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Biomedical subjects

Roger Strömberg

Publications and source records attributed to Roger Strömberg.

16 recordsLinked to original sources
PubMed2006

Biological activity and biotechnological aspects of peptide nucleic acid.

During the latest decades a number of different nucleic acid analogs containing natural nucleobases on a modified backbone have been synthesized. An example of this is peptide nucleic acid (PNA), a DNA mimic with a noncyclic peptide-like backbone, which was first synthesized in 1991. Owing to its flexible and neutral backbone PNA displays very good hybridization properties also at low-ion concentrations and has subsequently attracted large interest both in biotechnology and biomedicine. Numerous modifications have been made, which could be of value for particular settings. However, the original PNA does so far perform well in many diverse applications. The high biostability makes it interesting for in vivo use, although the very limited diffusion over lipid membranes requires further modifications in order to make it suitable for treatment in eukaryotic cells. The possibility to use this nucleic acid analog for gene regulation and gene editing is discussed. Peptide nucleic acid is now also used for specific genetic detection in a number of diagnostic techniques, as well as for site-specific labeling and hybridization of functional molecules to both DNA and RNA, areas that are also discussed in this chapter.

Animals↗
PubMed2005

A solid supported reagent for internucleoside H-phosphonate linkage formation.

A fast and convenient procedure for synthesis of dinucleoside H-phosphonates is obtained through use of the novel polystyrene supported 5-carboxy-5-methyl-2-oxo-2-chloro-1,3,2-diaoxaphosphorinane reagent. Virtually quantitative H-phosphonate condensations are obtained leading to excellent isolatedyields and with only a simple filtration as the purification procedure. This provides for a convenient and high-yielding procedure that should be suited for solution-phase synthesis of oligonucleotides.

Indicators and Reagents↗
PubMed2005

Studies in oligonucleotide-based artificial nuclease systems. intramolecular copper (II) complex formation in an oligonucleotide bis-phenanthroline conjugate.

We have recently developed oligonucleotide based artificial nuclease (OBAN) systems based on 2'-O-methyloligoribonucleotides carrying a 2,9-dimethylphenanthroline x Zn(II) complex. These hybridize to an RNA molecule with bulge formation in the central region of the target and cleave the RNA target in a catalytic manner. When studying an 11-mer 2'-O-methyloligoribonucleotide carrying two 2,9-dimethylphenanthroline moieties, located 5 base pairs apart from each other, we found that this forms a cyclic structure in the presence of Cu2+ ions. This is due to intramolecular Cu(2,9-dimethylphenanthroline)2 complex formation, i.e., with the two ligands conjugated to the same oligonucleotide.

Copper↗
PubMed2005

Di- and oligonucleotide synthesis using H-phosphonate chemistry.

In this chapter, a concise account of the synthesis of oligonucleotides using the H-phosphonate methodology is given. It includes various methods for the preparation of the starting material, nucleoside 3'-H-phosphonate monoesters, their conversion into dinucleoside H-phosphonate diesters, oxidative transformations of dinucleoside H-phosphonates into the corresponding phosphate and phosphorothioate derivatives, and protocols for the synthesis of oligonucleotides and their phosphorothio analogs.

Oligonucleotides↗
PubMed2004

Acidity of secondary hydroxyls in ATP and adenosine analogues and the question of a 2',3'-hydrogen bond in ribonucleosides.

As sugar-modified nucleosides, nucleotides, and oligonucleotides are much used in pharmacology and enzymatic studies, knowledge on the relative pKa values of the secondary hydroxyls can be of great help in design and interpretations. To obtain this, we have determined the acidity constants for ATP, dATP, 2'-F-dATP, 2'-NH2-ATP, and 2'-O-methyl ATP in aqueous solution. The influence of the relative acidities seem to be mainly from inductive effects since a good correlation between pKa values and group electronegativity is found. There is no clear energetic contribution from a much suggested H-bond between the 2'-OH and 3'-oxyanion in adenosine. To clarify if this kind of H-bond would make a more-prominent energetic contribution in less-polar solvents, we also determined the acidity of secondary hydroxyls in adenosine, 2'-O-methyladenosine, and 3'-O-methyladenosine in water, methanol, and DMSO. The relative differences in pKa values were, however, quite similar in all solvents, suggesting that no major energetic contribution is made by an intramolecular H-bond in adenosine.

