RNA molecule has on the inherent reactivity of its phosphodiester bonds, and on the catalytic activity of metal ion-based cleaving agents. The basic principles of the intramolecular transesterification of RNA phosphodiester bonds, particularly cleavage, are first briefly described. Studies of the structural effects on the cleavage, in the absence and in the presence of metal ion catalysts, are then reviewed, and the sources of the reactivity differences observed in different structures are discussed.

Base Sequence↗
PubMed2000

Sulfuric acid-catalyzed acetolysis of anomeric methyl 2,3,4,6-tetra-O-acetyl-D-mannopyranosides: kinetics and mechanism.

The kinetics of the acetolysis and accompanying anomerization of methyl 2,3,4,6-tetra-O-acetyl-alpha- and -beta-D-mannopyranosides at different concentrations of sulfuric acid in acetic anhydride-acetic acid mixtures were studied. The progress of the reactions was followed by gas chromatography, and the rate constants of the partial reactions were calculated on the basis of the time-dependent product distribution obtained. The mechanisms of the reactions involved are discussed. The involvement of unstable ionic intermediates is taken into account in the evaluation of the kinetic results, and simplified and extended models are used in the mathematical treatment of the results. A fourth-order Runge-Kutta algorithm is used to calculate rate constants. Acetolysis was found to be faster for mannosides than for glucosides relative to their anomerization. The beta-mannopyranoside prefers endocyclic CO-bond rupture, while in the alpha anomer the endocyclic and exocyclic cleavages are comparatively rapid.

Acetates↗
PubMed2000

Preparation, hydrolysis and intramolecular transesterification of 3'-deoxy-3'-thioinosine 3'-S-dimethylphosphorothiolate.

The hydrolytic reactions of the dimethyl ester of 3'-deoxy-3'-thioinosine 3'-S-phosphorothiolate have been followed over a wide aciditiy range by HPLC. At pH > 3, only hydroxide ion catalyzed isomerization to the 2'-dimethylphosphate takes place, whereas under more acidic conditions hydrolysis to the 2'-monomethylphosphate and 3'-S-monomethylphosphorothiolate competes. The latter is the only product accumulating in very acidic solutions (1 M hydrochloric acid). Mechanisms of the reactions are discussed.

Chromatography, High Pressure Liquid↗
PubMed1999

Mass spectrometric (HPLC/ESI--MS/MS) quantification of pyrimido[1,3-a]purin-10(3H)-one, a guanine adduct formed by reaction of malondialdehyde with DNA.

A high performance liquid chromatography/electrospray ionization tandem mass spectrometric (HPLC/ESI MS/MS) method has been developed for quantification of pyrimido[1,2-a]purin-10(3H)-one adducts from DNA. The method is based on acid-catalyzed cleavage of the adducts from DNA and the use of [2,3a,10-13C3]pyrimido[1,2-a]purin-10(3H)-one as an internal standard in the analysis. For this purpose the latter compound was prepared. Rate constants for the acid-catalyzed cleavage of pyrimido[1,2-a]purin-10(3H)-one from the corresponding 2'-deoxyribonucleoside were determined, and its hydrolytic stability and possible formation by a cross reaction between guanine and [2,3a,10]pyrimido[1,2-a]purin-10(3H)-one were studied.

Acids↗
PubMed1998

Simultaneous detection of several oligonucleotides by time-resolved fluorometry: the use of a mixture of categorized microparticles in a sandwich type mixed-phase hybridization assay.

