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At least 955 records · Page 53Linked to original sources
PubMed2000

Incorporation of a 4-hydroxy-N-acetylprolinol nucleotide analogue improves the 3'-exonuclease stability of 2'-5'-oligoadenylate-antisense conjugates.

Incorporation of a 4-hydroxy-N-acetylprolinol nucleotide analogue at the 3'-terminus of DNA or 2-5A-DNA sequences resulted in a significantly enhanced 3'-exonuclease resistance while the affinity for complementary RNA was only slightly decreased. Furthermore, the binding to and activation of human RNase L by thus modified 2-5A-DNA conjugates was not altered as compared to the parent unmodified 2-5A-DNAs.

Adenine Nucleotides↗
PubMed1999

Synthesis and hybridization property of an oligonucleotide containing a 3-thioformacetal linked pentathymidylate.

A pentathymidylate fully substituted with 3'-thioformacetal intemucleotidic linkages was synthesized and subsequently incorporated into an oligonucleotide (ON) 15mer. Tm analysis was performed on the resulting ON hybridized with its complementary RNA. This duplex demonstrated slightly improved binding affinity relative to the control phosphate diester ON/RNA hybrid.

Acetals↗
PubMed1999

Synthesis of a N-acylsulfamide linked dinucleoside and its incorporation into an oligonucleotide.

A N-acylsulfamide linked thymidine dinucleoside was synthesized and incorporated into an oligonucleotide (ON). The interest is in a linkage analog that has a higher pKa relative to a phosphodiester and when incorporated into ONs is capable of helix formation with complementary RNA. The hybridization property of the resultant ON with RNA was shown to result in significant destabilization.

Nucleic Acid Conformation↗
PubMed2000

Stereo-enriched phosphorothioate oligodeoxynucleotides: synthesis, biophysical and biological properties.

Stereo-enriched [Rp] and [Sp]-phosphorothioate oligodeoxynucleotides are synthesized using oxazaphospholidine derivatized monomers. Three different designs of phosphorothioate oligodeoxynucleotides (PS-oligos), (i) stereo-enriched all-[Rp] or all-[Sp] PS-linkages, (ii) stereo-random mixture of PS-linkages, and (iii) segments containing certain number of stereo-enriched [Rp] and [Sp] PS-linkages ([Sp-Rp-Sp] or [Rp-Sp-Rp]), have been studied. Thermal melting studies of these PS-oligos with RNA complementary strands showed that the binding affinities are in the order [Rp] > [Sp-Rp-Sp]-[Rp-Sp-Rp] > stereo-random > [Sp]. Circular dichroism (CD) studies suggest that the stereochemistry of the PS-oligo does not affect the global conformation of the duplex. The in vitro nuclease stability of these PS-oligos is in the order [Sp] > [Sp-Rp-Sp] > stereo-random > [Rp]. The RNase H activation is in the order [Rp] > stereo-random > [Rp-Sp-Rp] > [Sp] > [Sp-Rp-Sp]. Studies in a cancer cell line of PS-oligos targeted to MDM2 mRNA showed that all oligos had similar biological activity under the experimental conditions employed. Protein- and enzyme-binding studies showed insignificant stereo-dependent binding to proteins. The [Sp] and [Sp-Rp-Sp] chimeric and stereo-random PS-oligos that contained a CpG motif showed higher cell proliferation than [Rp] PS-oligo of the same sequence.

Animals↗
PubMedDate not supplied

Combined in situ hybridization and immunocytochemistry in the assay of pharmacological effects on tyrosine hydroxylase mRNA concentration.

An assay for tyrosine hydroxylase (TH) mRNA by in situ hybridization in combination with immunocytochemistry (ICC) for TH on the same section is described. The in situ hybridization protocol was optimized for [35S]cRNA (complementary RNA, i.e. anti-sense strand) probe concentration and time of hybridization. The specificity of hybridization was measured by several critera. The advantage of measuring grain density versus grains per cell is discussed for quantitation of in situ autoradiography. Finally, the reserpine-induced increase in adrenal TH mRNA was used to validate quantitative aspects of the in situ hybridization technique by comparison with blot hybridization. In contrast to the adrenal, reserpine did not increase TH mRNA in substantia nigra (s. nigra) neurons as measured by either technique.

