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Biomedical subjects

B S Sproat

Publications and source records attributed to B S Sproat.

At least 37 records · Page 2Linked to original sources
PubMed1992

Antisense probes targeted to an internal domain in U2 snRNP specifically inhibit the second step of pre-mRNA splicing.

Functional domains within the mammalian U2 snRNP particle that are required for pre-mRNA splicing have been analysed using antisense oligonucleotides. A comparison of the melting temperatures of duplexes formed between RNA and different types of antisense oligonucleotides has demonstrated that the most stable hybrids are formed with probes made of 2'-O-allyl RNA incorporating the modified base 2-aminoadenine. We have therefore used these 2'-O-allyl probes to target sequences within the central domain of U2 snRNA. Overlapping biotinylated 2'-O-allyloligoribonucleotides complementary to the stem loop Ila region of U2 snRNA (nucleotides 54-72) specifically affinity selected U2 snRNA from HeLa nuclear extracts. These probes inhibited mRNA production in an in vitro splicing assay and caused a concomitant accumulation of splicing intermediates. Little or no inhibition of spliceosome assembly and 5' splice site cleavage was observed for all pre-mRNAs tested, indicating that the oligonucleotides were specifically inhibiting exon ligation. This effect was most striking with a 2'-O-allyloligoribonucleotide complementary to U2 snRNA nucleotides 57-68. These results provide evidence for a functional requirement for U2 snRNP in the splicing mechanism occurring after spliceosome assembly.

Base Sequence↗
PubMed1992

Nuclease resistant ribozymes with high catalytic activity.

Hammerhead ribozymes are efficient RNA enzymes characterized by a typical hammerhead secondary structure and a number of conserved bases. Little is known about the role of the ribose-phosphate backbone, although it is obviously important since a DNA molecule with the same base sequence is not a catalyst. Here we describe the synthesis of artificial ribozymes where modified (2'-O-allyl- and 2'-O-methyl-) ribonucleotides substitute for the corresponding ribonucleotides. A systematic analysis of partially substituted polymers identified a minimum set of six non-contiguous positions where insertion of modified ribonucleotides strongly affects catalytic activity. Surprisingly, ribozymes completely substituted except for these six ribonucleotides are still very active. These molecules efficiently cleave in trans target RNAs in a sequence-specific way, but, unlike RNA ribozymes, are very resistant to nuclease degradation and are very stable in serum. These properties make such synthetic polymers potentially useful for in vivo gene expression studies and therapeutic applications.

Base Sequence↗
PubMed1991

Antisense probes containing 2-aminoadenosine allow efficient depletion of U5 snRNP from HeLa splicing extracts.

RNA duplexes containing the modified base 2-amino-adenine in place of adenine are stabilized through the formation of three hydrogen bonds in 2-amino A.U base pairs. Antisense 2'-O-alkyloligoribonucleotide probes incorporating 2-aminoadenosine are thus able to efficiently affinity select RNP particles which are otherwise inaccessible. This has allowed the efficient and specific depletion of U5 snRNP from HeLa cell nuclear splicing extracts. U5 snRNP is shown to be essential for spliceosome assembly and for both steps of pre-mRNA splicing. The absence of U5 snRNP prevents the stable association of U4/U6 but not U1 and U2 snRNPs with pre-mRNA.

Adenosine↗
PubMed1991

Site-directed ribose methylation identifies 2'-OH groups in polyadenylation substrates critical for AAUAAA recognition and poly(A) addition.

The importance of sugar contacts for the sequence-specific recognition that occurs during polyadenylation of mRNAs was investigated with chemically synthesized substrates containing 2'-O-CH3 groups at selected riboses. An RNA (5'-CUGCAAUAAACAAGU-UAA-3') with 2'-O-CH3 ribose at each nucleotide except for the AAUAAA sequence and 3'-terminal adenosine was efficiently polyadenylated in vitro. Methylation of single riboses within AAUAAA inhibited both poly(A) addition and binding of the specificity factor, but the magnitude of inhibition varied greatly at different nucleotides. Nucleotides that showed sensitivity to base substitutions did not necessarily show sensitivity to ribose methylation, and vice versa. The data indicate that the specificity factor interacts with AAUAAA through RNA-protein contacts involving essential recognition of both sugars and bases at different nucleotide positions.

Base Composition↗
PubMed1991

New synthetic routes to synthons suitable for 2'-O-allyloligoribonucleotide assembly.

