FRET-FCS as a tool to evaluate the stability of oligonucleotide drugs after intracellular delivery
FRET-FCS as a tool to evaluate the stability of oligonucleotide drugs after intracellular delivery
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DOI：
10.1016/j.jconrel.2004.11.019
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发表时间：
2005-03-02
影响因子：
10.8
通讯作者：
Demeester, J
中科院分区：
文献类型：
--
作者：
Remaut, K;Lucas, B;Demeester, J
研究了单链、双标记寡核苷酸(ONS)3‘端和5’端的荧光共振能量转移(FRET)消失后，单链、双标记ONS的细胞内降解情况。利用双色荧光相关光谱仪(FCS)的超灵敏探测器监测了罗丹明绿激发下的绿色和红色荧光强度。从这种FRET-FCS测量获得的红色和绿色荧光的比率(R/G比)表明，可以提供关于国家统计局完整性的准确信息，而不需要对登记的荧光强度波动进行额外的自相关或交叉相关分析。细胞内测量表明，大多数40聚体磷酸二酯ONS在进入细胞核之前就被降解了。对于20聚磷酸二酯ONS，这种降解发生的速度较慢，完整的和降解的ONS都进入了细胞核。对于20聚硫代ONS，在测量的时间段内没有观察到细胞内降解。通过FCS设置对细胞内荧光的灵敏检测将在预期荧光太低而无法通过FRET成像检测的情况下特别有用，例如通过阳离子载体在细胞内传递ONS之后可能发生的情况。(C)2004爱思唯尔B.V.保留所有权利。
The intracellular degradation of single-stranded, double-labeled oligonucleotides (ONs) was studied by following the disappearance of Fluorescence Resonance Energy Transfer (FRET) between the rhodamine green and Cy5 fluorophores attached to respectively the 3' and 5' end of the ONs. The green and red fluorescence intensities upon rhodamine green excitation were monitored using the ultra-sensitive detectors of a dual-color Fluorescence Correlation Spectroscopy (FCS) instrument. The ratio of the red to green fluorescence (R/G ratio) as obtained from such FRET-FCS measurements showed to give accurate information on the integrity of the ONs, without the need for additional auto- or cross-correlation analysis of the registered fluorescence intensity fluctuations. Intracellular measurements revealed that most of the 40mer phosphodiester ONs were degraded before they entered the nucleus. For the 20mer phosphodiester ONs, this degradation occurred more slowly, and both intact and degraded ONs entered the nucleus. For the 20mer phosphorothioate ONs, no intracellular degradation was observed during the measured time period. The sensitive detection of the intracellular fluorescence by the FCS setup will be particularly useful in situations where the expected fluorescence is too low to be detected by FRET-imaging as may occur after intracellular delivery of ONs by cationic carriers. (c) 2004 Elsevier B.V. All rights reserved.