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

F.-L Michael Lin,Alokes Majumdar,Lynn C Klotz,Anthony P Reszka,Stephen Neidle,Michael M Seidman

doi:10.1074/jbc.m005404200

Abstract

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.

Highlights

Introduction of TriplexPlasmid Complexes into Cells—In the following experiments, we monitored triplex stability after introduction into cells
Restriction Protection and Mutation Assays for Bound Triple helix-forming oligonucleotides (TFOs)— The assays for TFO binding are based on a shuttle vector plasmid described in previous publications [36, 37]
The requirement for a timed analysis of triplex stability following plasmid transfection dictated the choice of electroporation as a transfection method because it delivered the complex into the cell at a defined time [43]

Summary

Introduction

Plasmid Complexes into Cells—In the following experiments, we monitored triplex stability after introduction into cells. The requirement for a timed analysis of triplex stability following plasmid transfection dictated the choice of electroporation as a transfection method because it delivered the complex into the cell at a defined time [43]. We were initially concerned that the process of electroporation might destabilize the triplexes. We found severalfold losses of both TC301⁄7supF5 and Ag301⁄7supFG1 complexes when they were electroporated in the medium used for cell suspension. If the Mg2ϩ levels in the medium were adjusted to 10 mM, the triplexes were stable to electroporation. This condition was well tolerated by the cells and was maintained in all experiments with cells

Methods

Results

Conclusion