Batey Robert T, Kieft Jeffrey S
Department of Chemistry and BioChemistry, University of Colorado, Boulder, CO 80309-0215, USA.
RNA. 2007 Aug;13(8):1384-9. doi: 10.1261/rna.528007. Epub 2007 Jun 4.
RNA biochemical or structural studies often require an RNA sample that is chemically pure, and most protocols for its in vitro production use denaturing polyacrylamide gel electrophoresis to achieve this. Unfortunately, many RNAs do not quantitatively refold into an active conformation after denaturation, creating significant problems for downstream characterization or use. In addition, this traditional purification method is not amenable to studies demanding high-throughput RNA production. Recently, we presented the first general method for producing almost any RNA sequence that employs an affinity tag that is removed during the purification process. Because technical difficulties prevented application of this method to many RNAs, we have developed an improved version that utilizes a different activatable ribozyme and affinity tag that are considerably more robust, rapid, and broadly applicable.
RNA的生化或结构研究通常需要化学纯的RNA样本，并且大多数体外生产RNA的方案都使用变性聚丙烯酰胺凝胶电泳来实现这一目标。不幸的是，许多RNA在变性后不能定量地重新折叠成活性构象，给下游的表征或使用带来了重大问题。此外，这种传统的纯化方法不适用于需要高通量RNA生产的研究。最近，我们提出了第一种生产几乎任何RNA序列的通用方法，该方法采用了一种在纯化过程中被去除的亲和标签。由于技术困难阻碍了该方法在许多RNA上的应用，我们开发了一种改进版本，它使用了一种不同的可激活核酶和亲和标签，这些标签更加稳健、快速且具有广泛的适用性。
