The Experts below are selected from a list of 120 Experts worldwide ranked by ideXlab platform
Pamela A. Havre - One of the best experts on this subject based on the ideXlab platform.
-
Site-specific targeting of Psoralen photoadducts with a triple helix-forming oligonucleotide: characterization of Psoralen monoadduct and crosslink formation
Nucleic Acids Research, 1994Co-Authors: Francis P. Gasparro, Pamela A. Havre, Gerard A. Olack, Edward J. Gunther, Peter M. GlazerAbstract:Abstract A polypurine tract in the supF gene of bacteriophage lambda (base pairs 167-176) was selected as the target for triple helix formation and targeted mutagenesis by an oligopurine (5'-AGGAAGGGGG-3') containing a chemically linked Psoralen Derivative (4'-hydroxymethyl-4,5',8-trimethylPsoralen) at its 5' terminus (psoAG10). The thymines at base pairs 166 and 167, a 5'ApT site, were targeted for photomodification. Exposure of the triple helical complex to long wavelength ultraviolet radiation led to the covalent binding of psoAG10 to the targeted region in the supF gene and to the induction of site-specific mutations. We report here experiments to characterize the photomodification of the targeted region of the supF gene in the context of triple helix formation. An electrophoretic mobility-shift assay showed that, at low radiation doses, monoadducts at base pair 166 were the major photoadducts. At higher doses the monoadducts were converted to crosslinks between base pairs 166 and 167. HPLC analysis of enzymatically hydrolyzed photoreaction mixtures was used to confirm the electrophoresis results. A strong strand preference for specific photoadduct formation was also detected.
-
Targeted mutagenesis of simian virus 40 DNA mediated by a triple helix-forming oligonucleotide
1993Co-Authors: Pamela A. Havre, M. GlazerAbstract:Triple-helical DNA can be formed by oligonucleotides that bind as third strands of DNA in a sequence-specific manner in the major groove in homopurine/homopyrimidine stretches in duplex DNA. Such triple helix-forming oligonucleotides have been used to inhibit gene expression by blocking transcription factor access to promoter sites in transient expression assays. In an alternative approach to genetic manipulation using triplex DNA, we show that triplex-forming oligonucleotides can be used to produce site-specific, targeted mutations in a viral genome in order to achieve a permanent, heritable effect on gene function and expression. We use a triplex-forming oligonucleotide linked to a Psoralen Derivative at its 5 ' end to achieve targeted mutagenesis in a simian virus 40 (SV40) vector genome. Site-specific triplex formation delivers the Psoralen to the targeted site in the SV40 DNA. Photoactivation of the Psoralen yields adducts and thereby mutations at that site. Mutations were produced in the target gene in over 6 % of the viral genomes. DNA sequence analysis of the mutations in the target gene showed that all were in the targeted region, and 55 % were found to be the same T:A-to-A:T transversion precisely at the targeted base pair. In control experiments, no mutagenesis above the background frequency in the assay was produced by a non-triplex-forming, Psoralen-linked oligonucleotide unless a vast excess of this oligonucleotide was used, demonstrating the specificity of the targeted mutagenesis. This frequency of targeted mutagenesis of SV40 in monkey cells represents a 30-fold increase relative to simila
Timothy W. Nilsen - One of the best experts on this subject based on the ideXlab platform.
-
Detecting RNA–RNA Interactions Using Psoralen Derivatives
Cold Spring Harbor protocols, 2014Co-Authors: Timothy W. NilsenAbstract:Psoralens are tricyclic compounds that intercalate into double-stranded DNA or RNA and, on irradiation with long-wavelength (365-nm) UV light, covalently link pyrimidines on adjacent strands. More rarely, Psoralen cross-links can be observed at the ends of helices (i.e., double-stranded-single-stranded boundaries). Although Psoralens can, in some instances, cross-link protein to RNA, their primary application is to detect RNA-RNA base-pairing interactions. The most useful Psoralen Derivative is 4'-aminomethyl trioxsalen (AMT), which is soluble in H2O. This protocol describes the use of AMT to detect RNA-RNA interactions in tissue culture cells or in extracts. Cross-linked RNAs are detectable by their reduced mobility in polyacrylamide gels. Cross-links can be reversed by exposure to short-wavelength (254 nm) UV light.
Peter M. Glazer - One of the best experts on this subject based on the ideXlab platform.
