Biomimetic Remineralization of Carious Lesions by Self-Assembling Peptide.

Caries is the most common disease in the world. Great efforts have been undertaken for prevention and to identify a regenerative treatment solution for dental caries. Self-assembling ß-sheet forming peptides have previously shown to form 3-dimensional fiber networks supporting tissue regeneration. In particular, the self-assembling peptide P11-4 has shown potential in the treatment and prevention of dental caries. It has previously been shown that application of monomeric P11-4 solution to early carious lesions can increase net mineral gain by forming de novo hydroxyapatite crystals. The hypothesis for the mode of action was that monomeric self-assembling peptide P11-4 diffuses into the subsurface lesion body and assembles therein into higher order fibrils, facilitating mineralization of the subsurface volume by mimicking the natural biomineralization of the tooth enamel, and it remains within the lesion body as a scaffold built-in by the newly formed hydroxyapatite. The aim of the present study was to investigate the mechanism of action of the self-assembling peptide P11-4 supporting mineralization of carious enamel. By various analytical methods, it could be shown that the self-assembling peptide P11-4 diffuses into the subsurface lesion, assembles into higher formed aggregates throughout the whole volume of the lesion, and supports nucleation of de novo hydroxyapatite nanocrystals and consequently results in increased mineral density within the subsurface carious lesion. The results showed that the application of self-assembling peptide P11-4 can facilitate the subsurface regeneration of the enamel lesion by supporting de novo mineralization in a similar mode of action as has been shown for the natural formation of dental enamel.

Efficient sequence-specific cleavage of RNA using novel europium complexes conjugated to oligonucleotides.

BACKGROUND: A general method allowing the selective destruction of targeted mRNA molecules in vivo would have broad application in biology and medicine. Metal complexes are among the best synthetic catalysts for the cleavage of RNA, and covalent attachment of suitable metal complexes to oligonucleotides allows the cleavage of complementary single-stranded RNAs in a sequence-specific manner. RESULTS: Using novel europium complexes covalently linked to an oligodeoxyribonucleotide, we have achieved the sequence-specific cleavage of a complementary synthetic RNA. The complexes are completely resistant to chemical degradation under the experimental conditions. The cleavage efficiency of the conjugate strongly depends on the nature of the linker between the oligonucleotide and the complex. Almost complete cleavage of the RNA target has been achieved within 16 h at 37 degrees C. CONCLUSIONS: The results will be important for improving the efficacy of antisense oligonucleotides and will provide a basis for the design of synthetic RNA restriction enzymes. Conjugates of the kind described here may also find application as chemical probes for structural and functional studies of RNA.

Sequence analysis of phosphorothioate oligonucleotides via matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Modification of the natural phosphodiester backbone of deoxyribooligonucleotides can impart increased biostability via nuclease resistance. Further, uniform incorporation of phosphorothioate linkages renders oligonucleotides highly resistant to reagents traditionally used in sequencing reactions. As a consequence, analytical tests crucial for establishing the identity of such oligonucleotide drugs are less informative. To circumvent this problem, chemical oxidation has been employed for converting the phosphorothioate to the uniform phosphodiester, thereby facilitating enzymatic degradation. Following oxidation, exonucleases which sequentially cleave individual bases from the 3' or 5' terminus of the oligonucleotide or base-specific cleavage chemicals were used to facilitate sequence identification of the oligonucleotide. Matrix-assisted laser desorption ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS), previously used to sequence natural phosphodiester DNA, was then used to sequence the chemically oxidized phosphorothioate. Sequential enzymatic cleavage of desulphurized phosphorothioates in combination with MALDI analysis not only provides a viable alternative to radiolabeling as used in conventional sequencing approaches (e.g. Maxam-Gilbert), but also enables rapid sequencing of phosphorothioate oligonucleotides, for routine drug analysis.

Craniomaxillofacial fibrous dysplasia: a 10-year database 1996-2006.

Fibrous dysplasia is a rare bone disease caused by an abnormal proliferation of fibrous tissue in bone. We retrospectively evaluated eight patients (female to male ratio 3:1, mean age 22.5 years, range 10-32) with a monostotic form who were treated between 1996 and 2006. Two each were affected in the lower jaw, the upper jaw, the midface, and the frontoparietal region. Most patients were referred because of a painless swelling. Biopsy specimens from two patients were examined, six patients had modelling osteotomies, two of whom had further operations because of progressive enlargement. There was no visual impairment or malignant transformation. Fibrous dysplasia should be treated as conservatively as possible, but in cases of functional disturbance that results from malignant transformation, or from the involvement of the optic foramen or the foramen magnum, an immediate operation is needed. Disfigurement can be another reason for operation. When there is a risk of malignant transformation, follow-up of patients is recommended.

Psoralen covalently linked to oligodeoxyribonucleotides: synthesis, sequence specific recognition of DNA and photo-cross-linking to pyrimidine residues of DNA.

The psoralen derivative 4,5',8-trimethylpsoralen was covalently linked to the 5'-terminus of an 18mer oligodeoxyribonucleotide in the course of solid phase synthesis using phosphoroamidite chemistry. The derivative was introduced as a phosphitylation compound in the last cycle of the oligomer synthesis. The reagent was prepared by 4'-chloromethylation of 4,5',8-trimethylpsoralen, introduction of a linker by ethanediol and phosphitylation with chloro-[(beta-cyanoethoxy)-N,N-diisopropylamino]-phosphine. After oxydation and deprotection the 5'-psoralen modified oligodeoxyribonucleotide was characterised by HPLC. Hybridisation of the psoralen-modified oligomer to a complementary single stranded 21mer followed by irradiation at 350 nm revealed a photo-cross-linked double-stranded DNA fragment analysed on denaturing polyacrylamide gels. The cross-link could be reversed upon irradiation at 254nm.