Adenosine↗
PubMed2004

Characterization of an RNA bulge structure by Fourier transform infrared spectroscopy.

There may be several advantages associated with an antisense oligonucleotide that induces a bulged structure into its RNA target molecule. Many structures of RNA bulges are elucidated from single-stranded RNA models. However, a two-component system is the minimum requirement for a realistic antisense model. We have used Fourier transform infrared spectroscopy to investigate a single-stranded RNA oligonucleotide with known NMR solution structure, constructed to model a five nucleotide bulge, and its two-component oligonucleotide counterpart. The infrared spectra show A-helical base-paired stems and non-base-paired loops in both systems. The nucleosides are mainly in an anti-conformation. Both N-type and S-type of sugar puckers can be inferred from the infrared region sensitive to sugar conformations. The S-type of sugar pucker is likely to be associated with the nucleotides in the bulge. The FTIR results display an overall structural similarity between the two model systems.

Base Sequence↗
PubMed2004

A method for solid-phase synthesis of oligonucleotide 5'-peptide-conjugates using acid-labile alpha-amino protections.

We describe the development of a solid-phase technique for the synthesis of 5'-peptide-oligonucleotide conjugates (POCs) with a uniform protection strategy for the nucleic acid and the peptide fragments. On the alpha-amino function, the amino acid building blocks were protected with the 2-(biphenyl-4-yl)propan-2-yloxycarbonyl (Bpoc) group. This protection is removed during the stepwise peptide elongation by the same acidic conditions used for removal of the dimethoxytrityl (DMT) group used in the oligonucleotide assembly (3% trichloroacetic acid, 2 min). The 2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl (Ddz) group was also tested. With this somewhat more stable group, a prolonged contact with the acid (at least 16 min) was required for accomplishing complete alpha-amino deprotection, which resulted in some degree of depurination of the acid-sensitive DNA chain. Base-labile acyl protections were adopted for the side-chains of histidine, lysine, and the nucleobase amino functions. These were all removed in the final deblocking step by ammonolysis. This uniform protection scheme for the peptide and the oligonucleotide enabled the total stepwise synthesis of model conjugates in the 3' --> N direction with high efficiency and purity.

Acylation↗
PubMed2004

Synthesis of new OBAN's and further studies on positioning of the catalytic group.

Two new zinc ion dependent oligonucleotide based artificial nucleases (OBAN's) have been synthesized. These consist of 2'-O-methyl modified RNA oligomers conjugated to 5-amino-2,9-dimethylphenanthroline (neocuproine)via a urea linker. OBAN 4 carries the catalytic group on a linker extending from the C-4 of an internal cytosine moiety. OBAN 5 has two neocuproine units attached, each to linkers extending from the C-5 position of uridine moieties, one placed internally and the other at the at the 5'-end of the oligonucleotide. The key step in the synthesis of the OBAN systems is conjugation of the catalytic group to the respective amino linkers of the modified oligonucleotides. This is achieved by first converting the 5-amino-2,9-dimethylphenanthroline to the phenylcarbamate. The reaction of this neocuproine phenylcarbamate with the oligonucleotide carrying one or two primary aliphatic amines in aqueous buffer (at pH 8.5) leads to nearly quantitative formation of the urea-linked conjugates. Both OBAN systems were found to cleave RNA in the bulged out regions formed from the non-complementary part of the target sequences, in the presence of Zn(II) ions. Differences in efficiency between these and previously reported systems are discussed.

Amines↗
PubMed2003

Stabilisation of RNA bulges by oligonucleotide complements containing an adenosine analogue.