Animals↗
PubMed1998

Alternative splicing of insulin-like growth factor I mRNA is developmentally regulated in the rat and mouse with preferential exon 2 usage in the mouse.

The synthesis of an IGF-I complementary RNA (cRNA) standard containing the primer sequences for each of the four IGF-I mRNA alternative transcript forms (class 1Ea, 1Eb, 2Ea and 2Eb) and a polyA+ tail allowed the determination of the absolute abundance of each form in growing rats and mice by quantitative RT-PCR. In rat liver, class 1Ea mRNA was the most abundant form representing 90% of the total IGF-I mRNA. Though the relative proportion was maintained, the absolute abundance was maximal at 4 weeks of age and had declined by 6 weeks. In contrast, class 2Ea mRNA was the predominant transcript in mouse liver (70% of total IGF-I mRNA) and the 2Eb mRNA form was also 10-fold higher than that detected in rat liver. These results suggest that rats and mice differ both in their transcription initiation at two leader exons and in their alternative splicing activities for exon 5.

Alternative Splicing↗
PubMed2002

Distribution of serine proteinase inhibitor, clade B, member 6 (Serpinb6) in the adult mouse brain.

In the brain, Serpinb6 was identified as an endogenous inhibitor of neuropsin, a member of the S1 (clan SA) family of serine proteases [J. Biol. Chem. 276 (2001) 14562]. In the present study, we investigated the localization of Serpinb6 in the adult mouse brain using in situ hybridization histochemistry and immunohistochemistry. Region-specific patterns of expression were observed and two characteristics were recognized. First, the forebrain limbic area that expressed neuropsin mRNA contained Serpinb6 mRNA at moderate levels but not the lateral septum. On the other hand, Serpinb6 mRNA was also expressed moderately in the substantia nigra-ventral tegmental area system, whose fibers projected to the lateral septum. Additionally, Serpinb6 protein was detected in the lateral septum. Together, it was suggested that the expression of neuropsin in the brain is regulated entirely by Serpinb6. Second, Serpinb6 mRNA and the protein were strongly expressed in most somatic and visceral motoneurons among cranial nerve nuclei. This suggests that another serine protease is regulated by Serpinb6 in motoneurons and/or fibers.

Animals↗
PubMedDate not supplied

4-(2-aminooxyethoxy)-2-(ethylureido)quinoline-oligonucleotide conjugates: synthesis, binding interactions, and derivatization with peptides.

Oligo-2'-O-methylribonucleotides conjugated with 4-(2-aminooxyethoxy)-2-(ethylureido)quinoline (AOQ) and 4-ethoxy-2-(ethylureido)quinoline (EOQ) were prepared by reaction of the AOQ or EOQ phosphoramidite with the protected oligonucleotide on a controlled pore glass support. Deprotection with ethylenediamine enabled successful isolation and purification of the highly reactive AOQ-conjugated oligomer. Polyacrylamide gel electrophoresis mobility shift experiments showed that the dissociation constants of complexes formed between an AOQ- or EOQ-conjugated 8-mer and complementary RNA or 2'-O-methyl-RNA targets (9- and 10-mers) were in the low nM concentration range at 37 degrees C, whereas no binding was observed for the corresponding nonconjugated oligomer, even at a concentration of 500 nM. Fluorescence studies suggested that this enhanced affinity is most likely due to the ability of the quinoline ring of the AOQ or EOQ group to stack on the last base pair formed between the oligomer and target, thus stabilizing the duplex. The binding affinity of a 2'-O-methyl RNA 15-mer, which contained an alternating methylphosphonate/phosphodiester backbone, for a 59-nucleotide stem-loop HIV TAR RNA target, increased 2.3 times as a consequence of conjugation with EOQ. The aminooxy group of AOQ-conjugated oligomers is a highly reactive nucleophile, which reacts readily with aldehydes and ketones to form stable oxime derivatives. This feature was used to couple an AOQ-oligomer with leupeptin, a tripeptide that contains a C-terminus aldehyde group. A simple method was developed to introduce a ketone functionality into peptides that contain a cysteine residue by reacting the peptide with bromoacetone. The resulting keto-peptide was then coupled to the AOQ-oligomer. This procedure was used to prepare oligonucleotide conjugates of a tetrapeptide, RGDC, and a derivative of HIV tat peptide having a C-terminus cysteine. The combination of the unique reactivity of the aminooxy group and enhanced binding affinity conferred by its quinoline ring suggests that AOQ may serve as a useful platform for the preparation of novel oligonucleotide conjugates.

