Strategies to Induce Transcriptional Arrest

An alternative to the inhibition of RNA metabolism by way of an antisense mechanism is to inhibit transcription by interacting with double-stranded DNA in chromatin. Of the two most obvious binding strategies for oligonucleotides binding to double-stranded nucleic acids, strand invasion and triple-strand formation, triple-stranding strategies, until recently, attracted essentially all of the attention.

Polynucleotides were reported to form triple helices as early as 1957. Triple strands can form by non-Watson-Crick hydrogen bonds between the third strand and purines involved in Watson-Crick hydrogen bonding with the complementary strand of the duplex (for review, see Ref. 34). Thus, triple-stranded structures can be formed between a third strand composed of pyrimidines or purines that interact with a homopurine strand in a homopu-rine-homopyrimidine strand in a duplex DNA. With the demonstration that homopyrimidine oligonucleotides could indeed form triplex structures (35-37), interest in triple-strand approaches to inhibit transcription heightened.

Although there was initially considerable debate about the value of triple-stranding strategies vs. antisense approaches (38), there was little debate that much work remained to be done to design oligonucleotides that could form triple-stranded structures with duplexes of mixed sequences. Pursuit of several strategies has resulted in significant progress (for review, see Ref. 34). Considerable progress in creating chemical motifs capable of binding to duplex DNA with high affinity and specifici ties supportive of binding to sequences other than polypyrimidine polypurine traits has been reported. For example, peptide nucleic acid (PNA) has been shown to form on triple-stranded structures in isolated DNA and in mouse cell chromosomal DNA (39). Modifications such as 7-deazaxanthine and 2'-amino-ethoxy were reported to enhance triplex formation (40, 41). Additionally, 5'-propionyl-modified nucleosides have been shown to enhance triplex formation (42) (for review, see Ref. 43).

In addition to advances in the chemistry of triplex formation, a number of studies have shewn results in cells consistent with triplex formation in chromosomal DNA (39, 44-46). Peptide nucleic acids have been shown to be of particular value for triplex interactions because of the relatively high affinity of this modification. They have been shown to inhibit transcription initiation and elongation, to block DNA polymerases (47-49), and to inhibit binary of a number of proteins to DNA (50).DimericPNAs have been created that are reported to form PNA/DNA/PNA triplexes and these have been shown to induce gene-targeted mutations in streptolysin-o perme-abilized cells (39).

Strand invasion, an alternative approach to obstructing transcription by formation of tri-plie strands, has been shown to be feasible if analogs with sufficient affinity can be synthesized. PNAs have been shown to have verv

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