Intercalating nucleic acids containing insertions of 1-O-(1-pyrenylmethyl)glycerol:: stabilisation of dsDNA and discrimination of DNA over RNA
Intercalating nucleic acids containing insertions of 1-O-(1-pyrenylmethyl)glycerol:: stabilisation of dsDNA and discrimination of DNA over RNA
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DOI：
10.1093/nar/gkf624
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发表时间：
2002-11-15
影响因子：
14.9
通讯作者：
Pedersen, EB
中科院分区：
文献类型：
--
作者：
Christensen, UB;Pedersen, EB
我们研究了嵌入剂修饰的寡核苷酸的杂交亲和力和荧光行为。合成了（S）-1-O-（4，4 ′-二甲氧基三苯甲基）-3-O-（1-芘甲基）甘油亚磷酰胺（IPN），并通过标准方法将其插入到7 mer和13 mer寡脱氧核苷酸（ODNs）中，得到插入核酸（INAs）。INA显示出对互补单链DNA（ssDNA）的亲和力大大增加，如通过所形成的DNA/INA双链体的热稳定性所确定的，当IPN作为悬挂末端添加时，每个修饰高达10.9 ℃，当IPN作为凸起插入时，每个修饰高达6.7 ℃。当两个IPN分开至少1bp时，所形成的DNA/INA双链体对在INA链中引入第二个IPN具有积极的稳定作用。两个IPN的间隔越大，效果越明显。与增强的对ssDNA的亲和力相反，IPN降低了对互补单链RNA（ssRNA）的亲和力，当与相应的DNA/INA双链体相比时，对于RNA/INA双链体中的两个IPN插入，产生高达25.8 ℃的解链温度差异。通过这种方式，INA能够区分具有相同序列的ssDNA和ssRNA。荧光测量显示，芘部分与DNA的相互作用比与RNA的相互作用更强，表明插入是DNA/INA双链体中的稳定因子。
We have studied hybridisation affinities and fluorescence behaviour of intercalator-modified oligonucleotides. The phosphoramidite of (S)-1-O-(4, 4'-dimethoxytriphenylmethyl)-3-O-(1-pyrenylmethyl)glycerol, an intercalating pseudo-nucleotide (IPN), was synthesised and by standard methods inserted into 7mer and 13mer oligodeoxyribonucleotides (ODNs) to generate intercalating nucleic acids (INAs). INAs showed greatly increased affinity for complementary single-stranded DNA (ssDNA), as determined by a thermal stabilisation of the formed DNA/INA duplex of up to 10.9degreesC per modification when the IPN was added as a dangling end and up to 6.7degreesC per modification when the IPN was inserted as a bulge. There was a positive stabilisation effect of the formed DNA/INA duplex on introducing a second IPN in the INA strand, when the two IPNs were separated by at least 1 bp. The effect is more pronounced the larger the separation of the two IPNs. Contrary to the enhanced affinity for ssDNA, the IPNs lower the affinity for complementary single-stranded RNA (ssRNA), giving rise to a difference in melting temperature of up to 25.8degreesC for two IPN insertions in an RNA/INA duplex when compared with the corresponding DNA/INA duplex. In this way INA is able to discriminate ssDNA over ssRNA with identical sequences. Fluorescence measurements show a stronger interaction of the pyrene moiety with DNA than with RNA, indicating intercalation as the stabilising factor in DNA/INA duplexes.