Quantitative hybridization kinetics of DNA probes to RNA in solution followed by diffusional fluorescence correlation analysis
Quantitative hybridization kinetics of DNA probes to RNA in solution followed by diffusional fluorescence correlation analysis
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DOI：
10.1021/bi960517g
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发表时间：
1996-08-06
期刊：
影响因子：
2.9
通讯作者：
Walter, NG
中科院分区：
文献类型：
--
作者：
Schwille, P;Oehlenschlager, F;Walter, NG
用荧光相关光谱法（FCS）研究了6种N，N，N 'N'-四甲基-5-羧基罗丹明标记的寡脱氧核糖核苷酸探针与含有HIV-1逆转录酶引物结合位点的101聚体靶RNA在溶液中的结合动力学。FCS允许在选择的缓冲液中灵敏的、非放射性的真实的时间观察探针与RNA靶的杂交，而不分离游离和结合的探针。结合过程可以通过17 mer至37 mer DNA探针与较大RNA靶特异性杂交后的翻译扩散时间的变化来直接监测。通过直径为0.5 μ m的激光照射开放体积元件的特征扩散时间从0.13-0.2 ms（自由）增加到0.37-0.50 ms（结合），这取决于探针。杂交近似为双相不可逆二级反应动力学，对于不同的探针，第一相结合速率常数在3 x 10（4）和1.5 x 10（6）M（-1）s（-1）之间。这些不同的初始速率反映了探针和靶位点的二级结构，与从接吻复合物中的环-环相互作用开始的假设结合途径一致，并且完成杂交需要涉及探针和靶的单链区域的额外相互作用。因此，FCS允许以低的探针和靶标消耗快速筛选针对重要靶标如HIV-1 RNA的合适的反义核酸。
Binding kinetics in solution of six N,N,N'N'-tetramethyl-5-carboxyrhodamine-labeled oligodeoxyribonucleotide probes to a 101mer target RNA comprising the primer binding site for HIV-1 reverse transcriptase were characterized using fluorescence correlation spectroscopy (FCS). FCS allows a sensitive, non-radioactive real time observation of hybridization of probes to the RNA target in the buffer of choice without separation of free and bound probe. The binding process could directly be monitored by the change in translational diffusion time of the 17mer to 37mer DNA probe upon specific hybridization with the larger RNA target. The characteristic diffusion time through a laser-illuminated open volume element with 0.5 mu m in diameter increased from 0.13-0.2 ms (free) to 0.37-0.50 ms (bound), depending an the probe. Hybridization was approximated by biphasic irreversible second-order reaction kinetics, yielding first-phase association rate constants between 3 x 10(4) and 1.5 x 10(6) M(-1) s(-1) for the different probes. These varying initial rates reflected the secondary structures of probes and target sites, being consistent with a hypothetical binding pathway starting from loop-loop interactions in a kissing complex, and completion of hybridization requiring an additional interaction involving single-stranded regions of both probe and target. FCS thus permits rapid screening for suitable antisense nucleic acids directed against an important target like HIV-1 RNA with low consumption of probes and target.