The chemical cross-linking of complexes of proteins with nucleic acids is often used in structural and mechanistic studies of these oftentimes unstable and transient complexes. To date, no method has been reported for the thiol-based conjugation of proteins with an RNA backbone, mainly because of instability of the modified ribonucleic acid that is functionalized at the phosphodiester and its rapid hydrolysis. Here, we report the site-specific synthesis of stable RNA oligonucleotides with a thiol-bearing linker that was attached to the phosphodiester backbone, where the ribonucleotide at the cross-linking site was either replaced with 2′-deoxy- or 2′-fluororibonucleotide. The utility of this approach was validated in cross-linking tests with RNase H1, a model protein for RNA/DNA binding and key effector in DNA-like antisense drug therapy. Furthermore, scale-up cross-linking and purification of the complexes confirmed that the method is useful for obtaining preparations of protein-RNA/DNA complexes with purity and stability that are suitable for further biochemical and structural studies. The present approach broadens the repertoire of disulfide-based cross-linking strategies and is a novel tool for the stabilization of protein-RNA complexes in which the interaction occurs via the RNA backbone. This methodology may be broadly applicable to studies of otherwise unstable or transient complexes of proteins with RNA and RNA/DNA.

蛋白质复合物与核酸的化学交联通常用于这些经常不稳定和短暂的复合物的结构和机理研究中。迄今为止，尚未报道将蛋白质与RNA主链进行基于硫醇的缀合的方法，这主要是由于在磷酸二酯中官能化的修饰核糖核酸的不稳定性及其快速水解。在这里，我们报告稳定的RNA寡核苷酸的位点特异性合成，该寡核苷酸带有连接到磷酸二酯主链上的带有硫醇的连接子，其中交联位点的核糖核苷酸被2'-脱氧或2'-氟核糖核苷酸取代。该方法的实用性已在与RNase H1的交联测试中得到验证，RNase H1是RNA / DNA结合的模型蛋白，是类似DNA的反义药物治疗中的关键效应物。此外，复合物的放大交联和纯化证实了该方法可用于获得具有纯度和稳定性的蛋白质-RNA / DNA复合物的制备物，其适合于进一步的生物化学和结构研究。本方法拓宽了基于二硫键的交联策略的范围，并且是用于稳定蛋白质-RNA复合物的新工具，其中相互作用通过RNA主链发生。该方法可广泛应用于研究蛋白质与RNA和RNA / DNA的不稳定或瞬时复合物。本方法拓宽了基于二硫键的交联策略的范围，并且是用于稳定蛋白质-RNA复合物的新工具，其中相互作用通过RNA主链发生。该方法可广泛应用于研究蛋白质与RNA和RNA / DNA的不稳定或瞬时复合物。本方法拓宽了基于二硫键的交联策略的范围，并且是用于稳定蛋白质-RNA复合物的新工具，其中相互作用通过RNA主链发生。该方法可广泛应用于研究蛋白质与RNA和RNA / DNA的不稳定或瞬时复合物。