Mutagenesis Mediated by Triple Helix–Forming Oligonucleotides Conjugated to Psoralen: Effects of Linker Arm Length and Sequence Context

Raha, Manidipa; Lacroix, Laurent; Glazer, Peter M.

doi:10.1111/j.1751-1097.1998.tb05201.x

Cited by 22 publications

(13 citation statements)

References 17 publications

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“…Although the 2′- O -methyl modified TFOs were expected to bind with higher affinity to the duplex DNA target [1, 5, 8], they actually exhibited binding affinities quite similar to that of TFO DNA-C2 (Figure 2, Table 1). While TFO RNA-C2 had a K d that was not significantly different from that of TFO DNA-C2, a modest reduction in K d was observed for TFO RNA-C6, suggesting that the 6-carbon linker further stabilizes binding by allowing better psoralen intercalation, or less likely through direct interactions with the triplex structure, consistent with observations in DNA TFOs [19]. This lack of dramatic enhancement in binding affinity of RNA third strands binding to DNA duplexes has also been observed previously by Han and Dervan [20] and may be due to the RNA TFO's lack of the C-5 methyl group in uridine which likely enhances base stacking and triplex stability [1–3, 18, 21, 22].…”

Section: Resultssupporting

confidence: 77%

Structural Determinants of Photoreactivity of Triplex Forming Oligonucleotides Conjugated to Psoralens

Krishnan

2010

Journal of Nucleic Acids

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Triplex-forming oligonucleotides (TFOs) with both DNA and 2′-O-methyl RNA backbones can direct psoralen photoadducts to specific DNA sequences. However, the functional consequences of these differing structures on psoralen photoreactivity are unknown. We designed TFO sequences with DNA and 2′-O-methyl RNA backbones conjugated to psoralen by 2-carbon linkers and examined their ability to bind and target damage to model DNA duplexes corresponding to sequences within the human HPRT gene. While TFO binding affinity was not dramatically affected by the type of backbone, psoralen photoreactivity was completely abrogated by the 2′-O-methyl RNA backbone. Photoreactivity was restored when the psoralen was conjugated to the RNA TFO via a 6-carbon linker. In contrast to the B-form DNA of triplexes formed by DNA TFOs, the CD spectra of triplexes formed with 2′-O-methyl RNA TFOs exhibited features of A-form DNA. These results indicate that 2′-O-methyl RNA TFOs induce a partial B-to-A transition in their target DNA sequences which may impair the photoreactivity of a conjugated psoralen and suggest that optimal design of TFOs to target DNA damage may require a balance between binding ability and drug reactivity.

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Section: Resultssupporting

confidence: 77%

Structural Determinants of Photoreactivity of Triplex Forming Oligonucleotides Conjugated to Psoralens

Krishnan

2010

Journal of Nucleic Acids

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“…We addressed this issue by analyzing the sequence of mutant supF genes isolated from experiments in which the cells were treated with UVA 3 h after transfection. We found that the pattern of mutations with the supFG1⅐AG30 triplexes was the same as that reported earlier from experiments with the complexes that had been cross-linked in vitro or in vivo (33,44). Plasmids with mutant supFG1 genes contained single base substitutions located at the positions of the psoralen cross-link as well as deletions of 5-75 bases of sequence in the triplex target region.…”

Section: Stability Of Triplexes On Total Transfectedsupporting

confidence: 82%

Stability of DNA Triplexes on Shuttle Vector Plasmids in the Replication Pool in Mammalian Cells

Liu¹,

Majumdar²,

Klotz³

et al. 2000

Journal of Biological Chemistry

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Triple helix-forming oligonucleotides may be useful as gene-targeting reagents in vivo, for applications such as gene knockout. One important property of these complexes is their often remarkable stability, as demonstrated in solution and in cells following transfection. Although encouraging, these measurements do not necessarily report triplex stability in cellular compartments that support DNA functions such as replication and mutagenesis. We have devised a shuttle vector plasmid assay that reports the stability of triplexes on DNA that undergoes replication and mutagenesis. The assay is based on plasmids with novel variant supF tRNA genes containing embedded sequences for triplex formation and psoralen cross-linking. Triple helix-forming oligonucleotides were linked to psoralen and used to form triplexes on the plasmids. At various times after introduction into cells, the psoralen was activated by exposure to long wave ultraviolet light (UVA). After time for replication and mutagenesis, progeny plasmids were recovered and the frequency of plasmids with mutations in the supF gene determined. Site-specific mutagenesis by psoralen cross-links was dependent on precise placement of the psoralen by the triple helix-forming oligonucleotide at the time of UVA treatment. The results indicated that both pyrimidine and purine motif triplexes were much less stable on replicated DNA than on DNA in vitro or in total transfected DNA. Incubation of cells with amidoanthraquinone-based triplex stabilizing compounds enhanced the stability of the pyrimidine triplex.Triple helix-forming oligonucleotides (TFOs), 1 or third strands, have received considerable attention because of their potential for intracellular gene targeting (1-11). Many of the biochemical and biophysical properties of triplexes appear to support this application. Triplex formation following the encounter between an appropriate third strand and duplex target has long been recognized as a fundamental structural option of nucleic acids and is not dependent on enzymes or proteins (12). The most stable triplexes are formed on polypurine:polypyrimidine sequences. However, there is considerable stringency with respect to the specific sequence such that single interruptions in a polypurine run can be destabilizing (13-15). Depending on the actual sequence of the purine:pyrimidine run, triplexes can be formed by third strands composed of either purines or pyrimidines (16), which offers some flexibility in TFO design.Once formed, triplexes can be quite stable under appropriate conditions. This was demonstrated some time ago with a pyrimidine triplex with a dissociation half-life of many hours (17). Some purine TFOs form even more stable complexes, with melting temperatures equivalent to or greater than the target duplex (18), and with half-lives of days under optimal conditions (19,20). The persistence of purine motif triplexes formed on DNA fragments and then introduced into mammalian cells has also been measured. A footprinting assay to measure triplexes on transfected...