-
Site-specific targeting of Psoralen photoadducts with a triple helix-forming oligonucleotide: characterization of Psoralen monoadduct and crosslink formation
Nucleic Acids Research, 1994Co-Authors: Francis P. Gasparro, Pamela A. Havre, Gerard A. Olack, Edward J. Gunther, Peter M. GlazerAbstract:Abstract A polypurine tract in the supF gene of bacteriophage lambda (base pairs 167-176) was selected as the target for triple helix formation and targeted mutagenesis by an oligopurine (5'-AGGAAGGGGG-3') containing a chemically linked Psoralen Derivative (4'-hydroxymethyl-4,5',8-trimethylPsoralen) at its 5' terminus (psoAG10). The thymines at base pairs 166 and 167, a 5'ApT site, were targeted for photomodification. Exposure of the triple helical complex to long wavelength ultraviolet radiation led to the covalent binding of psoAG10 to the targeted region in the supF gene and to the induction of site-specific mutations. We report here experiments to characterize the photomodification of the targeted region of the supF gene in the context of triple helix formation. An electrophoretic mobility-shift assay showed that, at low radiation doses, monoadducts at base pair 166 were the major photoadducts. At higher doses the monoadducts were converted to crosslinks between base pairs 166 and 167. HPLC analysis of enzymatically hydrolyzed photoreaction mixtures was used to confirm the electrophoresis results. A strong strand preference for specific photoadduct formation was also detected.
Glazer P M - One of the best experts on this subject based on the ideXlab platform.
-
Targeted mutagenesis of simian virus 40 DNA mediated by a triple helix-forming oligonucleotide.
1993Co-Authors: Havre P A, Glazer P MAbstract:Triple-helical DNA can be formed by oligonucleotides that bind as third strands of DNA in a sequence-specific manner in the major groove in homopurine/homopyrimidine stretches in duplex DNA. Such triple helix-forming oligonucleotides have been used to inhibit gene expression by blocking transcription factor access to promoter sites in transient expression assays. In an alternative approach to genetic manipulation using triplex DNA, we show that triplex-forming oligonucleotides can be used to produce site-specific, targeted mutations in a viral genome in order to achieve a permanent, heritable effect on gene function and expression. We use a triplex-forming oligonucleotide linked to a Psoralen Derivative at its 5' end to achieve targeted mutagenesis in a simian virus 40 (SV40) vector genome. Site-specific triplex formation delivers the Psoralen to the targeted site in the SV40 DNA. Photoactivation of the Psoralen yields adducts and thereby mutations at that site. Mutations were produced in the target gene in over 6% of the viral genomes. DNA sequence analysis of the mutations in the target gene showed that all were in the targeted region, and 55% were found to be the same T:A-to-A:T transversion precisely at the targeted base pair. In control experiments, no mutagenesis above the background frequency in the assay was produced by a non-triplex-forming, Psoralen-linked oligonucleotide unless a vast excess of this oligonucleotide was used, demonstrating the specificity of the targeted mutagenesis. This frequency of targeted mutagenesis of SV40 in monkey cells represents a 30-fold increase relative to similar experiments using lambda phage in bacteria, suggesting that fixation of the triplex-directed lesion into a mutation occurs more efficiently in mammalian cells. If the ability to reproducibly and predictably target mutations to sites in viral DNA in vitro by using modified oligonucleotides can be extended to DNA in vivo, this approach may prove useful as a technique for gene therapy, as a strategy for antiviral therapeutics, and as a tool for genetic engineering
-
Targeted mutagenesis of DNA using triple helix-forming oligonucleotides linked to Psoralen.
1993Co-Authors: Havre P A, Gasparro F P, Gunther E J, Glazer P MAbstract:Oligonucleotides can bind as third strands of DNA in a sequence-specific manner in the major groove in homopurine/homopyrimidine stretches in duplex DNA. Here we use a 10-base triplex-forming oligonucleotide linked to a Psoralen Derivative at its 5' end to achieve site-specific, targeted mutagenesis in an intact, double-stranded lambda phage genome. Site-specific triplex formation delivers the Psoralen to the targeted site in the lambda DNA, and photoactivation of the Psoralen produces adducts and thereby mutations at that site. Mutations in the targeted gene were at least 100-fold more frequent than those in a nontargeted gene, and sequence analysis of mutations in the targeted gene showed that 96% were in the targeted region and 56% were found to be the same T.A to A.T transversion precisely at the targeted base pair. The ability to reproducibly and predictably target mutations to sites in intact duplex DNA by using modified oligonucleotides may prove useful as a technique for gene therapy, as an approach to antiviral therapeutics, and as a tool for genetic engineering
Hélène C - One of the best experts on this subject based on the ideXlab platform.
