Electrical and Computer Engineering Department, Concordia University, Montreal, Quebec, Canada H3G 1M8.
INRS-Centre Armand-Frappier Santé Biotechnologie, Laval, Quebec, Canada H7V 1B7.
RNA. 2022 Feb;28(2):263-273. doi: 10.1261/rna.078823.121. Epub 2021 Dec 3.
This paper presents a probe comprising a fluorophore and a quencher, enabling measurement of released product from self-cleaving hammerhead ribozyme, without labeled RNA molecules, regular sampling or use of polyacrylamide gels. The probe is made of two DNA strands; one strand is labeled with a fluorophore at its 5'-end, while the other strand is labeled with a quencher at its 3'-end. These two DNA strands are perfectly complementary, but with a 3'-overhang of the fluorophore strand. These unpaired nucleotides act as a toehold, which is utilized by a detached cleaved fragment (coming from a self-cleaving hammerhead ribozyme) as the starting point for a strand displacement reaction. This reaction causes the separation of the fluorophore strand from the quencher strand, culminating in fluorescence, detectable in a plate reader. Notably, the emitted fluorescence is to the amount of detached cleaved-off RNAs, displacing the DNA quencher strand. This method can replace or complement radio-hazardous unstable P as a method of measurement of the product release from ribozyme cleavage reactions; it also eliminates the need for polyacrylamide gels, for the same purpose. Critically, this method allows to distinguish between the total amount of cleaved ribozymes and the amount of fragments, resulting from that cleavage reaction.
本文提出了一种探针，由荧光团和猝灭剂组成，可在无需标记 RNA 分子、定期采样或使用聚丙烯酰胺凝胶的情况下，测量自切割锤头核酶释放的产物。该探针由两条 DNA 链组成；一条链在 5'端标记有荧光团，另一条链在 3'端标记有猝灭剂。这两条 DNA 链完全互补，但荧光团链有 3'突出。这些未配对的核苷酸充当一个结合点，一个从自切割锤头核酶上脱离的切割片段将其作为链置换反应的起点。该反应导致荧光团链与猝灭剂链分离，最终在平板读数器中可检测到荧光。值得注意的是，发出的荧光与脱离的切割 RNA 的量成正比，从而置换了 DNA 猝灭剂链。这种方法可以替代或补充放射性不稳定 P，作为测量核酶切割反应产物释放的方法；它还消除了为达到相同目的使用聚丙烯酰胺凝胶的需要。至关重要的是，该方法允许区分总切割核酶量和来自该切割反应的片段量。
