PubMed Health⌕ Search

Biomedical subjects

B Sproat

Publications and source records attributed to B Sproat.

15 recordsLinked to original sources
PubMed2000

Secondary structure prediction and in vitro accessibility of mRNA as tools in the selection of target sites for ribozymes.

We have investigated the relative merits of two commonly used methods for target site selection for ribozymes: secondary structure prediction (MFold program) and in vitro accessibility assays. A total of eight methylated ribozymes with DNA arms were synthesized and analyzed in a transient co-transfection assay in HeLa cells. Residual expression levels ranging from 23 to 72% were obtained with anti-PSKH1 ribozymes compared to cells transfected with an irrelevant control ribozyme. Ribozyme efficacy depended on both ribozyme concentration and the steady state expression levels of the target mRNA. Allylated ribozymes against a subset of the target sites generally displayed poorer efficacy than their methylated counterparts. This effect appeared to be influenced by in vivo accessibility of the target site. Ribozymes designed on the basis of either selection method displayed a wide range of efficacies with no significant differences in the average activities of the two groups of ribozymes. While in vitro accessibility assays had limited predictive power, there was a significant correlation between certain features of the predicted secondary structure of the target sequence and the efficacy of the corresponding ribozyme. Specifically, ribozyme efficacy appeared to be positively correlated with the presence of short stem regions and helices of low stability within their target sequences. There were no correlations with predicted free energy or loop length.

Algorithms↗
PubMed1995

RNase H is responsible for the non-specific inhibition of in vitro translation by 2'-O-alkyl chimeric oligonucleotides: high affinity or selectivity, a dilemma to design antisense oligomers.

Ribonuclease H (RNase H) which recognizes and cleaves the RNA strand of mismatched RNA-DNA heteroduplexes can induce non-specific effects of antisense oligonucleotides. In a previous paper [Larrouy et al. (1992), Gene, 121, 189-194], we demonstrated that ODN1, a phosphodiester 15mer targeted to the AUG initiation region of alpha-globin mRNA, inhibited non-specifically beta-globin synthesis in wheat germ extract due to RNase H-mediated cleavage of beta-globin mRNA. Specificity was restored by using MP-ODN2, a methylphosphonate-phosphodiester sandwich analogue of ODN1, which limited RNase H activity on non-perfect hybrids. We report here that 2'-O-alkyl RNA-phosphodiester DNA sandwich analogues of ODN1, with the same phosphodiester window as MP-ODN2, are non-specific inhibitors of globin synthesis in wheat germ extract, whatever the substituent (methyl, allyl or butyl) on the 2'-OH. These sandwich oligomers induced the cleavage of non-target beta-globin RNA sites, similarly to the unmodified parent oligomer ODN1. This is likely due to the increased affinity of 2'-O-alkyl-ODN2 chimeric oligomers for both fully and partly complementary RNA, compared to MP-ODN2. In contrast, the fully modified 2'-O-methyl analogue of ODN1 was a very effective and highly specific antisense sequence. This was ascribed to its inability (i) to induce RNA cleavage by RNase H and (ii) to physically prevent the elongation of the polypeptide chain.

Alkylation↗
PubMed1994

Relative contribution of photo-addition, helper oligonucleotide and RNase H to the antisense effect of psoralen-oligonucleotide conjugates, on in vitro translation of Leishmania mRNAs.

We investigated the properties of two antisense oligonucleotides, 11 alpha Pso and 14TMP, 11 and 14 nucleotides long, respectively, and conjugated to psoralen derivatives. These oligonucleotides were complementary to the mini-exon sequence of Leishmania amazonensis. Upon ultraviolet (UV) irradiation these oligomers were selectively cross-linked to DNA or RNA target sequences, either 14 or 35 nucleotides long. The yield of photo-addition was much lower on the longer targets than on the shorter ones, due to the presence of a hairpin structure. The co-addition of a helper oligonucleotide, whose binding site, on the 35-mer, was adjacent to that of the psoralen-derivatized antisense oligomer, improved the cross-linking efficiency. We then determined the effect of 14TMP on in vitro translation of Leishmania mRNA in cell-free extracts. Non-irradiated antisense oligonucleotide/mRNA complexes reduced the protein synthesis in wheat germ extract but not in rabbit reticulocyte lysate. Conversely, UV irradiation induced a 14TMP-dependent reduction of translation in reticulocyte lysate whereas the inhibition was not improved in the wheat germ extract. These results are discussed with respect to the involvement of RNase-H in the oligonucleotide-mediated effect on protein synthesis.

Animals↗
PubMed1994

Crystal structures at 2.5 angstrom resolution of seryl-tRNA synthetase complexed with two analogs of seryl adenylate.

