Minimum Number of 2‘-<i>O</i>-(2-Aminoethyl) Residues Required for Gene Knockout Activity by Triple Helix Forming Oligonucleotides

Puri, Nitin; Majumdar, Angshuman; Cuenoud, Bernard; Natt, François; Martin, Pierre; Boyd, A. Christopher; Miller, Paul S.; Seidman, Michael M.

doi:10.1021/bi025734y

Cited by 52 publications

(88 citation statements)

References 62 publications

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“…1, a and b). We have described the synthesis and characterization of TFOs containing 2Ј-AE substitutions in previous publications (17,18). The 2Ј-AE residues are protonated at physiological pH.…”

Section: Resultsmentioning

confidence: 99%

“…They also establish a stabilizing interaction with phosphates in the purine strand of the duplex target (36). Psoralenlinked TFOs containing the appropriate amount and distribution of this modification are active in the Hprt assay (18).…”

Section: Resultsmentioning

confidence: 99%

“…The assay reflects the binding of the target sequence by the TFO and, following photoactivation of the psoralen, the generation of an inactivating mutation as a result of cellular processing of the cross-link. We have used this approach to measure the activity of TFOs carrying sugar and base modifications and have shown that TFOs with 2Ј-O-aminoethyl (2Ј-AE) substitutions are bioactive (18). We have now asked whether the biological state of the cell could influence TFO activity.…”

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confidence: 99%

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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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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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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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“…Activity of SSO Donors-In earlier publications we described the development of biologically active triple helix forming oligonucleotides (TFOs) linked to psoralen, a photoactive crosslinking agent (67,68). Some experiments presented in this report employed a psoralen-linked TFO (pso-TFO) designed to introduce a cross-link into a site immediately adjacent to exon 5 in the Chinese hamster Hprt gene.…”

Section: Resultsmentioning

confidence: 99%

Sequence Conversion by Single Strand Oligonucleotide Donors via Non-homologous End Joining in Mammalian Cells

Liu

Majumdar

Liu

et al. 2010

Journal of Biological Chemistry

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Double strand breaks (DSBs) can be repaired by homology independent nonhomologous end joining (NHEJ) pathways involving proteins such as Ku70/80, DNAPKcs, Xrcc4/Ligase 4, and the Mre11/Rad50/Nbs1 (MRN) complex. DSBs can also be repaired by homology-dependent pathways (HDR), in which the MRN and CtIP nucleases produce single strand ends that engage homologous sequences either by strand invasion or strand annealing. The entry of ends into HDR pathways underlies protocols for genomic manipulation that combine site-specific DSBs with appropriate informational donors. Most strategies utilize long duplex donors that participate by strand invasion. Work in yeast indicates that single strand oligonucleotide (SSO) donors are also active, over considerable distance, via a single strand annealing pathway. We examined the activity of SSO donors in mammalian cells at DSBs induced either by a restriction nuclease or by a targeted interstrand cross-link. SSO donors were effective immediately adjacent to the break, but activity declined sharply beyond ϳ100 nucleotides. Overexpression of the resection nuclease CtIP increased the frequency of SSO-mediated sequence modulation distal to the break site, but had no effect on the activity of an SSO donor adjacent to the break. Genetic and in vivo competition experiments showed that sequence conversion by SSOs in the immediate vicinity of the break was not by strand invasion or strand annealing pathways. Instead these donors competed for ends that would have otherwise entered NHEJ pathways. Non-homologous end joining (NHEJ)3 repair is un-templated, as there is no requirement, or even apparent use, for an informational reference sequence acting in trans. NHEJ might be regarded as a collection of operations (end processing by nucleases, limited single strand exposure of the ends, fill in by polymerases, ligation), without requirement for a specific order of events (1). In mammalian cells the best characterized version of NHEJ, often termed "classical" (C-NHEJ) (2, 3), utilizes several factors, including Ku70/80, Artemis, XLF, DNAPKcs, Xrcc4/DNA ligase 4, pol, pol, and XLF/Cernunnos (4 -8).Although precise joining of restriction enzyme cleaved ends can occur, the pathway is frequently mutagenic, resulting in the introduction of short deletions at the break site (9 -11).Deletions at the DSB are sometimes bounded by short homology elements located on either side of the break. These have been described as the product of a microhomology mediated end joining (MMEJ) pathway (5, 12). Deletions without microhomologies are also recovered and recent work suggests that the MRN complex plays an important role in both versions of deletional NHEJ (2, 13-15). A backup pathway involving poly(ADP-ribose) polymerase 1 and ligase III has been described as well (16,17). It should be noted that whether end joining in the absence of one of the canonical factors is evidence of different discrete pathways, or simply a variation on a fundamental scheme, is a matter for discussion (7). Nonetheless, NHEJ is the dom...

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“…40) More recently, psoralen-TFOs containing several 2Ј-aminoethyl (2Ј-AE) residues as well as 2Ј-OMe substitutions showed the highest HPRT gene knockout activity. 41,42) The mutagenesis frequencies using psoralen-TFOs with three or four 2Ј-AE residues at terminal positions were 20-30 fold greater than those of the uniformly 2Ј-O-Me-substituted TFOs. These mixed substitution TFOs formed stable triplexes at lower levels of Mg 2ϩ , even at 1 mM Mg 2ϩ concentration, and at physiologic pH in vitro.…”

Section: Targeted Mutagenesis By Modified Tfomentioning

confidence: 99%

Chemical Tools for Targeted Mutagenesis of DNA Based on Triple Helix Formation

Nagatsugi

Sasaki

2004

Biological & Pharmaceutical Bulletin

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Minimum Number of 2‘-O-(2-Aminoethyl) Residues Required for Gene Knockout Activity by Triple Helix Forming Oligonucleotides

Cited by 52 publications

References 62 publications

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

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

Sequence Conversion by Single Strand Oligonucleotide Donors via Non-homologous End Joining in Mammalian Cells

Chemical Tools for Targeted Mutagenesis of DNA Based on Triple Helix Formation

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