Triplex-Forming Twisted Intercalating Nucleic Acids (TINAs): Design Rules, Stabilization of Antiparallel DNA Triplexes and Inhibition of G-Quartet-Dependent Self-Association

The majority of studies on DNA triple helices have been focused on pH-sensitive parallel triplexes with Hoogsteen CTcontaining third strands that require protonation of cytosines. Reverse Hoogsteen GT/GA-containing antiparallel triplex-forming oligonucleotides (TFOs) do not require an acidic pH but their applicability in triplex technology is limited because of their tendency to form undesired highly stable aggregates such as G-quadruplexes. In this study, G-rich oligonucleotides containing 2-4 insertions of twisted intercalating nucleic acid (TINA) monomers are demonstrated to disrupt the formation of G-quadruplexes and form stable antiparallel triplexes with target DNA duplexes. The structure of TINA-incorporated oligo-nucleotides was optimized, the rules of their design were established and the optimal triplex-forming oligonucleotides were selected. These oligonucleotides show high affinity towards a 16 bp homopurine model sequence from the HIV-1 genome; dissociation constants as low as 160 nm are observed whereas the unmodified TFO does not show any triplex formation and instead forms an intermolecular G-quadruplex with T-m exceeding 90 degrees C in the presence of 50 mm NaCl. Here we present a set of rules that help to reach the full potential of TINA-TFOs and demonstrate the effect of TINA on the formation and stability of triple helical DNA.