Porous, uniformly sized (50 micrometer) glycidyl methacrylate/ethylene dimethacrylate particles (SINTEF) were used as a solid phase to construct a sandwich type hybridization assay that allowed simultaneous detection of up to six oligonucleotides from a single sample. The assay was based on categorization of the particles by two organic prompt fluorophores, viz. fluorescein and dansyl, and quantification of the oligonucleotide hybridization by time-resolved fluorometry. Accordingly, allele-specific oligodeoxyribonucleotide probes were assembled on the particles by conventional phosphoramidite strategy using a non-cleavable linker, and the category defining fluorescein and/or dansyl tagged building blocks were inserted in the 3'-terminal sequence. An oligonucleotide bearing a photoluminescent europium(III) chelate was hybridized to the complementary 3'-terminal sequence of the target oligonucleotide, and the resulting duplex was further hybridized to the particle-bound allele-specific probes via the 5'-terminal sequence of the target. After hybridization each individual particle was subjected to three different fluorescence intensity measurements. The intensity of the prompt fluorescence signals of fluorescein and dansyl defined the particle category, while the europium(III) chelate emission quantified the hybridization. The length of the complementary region between the target oligonucleotide and the particle-bound probe was optimized to achieve maximal selectivity. Furthermore, the kinetics of hybridization and the effect of the concentration of the target oligomer on the efficiency of hybridization were evaluated. By this approach the possible presence of a three base deletion (DeltaF508), point mutation (G542X) and point deletion (1078delT) related to cystic fibrosis could unequivocally be detected from a single sample.

Fluorometry↗
PubMed1998

Metal ion-dependent hydrolysis of RNA phosphodiester bonds within hairpin loops. A comparative kinetic study on chimeric ribo/2'-O-methylribo oligonucleotides.

Several chimeric ribo/2'- O -methylribo oligonucleotides were synthesized and their hydrolytic cleavage studied in the presence of Mg2+, Zn2+, Pb2+and the 1,4,9-triaza-cyclododecane chelate of Zn2+(Zn2+[12]aneN3) to evaluate the importance of RNA secondary structure as a factor determining the reactivity of phosphodiester bonds. In all the cases studied, a phosphodiester bond within a 4-7 nt loop was hydrolytically more stable than a similar bond within a linear single strand, but markedly less stable than that in a double helix. With Zn2+and Zn2+[12]aneN3, the hydrolytic stability of a phosphodiester bond within a hairpin loop gradually decreased on increasing the distance from the stem. A similar but less systematic trend was observed with Pb2+. Zn2+- and Pb2+-promoted cleavage was observed to be considerably more sensitive to the secondary structure of the chain than that induced by Zn2+[12]aneN3. This difference in behaviour may be attributed to bidentate binding of uncomplexed aquo ions to two different phosphodiester bonds. Mg2+was observed to be catalytically virtually inactive compared with the other cleaving agents studied.

Base Sequence↗
PubMed1998

A fluorescence spectroscopic study on the binding of mRNA 5'-cap-analogs to human translation initiation factor eIF4E: a critical evaluation of the sources of error.

Equilibrium constants for the association of human protein translation initiation factor eIF4E with two mRNA 5'-cap analogs, namely 7-methylguanosine 5'-triphosphate and P1-(7-methylguanosine-5') P3-(guanosine-5') triphosphate, and with guanosine 5'-monophosphate have been redetermined by the fluorescence quenching method taking the inner filter effect of the cap-analog into account. It has been shown that neglecting the latter correction may lead to either underestimation or overestimation of the association constant obtained by applying the Eadie-Hofstee plot: the reasonably firm binding of 7-methylated cap-analogs becomes underestimated, while the weak binding of non-methylated nucleotides becomes overestimated.

Binding Sites↗
PubMed1997

Determination of the nucleotide conformation in the productive enzyme-substrate complexes of RNA-depolymerases.

The aim of this work is to determine the conformation of the nucleobase adjacent to the cleavable phosphodiester bond in the productive enzyme-substrate complex of RNA-depolymerizing enzymes. To this end the kinetic parameters of hydrolysis of UpA, 2'-C-Me- and 3'-C-Me-UpA were determined for RNase A, RNase Pb2, nuclease S1 and snake venom phosphodiesterase. In these derivatives the ranges of the allowed orientation of uridine residues are restricted due to the substitution of methyl groups for the ribose hydrogen atoms. The results described demonstrate that the proposed method is of general value for the estimation of the nucleotide glycoside angles in the productive enzyme-substrate complexes.

Animals↗
PubMed1997

Sensitive bioaffinity assays with individual microparticles and time-resolved fluorometry.