Base Sequence↗
PubMedDate not supplied

Modulation of the pharmacokinetic properties of PNA: preparation of galactosyl, mannosyl, fucosyl, N-acetylgalactosaminyl, and N-acetylglucosaminyl derivatives of aminoethylglycine peptide nucleic acid monomers and their incorporation into PNA oligomers.

A series of N-(2-aminoethyl)-alpha-amino acid thymine peptide nucleic acid (PNA) monomers bearing glycosylated side chains in the alpha-amino acid position have been synthesized. These include PNA monomers where glycine has been replaced by serine and threonine (O-glycosylated), derivatives of lysine and nor-alanine (C-glycosylated), and amide derivatives of aspartic acid (N-glycosylated). The Boc and Fmoc derivatives of these monomers were used for incorporation in PNA oligomers. Twelve PNA decamers containing the glycosylated units in one, two, or three positions were prepared, and the thermal stability (T(m)) of their complexes with a complementary RNA was determined. Incorporation of the glycosyl monomers reduced the duplex stability by 0-6 degrees C per substitution. A cysteine was attached to the amino terminus of eight of the PNA decamers (Cys-CTCATACTCT-NH(2)) for easy conjugation to a [(18)F]radiolabeled N-(4-fluorobenzyl)-2-bromoacetamide. The in vivo biodistribution of these PNA oligomers was determined in rat 2 h after intravenous administration. Most of the radioactivity was recovered in the kidneys and in the urine. However, N-acetylgalactosamine (and to a lesser extent galactose and mannose)-modified PNAs were effectively targeting the liver (40-fold over unmodified PNA). Thus, the pharmacodistribution in rats of PNA oligomers can be profoundly changed by glycosylation. These results could be of great significance for PNA drug development, as they should allow modulation and fine-tuning of the pharmacokinetic profile of a drug lead.

Animals↗
PubMedDate not supplied

Design of phosphoramidite monomer for optimal incorporation of functional intercalator to main chain of oligonucleotide.

Chirally pure phosphoramidite monomers bearing 9-amino-6-chloro-2-methoxyacridine were synthesized from D- or L-threoninol and omega-aminocarboxylic acid, and incorporated into oligonucleotides. These acridine-DNA conjugates formed stable duplexes with complementary RNA because of intercalation of the acridine to DNA/RNA heteroduplexes. The stability of duplexes was not very dependent on either the chirality of the central carbon bearing the acridine or the length of the side chain. However, the ability for site-selective activation of the phosphodiester linkage in front of the acridine, which induced Lu(III)-promoted RNA scission, was strongly dependent on these two factors. The largest activation was achieved when the monomer unit was prepared from L-threoninol and 4-aminobutyric acid and the acridine was bound to the amino group. By attaching the more acidic 9-amino-2-methoxy-6-nitroacridine to this optimized scaffold, a quite effective acridine-DNA conjugate for site-selective RNA scission was obtained.

