Complex (RLC) containing double stranded siRNA, RNA binding protein (known as R2D2), and Dicer (Dcr-2) acts to identify siRNA asymmetry, recognize guide versus passenger strand, initiate unwinding of siRNA, and associate the guide strand with Argonaute. Previous studies in vitro have shown that recombinant Dcr-2/R2D2 heterodimer or Dcr-2 alone cannot catalyze siRNA unwinding as it binds to the RLC. Studies were carried out using Drosophila embryo lysates, and by photo-crosslinking a series of 5IU labeled siRNAs, it was shown that fully assembled RLC initiates unwinding of bound siRNA through an ordered stepwise process. Previous researchers found that photo-crosslinking of siRNA to RISC was highly dependent on the position of 5IU in the sequence.910463-68-2 SMILES 6 In this work, siRNAs 1 and 2 were used to target the luciferase gene (Table 1), where red U indicates the presence of 5IU and asterisks show the position of 32P. Labeled siRNA was pre-incubated with lysate in microwells and then photo-cross-linked by irradiation for 105 min at 302 nm using an inverted transilluminator.
Figure 2. Cross-linking to RBP or Dicer-2
PAGE autoradiography showed that R2D2 was more efficiently cross-linked to oligo when 5IU was on the siRNA antisense strand. Alternately, when 5IU was on the sense strand, it more efficiently cross-linked to Dicer-2 (Figure 2). Thus, Dicer-2 is adjacent to this siRNA sense strand’s 3′-end on entering the RISC. Additional siRNAs a through g were prepared to target human superoxide dismutase gene (sod1), where the first listed, siRNA a, is nearly symmetric, while the remainder, b through g, are highly asymmetric due to a 5′-base mismatch (Table 2).80451-05-4 IUPAC Name Note that when there is a 5′-base mismatch, that strand is loaded into the RISC nearly exclusively.PMID:31078607 Thus b-d have a single mismatch at the 5′-end of the upper strand, while e-g have a single mismatch at the 5′-end of the lower strand (Figure 3, C).
In duplexes b and c, siRNA b contains 5IU near the 3′-end of the antisense strand (one base in from the terminus), while in duplex c, 5IU is positioned next to the 3′-end of the sense strand, respectively. The siRNA d contains both phosphorylated strands of b and c. The gel reveals that siRNA b crosslinks predominantly to Dcr-2 and contains only double-stranded RNA (Figure 3, D, lane b). In contrast, siRNA c crosslinks to RBP and to Argonaute protein, and not at all to Dcr-2. It also contains significant single-stranded RNA, due to siRNA unwinding. siRNA d crosslinks to proteins with a combination of the results in b and c, which is expected since 5IU is present in both termini of corresponding b and c strands. The reader can follow the gel data in the similar series of siRNAs e, f, and g, in which asymmetry of the single 5′-mismatch is positioned on the opposite strand to siRNAs
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Table 2. siRNAs Targeting Human sod1
b, c, and d, while keeping the 5IU at the corresponding location, respectively, adjacent to the terminus of each corresponding oligo. The data are consistent with analogous positioning of the duplex and the less-stable mismatched siRNA end preferentially loading into the RISC. The authors propose that “R2D2 orients the Dcr-2/R2D2 heterodimer on the siRNA within the RLC. As siRNA unwinding proceeds, the heterodimer is exchanged for Ago2, the core component of the RISC,” and siRNA unwinding only occurs when the RISC, containing Argonaute, is assembled. Further studies support and expand these conclusions and readers are directed to recent revie.MedChemExpress (MCE) offers a wide range of high-quality research chemicals and biochemicals (novel life-science reagents, reference compounds and natural compounds) for scientific use. We have professionally experienced and friendly staff to meet your needs. We are a competent and trustworthy partner for your research and scientific projects.Related websites: https://www.medchemexpress.com
