Triple helix formation by purine-rich oligonucleotides targeted to the human dihydrofolate reductase promoter

Abstract

The ability of oligodeoxynucleotides to form specific triple helical structures with critical regulatory sequences in the human dihydrofolate reductase (DHFR) promoter was investigated. A battery of purine-rich oligonucleotides targeted to the two purine.pyrimidine strand biased regions near the DHFR transcription initiation site was developed. The stable triple helical structures formed by binding of the oligonucleotides to the native promoter double helix were dominated by G*G.C triplets, with interspersed C*C.G and A*A.T alignments. Mismatches between the oligonucleotide and the purine-rich strand of the target significantly destabilized third strand binding, and a G*A.T alignment was particularly unfavorable. Formation of a pur.pur.pyr triple helical structure results in a localized limitation of access to the native double helical DNA and produces sequence dependent conformational alterations extending several nucleotides beyond the triplex-duplex boundary. Although they differ only by the insertion of two A.T base pairs, the distal and proximal purine.pyrimidine regions can be targeted individually due to the high degree of sequence specificity of triple helical alignment. Triplex formation overlapping any of three consensus transcriptional regulatory elements and collectively covering 50% of the DHFR core promoter is now possible with this set of oligonucleotides.

Effect of unmodified triple helix‐forming oligodeoxyribonucleotide targeted to human multidrug‐resistance gene mdr1 in MDR cancer cells

“…The potential selective interaction within megabase DNA has suggested the possibility of exogenously modulating gene expression by intermolecular triplex formation [5,6]. Many in vitro experiments have demonstrated that triple helix-forming oligodeoxyribonucleotides (ODNs) can selectively inhibit gene transcription, either by preventing transcription factor binding to promoters [6,7] or by blocking transcription elongation [8,9]. This has led to consideration of the triple helix 'anti-gene' strategy as a new therapeutic approach to selectively suppress unwanted gene expression in vivo.…”

Triplex formation inhibits HER-2/neu transcription in vitro.

“…The promoters of the human c-myc, EGF-R, dihydrofolate reductase (DHFR), and IL-2 receptor (IL-2-R) genes have been successfully targeted for site-specific triplex formation (14)(15)(16)(17)(18). In addition, sequences in the HIV long terminal repeat (LTR) ( 19) and coding sequence (20) are targets for triplex formation.…”

“…Interstrand DNA triplex formation typically occurs at polypurine/polypyrimidine tracts (14)(15)(16)(17)(18)(19)(20)(21)(22)(23)). An exogenous polypurine oligonucleotide forms the third strand by occupying the major groove of the native duplex and forming Hoogsteen hydrogen bonds with the purine bases ofthe duplex (23)(24)(25) base triplets in this purine:purine:pyrimidine (R:R:Y) triplex include G:G:C and A:A:T (third strand bases in bold), and the Hoogsteen bonding makes the triplex sequence specific (24,25).…”

“…While purine-rich sequences occur disproportionately in the promoters ofeukaryotic genes (23), there are often pyrimidines interspersed in the polypurine tract that make triplex formation less favorable. Nonetheless, triplex formation has been shown in several naturally occurring sequences that are not strictly homopurine:homopyrimidine (14)(15)(16)(17)(18)(19)(20). Pyrimidine interruptions of the polypurine tract potentially disrupt the ability ofthese sequences to form triplex because ofthe loss ofHoogsteen hydrogen bonds (25,26).…”

Noncanonical DNA Elements in the Lamin B2 Origin of DNA Replication

“…In addition to chemical assays, origin fragment was also subjected to limited hydrolysis using DNase I (Fig. 1); DNase was expected to strongly react with double stranded DNA and to be significantly less reactive with single or multistranded regions if present in the origin (27,28).…”