New England BioLabs, Ipswich, MA 01938, USA.
Biotechniques. 2010 Jun;48(6):xvii-xxiii. doi: 10.2144/000113364.
p19 RNA binding protein from the Carnation Italian ringspot virus (CIRV) is an RNA-silencing suppressor that binds small interfering RNA (siRNA) with high affinity. We created a bifunctional p19 fusion protein with an N-terminal maltose binding protein (MBP), for protein purification, and a C-terminal chitin binding domain (CBD) to bind p19 to chitin magnetic beads. The fusion protein binds dsRNAs in the size range of 20-23 nucleotides, but does not bind ssRNA or dsDNA. Relative affinities of the p19 fusion protein for different-length RNA and DNA substrates were determined. Binding specificity of the p19 fusion protein for small dsRNA allows detection of miRNA:RNA probe duplexes. Using radioactive RNA probes, we were able to detect low levels of miRNAs in the sub-femtomole range and in the presence of a million-fold excess of total RNA. Detection is linear over three logs. Unlike most nucleic acid detection methods, p19 selects for RNA hybrids of correct length and structure. Rules for designing optimal RNA probes for p19 detection of miRNAs were determined by in vitro binding of 18 different dsRNA oligos to p19. These studies demonstrate the potential of p19 fusion protein to detect miRNAs and isolate endogenous siRNAs.
香石竹斑驳病毒（CIRV）的 p19 RNA 结合蛋白是一种 RNA 沉默抑制子，能高亲和力地结合小干扰 RNA（siRNA）。我们构建了一个具有 N 端麦芽糖结合蛋白（MBP）的两用 p19 融合蛋白，用于蛋白纯化，以及 C 端几丁质结合域（CBD），以将 p19 结合到几丁质磁珠上。融合蛋白可结合大小在 20-23 个核苷酸范围内的 dsRNA，但不结合 ssRNA 或 dsDNA。测定了 p19 融合蛋白对不同长度 RNA 和 DNA 底物的相对亲和力。p19 融合蛋白对小 dsRNA 的结合特异性允许 miRNA:RNA 探针双链的检测。使用放射性 RNA 探针，我们能够在存在百万倍过量总 RNA 的情况下，在亚 femtomole 范围内检测到低水平的 miRNA。检测呈线性，对数范围达三个数量级。与大多数核酸检测方法不同，p19 选择正确长度和结构的 RNA 杂交体。通过体外结合 18 种不同的 dsRNA 寡核苷酸到 p19，确定了用于 p19 检测 miRNA 的最佳 RNA 探针设计规则。这些研究表明，p19 融合蛋白具有检测 miRNA 和分离内源性 siRNA 的潜力。
