With the goal of expanding the very limited toolkit of cross-linking agents available for nucleic acids and their protein complexes, we evaluated the merits of a wide range of bifunctional agents that may be capable of reacting with the functional groups characteristic of these types of biopolymers. The survey specifically focused on the ability of test reagents to produce desirable inter-molecular conjugates, which could reveal the identity of interacting components and the position of mutual contacts, while also considering a series of practical criteria for their utilization as viable nucleic acid probes. The survey employed models consisting of DNA, RNA, and corresponding protein complexes to mimic as close as possible typical applications. Denaturing polyacrylamide gel electrophoresis (PAGE) and mass spectrometric (MS) analyses were implemented in concert to monitor the formation of the desired conjugates. In particular, the former was used as a rapid and inexpensive tool for the efficient evaluation of cross-linker activity under a broad range of experimental conditions. The latter was applied after preliminary rounds of reaction optimization to enable full-fledged product characterization and, more significantly, differentiation between mono-functional and intra- versus inter-molecular conjugates. This information provided the feedback necessary to further optimize reaction conditions and explain possible outcomes. Among the reagents tested in the study, platinum complexes and nitrogen mustards manifested the most favorable characteristics for practical cross-linking applications, whereas other compounds provided inferior yields, or produced rather unstable conjugates that did not survive the selected analytical conditions. The observed outcomes will help guide the selection of the most appropriate cross-linking reagent for a specific task, whereas the experimental conditions described here will provide an excellent starting point for approaching these types of applications. As a whole, the results of the survey clearly emphasize that finding a universal reagent, which may afford excellent performance with all types of nucleic acid substrates, will require extending the exploration beyond the traditional chemistries employed to modify the constitutive functional groups of these vital biopolymers.

中文翻译：

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基于质谱的核酸和蛋白质-核酸组装研究的双功能交联方法

为了扩展可用于核酸及其蛋白质复合物的非常有限的交联剂工具包，我们评估了各种双功能试剂的优点，这些试剂可能能够与这些类型的生物聚合物的特征官能团反应. 该调查特别关注测试试剂产生理想分子间偶联物的能力，这可以揭示相互作用成分的身份和相互接触的位置，同时还考虑了一系列将其用作可行核酸探针的实用标准。该调查采用由 DNA、RNA 和相应蛋白质复合物组成的模型来模拟尽可能接近的典型应用。同时进行变性聚丙烯酰胺凝胶电泳 (PAGE) 和质谱 (MS) 分析以监测所需缀合物的形成。特别是，前者被用作在广泛的实验条件下有效评估交联剂活性的快速且廉价的工具。后者在初步反应优化后应用，以实现全面的产品表征，更重要的是，区分单功能和分子内与分子间共轭物。该信息为进一步优化反应条件和解释可能的结果提供了必要的反馈。在研究中测试的试剂中，铂络合物和氮芥表现出最适合实际交联应用的特性，而其他化合物的产率较低，或者产生了相当不稳定的偶联物，无法在选定的分析条件下存活。观察到的结果将有助于指导为特定任务选择最合适的交联试剂，而此处描述的实验条件将为接近这些类型的应用提供一个极好的起点。总的来说，调查结果清楚地强调，要找到一种通用试剂，可以对所有类型的核酸底物提供出色的性能，需要将探索扩展到用于修饰这些重要生物聚合物的组成官能团的传统化学之外。 . 或产生相当不稳定的偶联物，无法在选定的分析条件下存活。观察到的结果将有助于指导为特定任务选择最合适的交联试剂，而此处描述的实验条件将为接近这些类型的应用提供一个极好的起点。总的来说，调查结果清楚地强调，要找到一种通用试剂，可以对所有类型的核酸底物提供出色的性能，需要将探索扩展到用于修饰这些重要生物聚合物的组成官能团的传统化学之外。 . 或产生相当不稳定的偶联物，无法在选定的分析条件下存活。观察到的结果将有助于指导为特定任务选择最合适的交联试剂，而此处描述的实验条件将为接近这些类型的应用提供一个极好的起点。总的来说，调查结果清楚地强调，要找到一种通用试剂，可以对所有类型的核酸底物提供出色的性能，需要将探索扩展到用于修饰这些重要生物聚合物的组成官能团的传统化学之外。 . 而这里描述的实验条件将为接近这些类型的应用提供一个很好的起点。总的来说，调查结果清楚地强调，要找到一种通用试剂，可以对所有类型的核酸底物提供出色的性能，需要将探索扩展到用于修饰这些重要生物聚合物的组成官能团的传统化学之外。 . 而这里描述的实验条件将为接近这些类型的应用提供一个很好的起点。总的来说，调查结果清楚地强调，要找到一种通用试剂，可以对所有类型的核酸底物提供出色的性能，需要将探索扩展到用于修饰这些重要生物聚合物的组成官能团的传统化学之外。 .