Crystal structures of seryl-tRNA synthetase from Thermus thermophilus complexed with two different analogs of seryl adenylate have been determined at 2.5 A resolution. The first complex is between the enzyme and seryl-hydroxamate-AMP (adenosine monophosphate), produced enzymatically in the crystal from adenosine triphosphate (ATP) and serine hydroxamate, and the second is with a synthetic analog of seryl adenylate (5'-O-[N-(L-seryl)-sulfamoyl]adenosine), which is a strong inhibitor of the enzyme. Both molecules are bound in a similar fashion by a network of hydrogen bond interactions in a deep hydrophilic cleft formed by the antiparallel beta sheet and surrounding loops of the synthetase catalytic domain. Four regions in the primary sequence are involved in the interactions, including the motif 2 and 3 regions of class 2 synthetases. Apart from the specific recognition of the serine side chain, the interactions are likely to be similar in all class 2 synthetases.

Adenosine↗
PubMed1993

Efficient cleavage of pre-tRNAs by E. coli RNAse P RNA requires the 2'-hydroxyl of the ribose at the cleavage site.

RNAse P cleaves pre-tRNAs to liberate 5'-flanks and 5'-matured, 5'-phosphorylated tRNAs. It is not evident if the 2'-hydroxyls of the ribose moieties in the substrate are involved in the reaction. To study their influence in two different pre-tRNAs, we have modified specifically the 2'-hydroxyl groups at the cleavage site and in neighbouring positions. We have shown that these hydroxyls are important but not essential for the processing of these substrates by E. coli RNase P RNA (M1 RNA). The reduction in the catalytic efficiency was moderate for 2'-deoxy and severe for 2'-methoxy substitutions at the cleavage site. Additional effects of modifications in neighbouring positions were smaller. Based on our data we suggest that the modifications do not interfere with binding of the substrate, whereas they prevent an optimal steric arrangement for the hydrolysis reaction.

Base Sequence↗
PubMed1992

Initiator oligonucleotides for the combination of chemical and enzymatic RNA synthesis.

Transcription reactions with T7 RNA polymerase were performed in the presence of short oligonucleotides (oligos) with guanosine at the 3'-end. We obtained transcripts which had included these 'initiator oligos' at their 5'-termini. The oligos could contain mixtures of deoxyribo-, ribo-, 2'-O-methylated and biotinylated nucleotides. Only the 3'-terminal guanosine of these oligos was encoded in the template DNA at the transcription start point, in contrast to the remainder of the sequence. This 5'-terminal sequence is variable and eliminates the limitation that transcripts must start with a 5'-terminal guanosine. With a 5'-biotinylated dinucleotide, we obtained end-labeled RNAs suitable for nonradioactive RNA sequencing.

Biotin↗
PubMed1990

Automated sequencing of fluorescently labelled DNA by chemical degradation.

A new general method for sequencing fluorescently labelled DNA by chemical degradation has been developed. It is based on the observation that fluorescein attached via a mercaptopropyl or aminopropyl linker arm to the 5'-phosphate of an oligonucleotide is stable during the reactions commonly used in chemical cleavage procedures. DNA to be degraded is first enzymatically synthesized in vitro by annealing and extending a fluorescently labelled primer thereby introducing the fluorescent label at the 5'-end of the fragment. The newly synthesized fluorescently labelled DNA is then chemically degraded using: (a) a set of four different cleavage reactions; or (b) only one reaction comprising methylation of G-residues followed by a partial cleavage with piperidine in the presence of sodium chloride. The fluorescent degradation products are loaded on either four lanes or one lane of the gel, respectively, and the emitted fluorescence detected online during electrophoresis. In the 'four reactions/four lanes' method 200-350 bp (base pairs) can be read from the labelled end. The 'one reaction/one lane' method, in which the nucleotide sequence is determined by measuring different signal intensities following the rule G greater than A greater than C greater than T, currently yields around 100-200 bp of sequence per sample.

Automation↗
PubMed1989

Probing the structure and function of U2 snRNP with antisense oligonucleotides made of 2'-OMe RNA.

We have used oligonucleotides made of 2'-OMe RNA to analyze the role of separate domains of U2 snRNA in the splicing process. We show that antisense 2'-OMe RNA oligonucleotides bind efficiently and specifically to U2 snRNP and demonstrate that masking of two separate regions of U2 snRNA can inhibit splicing by affecting different steps in the spliceosome assembly pathway. Masking the 5' terminus of U2 snRNA does not prevent U2 snRNP binding to pre-mRNA but blocks subsequent assembly of a functional spliceosome. By contrast, masking of U2 sequences complementary to the pre-mRNA branch site completely inhibits binding of pre-mRNA. Hybrid formation at the branch site complementary region also triggers a specific change which affects the 5' terminus of U2 snRNA.