-
Oligonucleotide clamps arrest DNA synthesis on a single-stranded DNA target.
1993Co-Authors: Giovannangéli C, Thuong N T, Hélène CAbstract:Triple helices can be formed on single-stranded oligopurine target sequences by composite oligonucleotides consisting of two oligonucleotides covalently linked by either a hexaethylene glycol linker or an oligonucleotide sequence. The first oligomer forms Watson-Crick base pairs with the target, while the second oligomer engages in Hoogsteen base pairing, thereby acting as a molecular clamp. The triple-helical complex formed by such an oligonucleotide clamp, or "oligonucleotide-loop-oligonucleotide" (OLO), is more stable than either the corresponding trimolecular triple helix or the double helix formed upon binding of the oligopyrimidine complement to the same oligopurine target. Attaching a Psoralen Derivative to the 5' end of the OLO allowed us to photoinduce a covalent linkage to the target sequence. The Psoralen moiety became covalently linked to all three portions of the triplex, thereby making the oligonucleotide clamp irreversible. These crosslinking reactions introduced strong stop signals during DNA replication, as shown on a plasmid containing a portion of the HIV proviral sequence of human immunodeficiency virus. A 16-mer oligopurine sequence corresponding to the "polypurine tract" of human immunodeficiency virus was chosen as a target for a Psoralen-OLO conjugate. Three different stop signals for DNA polymerase were observed, corresponding to different sites of polymerase arrest on its template. Even in the absence of photoinduced crosslinking, the Psoralen-OLO conjugate was able to arrest DNA replication. The formation of triple-helical structures on single-stranded targets may provide an alternative to the antisense strategy for the control of gene expression
-
Triple-helix formation by oligonucleotides containing the three bases thymine, cytosine, and guanine.
1992Co-Authors: Giovannangéli C, Thuong N T, Rougée M, Garestier T, Hélène CAbstract:A homopurine-homopyrimidine sequence of human immunodeficiency virus (HIV) proviral DNA was chosen as a target for triple-helix-forming oligonucleotides. An oligonucleotide containing three bases (thymine, cytosine, and guanine) was shown to bind to its target sequence under physiological conditions. This oligonucleotide is bound in a parallel orientation with respect to the homopurine sequence. Thymines recognize A.T base pairs to form T.A.T base triplets and guanines recognize a run of G.C base pairs to form G.G.C base triplets. A single 5-methylcytosine was shown to stabilize the triple helix when incorporated in a stretch of thymines; it recognizes a single G.C base pair in a run of A.T base pairs. These results provide some of the rules required for choosing the more appropriate oligonucleotide sequence to form a triple helix at a homopurine-homopyrimidine sequence of duplex DNA. A Psoralen Derivative attached to the oligonucleotide containing thymine, 5-methylcytosine, and guanine was shown to photoinduce cross-linking of the two DNA strands at the target sequence in a plasmid containing part of the HIV proviral DNA sequence. Triplex formation and cross-linking were monitored by inhibition of Dra I restriction enzyme cleavage. The present results provide a rational basis for the development of triplex-forming oligonucleotides targeted to specific sequences of the HIV provirus integrated in its host genome
-
Specific inhibition of transcription by triple helix-forming oligonucleotides.
1992Co-Authors: Duval-valentin G, Thuong N T, Hélène CAbstract:Homopyrimidine oligonucleotides bind to the major groove of a complementary homopyrimidine.homopurine stretch by triple helix formation. The bla gene from transposon Tn3 contains a homopyrimidine.homopurine sequence of 13 base pairs located just downstream of the RNA polymerase binding site. A 13-mer homopyrimidine oligonucleotide targeted to this sequence was tested for its effect on transcription of the bla gene in vitro. We show that the consequence of triple helix formation in front of the Escherichia coli RNA polymerase-promoter complex is to block the holoenzyme at its start site during a period that is dependent on temperature. The temperature dependence of transcription inhibition shows a direct correlation between this effect and the stabilization of the triple helix. Substitution of 5-methylcytosine to cytosine in the 13-mer oligonucleotide enhances triplex stability and transcription inhibition. Transcription inhibition by this synthetic repressor was also confirmed by footprinting studies demonstrating its specificity of action. The 13-mer oligonucleotide containing a Psoralen Derivative covalently linked to its 5' end shows an irreversible and specific inhibition of transcription initiation after exposure to light of wavelength greater than 310 nm
