Triplex-forming oligonucleotides: principles and applications

Abstract

1. Triple-helical nucleic acids 891.1 History 891.2 Use of oligomers in triplex formation 902. Modes of triplex formation 902.1 Intermolecular triplexes 902.2 Intramolecular triplexes (H-DNA) 922.3 R-DNA (recombination DNA) 922.4 PNA (peptide nucleic acids) 933. Triplex structural models 933.1 YR-Y triplexes 943.2 GT-A base triplets 943.3 TC-G base triplets 943.4 TA-T and C+G-C base triplets 943.5 RR-Y triplexes 944. Modifications of TFOs 954.1 Backbone modification of oligonucleotides 954.2 Modification of the ribose in oligonucleotides 964.3 Base modification of oligonucleotides 975. Gene targeting and modification via triplex technology 985.1 Transcription and replication inhibition 995.2 TFO-directed mutagenesis 995.3 TFO-induced recombination 1005.4 Future challenges in triplex-directed genome modification 1006. References 101The 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.