Sequence-selective compounds that target chromosomal DNA might be used to regulate gene expression, probe the structural and functional importance of target sequences, direct sequence-specific mutations, and induce gene corrections (1). Difficult challenges, however, confront any scheme for developing compounds that recognize chromosomal DNA.
Before a synthetic antigene agent can bind a target sequence within a chromosome, it must cross both the outer cell and nuclear membranes. These agents must compete with chromatin structure and bound proteins to recognize and bind specific sequences within chromosomal DNA. Once bound, antigene agents must resist displacement by polymerases, helicases, and other proteins that participate in promoter recognition and transcription.
Several approaches have been developed for recognition of chromosomal DNA inside cells. Oligonucleotides can bind to duplex DNA by Hoogsteen base-pairing in the major groove to form triple helices (2,3). Alternatively, synthetic pyrimidine polyamides can recognize DNA through binding in the minor groove (4,5). More recently, duplex RNA targeting CpG islands within promoter DNA has been reported to silence gene expression in mammalian cells by inducing methylation (6,7), although negative results have also been reported (16). RNA-directed methylation had been well-studied in plants (Matzke et al, 2004; Chan et al, 2004) and yeast (Sugiyama et al, 2004; Sigova et al, 2004; and Motamedi et al, 2004), but these two positive recent reports were the first indication that RNA could also recognize sequences within the chromosomal DNA of mammalian cells. However, efficiency inhibition required delivering the RNA using lentiviral transduction (7), or simultaneously targeting multiple (ten) genomic regions (6). A recent review (Kawasaki et al, 2005) discusses the mechanism and implications of methylase-dependent, chromosome-targeted RNA interference (RNAi). Another review (Paroo and Corey, 2004) summarizes challenges for RNAi in vivo.
Peptide nucleic acids (PNAs) are nonionic DNA mimics that offer advantages for recognition of duplex DNA because hybridization is not hindered by phosphate-phosphate repulsion (1,8). Applications for strand invasion by PNAs include: creation of artificial primosomes (9), inhibition of transcription (10), activation of transcription (11), and directed mutagenesis (12). Several references report inhibition of gene transcription in human cells using antigene PNAs to target chromosomal DNA including coding regions (Boffa et al, 1996) and enhancer regions (Cutrona et al, 2003) of the targeted genes. Antigene PNAs targeting coding regions have also been reported to inhibit gene transcription when injected into rats (McMahon et al, 2002; Tyler et al, 1999). Transcription of a DNA restriction fragment in vitro has been reported to promote PNA binding about 50 bp downstream from phage promoters (10). A recent review summarizes use of PNAs for recognition of chromosomal DNA, and suggests that partially single-stranded regions like non-B-type structures and the open complex of transcription initiation might be susceptible to binding synthetic oligomers (1).
Strand invasion by PNAs in cell-free systems is most efficient at sequences that are partially single-stranded (13,14). Assembly of RNA polymerase and transcription factors into the pre-initiation complex on DNA induces the formation of a structure known as the open complex that contains several bases of unwound single-stranded DNA (15,16). Working in a cell-free prokaryotic system, Sigman and coworkers demonstrated that this single-stranded region of DNA was accessible to binding by short RNAs and that transcription could be inhibited (17). Inhibition was almost completely abolished by oligomers of 5 bases, whereas 8-mer sequences were much less effective, and there was no inhibition with 11-mer sequences. The authors concluded that RNA polymerase enforces rigid length and position constraints on open complex-targeted oligoribonucleotides.