Kinetics of spontaneous displacement of RNA from heteroduplexes by DNA.
Kinetics of spontaneous displacement of RNA from heteroduplexes by DNA.
复制标题
DNA 从异源双链体中自发置换 RNA 的动力学。
DOI：
10.1093/nar/24.16.3246
复制
发表时间：
1996
影响因子：
14.9
通讯作者：
Sigman,DS
中科院分区：
文献类型：
--
作者：
Landgraf,R;Ramamurthi,KS;Sigman,DS
我们已经使用R环的形成和直接杂交技术来分析动力学，其中RNA是由相同序列的DNA从异源双链体置换。使用随机行走模拟，我们能够计算单个位移反应的步长。对于GC含量为57-60%的RNA，数据表明RNA交换概率为50.06%，这表明与镁存在下的DNA双链体相比，异源双链体适度不稳定。单核苷酸可逆交换的平均步骤时间为345.0（± 1.3）ms/步。在不存在镁的情况下观察到置换反应的加速。与延伸的步骤时间的比较显示，在以下两种条件下，RNA置换不会限制转录延伸的速率：（i）如果从新合成的异源双链体中消除镁;（ii）如果置换通过结合出现的RNA保持在仅正向交换模式。Distamycin是一种小沟结合药物，作为RNA置换的“催化剂”非常有效。这种效应可能是由于偏端霉素优先结合DNA双链体的小沟而不是异源双链体。因此，该动力学测定可用作用于确定核酸配体的结合偏好的方便测定。
We have used R-loop formation and direct hybridization techniques to analyze the kinetics by which RNA is displaced from a heteroduplex by DNA of identical sequence. Using random walk simulations we were able to calculate the step times for a single displacement reaction. For RNA with a GC content of 57–60% the data indicate an RNA exchange probability of 50.06%, which is indicative of a modest destabilization of the hetero-duplex compared with a DNA duplex in the presence of magnesium. The average step time for the reversible exchange of a single nucleotide is 345.0 (± 1.3) ms/step. An acceleration of the displacement reaction was observed in the absence of magnesium. A comparison with step times for elongation shows that RNA displacement would not be rate limiting to transcription elongation under two conditions: (i) if magnesium is eliminated from the newly synthesized heteroduplex; (ii) if displacement is kept in a forward only exchange mode through binding of the emerging RNA. Distamycin, a minor groove binding drug, is very effective as a ‘catalyst’ of RNA displacement. This effect is likely to be due to preferential binding of distamycin to the minor groove of the DNA duplex as opposed to the hetero-duplex. This kinetic assay could therefore serve as a convenient assay for the determination of binding preferences of nucleic acid ligands.