QuantiBact Inc, Department of Clinical Microbiology, Hvidovre Hospital, Kettegaards Alle 30, 2650 Hvidovre, Denmark.
Nucleic Acids Res. 2010 Jul;38(13):4394-403. doi: 10.1093/nar/gkq188. Epub 2010 Mar 24.
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.
扭曲嵌入核酸（TINA）是一种新型嵌入剂和 Hoogsteen 型平行三聚体形成物（PT）的稳定剂。TFO 中 TINA 的位置没有以前提出的特定设计规则。我们根据超过 2500 个通过 FRET 确定的熔点（Tm）描述了一套完整的简单而强大的设计规则。为了提高 PT 的灵敏度，应该在 TFO 之间至少间隔 3 个核苷酸，或者最好在每个半螺旋转弯和/或整个螺旋转弯处放置一个 TINA。我们发现，与反平行双链体（3.8 至 9.4 摄氏度）相比，PT 上碱基错配的ΔTm 非常高（7.4 至 15.2 摄氏度）。当选择较短的 TFO 和较高的 pH 值时，PT 的特异性通过ΔTm 增加。为了增加ΔTm，碱基错配应放置在 TFO 的中心，并且在可行的情况下，应避免 A、C 或 T 到 G 的碱基错配。碱基错配可以通过在碱基错配的每一侧嵌入一个 TINA 来中和，并通过在其 3'（优选）或 5'处嵌入一个 TINA 来掩盖。我们预测 TINA 稳定的 PT 将提高基于 DNA 的临床诊断测定的灵敏度和特异性。
