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Optimal design of parallel triplex forming oligonucleotides containing Twisted Intercalating Nucleic Acids--TINA.

Nucleic acids research (2010-03-27)
Uffe V Schneider, Nikolaj D Mikkelsen, Nina Jøhnk, Limei M Okkels, Henrik Westh, Gorm Lisby
ABSTRACT

Twisted intercalating nucleic acid (TINA) is a novel intercalator and stabilizer of Hoogsteen type parallel triplex formations (PT). Specific design rules for position of TINA in triplex forming oligonucleotides (TFOs) have not previously been presented. We describe a complete collection of easy and robust design rules based upon more than 2500 melting points (T(m)) determined by FRET. To increase the sensitivity of PT, multiple TINAs should be placed with at least 3 nt in-between or preferable one TINA for each half helixturn and/or whole helixturn. We find that Delta T(m) of base mismatches on PT is remarkably high (between 7.4 and 15.2 degrees C) compared to antiparallel duplexes (between 3.8 and 9.4 degrees C). The specificity of PT by Delta T(m) increases when shorter TFOs and higher pH are chosen. To increase Delta Tms, base mismatches should be placed in the center of the TFO and when feasible, A, C or T to G base mismatches should be avoided. Base mismatches can be neutralized by intercalation of a TINA on each side of the base mismatch and masked by a TINA intercalating direct 3' (preferable) or 5' of it. We predict that TINA stabilized PT will improve the sensitivity and specificity of DNA based clinical diagnostic assays.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Atto 495 NHS ester, BioReagent, suitable for fluorescence, ≥90% (coupling to amines)
Sigma-Aldrich
Atto 590-Biotin, BioReagent, suitable for fluorescence, ≥90.0% (HPCE)
Supelco
Atto 590 NHS ester, BioReagent, suitable for fluorescence, ≥60% (coupling to amines)
Sigma-Aldrich
Atto 495, BioReagent, suitable for fluorescence, ≥98.0% (HPCE)
Sigma-Aldrich
Atto 495 maleimide, BioReagent, suitable for fluorescence
Sigma-Aldrich
Atto 590, BioReagent, suitable for fluorescence, ≥90% (HPCE)

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