Cell Nucleus↗
PubMed1989

Direct genomic fluorescent on-line sequencing and analysis using in vitro amplification of DNA.

In vitro amplification of genomic DNA and total RNA, as well as recombinant DNA, using one fluorescently labelled and one unlabelled primer during amplification, together with on-line analysis of the products on the EMBL fluorescent DNA sequencer, is described. Further is reported direct sequencing of fluorescently labelled amplified probes by solid-phase chemical degradation, without subcloning and purification steps involved. At present up to 350 bases in 4 hours are determined with this technique. The fluorescent dye and its bond to the oligonucleotide are stable during the amplification cycles, and do not interfere with the enzymatic polymerization. High sensitivity of the detection device, down to 10(-18) moles, corresponding to less than 10(6) molecules makes possible analyses of the non-radioactive amplified probes after only 10 amplification cycles, starting with about 5 x 10(4) copies of recombinant DNA.

Autoanalysis↗
PubMed1988

T7 DNA polymerase in automated dideoxy sequencing.

T7 DNA polymerase with chemically inactivated 3'-5' exo-nuclease activity, as well as unmodified T7 DNA polymerase, were used for sequencing by the dideoxy method in an automated system with fluorescence labelled primer and on-line detection of laser-excited reaction products. An analysis of signal intensity variations in the C track revealed that low C signals were usually preceded by a T in the sequence. This effect was modified by surrounding nucleotides. Signal intensities were more uniform with T7 polymerase than with the Klenow fragment of DNA polymerase I. Some sequences ambiguous with the Klenow enzyme could easily be evaluated with the T7 enzyme. One sequence could only be read by the unmodified T7 polymerase, while both the Klenow fragment and the chemically modified T7 enzyme gave uninterpretable data.

Base Sequence↗
PubMed1988

Non-radioactive automated sequencing of oligonucleotides by chemical degradation.

A non-radioactive sequencing of fluorescently labelled oligonucleotides by solid-phase chemical degradation is described. Although non-radioactive methods have been reported for the dideoxy chain termination technique, such a method has not yet been developed for the chemical degradation sequencing of DNA fragments. A 21-mer fluorescein labelled M13 sequencing primer was sequenced in an on-line automated system in about 30 minutes. The fluorescent dye and its bond to the oligonucleotide were stable during the chemical reactions used for the base specific degradations. As the sequence is determined on-line during electrophoresis, reloading and running 10 fragments simultaneously allows us to use one gel for sequencing of about 50 different oligonucleotides.

Automation↗
PubMed1987

Automated DNA sequencing: ultrasensitive detection of fluorescent bands during electrophoresis.

A simple system has been designed enabling ultrasensitive on-line detection of fluorescently labelled macromolecules, e.g. nucleic acids, proteins and peptides during electrophoretic separations in gels. An important application is the automated DNA sequence determination without radioactivity. Drying of gels, film exposure and handling are not necessary. A sulphydryl containing M13 sequencing primer has been synthesised and end-labelled in a reaction with fluorescein iodoacetamide. This is then used in the dideoxy reactions. In particular no moving parts or complicated software are required for data collection and analysis. Compared to our first automated device detection sensitivity has been improved by a factor of thirty to about 3 X 10(-18) mol per band. The resolution has increased to about 400 bases in 5 hours, with the possibility to read up to about 500 bases when they are properly labelled. Gels shorter than 20 cm may be used for resolution of about 300 bases. The single gel system may be upgraded for simultaneous running and reading of six or ten sequencing samples.

Autoanalysis↗
PubMedDate not supplied

High-throughput automated DNA sequencing facility with fluorescent labels at the European Molecular Biology Laboratory.

One of the aims of the facility is to develop and push the automated on-line DNA sequencing gel technology to its limit in sequence throughput, which may be somewhere around 100 kilobases of sequence per device per day. Key new developments were initiated and applied in operation on the European Molecular Biology Laboratory (EMBL) automated sequencer and its commercial version A.L.F. (Pharmacia). Sequencing speed was increased by a factor of 10-20, up to 1500 bases per hour per clone on ultrathin (about 100 microns) gels, while the resolution and reading length were extended to 1000 bases on gels with 50 cm separation length, using fluorescein-15-*dATP as the internal label. With our sequencing strategy, closing about 40% of the sequence with "walking" primers and F-15-*dATP as internal label, we sequenced both strands of a cosmid insert of 38.5 kb in length, each strand twice, in only 430 sequencing reactions and with average reading of 380 bases per reaction.

Base Sequence↗