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“…17). Targeted point mutations were also the major event in earlier studies (39,40) with psoralen-linked TFOs and supF reporter genes. However, in our analysis of pso-TFO-targeted mutations at the endogenous chromosomal Hprt target site, we found that ϳ90% thioguanine-resistant clones carried deletions in the target region extending into Exon 5 (14,17) and no point mutations were observed at the T of the 5Ј-TA cross-link site.…”

Section: Kinds and Frequency Of Mutations Induced By Pso-tfo-mentioning

confidence: 99%

Cell Cycle Modulation of Gene Targeting by a Triple Helix-forming Oligonucleotide

Majumdar¹,

Puri²,

Cuenoud³

et al. 2003

Journal of Biological Chemistry

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Successful gene-targeting reagents must be functional under physiological conditions and must bind chromosomal target sequences embedded in chromatin. Triple helix-forming oligonucleotides (TFOs) recognize and bind specific sequences via the major groove of duplex DNA and may have potential for gene targeting in vivo. We have constructed chemically modified, psoralenlinked TFOs that mediate site-specific mutagenesis of a chromosomal gene in living cells. Here we show that targeting efficiency is sensitive to the biology of the cell, specifically, cell cycle status. Targeted mutagenesis was variable across the cycle with the greatest activity in S phase. This was the result of differential TFO binding as measured by cross-link formation. Targeted cross-linking was low in quiescent cells but substantially enhanced in S phase cells with adducts in ϳ20 -30% of target sequences. 75-80% of adducts were repaired faithfully, whereas the remaining adducts were converted into mutations (>5% mutation frequency). Clones with mutations could be recovered by direct screening of colonies chosen at random. These results demonstrate high frequency target binding and target mutagenesis by TFOs in living cells. Successful protocols for TFOmediated manipulation of chromosomal sequences are likely to reflect a combination of appropriate oligonucleotide chemistry and manipulation of the cell biology.Effective gene-targeting reagents would have a broad application as probes of chromatin structure and in a variety of genomic manipulations, e.g. gene knock-out, targeted gene conversion and/or recombination, and gene therapy. Successful constructs must be functional in the nuclear environment of living cells. Furthermore, their intended targets must be accessible despite the packaging and condensation of nuclear DNA by chromosomal proteins. One targeting strategy that has been of interest for many years is based on triple-helix forming oligonucleotides (TFOs). 1A DNA triple helix can form when a TFO lies in the major groove of intact duplex DNA (1-5). The most stable structures assemble on polypurine:polypyrimidine sequences with hydrogen bonds formed between the bases in the third strand and those in the purine strand of the duplex. The purine or pyrimidine motif third strands may be involved in triplex formation depending on the target sequence, and a binding code for the design of the third strands has been defined (6). Although conventional oligonucleotides have features that limit their activity under physiological conditions, there is a variety of chemical modifications that ameliorate these limitations (7). Thus, for pyrimidine TFOs, the replacement of cytosine by 5-methylcytosine and the use of 2Ј-O-methyl (2Ј-OMe)-modified sugars permit stable triplex formation at physiological pH in vitro (8 -13). Other derivatives contribute to the bioactivity of TFOs of both motifs as shown by us and others in recent publications (14 -18).Although chemical modifications can enhance TFO affinity and triplex stability, they obviously cannot add...

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Mutagenesis Mediated by Triple Helix–Forming Oligonucleotides Conjugated to Psoralen: Effects of Linker Arm Length and Sequence Context

Cited by 22 publications

References 17 publications

Structural Determinants of Photoreactivity of Triplex Forming Oligonucleotides Conjugated to Psoralens

Structural Determinants of Photoreactivity of Triplex Forming Oligonucleotides Conjugated to Psoralens

Stability of DNA Triplexes on Shuttle Vector Plasmids in the Replication Pool in Mammalian Cells

Cell Cycle Modulation of Gene Targeting by a Triple Helix-forming Oligonucleotide

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