Triplex-forming oligonucleotides: principles and applications

1. Triple-helical nucleic acids 89 1.1 History 89 1.2 Use of oligomers in triplex formation 90 2. Modes of triplex formation 90 2.1 Intermolecular triplexes 90 2.2 Intramolecular triplexes (H-DNA) 92 2.3 R-DNA (recombination DNA) 92 2.4 PNA (peptide nucleic acids) 93 3. Triplex structural models 93 3.1 YR-Y triplexes 94 3.2 GT-A base triplets 94 3.3 TC-G base triplets 94 3.4 TA-T and C+G-C base triplets 94 3.5 RR-Y triplexes 94 4. Modifications of TFOs 95 4.1 Backbone modification of oligonucleotides 95 4.2 Modification of the ribose in oligonucleotides 96 4.3 Base modification of oligonucleotides 97 5. Gene targeting and modification via triplex technology 98 5.1 Transcription and replication inhibition 99 5.2 TFO-directed mutagenesis 99 5.3 TFO-induced recombination 100 5.4 Future challenges in triplex-directed genome modification 100 6. References 101 The first description of triple-helical nucleic acids was by Felsenfeld and Rich in 1957 (Felsenfeld et al. 1957). While studying the binding characteristics of polyribonucleotides by fiber diffraction studies, they determined that polyuridylic acid [poly(U)] and polyadenylic acid [poly(A)] strands were capable of forming a stable complex of poly(U) and poly(A) in a 2:1 ratio. It was therefore concluded that the nucleic acids must be capable of forming a helical three-stranded structure. The formation of the three-stranded complex was preferred over duplex formation in the presence of divalent cations (e.g. 10 mm MgCl2). The reaction was quite specific, since the (U-A) molecule did not react with polycytidylic acid [(poly(C)], polyadenylic acid or polyinosinic acid [(poly(I)] (Felsenfeld et al. 1957). It was later found that poly(dT-dC) and poly(dG-dA) also have the capacity to form triple-stranded structures (Howard & Miles, 1964; Michelson & Monny, 1967). Other triple helical combinations of polynucleotide strands were identified from X-ray fiber-diffraction studies including, (A)n.2(I)n and (A)n.2(T)n (Arnott & Selsing, 1974). X-ray diffraction patterns of triple-stranded fibers of poly(A).2poly(U) and poly(dA).2poly(dT) showed an A-form conformation of the Watson–Crick strands. The third strand was bound in a parallel orientation to the purine strand by Hoogsteen hydrogen bonds (Hoogsteen, 1959; Arnott & Selsing, 1974). In 1968, the first potential biological role of these structures was identified by Morgan & Wells (1968). Using an in vitro assay, they found that transcription by E. coli RNA polymerase was inhibited by an RNA third strand. Thus, the recent developments identifying the potential of triplex formation for gene regulation and genome modification came more than 20 years after this first study of transcription inhibition by triplex formation.