Incorporation of 2'-deoxy-2'-beta-(1-naphthylmethyl)tubercidin into an oligodeoxyribonucleotide mostly has little or a slightly negative effect on the T(m) values of complexes with DNA complements. With the same naphthylmethyl-substituted nucleoside at the 3'-end of a 2'-O-methyloligoribonucleotide, however, a stabilisation of 1-2 degrees C in the corresponding complexes with both DNA and RNA is observed. When the target sequence is an RNA fragment forming a two- or three-nucleotide bulge, complexes with (naphthylmethyl)tubercidin-modified oligodeoxyribonucleotides, as well as with the corresponding 2'-O-methyloligoribonucleotides, give stabilisations of 1-2 degrees C for the three-nucleotide bulge and of almost 4 degrees C for the two-nucleotide bulge. This stabilisation is specific to RNA, since the corresponding complexes with the DNA fragments do not display this effect. Thus, the (naphthylmethyl)tubercidin-containing oligonucleotides are the first reported oligonucleotide modifications that specifically stabilise bulged RNA.

Adenosine↗
PubMed2003

Synthesis and properties of RNA analogues having amides as interuridine linkages at selected positions.

Oligoribonucleotide analogues having amide internucleoside linkages (AM1: 3'-CH(2)CONH-5' and AM2: 3'-CH(2)NHCO-5') at selected positions have been synthesized and the thermal stability of duplexes formed by these analogues with complementary RNA fragments has been evaluated by UV melting experiments. Two series of oligomers with either 2'-OH or 2'-OMe vicinal to the amide linkages were studied. Monomeric synthons (3' and 5'-C amines and carboxylic acids) were synthesized as follows: For synthesis of the AM1 analogue, the known sequence of radical allylation followed by the cleavage of the double bond was adopted. For synthesis of the AM2 analogue, novel routes via addition of nitromethane followed by conversion of the nitro function to either amino or carboxyl groups were developed. Coupling of monomeric amines and carboxylic acids followed by protecting group manipulation and phosphonylation gave dimeric 3'-hydrogenphosphonate building blocks for oligonucleotide synthesis. Monomeric model compounds having 3'-amide and 2'-OH or 2'-OMe groups were also prepared and their conformational equilibrium was determined by (1)H NMR. The AM1 and AM2 models showed equal preferences for the North conformers (at 40 degrees C, 88-89% with 2'-OH, and 92-93% with 2'-OMe). At physiological salt concentration (0.1 M NaCl) the duplexes between AM1 modified oligonucleotides and RNA had stability similar to unmodified RNA-RNA duplexes (Delta t(m)= -0.2 to +0.7 degrees C per modification). However, the AM2 modification resulted in substantial stabilization of duplexes: Delta t(m)= +1 to +2.4 degrees C per modification compared to all RNA. A 2'-O-methyl vicinal to the AM2 linkage further increased the duplex stability. Our results suggest that RNA analogues having amide internucleoside bonds are very promising candidates for medicinal applications.

Amides↗
PubMed2003

Facile determination of the protecting group location of Nim-protected histidine derivatives by 1H-15N heteronuclear correlation NMR.

The positioning of the imidazole protecting group of several histidine derivatives was determined by means of (1)H-(15)N heteronuclear multiple-bond correlation NMR experiments. The cross-peak originated from the three-bond correlation between the histidine side-chain H(beta) and the imidazole N(pi) was used for the identification of the N(pi) signal in the (15)N spectrum. Therefore, based on the fact that the signal of the substituted imidazole nitrogen appears always at lower chemical shift (delta) than the unsubstituted one, the position of the blocking group could easily be inferred. The obtained data confirmed previous findings that were accomplished with other less generally applicable spectroscopic or crystallographic techniques.

Combinatorial Chemistry Techniques↗
PubMed2003

Oligonucleotide based artificial nuclease (OBAN) systems. Bulge size dependence and positioning of catalytic group in cleavage of RNA-bulges.

Three zinc ion dependent oligonucleotide based artificial nucleases (OBANs) have been synthesized. These consist of 2'-O-methyloligoribonucleosides connected to 5-amino-2,9-dimethylphenanthroline via a urea function to a linker extending either from C-5 of deoxyuridine or from the 2'-position of uridine moieties. Both types of linkers are placed centrally in the modified sequence and in addition one OBAN carries the C-5 modified dU as an additional nucleoside unit at the 5'-end. All three OBANs are shown to cleave target oligoribonucleotides selectively. The target RNA's are varied to form differently sized bulges (0-5 nucleotides (nt)) and the different OBANs have different preferences for which sizes are preferentially cleaved. The OBAN with the centrally positioned C-5 linked zinc chelate preferentially cleaves 3 and 4-nt bulges, the OBAN with the 2'-linked chelate has a preference for slightly smaller bulges and the OBAN with a 5'-end chelate is more efficient the larger the bulge is. In addition the OBAN with the centrally positioned C-5 linked zinc chelate is shown to be a real enzyme, capable of turnover of substrate and displaying Michaelis-Menten behaviour. The main differences in efficiency of cleavage between the different OBAN-RNA substrate combinations are likely to be due to proximity factors i.e. the positioning of a catalytic group relative to cleaved phosphodiester functions. The model systems investigated partially display the importance of catalytic group positioning and should be useful in future development of more efficient OBANs.

