Afonin Kirill A, Cieply Dennis J, Leontis Neocles B
Department of Chemistry and Center for Photochemical Sciences, Bowling Green State University, Bowling Green, Ohio 43403, USA.
J Am Chem Soc. 2008 Jan 9;130(1):93-102. doi: 10.1021/ja071516m. Epub 2007 Dec 12.
The paranemic crossover (PX) is a motif for assembling two nucleic acid molecules using Watson-Crick (WC) basepairing without unfolding preformed secondary structure in the individual molecules. Once formed, the paranemic assembly motif comprises adjacent parallel double helices that crossover at every possible point over the length of the motif. The interaction is reversible as it does not require denaturation of basepairs internal to each interacting molecular unit. Paranemic assembly has been demonstrated for DNA but not for RNA and only for motifs with four or more crossover points and lengths of five or more helical half-turns. Here we report the design of RNA molecules that paranemically assemble with the minimum number of two crossovers spanning the major groove to form paranemic motifs with a length of three half turns (3HT). Dissociation constants (Kd's) were measured for a series of molecules in which the number of basepairs between the crossover points was varied from five to eight basepairs. The paranemic 3HT complex with six basepairs (3HT_6M) was found to be the most stable with Kd = 1 x 10-8 M. The half-time for kinetic exchange of the 3HT_6M complex was determined to be approximately 100 min, from which we calculated association and dissociation rate constants ka = 5.11 x 103 M-1s-1 and kd = 5.11 x 10-5 s-1. RNA paranemic assembly of 3HT and 5HT complexes is blocked by single-base substitutions that disrupt individual intermolecular Watson-Crick basepairs and is restored by compensatory substitutions that restore those basepairs. The 3HT motif appears suitable for specific, programmable, and reversible tecto-RNA self-assembly for constructing artificial RNA molecular machines.
平行排列交叉（PX）是一种利用沃森-克里克（WC）碱基配对来组装两个核酸分子的基序，无需展开单个分子中预先形成的二级结构。一旦形成，平行排列组装基序由相邻的平行双螺旋组成，这些双螺旋在基序长度上的每个可能点处交叉。这种相互作用是可逆的，因为它不需要每个相互作用分子单元内部的碱基对变性。平行排列组装已在DNA中得到证实，但在RNA中尚未得到证实，并且仅适用于具有四个或更多交叉点以及五个或更多螺旋半圈长度的基序。在这里，我们报告了RNA分子的设计，这些RNA分子通过跨越主沟的最少两个交叉点进行平行排列组装，形成长度为三个半圈（3HT）的平行排列基序。测量了一系列分子的解离常数（Kd），其中交叉点之间的碱基对数从五个到八个碱基对不等。发现具有六个碱基对的平行排列3HT复合物（3HT_6M）最稳定，Kd = 1×10^-8 M。确定3HT_6M复合物的动力学交换半衰期约为100分钟，由此我们计算出缔合和解离速率常数ka = 5.11×10^3 M^-1s^-1和kd = 5.11×10^-5 s^-1。3HT和5HT复合物的RNA平行排列组装被破坏单个分子间沃森-克里克碱基对的单碱基取代所阻断，并通过恢复这些碱基对的补偿性取代得以恢复。3HT基序似乎适用于特定的、可编程的和可逆的构造RNA自组装，以构建人工RNA分子机器。