Aminoacridines↗
PubMed1992

Effects of the level of mRNA expression on biophysical properties, sensitivity to neurotoxins, and regulation of the brain delayed-rectifier K+ channels Kv1.2.

Injection of 0.2 ng of cRNA encoding the brain Kv1.2 channel into Xenopus oocytes leads to the expression of a very slowly inactivating K+ current. Inactivation is absent in oocytes injected with 20 ng of cRNA although activation remains unchanged. Low cRNA concentrations generate a channel which is sensitive to dendrotoxin I (IC50 = 2 nM at 0.2 ng of cRNA/oocyte) and to less potent analogs of this toxin from Dendroaspis polylepis venom. A good correlation is found between blockade of the K+ current and binding of the different toxins to rat brain membranes. High cRNA concentrations generate another form of the K+ channel which is largely insensitive to dendrotoxin I (IC50 = 200 nM at 20 ng of cRNA per oocyte). At low cRNA concentrations, the expressed Kv1.2 channel is also blocked by other polypeptide toxins such as MCD peptide (IC50 = 20 nM), charybdotoxin (IC50 = 50 nM), and beta-bungarotoxin (IC50 = 50 nM), which bind to distinct and allosterically related sites on the channel protein. The pharmacologically distinct type of K+ channel expressed at high cRNA concentrations (20 ng of cRNA/oocyte) is nearly totally resistant to 100 nM MCD peptide and hardly altered by charybdotoxin and beta-bungarotoxin at concentrations as high as 1 microM. Both at low and at high cRNA concentrations, the expressed Kv1.2 channel is blocked by an increase in intracellular Ca2+ from the inositol trisphosphate sensitive pools and by the phorbol ester PMA that activates protein kinase C.

Allosteric Site↗
PubMed1996

HIV-1 genome dimerization: formation kinetics and thermal stability of dimeric HIV-1Lai RNAs are not improved by the 1-232 and 296-790 regions flanking the kissing-loop domain.

The genome of all retroviruses consists of two identical RNAs noncovalently linked near their 5' end. Dimerization of genomic RNA is thought to modulate several steps in the retroviral life cycle, such as recombination, translation, and encapsidation. The kissing-loop model of HIV-1 genome dimerization posits that the 233-285 region of the HIV-1 genome, by forming a hairpin and initiating dimerization through a loop-loop interaction, is at least the core dimerization domain of HIV-1 RNA. This region is called the kissing-loop domain. In addition, it can be argued that sequences within the 296-401 region [Paillart et al. (1994) J. Biol. Chem. 269, 27486-27493] or 5' of the primer binding site [Laughrea & Jetté (1996) Biochemistry 35, 1589-1598] might play some role in the dimerization process. Accordingly, we have studied the effect of regions 1-232 and 296-790 on the dimerization kinetics and thermal stability of HIV-1Lai RNAs containing the kissing-loop domain (HIV-1Lai is a typical representative of North American and European HIV-1 viruses). Experiments conducted at high and low ionic strength indicate that these regions have no strongly positive effect on the dimerization process. Our experiments also indicate that the kissing-loop domain of HIV-1Lai has an apparent dissociation temperature 13 degrees C higher than that of the HIV-1Mal kissing-loop domain (HIV-1Mal is a Central African virus whose kissing-loop domain has a "weak" GUGCAC autocomplementary sequence). Because the 296-401 region of HIV-1Mal RNA stabilizes dimeric RNAs by < or = 12 degrees C (Paillart et al., 1994), we infer that the contributions of sequences downstream of U295 are (at best) concealed in HIV-1Lai and in most American and European HIV-1 viruses, i.e., in viruses whose kissing-loop domain is characterized by a "strong" GCGCGC autocomplementary sequence.

Base Sequence↗
PubMed2006

Synthesis and biochemical evaluation of phosphonoformate oligodeoxyribonucleotides.