Base Sequence↗
PubMed2003

Side reactions in the H-phosphonate approach to oligonucleotide synthesis: a kinetic investigation on bisacylphosphite formation and 5'-O-acylation.

A kinetic study on the reactions of pivaloyl chloride with a nucleoside H-phosphonate (bisacylphosphite formation), or a protected nucleoside with a free 5'-OH (5'-O-pivaloylation) has been carried out in the presence of differently substituted pyridines. The bisacylphosphite formation is a result of acylation of the mixed carboxylic-phosphonic acid anhydride to give the corresponding bispivaloylphosphite derivative. The rate of this reaction is dependent on the concentration of pyridine derivative and pivaloyl chloride. The 5'-O-pivaloylation reaction is also dependent on the concentration of pyridine derivative and pivaloyl chloride. The rate of both reactions is dependent on the basicity of the pyridine derivative, displaying a higher rate with more basic pyridines. The dependence of log k vs. pKa of pyridine is linear but the slopes are quite different for the two reactions (0.84 for bisacylation and 0.21 for 5'-O-pivaloylation). For both reactions considerably lower rates in the presence of sterically hindered pyridines suggest the existence of nucleophilic catalysis on pivaloyl chloride in reactions with non-hindered pyridines.

Acetonitriles↗
PubMedDate not supplied

Investigation of potential RNA bulge stabilizing elements.

As a part of our interest in recognition and cleavage of RNA we carried out thermal melting studies with the aim of screening a number of simple oligonucleotide modifications for their potential as modifying elements for RNA bulge stabilizing oligonucleotides. A specific model system from our studies on oligonucleotide-based artificial nuclease (OBAN) systems was chosen and the bulge size was varied from one to five nucleotides. Introduction of single 2'-modified nucleoside moieties (2'-O-methyl, 2'-deoxy and 2'-deoxy-2'-amino) with different conformational preferences adjacent to the bulge revealed that a higher preference for the north conformers gave more stable bulges across the whole range of bulge sizes. Changing a bulge closing a G-U wobble base pair to a G-C pair resulted in the interesting observation that, although the fully complementary complex and small bulges were highly stabilized, there was little difference in the stability of the larger bulges. The wobble base pair even gave a slight stabilization of the 5 nt bulge system. Introduction of a uridine C-5 linker with a single ammonium group was clearly bulge stabilizing (DeltaT(m) + 4.6 to + 5.4 degrees C for the three most stabilized bulges), although with limited selectivity for different bulge sizes since the fully complementary duplex was also stabilized. Introduction of a naphthoyl group on a 2'-aminolinker mostly gave a destabilizing effect, while introduction of a 5-aminoneocuproine moiety on the same linker resulted in stabilization of all bulges, in particular those with two or four unpaired nucleotides (DeltaT(m) + 3.6 and + 2.9 degrees C respectively). The aromatic groups destabilize the fully complementary duplex, resulting in higher selectivity towards stabilization of bulges. A combination of the studied partial element should be suitable for future designs of modified oligonucleotides that, apart from standard base pairing, can also provide additional non-Watson-Crick recognition of RNA.

Base Pairing↗
PubMedDate not supplied

Synthesis of 2'-deuterio and 3'-deuterio cytidine 5'-diphosphate.

2'-2H- and 3'-2H-CDP were synthesized from 5'-MMT-3'-O-TBDMS and 2',5'-O-diTBDMS cytidine derivatives, respectively, by oxidation followed by acidic removal of 5'-protection, reduction with [NaBD(OAc)3] and finally displacement of a tosyl group by pyrophosphate.

Cytidine Diphosphate↗