New synthetic routes have been devised for the high yield preparation of protected 2'-O-allylribonucleoside-3'-O-phosphoramidites, exemplified by the ribonucleosides guanosine and 2,6-diaminopurine riboside (2-aminoadenosine). Key features are the use of versatile intermediates and an easy allylation step. The development of a novel synthon based on 2'-O-allyl-2,6-diaminopurine riboside enables short 2'-O-allyl-oligoribonucleotide probes to be synthesized with adenine replaced by 2-aminoadenine. Thus very stable hybrids with complementary RNA target sequences can be formed due to the formation of the three hydrogen bond 2-amino A.U base pairs.

Allyl Compounds↗
PubMed1991

In vivo detection of snRNP-rich organelles in the nuclei of mammalian cells.

The in vivo distribution of snRNPs has been analysed by microinjecting fluorochrome-labelled antisense probes into the nuclei of live HeLa and 3T3 cells. Probes for U2 and U5 snRNAs specifically label the same discrete nuclear foci while a probe for U1 snRNA shows widespread nucleoplasmic labelling, excluding nucleoli, in addition to labelling foci. A probe for U3 snRNA specifically labels nucleoli. These in vivo data confirm that mammalian cells have nuclear foci which contain spliceosomal snRNPs. Co-localization studies, both in vivo and in situ, demonstrate that the spliceosomal snRNAs are present in the same nuclear foci. These foci are also stained by antibodies which recognize snRNP proteins, m3G-cap structures and the splicing factor U2AF but are not stained by anti-SC-35 or anti-La antibodies. U1 snRNP and the splicing factor U2AF closely co-localize in the nucleus, both before and after actinomycin D treatment, suggesting that they may both be part of the same complex in vivo.

Animals↗
PubMed1991

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.

Antisense Elements (Genetics)↗
PubMed1990

Sequence-specific affinity selection of mammalian splicing complexes.

Antisense oligonucleotides made of 2'-OMe RNA are shown to bind specifically and efficiently to targeted sites on pre-mRNA substrates, allowing affinity selection of splicing complexes using streptavidin/biotin chromatography. The position of probe binding to the pre-mRNA influences which type of splicing complex can be selected. The accessibility of pre-mRNA sequences to antisense probes changes during the course of the splicing reaction. U1, U2, U4, U5 and U6 snRNAs are all detected in affinity-selected mammalian splicing complexes. However, antisense oligonucleotides targeted to snRNAs can block the binding of specific snRNPs to pre-mRNA. Quantitative affinity selection analyses show that only a small fraction of snRNPs in a HeLa nuclear splicing extract participate in spliceosome formation.

Base Sequence↗
PubMed1990

Targeted snRNP depletion reveals an additional role for mammalian U1 snRNP in spliceosome assembly.

HeLa cell nuclear splicing extracts have been prepared that are specifically and efficiently depleted of U1, U2, or U4/U6 snRNPs by antisense affinity chromatography using biotinylated 2'-OMe RNA oligonucleotides. Removal of each snRNP particle prevents pre-mRNA splicing but arrests spliceosome formation at different stages of assembly. Mixing extracts depleted for different snRNP particles restores formation of functional splicing complexes. Specific binding of factors to the 3' splice site region is still detected in snRNP-depleted extracts. Depletion of U1 snRNP impairs stable binding of U2 snRNP to the pre-mRNA branch site. This role of U1 snRNP in promoting stable preslicing complex formation is independent of the U1 snRNA-5' splice site interaction.

Base Sequence↗
PubMed1990

Synthesis and applications of oligoribonucleotides with selected 2'-O-methylation using the 2'-O-[1-(2-fluorophenyl)-4-methoxypiperidin-4-yl] protecting group.

The synthesis of base protected 5'-O-dimethoxytrityl-2'-O-[1-(2- fluorophenyl)-4-methoxypiperidin-4-yl]-3'-O-(2-cyanoethyl N,N-diisopropylphosphoramidites) is described, using phenoxyacetyl protection for the exocyclic amino groups of guanosine and adenosine and acetyl protection of the amino group of cytidine. High yield assembly of these building blocks into oligoribonucleotides on aminopropyl controlled pore glass was achieved using 5-(4-nitrophenyl)-1H-tetrazole as activator. Mixed sequences containing selected 2'-O-methylation were also synthesised and their significance for the study of RNA biochemistry is discussed.

Base Sequence↗
PubMed1990

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.

Alkylation↗
PubMed1990

New synthetic routes to protected purine 2'-O-methylriboside-3'-O-phosphoramidites using a novel alkylation procedure.

A highly selective alkylation procedure has been developed enabling new synthetic routes to be established for protected purine 2'-O-methylriboside-3'-O-phosphoramidites; building blocks for the assembly of 2'-O-methyloligoribonucleotides. The new procedure avoids the use of the highly toxic and potentially explosive reagent diazomethane and is far superior to the use of silver oxide/methyl iodide. Moreover, the use of highly versatile key intermediates will enable the synthesis of a wide variety of base modified analogues as well as other 2'-O-alkylriboside derivatives.