Phosphonoformate oligodeoxyribonucleotides were prepared via a solid phase synthesis strategy. The first step in the preparation of appropriate synthons was condensation of bis(N,N-diisopropylamino)phosphine and diphenylmethylsilylethyl chloroformate in the presence of sodium metal to yield formic acid, [bis(N,N-diisopropylamino)phosphino]-beta-(diphenylmethylsilylethyl) ester. The product of this reaction was then condensed with appropriately protected 2'-deoxynucleosides using 4,5-dicyanoimidazole to yield the 3'-O-phosphinoamidite reactive monomers. The exocyclic amines of cytosine, adenine, and guanine were protected with 9-fluorenylmethyloxycarbonyl, and oligodeoxyribonucleotides were synthesized on controlled pore glass using the hydroquinone-O,O'-diacetic acid linker. Synthons were sequentially added to this support using tetrazole as an activator, oxidized to phosphonoformate, and the transient 5'-protecting group was removed with acid. Following total synthesis of an oligomer, protecting groups were removed with TEMED.HF and products purified by HPLC. These analogues were resistant to nucleases, formed duplexes with complementary RNA (A-form), and, as chimeric oligomers containing phosphate at selected sites, stimulated RNase H1 activity.

Biophysical Phenomena↗
PubMed2000

Synthesis of aminoglycoside-modified oligonucleotides.

[structure: see text] To study the structural requirements of aminoglycoside binding to nucleic acids, compound 1-an analogue of the naturally occurring nucleoside J-was synthesized. When incorporated into oligodeoxynucleotides, 1 leads to thermal stabilization of the resulting duplexes. The increase in pairing affinity is stronger with complementary RNA than with DNA.

Aminoglycosides↗
PubMed1997

Rat hypothalamus neuron-like cells in primary culture accumulate and translate mRNA coding for the amphibian P-domain peptide xP1.

1. Neurons seem to possess the intrinsic capability to incorporate and translate exogenous RNA. For further evaluation of this phenomenon, we wanted to study the uptake and processing capacity of rat hypothalamic neurons for species-unspecific heterologous cRNA under in vitro conditions. 2. cRNA coding for the amphibian p-domain peptide xP1 was prepared by in vitro transcription and added to the culture medium of rat hypothalamic cells, derived from E18 fetuses. 3. After 2 hr, a fraction of the hypothalamic neuron-like cells had accumulated the radiolabeled transcripts, as could be demonstrated by autoradiographic assessment. Specific immunostaining for xP1 could be demonstrated 18 hr after incubation with the cRNA. 4. Our findings indicate that hypothalamic neuron-like cells are capable of accumulating and translating nonmammalian transcripts. Since it was only a portion of hypothalamic cells that showed this effect, specific recognition sites for RNA may be presented by certain neurons, further supporting the assumption that binding, uptake, and translation of cRNA transcripts represent a general neural property which is malleable to functional status.

Animals↗
PubMed1997

Simultaneous in situ hybridization and TUNEL to identify cells undergoing apoptosis.

Apoptotic cells in tissue sections can be localized by in situ labelling of partly degraded DNA. In a heterogeneous population of cells, however, the specific identity of cell types undergoing apoptosis often cannot be reliably achieved at the light microscope level because of the marked alterations in cellular morphology that characterize apoptosis. In order to clearly specify cell types undergoing apoptosis, in situ end labelling has been coupled to immunohistochemistry. This method is limited by the availability of antibodies that bind to cell-specific protein markers in tissue sections. In contrast, we describe a method that combines in situ end labellin with in situ hybridization, a technique that specifies cell types based on mRNA expression. Taking advantage of the specific expression of surfactant protein C mRNA in type II alveolar epithelial cells, we demonstrate that this technique has the ability to localize alveolar type II cells undergoing apoptosis in vivo after the intratracheal instillation of an antibody that activates the cell surface Fas protein. The wide availability of cell-specific gene markers suggests that this method can be adapted to define cell types that undergo apoptosis during various physiological and pathological states in vivo.

Animals↗