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ortho-TINA for PCR Primers

ortho-Twisted Intercalating Nucleic Acid (oTINA) or (S)-1-O-[2-(1-pyrenylethynyl)phenylmethyl]glycerol was synthesized in 2008 as part of a structural study on TINA molecules(1).

A single oTINA molecule inserted in the middle of a 14-mer homopyrimidine hybridization oligonucleotide increased the annealing temperature (Tm) of an antiparallel DNA duplex by 3 °C in the presence of 120 mM Na⁺ and 10 mM Mg²⁺ (1). A later thermal stability study identified the 5' and 3' terminal positions as the optimal positions in oligonucleotides for placement of oTINA molecules to increase the thermal stability of antiparallel DNA duplexes (2). When placed at the 5' position / 3' position / 5' and 3' positions, the oTINA molecule increased Tm by 3.9 / 1.3 / 5.7 °C for a 18-mer DNA oligonucleotide or 4.3 / 3.0 / 7.9 °C for a 16-mer DNA oligonucleotide in the presence of 150 mM Na⁺ (2).

oTINA modified oligonucleotides increase the analytical sensitivity, when placed at the 5' and 3' terminal position of hybridization probes. A 27-fold increase in analytical sensitivity was demonstrated in buffer with 150 mM monovalent cation. In buffer with 300 mM monovalent cation the analytical sensitivity is increased 11-fold and even in buffer with 1 M of monovalent cation a 4-fold increase in analytical sensitivity is seen making oTINA modified oligonucleotides well-suited for antiparallel duplex hybridization assays(2).

Figure legend: To the left a 5' placed oTINA molecule and to the right an internally inserted oTINA molecule

In a PCR primer, the optimal position of an oTINA modification is at the 5' terminal position. This has been established as the oTINA modification allows enzymes to read up to the modification, but blocks the read-through by exonucleases and polymerases. The blocking activity of the oTINA modification is actively used to generate the single stranded DNA overhang introduced by Cliffhanger primers in PCR. To learn more about the use of oTINA modified primers in PCR and Cliffhanger PCR, please refer to the specific sections on these topics on this site.

To summarize, the oTINA modification is well-suited for experimental set-ups involving the formation of DNA antiparallel duplexes.


(1) Filichev VV, Astakhova IV, Malakhov AD, Korshun VA and Pedersen EB (2008) 1-, 2-, and 4-Ethynylpyrenes in the Structure of Twisted Intercalating Nucleic Acids: Structure, Thermal Stability, and Fluorescence Relationship. Chem. Eur. J., 14, 9968-9980.
(2) Schneider UV, Géci I, Jøhnk N, Mikkelsen ND, Pedersen EB and Lisby G (2011) Increasing the Analytical Sensitivity by Oligonucleotides Modified with Para- and Ortho-Twisted Intercalating Nucleic Acids - TINA. PLoS ONE, 6(6): e20565.