A SYBR Gold-based Label-free in vitro Dicing Assay

Background

Dicer family endonucleases play critical roles in small RNA (sRNA) biogenesis by cutting the substrate double-stranded RNA (dsRNA) into product dsRNA with defined lengths, of approximately 19–30 bp (Chen et al., 2010; Borges and Martienssen, 2015). The substrate recognition of Dicer depends on the termini features of the dsRNA. Typically, Dicers prefer substrate dsRNA, with the guide strand possessing 5’-phosphate, and the complementary strand possessing a 1- or 2-nt 3’-overhang. The cutting of the two RNA strands is simultaneously conducted by the paired RNaseIII domains, towards the 3’ of the guide strand and the 5’ of the complementary strand, yielding asymmetric dicing. Depending on the different 3’-overhang lengths, the two strands of the product dsRNA could be with the same length or with minor differences in length (usually, only ~1–2-nt). In addition, the cleavage efficiencies of different Dicers vary considerably (Fukudome et al., 2011; Hung and Slotkin, 2021). Therefore, a bench-based simple and easy-to-use method to monitor both the lengths and quantity of the single-stranded RNA (ssRNA) strands of the siRNA duplex is required. The method should be efficient in separating different 19–30-nt ssRNAs with single base resolution, and could be adapted to a quantifying system. In past decades, the dicing assay was usually conducted by using phosphorus-32 (³²P) to label the RNA termini, denaturing urea polyacrylamide gel electrophoresis (urea PAGE) to separate ssRNA strands, and autoradiography to visualize the ssRNA on PAGE, which is expensive, hazardous, and time-consuming (Adachi and Yu, 2014). To prevent the hazards linked to ³²P and to make the assay more convenient in a small-scale lab, we developed this label-free, fluorescent dye SYBR Gold-based in vitro dicing assay, based on several previously published protocols (Tuma et al., 1999; Seo et al., 2019). In this protocol, we firstly listed the details for the dicing reaction (Nagano et al., 2014), using a plant dicer called DICER-LIKE PROTEIN 3 (DCL3), and for running urea PAGE (Adachi and Yu, 2014), which employs 6–8 M urea to denature secondary RNA structures, enables the distribution of the ssRNA on the gel according to molecular weight (Dodd and Hudson, 2007; Ellington and Chory, 2001), and is convenient for monitoring the length differences in ssRNAs (Summer et al., 2009; Petrov et al., 2013; Green and Sambrook, 2021). The bands of the ssRNAs are further stained by SYBR Gold, which is a label-free fluorescent dye (Tuma et al., 1999; Seo et al., 2019), scanned by an image recorder, quantified using the ImageJ software (Seo et al., 2019; Gassmann et al., 2009; Schneider et al., 2012), and statistically analyzed by the Origin 7.0 software. Considering the conserved mechanism of Dicer family enzymes and length distributions of sRNA, the protocol presented here can be adapted to dicing assays for most Dicer proteins, as well as other analysis regarding the length and quantification of sRNAs.