Future immunoassays and nucleic acid hybridization assays will be performed in miniaturized formats that utilize microchips or microparticles. This will require a sensitive detection technology that allows spatial resolution. By using fluorescent europium chelates and time-resolved microfluorometry, one can detect 11,000 europium molecules on individual microparticles. In a miniaturized noncompetitive immunoassay of prostate-specific antigen (PSA), we quantitatively detected 5 ng/L (0.05 amol per particle) of the analyte on an individual microparticle with excellent precision over the whole measurement range (CV <10%). Using a hybridization assay, we also could detect the deltaF508 mutation for cystic fibrosis on individual microparticles. Consequently, fluorescent lanthanide chelate labels and time-resolved microfluorometry qualify as the next generation of technology in this field.

Chelating Agents↗
PubMed1996

1H-NMR studies on association of mRNA cap-analogues with tryptophan-containing peptides.

1H-NMR spectroscopy was applied to a study of the mode of interaction, in aqueous medium in the pH range 5.2-8.5 and at low and high temperatures, between several mono- and dinucleotide analogues of the mRNA cap m7GpppG and a selected tripeptide Trp-Leu-Glu, and a tetrapeptide Trp-Glu-Asp-Glu, the sequence of which corresponds to one of the suspected binding sites in the mRNA cap-binding protein (CBP). A program, GEOSHIFT, was developed, based on ring-current anisotropy theory, for analysis of experimentally observed changes in chemical shifts accompanying interactions between aromatic heterocyclic rings. This permitted quantitative evaluation of stacking interactions between the m7G cap and the tryptophan indole ring, and the relative orientations of the planes of the two rings, spaced about 3.2 angstroms apart. The structures of the stacked complexes were determined. In particular, stacking between m(2,2,7)3G (which has no free amino group for hydrogen bonding) and the indole ring is weaker and quite different from that between m7G and m(2,7)2G and indole. With the dinucleotide cap-analogues, only the m7G component stacks with the indole ring, without disruption of intramolecular stacking. In contrast to numerous earlier reports, the calculated stacking interactions are quantitatively in accord with the values derived from fluorescence measurements. It also has been shown that the positively charged (cationic) form of m7G stacks much more efficiently with the indole ring than the zwitterionic form resulting from dissociation of the guanine ring N1H (pKa approximately 7.3).

Amino Acid Sequence↗
PubMed1996

Towards genomic drug therapy with antisense oligonucleotides.

Antisense oligonucleotides represent a novel class of potential drugs for highly selective blocking of genes. The basic concept of antisense strategy is simple: an antisense molecule recognizes a complementary mRNA (or DNA) by sequence-specific base pairing, and hence prevents translation (or transcription), resulting in a selective inhibition of protein synthesis. Because of these properties, antisense oligonucleotides have great potential as therapeutic agents in several human diseases, such as viral diseases, malignancies and dominant hereditary diseases. However, technical difficulties have slowed down their use as drugs: structural modifications are needed to increase the stability and potency of synthetic oligonucleotides, specific delivery systems are required to facilitate their entry into target cells, and more information is needed to their mechanism of action. Much of the current research on antisense oligonucleotides takes place at the interface of chemistry and biomedical sciences, a multidisciplinary field where finding a common language is sometimes difficult. The aim of this review is to present an overview of the antisense strategy in terms which should be understandable for chemists, biologists and physicians.

Animals↗
PubMed1995

Selective binding of looped oligonucleotides to a single-stranded DNA and its influence on replication in vitro.