Adenine Nucleotides↗
PubMed1990

2'-O-alkyl oligoribonucleotides as antisense probes.

2'-O-Methyl oligoribonucleotides have recently been introduced as antisense probes for studying RNA processing and for affinity purification of RNA-protein complexes. To identify RNA analogues with improved properties for antisense analysis, 2'-O-alkyl oligoribonucleotides were synthesized in which the alkyl moiety was either the three-carbon linear allyl group or the five-carbon branched 3,3-dimethylallyl group. Both these analogues were found to be completely resistant to degradation by either DNA- or RNA-specific nucleases. Use of biotinylated derivatives of the probes to affinity-select ribonucleoprotein particles from crude HeLa cell nuclear extracts showed that the presence of the bulky 3,3-dimethylallyl group significantly reduces affinity selection, whereas the allyl derivative binds rapidly and stably to targeted sequences and affinity-selects efficiently. The allyl derivatives also showed an increase in the level of specific binding to targeted sequences compared with 2'-O-methyl probes of identical sequence. These properties indicate that the 2'-O-allyl oligoribonucleotides are particularly well suited for use as antisense probes.

Alkylation↗
PubMed1989

Mapping U2 snRNP--pre-mRNA interactions using biotinylated oligonucleotides made of 2'-OMe RNA.

Biotinylated 2'-OMe RNA oligonucleotides complementary to two separate regions of human U2 snRNA have been used as affinity probes to study U2 snRNP--pre-mRNA interactions. Both oligonucleotides bind specifically and allow highly selective removal of U2 snRNP from HeLa cell nuclear extracts. Pre-mRNA substrates can also be specifically affinity selected through oligonucleotides binding to U2 snRNP particles in splicing complexes. Stable binding of U2 snRNP to pre-mRNA is blocked by the pre-binding of an oligonucleotide to the branch site complementary region of U2 snRNA, but not by an oligonucleotide binding to the 5' terminus of U2. Both oligonucleotides affinity select the intron product, but not the intron intermediate, when added after spliceosome assembly has taken place. The effect of 2'-OMe RNA oligonucleotides on splicing complex formation has been used to demonstrate that complexes containing U2 snRNP and unspliced pre-mRNA are precursors to functional spliceosomes.

Base Sequence↗
PubMed1989

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.

Base Sequence↗
PubMed1989

Antisense probing of the human U4/U6 snRNP with biotinylated 2'-OMe RNA oligonucleotides.

We have used antisense 2'-OMe RNA oligonucleotides carrying four 5'-terminal biotin residues to probe the structure and function of the human U4/U6 snRNP. Nine oligonucleotides, complementary to multiple regions of U4 and U6 snRNAs, bound stably and specifically to U4/U6 snRNP. This allowed for efficient and selective removal of U4/U6 from HeLa cell nuclear extracts. Binding of oligonucleotides to certain snRNA domains inhibited splicing and affected the U4-U6 interaction. Pre-mRNA and splicing products could also be affinity-selected through binding of the oligonucleotides to U4/U6 snRNPs in splicing complexes. The results suggest that U4 snRNP is not released during spliceosome assembly.

Base Sequence↗
PubMed1989

Highly efficient chemical synthesis of 2'-O-methyloligoribonucleotides and tetrabiotinylated derivatives; novel probes that are resistant to degradation by RNA or DNA specific nucleases.

2'-O-Methyloligoribonucleotides have been synthesised on solid phase from base protected 5'-O-dimethoxytrityl-2'-O-methylribonucleoside-3'-O-(2-cyanoethyl N,N-diisopropylphosphoramidites) using 5-(4-nitrophenyl)-1H-tetrazole as activator. Coupling yields greater than 99% were achieved, as judged by trityl cation release. The preparation of a modified 2'-deoxycytidine building block bearing an N4-(5-trifluoroacetylaminopentyl) spacer is also described. The latter compound enabled the chemical synthesis of 2'-O-methyloligoribonucleotide probes carrying several 5'- terminal biotinylation sites (in general four modified residues were used), which can be conveniently 32P end-labelled enzymatically using polynucleotide kinase. Used in conjunction with streptavidin-containing derivatives, such biotinylated probes have important applications in biochemical purification and electron microscopy of RNA-protein complexes. The 2'-O-methyloligoribonucleotides are completely resistant to degradation by either RNA or DNA specific nucleases. In contrast, nucleases with dual RNA/DNA specificity show a complete spectrum of cleavage rates.

Biotin↗