A protected biotin containing deoxycytidine building block for solid phase synthesis of biotinylated oligonucleotides.

The synthesis of a modified 2'-deoxycytidine-3'-O-phosphoramidite carrying an N-t-butylbenzoyl protected biotin on a long polar spacer arm attached to the 4-N position is described. The presence of the bulky lipophilic t-butylbenzoyl protecting group enables the direct solid phase synthesis of biotinylated oligoribonucleotides and a variety of analogues in high yield without modification of the biotin moiety. Biotinylated antisense oligonucleotides incorporating this new derivative allow convenient isolation and purification of ribonucleic acid-protein complexes. The kinetics of biotin binding to streptavidin agarose is facilitated by the long polar spacer arm.

An approach to the structure determination of nucleic acid analogues hybridized to RNA. NMR studies of a duplex between 2'-OMe RNA and an oligonucleotide containing a single amide backbone modification.

The backbone modification amide-3, in which -CH2-NH-CO-CH2- replaces -C5'H2-O5'-PO2-O3'-, is studied in the duplex d(G1-C2-G3-T4.T5-G6-C7-G8)*mr(C9-G10-C11-A12-A13-C14-G15+ ++-C16) where . indicates the backbone modification and mr indicates the 2'-OMe RNA strand. The majority of the exchangeable and non-exchangeable resonances have been assigned. The assignment procedure differs from standard methods. The methyl substituent of the 2'-OMe position of the RNA strand can be used as a tool in the interpretation. The duplex structure is a right-handed double helix. The sugar conformations of the 2'-OMe RNA strand are predominantly N-type and the 2'-OMe is positioned at the surface of the minor groove. In the complementary strand, only the sugar of residue T4 is found exclusively in N-type conformation. The incorporation of the amide modification does not effect very strongly the duplex structure. All bases are involved in Watson-Crick base pairs.

Preparation of a novel psoralen containing deoxyadenosine building block for the facile solid phase synthesis of psoralen-modified oligonucleotides for a sequence specific crosslink to a given target sequence.

4,5',8-Trimethylpsoralen was attached to the C8-position of deoxyadenosine via a sulfur atom and a five carbon atom linker. The modified deoxyadenosine was then converted to a protected phosphoramidite and used as unusual as a building block for solid phase oligodeoxyribonucleotide synthesis. The efficiency of the photoreaction of a psoralen-modified oligonucleotide to a complementary matrix strand reached more than 90% within a 1 hour irradiation time at a wavelength of 345 nm.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry: a powerful tool for the mass and sequence analysis of natural and modified oligonucleotides.

We report the analysis and characterization of natural and modified oligonucleotides by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The present technology was highly improved for this class of compounds by using a new matrix, 2,4,6-trihydroxy acetophenone, together with di- and triammonium salts of organic or inorganic acids to suppress peak broadening due to multiple ion adducts. This methodology can be used in combination with time dependent degradation of oligonucleotides by exonucleases as powerful tool to determine sequence compositions.

A novel fluorogenic substrate for ribonucleases. Synthesis and enzymatic characterization.

The synthesis and enzymatic characterization of DUPAAA, a novel fluorogenic substrate for RNases of the pancreatic type is described. It consists of the dinucleotide uridylyl-3',5'-deoxyadenosine to which a fluorophore, o-aminobenzoic acid, and a quencher, 2,4-dinitroaniline, have been attached by means of phosphodiester linkages. Due to intramolecular quenching the intact substrate displayed very little fluorescence. Cleavage of the phosphodiester bond at the 3'-side of the uridylyl residue by RNase caused a 60-fold increase in fluorescence. This allowed the continuous and highly sensitive monitoring of enzyme activity. The substrate was turned over efficiently by RNases of the pancreatic type, but no cleavage was observed with the microbial RNase T1. Compared to the dinucleotide substrate UpA, the specificity constant with RNase A, RNase PL3 and RNase U(s) increased 6-, 18-, and 29-fold, respectively. These differences in increased catalytic efficiency most likely reflect differences in the importance of subsites on the enzyme in the binding of elongated substrates. Studies on the interactions of RNase inhibitor with RNase A using DUPAAA as a reporter substrate showed that it was well suited for monitoring this very tight protein-protein interaction using pre-steady-state kinetic methods.

2'-O-alkyloligoribonucleotides, synthesis and applications in molecular biology.

Oligo(2'-O-alkylribonucleotides) have been synthesized in which alkyl is methyl, allyl and butyl. The various phosphoramidite monomers of 2'-O-alkyl uridine, cytidine, adenosine, guanosine, inosine and 2,6-diaminopurine riboside have been synthesized from a minimum of key intermediates. Extra protection of the lactam function in uracil and hypoxanthine proves useful. The high stability of the oligomers combined with incorporation of non-radioactive reporter groups such as fluorophores, biotin and 2,4-dinitrophenylamino (DNP) moities renders them as excellent antisense tools for studying RNA processing, for locating and visualising RNA and RNP complexes in cells, for examining splicing complexes by electron microscopy and for the affinity chromatography of RNA or RNP complexes.