Complexing of looped and circular oligonucleotides, composed of either 2'-deoxyribo- or 2'-O-methylribonucleoside units, with completely matching or partially mismatching complementary DNA sequences was studied. Melting experiments revealed considerable differences among the stabilities of these hybrid complexes. Maximum stability and selectivity was displayed by oligomers 2 and 5. It was concluded that a linear stretch, attached to 1'-O- of 3'-deoxypsicothymidine unit (Z) increases the selectivity of hybridisation and stability of the complex as a whole. This allows one to aim the target DNA very precisely at its polyadenine part as well as at adjacent sequence simultaneously. Experiments on termination of primer extension catalysed by different DNA-polymerases--Sequenase, Klenow fragment and Tth--have demonstrated that looped oligomer 5, composed of 2'-O-methylribonucleosides appears to be a highly selective and potent inhibitor of replication in vitro. Features of looped oligonucleotides, composed of 2'-O-methylribonucleosides seem to be useful for design of highly specific antigene oligonucleotides.

Base Sequence↗
PubMed1995

Looped oligonucleotides form stable hybrid complexes with a single-stranded DNA.

Several new branched (1, 2), circular (9) and looped oligonucleotides (14-17) were synthesized. 3'-Deoxypsicothymidine was employed to create the site of branching when required. The circular and looped structures were obtained by oxidative disulfide bond formation between mercaptoalkyl tether groups. All the oligonucleotides prepared contained two T11 sequences, and the branched and looped oligomers an additional alternating CT sequence. The melting experiments revealed that the branched oligonucleotides form relatively weak hybrid (double/triple helix) complexes with the single-stranded oligodeoxyribonucleotide, showing a considerable destabilizing effect produced by the structure at the point of branching. The data obtained with looped oligonucleotides demonstrated considerable stabilization of the hybrid (double/triple helix) complexes with the complement. The data reported may be useful in attempting to design new antisense or antigene oligonucleotides capable of forming selective and stable bimolecular hybrid complexes with nucleic acids.

Base Sequence↗
PubMed1995

Fluorescence and absorption spectroscopic properties of RNA 5'-cap analogues derived from 7-methyl-, N2,7-dimethyl- and N2,N2,7-trimethyl-guanosines.

Absorption and fluorescence properties of several cap analogues, namely nucleosides, nucleoside 5'-monophosphates and P1,P3-dinucleoside triphosphates derived from 7-methylguanine, N2,7-dimethylguanine and N2,N2,7-trimethylguanine, have been studied. The data obtained include the absorption and fluorescence spectra of the cationic (N1-protonated) and zwitterionic (N1-deprotonated) species, the pKa values of the ground and excited states of the methylated base moiety and the effect of temperature and solvent composition (mixtures of water and 1,4-dioxane) on the fluorescence intensity. Furthermore, the fluorescence lifetimes of N2,N2,7-trimethylguanosine 5'-triphosphate and P1-guanosine(5')-P3-[N2,N2,7-trimethylguanosine(5')] triphosphate have been determined as a function of temperature. These data clearly indicate that dynamic quenching must be taken into account when the extent of the intramolecular stacking of the latter compound is estimated by fluorescence spectroscopy.

Guanosine↗
PubMed1993

Association of nucleosides and their 5'-monophosphates with a tryptophan containing tripeptide, Trp-Leu-Glu: the source of an overestimation by fluorescence spectroscopy.

Association of 7-methylguanosine 5'-monophosphate with a tryptophan containing tripeptide, Trp-Leu-Glu, has been studied by fluorescence titration using two different geometries of detection, viz. right angle and front surface geometry. The applicability of these two techniques to determine the stability constant of the nucleotide-peptide adduct is discussed. Evidence is presented that fluorescence titration based on right angle detection may lead to considerable overestimation of the strength of interaction.

Amino Acid Sequence↗
PubMed1991

Interaction of pyrophosphate ion with di-, tri- and tetra-amines in aqueous solution: a potentiometric and calorimetric study.

Successive macroscopic pKa values have been determined potentiometrically for the conjugate acids of spermidine (4-azaoctane-1,8-diamine), spermine (4,9-diazadodecane-1,12-diamine) and some acylic diamines at various ionic strengths in the absence and presence of tetrasodium pyrophosphate. The stability of the amine/pyrophosphate adducts have been estimated on the basis of effects of the pyrophosphate ion on the apparent acidity constants of the amines. Stoichiometry and thermodynamics of adduct formation have been elucidated from microcalorimetric measurements.

Amines↗