Properties of short double-stranded RNAs carrying randomized base pairs: toward better controls for RNAi experiments

Abstract

Short interfering RNAs (siRNAs) are mediators of RNA interference (RNAi), a commonly used technique for selective downregulation of target gene expression. Using an equimolar mixture of A, G, C, and U phosphoramidites during solid-phase synthesis, we introduced degenerate positions into RNA guide and passenger strands so that, when annealed, a large pool of distinct siRNA duplexes with randomized base pairs at defined sites was created. We assessed the randomization efficiency by deep sequencing one of the RNAs. All possible individual sequences were present in the pool with generally an excellent distribution of bases. Melting temperature analyses suggested that pools of randomized guide and passenger strands RNAs with up to eight degenerate positions annealed so that mismatched base-pairing was minimized. Transfections of randomized siRNAs (rnd-siRNAs) into cells led to inhibition of luciferase reporters by a miRNA-like mechanism when the seed regions of rnd-siRNA guide strands were devoid of degenerate positions. Furthermore, the mRNA levels of a select set of genes associated with siRNA off-target effects were measured and indicated that rnd-siRNAs with degenerate positions in the seed likely show typical non-sequence-specific effects, but not miRNA-like off-target effects. In the wake of recent reports showing the preponderance of miRNA-like off-target effects of siRNAs, our findings are of value for the design of a novel class of easily prepared and universally applicable negative siRNA controls.

Design and Application of Mini‐libraries of miRNA Probes for an Efficient and Versatile miRNA‐mRNA Cross‐linking

“…For this purpose, a fully complementary binding site to miR‐124 was cloned into the 3’‐UTR of the Renilla luciferase gene, encoded on a dual reporter plasmid. Next, HEK293T cells were co‐transfected with the plasmid and with duplexes containing probes (ORN‐ 4b to ORN‐ 4e and ORN‐ 5a to ORN‐ 5d ), native miR‐124 (ORN‐ 4 ) and a positive control (siRNA targeting Renilla gene) or a negative control (randomized duplex) [26] . After readout, all of the trioxsalen‐bearing probes showed similar concentration‐dependent inhibition of reporter gene expression, with effects comparable to the positive controls (Figure 5).…”

“…After the solid-phase synthesis, the CPG with the synthesized oligoribonucleotides was treated with gaseous methylamine for 1.5 h at 70°C (unmodified oligoribonucleotides) or with a mixture of 200 μL of ammonia solution (25 % in water) and 200 μL of HEK293T cells were transfected with the fully complementary reporter plasmid and increasing concentrations (0, 2.5, 10 and 40 nM) of one of the following duplexes: siRenilla, unmodified miR-124 duplex (ORN-4), trioxsalen-modified miR-124 duplexes ORN-4b to ORN-4e and ORN-5a to ORN-5d, or a randomized negative control. [26] A fullycomplementary counter-strand (ORN-2) was used as the passenger strand to create duplexes with ORN-4, ORN-4b to ORN-4e and ORN-5a to ORN-5d. Significance calculated by 2-way ANOVA followed by Dunnett's test: * (P � 0.05), ** (P � 0.01), *** (P � 0.001), **** (P � 0.0001).…”

MiR-CLIP reveals iso-miR selective regulation in the miR-124 targetome

“…( E and F ) Transcript levels of literature-reported miR-124 (E) and miR-132 (F) targets after transfection with 40 nM of the indicated RNA. Transcript levels were compared to transfection with negative control RNA, siCon ( 40 ); N = 3. Error bars indicate standard deviations.…”

Site‐Specific Labeling of MicroRNA Precursors: A Structure–Activity Relationship Study

“… Cy3‐labeled pre‐miRNAs in dual‐luciferase reporter assays with different pre‐miRNAs, as described in Figure except the negative control (negCON) was siRND2, and pre‐miR‐32 was at 1.5, 6, 25, 100 n m .…”

“…The sequence of the positive control siREN was GAGCG AAGAG GGCGA GAAAU U; GUGXX UAAXX AACXC AXACT T was the negative control siRND2 (X: random nucleotide) . Read‐out was performed with the Dual‐Glo Luciferase Assay System (E2980, Promega), according to manufacturer's protocol, 48 h after plasmid transfection.…”