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para-TINA for Hybridization Probes

para-Twisted Intercalating Nucleic Acid (pTINA) or (S)-1-O-[4-(1-pyrenylethynyl)phenylmethyl]glycerol was introduced by Vyacheslav V. Filichev and Erik B. Pedersen in 2005 and is to date the most powerful melting point increasing molecule developed to stabilize Hoogsteen-type DNA triplexes and G-quadruplex formations(1).

Hoogsteen-type DNA triplexes are characterised by a very high change in melting point (ΔTm) per single nucleotide polymorphism (SNP), which is in contrast to the relatively low ΔTm per SNP seen in antiparallel duplexes. Triplexes are divided into parallel and antiparallel triplexes by the orientation of the Triplex Forming Oligonucleotide (TFO). The TFO of parallel triplexes follow the orientation of the homopurine target strand and consist of CT nucleobase sequences, whereas antiparallel triplexes follow the orientation of the homopyrimidine antiparallel duplex (AD) strand and consist of GT or GA nucleobase sequences. The formation of triplexes is limited by 1) dependence on DNA homopurine stretches in the target sequence, 2) acidic pH for parallel triplexes due to the need of protonated cytosine in the TFO  and 3) quadruplex formation of guanosine-rich TFO in antiparallel triplexes.

Despite the remarkable ΔTm of parallel triplexes, their use is still restricted by the need of DNA homopurine stretches in the target sequence and protonation of cytosine in the TFO at acidic pH. Since incorporation of pTINA decrease the pH dependence of parallel triplexes they are an obvious choice for stabilisation of triplexes, especially since ΔTm of triplexes in general is not changed by introduction of pTINAs in the TFO. One exception from this rule is that pTINA minimize the ΔTm of a SNP, when pTINA in the TFO is placed adjacent to a SNP in the target strand(2).

 

Figure legend: An internally inserted pTINA molecule
 

Optimal position of pTINAs is with 5-7 or 10-14 nucleotides in-between, equaling a half to one helix turn of the DNA double helix (2). By optimal placement of pTINAs, the Tm, and thereby the stability of the parallel triplex, is markedly increased and will most likely enable an increase in sensitivity of clinical assays. Please feel free to contact us, if you plan to use pTINA modified oligonucleotides in your research.

 

(1) Filichev VV and Pedersen EB (2005) Stable and Selective Formation of Hoogsteen-Type Triplexes and Duplexes Using Twisted Intercalating Nucleic Acids (TINA) Prepared via Postsynthetic Sonogashira Solid-Phase Coupling Reactions. J. Am. Chem. Soc., 127 (42), 14849-14858.
 
(2) Schneider UV, Mikkelsen ND, Jøhnk N, Okkels LM, Westh H and Lisby G (2010) Optimal design of parallel triplex forming oligonucleotides containing Twisted Intercalating Nucleic Acids - TINA. Nucleic Acids Research, 38 (13), 4394-4403.