Pontius B W, Berg P
Biochemistry Department, Stanford University Medical Center, CA 94305.
Proc Natl Acad Sci U S A. 1991 Sep 15;88(18):8237-41. doi: 10.1073/pnas.88.18.8237.
The rate of renaturation for complementary DNA strands can be enhanced greater than 10(4)-fold by the addition of simple cationic detergents, and the reaction is qualitatively and quantitatively very similar to that found with purified heterogeneous nuclear ribonucleoprotein A1 protein. Under optimal conditions, renaturation rates are greater than 2000-fold faster than reactions run in 1 M NaCl at 68 degrees C. The reaction is second-order with respect to DNA concentration, and reaction rates approach or equal the rate with which complementary strands are expected to encounter each other in solution. Renaturation can even be observed well above the expected melting temperature of the duplex DNA, demonstrating that some cationic detergents have DNA double-helix-stabilizing properties. The reaction is also extremely rapid in the presence of up to a 10(6)-fold excess of noncomplementary sequences, establishing that renaturation is specific and relatively independent of heterologous DNA. This finding also implies that up to several thousand potential target sequences can be sampled per strand per second. Such reagents may be useful for procedures that require rapid nucleic acid renaturation, and these results suggest ways to identify and design other compounds that increase the kinetics of association reactions. Moreover, this work provides further support for a model relating the existence of flexible, weakly interacting, repeating domains to their function in rapid molecular assembly processes in vitro and in vivo.
通过添加简单的阳离子去污剂,互补DNA链的复性速率可提高超过10^4倍,并且该反应在定性和定量上与用纯化的不均一核核糖核蛋白A1蛋白所发现的反应非常相似。在最佳条件下,复性速率比在68℃的1M NaCl中进行的反应快2000倍以上。该反应对DNA浓度呈二级反应,反应速率接近或等于互补链在溶液中相互相遇的速率。甚至在远高于双链DNA预期解链温度的情况下也能观察到复性,这表明一些阳离子去污剂具有稳定DNA双螺旋的特性。在存在高达10^6倍过量的非互补序列的情况下,反应也极其迅速,这表明复性是特异性的,并且相对独立于异源DNA。这一发现还意味着每条链每秒可以对多达数千个潜在靶序列进行采样。此类试剂可能对需要快速核酸复性的程序有用,并且这些结果提示了鉴定和设计其他可提高缔合反应动力学的化合物的方法。此外,这项工作为一个模型提供了进一步的支持,该模型将灵活的、弱相互作用的重复结构域的存在与其在体外和体内快速分子组装过程中的功能联系起来。