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Triplex formation by oligonucleotides containing 5-(1-propynyl)-2'-deoxyuridine: decreased magnesium dependence and improved intracellular gene targeting.

Abstract：

:Oligonucleotides capable of sequence-specific triple helix formation have been proposed as DNA binding ligands useful for modulation of gene expression and for directed genome modification. However, the effectiveness of such triplex-forming oligonucleotides (TFOs) depends on their ability to bind to their target sites within cells, and this can be limited under physiologic conditions. In particular, triplex formation in the pyrimidine motif is favored by unphysiologically low pH and high magnesium concentrations. To address these limitations, a series of pyrimidine TFOs were tested for third-strand binding under a variety of conditions. Those containing 5-(1-propynyl)-2'-deoxyuridine (pdU) and 5-methyl-2'-deoxycytidine (5meC) showed superior binding characteristics at neutral pH and at low magnesium concentrations, as determined by gel mobility shift assays and thermal dissociation profiles. Over a range of Mg2+ concentrations, pdU-modified TFOs formed more stable triplexes than did TFOs containing 2'-deoxythymidine. At 1 mM Mg2+, a DeltaTm of 30 degreesC was observed for pdU- versus T-containing 15-mers (of generic sequence 5' TTTTCTTTTTTCTTTTCT 3') binding to the cognate A:T bp rich site, indicating that pdU-containing TFOs are capable of substantial binding even at physiologically low Mg2+ concentrations. In addition, the pdU-containing TFOs were superior in gene targeting experiments in mammalian cells, yielding 4-fold higher mutation frequencies in a shuttle vector-based mutagenesis assay designed to detect mutations induced by third-strand-directed psoralen adducts. These results suggest the utility of the pdU substitution in the pyrimidine motif for triplex-based gene targeting experiments.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Lacroix L,Lacoste J,Reddoch JF,Mergny JL,Levy DD,Seidman MM,Matteucci MD,Glazer PM

doi

10.1021/bi982290q

subject

Has Abstract

pub_date

1999-02-09 00:00:00

pages

1893-901

issue

eissn

0006-2960

issn

1520-4995

pii

bi982290q

journal_volume

pub_type

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Triplex formation by oligonucleotides containing 5-(1-propynyl)-2'-deoxyuridine: decreased magnesium dependence and improved intracellular gene targeting.