Hybridization-sensitive fluorescent DNA probe with self-avoidance ability
Hybridization-sensitive fluorescent DNA probe with self-avoidance ability
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DOI：
10.1039/b917321h
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发表时间：
2010-01-01
影响因子：
3.2
通讯作者：
Okamoto, Akimitsu
中科院分区：
文献类型：
--
作者：
Ikeda, Shuji;Kubota, Takeshi;Okamoto, Akimitsu
杂交敏感的荧光探针具有固有的缺点：探针的自二聚化防止在与靶杂交之前荧光淬灭，导致高背景信号。为了避免探针的自身二聚化，我们集中于由2 '-脱氧肌苷（I）和N-4-乙基-2'-脱氧胞苷（E）形成的碱基对。与I/E碱基对相比，I和E碱基分别与胞嘧啶和鸟嘌呤形成更稳定的碱基对。制备了一种新的杂交敏感的荧光探针IE探针，该探针含有三个非天然核苷酸I、E和作为双噻唑橙标记核苷酸的D-514。IE探针具有低的热稳定性，足以避免自二聚化。IE探针的吸收光谱显示出最大吸收的杂交依赖性位移，表明激发子相互作用在探针中的噻唑橙子染料之间起作用。IE探针的染料间激子相互作用是非常有效的，因此，鸟嘌呤和胞嘧啶与I和E的替换提高了杂交前后的荧光强度的比率（I-杂交/I-非杂交）。尽管当与D-514相邻的两个或一个碱基是E时，在与靶序列杂交后观察到几个IE探针的荧光强度显著减弱，但当与D-514相邻的任何E被胞嘧啶取代时，观察到杂交体的荧光强度显著恢复。通过掺入I和E改善I-杂交/I-非杂交值有助于设计用于mRNA成像的长探针序列。
Hybridization-sensitive fluorescent probes have an inherent disadvantage: self-dimerization of the probe prevents the fluorescence quenching prior to hybridization with the target, resulting in a high background signal. To avoid self-dimerization of probes, we focused on a base pair formed by 2'-deoxyinosine (I) and N-4-ethyl-2'-deoxycytidine (E). I and E bases form more stable base pairs with cytosine and guanine, respectively, compared with an I/E base pair. New hybridization-sensitive fluorescent probes, IE probes, were prepared containing three unnatural nucleotides, I, E and D-514 as a doubly thiazole orange-labeled nucleotide. The IE probes had low thermostability, sufficient to avoid self-dimerization. Absorption spectra of the IE probes exhibited a hybridization-dependent shift of the absorption maximum, suggesting that excitonic interaction was working between the thiazole orange dyes in the probe. Interdye excitonic interaction of IE probes was very effective; thus, replacement of guanine and cytosine with I and E improved the ratio of fluorescence intensities after and before hybridization (I-hybrid/I-nonhybrid). Although a significant weakness in fluorescence intensity was observed for several IE probes after hybridization with the target sequence when both or either of the bases adjacent to D-514 is E, a dramatic recovery of the fluorescence intensity of hybrids was observed when any E adjacent to D-514 was replaced with cytosine. Improvement of the I-hybrid/I-nonhybrid value by incorporation of I and E helped the design of a long probe sequence for mRNA imaging.