Departmens of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
J Biol Chem. 2012 May 25;287(22):18843-53. doi: 10.1074/jbc.M111.316596. Epub 2012 Mar 26.
Most cellular RNAs engage in intrastrand base-pairing that gives rise to complex three-dimensional folds. This self-pairing presents an impediment toward binding of the RNA by nucleic acid-based ligands. An important step in the discovery of RNA-targeting ligands is therefore to identify those regions in a folded RNA that are accessible toward the nucleic acid-based ligand. Because the folding of RNA targets can involve interactions between nonadjacent regions and employ both Watson-Crick and non-Watson-Crick base-pairing, screening of candidate binder ensembles is typically necessary. Microarray-based screening approaches have shown great promise in this regard and have suggested that achieving complete sequence coverage would be a valuable attribute of a next generation system. Here, we report a custom microarray displaying a library of RNA-interacting polynucleotides comprising all possible 2'-OMe RNA sequences from 4- to 8-nucleotides in length. We demonstrate the utility of this array in identifying RNA-interacting polynucleotides that bind tightly and specifically to the highly conserved, functionally essential template/pseudoknot domain of human telomerase RNA and that inhibit telomerase function in vitro.
大多数细胞 RNA 都会进行链内碱基配对，从而形成复杂的三维折叠。这种自我配对会阻碍 RNA 与基于核酸的配体结合。因此，发现靶向 RNA 的配体的重要步骤是确定折叠 RNA 中那些可接近基于核酸的配体的区域。由于 RNA 靶标的折叠可能涉及非相邻区域之间的相互作用，并采用 Watson-Crick 和非 Watson-Crick 碱基配对，因此通常需要筛选候选结合物的集合。基于微阵列的筛选方法在这方面显示出巨大的潜力，并表明实现完全序列覆盖将是下一代系统的有价值的属性。在这里，我们报告了一种定制的微阵列，该阵列显示了一个由 RNA 相互作用多核苷酸组成的文库，这些多核苷酸包含长度为 4 至 8 个核苷酸的所有可能的 2'-OMe RNA 序列。我们证明了该阵列在识别与人类端粒酶 RNA 的高度保守、功能必需的模板/假结结构域紧密且特异性结合的 RNA 相互作用多核苷酸，并在体外抑制端粒酶功能方面的效用。
