INTRACELLULAR OLIGONUCLEOTIDE HYBRIDIZATION DETECTED BY FLUORESCENCE RESONANCE ENERGY-TRANSFER (FRET)
INTRACELLULAR OLIGONUCLEOTIDE HYBRIDIZATION DETECTED BY FLUORESCENCE RESONANCE ENERGY-TRANSFER (FRET)
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DOI：
10.1093/nar/22.4.662
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发表时间：
1994-02-25
影响因子：
14.9
通讯作者：
CHIN, DJ
中科院分区：
文献类型：
--
作者：
SIXOU, S;SZOKA, FC;CHIN, DJ
荧光共振能量转移（FRET）技术用于研究微注射寡核苷酸（ODNs）进入活细胞后杂化的形成和解离。合成了一个28聚磷酸二酯ODN (+ PD)，并用3'罗丹明（+ PD- r）标记。互补的反义5'-荧光素标记的硫代ODN （- PT-F）通过添加+ PD-R特异性猝灭。在溶液中，通过吸光度和FRET检测，- PT-F/+ PD-R杂化物的变性温度为65 +/- 3℃。在17 μ M下，odn之间的杂交在1分钟内发生，并且不受非特异性DNA存在的明显影响。在未标记的互补ODN超过300倍的情况下，预形成的杂交体缓慢解离（T-1/2约为3小时），并可被DNAse i降解。将微注射到细胞细胞质中后，预形成的荧光杂交体解离的半衰期为15分钟，这归因于磷酸二酯的降解。通过FRET检测，顺序注射的odn在细胞质中短暂形成，随后在细胞核中形成，几乎所有注射的odn都在细胞核中积累。这表明反义odn可以在细胞质和细胞核中与外源的细胞内靶标杂交。
Fluorescence resonance energy transfer (FRET) was used to study hybrid formation and dissociation after microinjection of oligonucleotides (ODNs) into living cells. A 28-mer phosphodiester ODN (+ PD) was synthesized and labeled with a 3' rhodamine (+ PD-R). The complementary, antisense 5'-fluorescein labeled phosphorothioate ODN (- PT-F) was specifically quenched by addition of the + PD-R. In solution, the - PT-F/+ PD-R hybrid had a denaturation temperature of 65 +/- 3 degrees C detected by both absorbance and FRET. Hybridization between the ODNs occurred within 1 minute at 17 mu M and was not appreciably affected by the presence of non-specific DNA. The pre-formed hybrid slowly dissociated (T-1/2 approximate to 3 h) in the presence of a 300-fold excess of the unlabeled complementary ODN and could be degraded by DNAse I. Upon microinjection into the cytoplasm of cells, pre-formed fluorescent hybrids dissociated with a half-time of 15 minutes, which is attributed to the degradation of the phosphodiester. Formation of the hybrid from sequentially injected ODNs was detected by FRET transiently in the cytoplasm and later in the cell nucleus, where nearly all injected ODNs accumulate. This suggests that antisense ODNs can hybridize to an intracellular target, of exogenous origin in these studies, in both the cytoplasm and the nucleus.