Improved knockdown from artificial microRNAs in an enhanced miR-155 backbone: a designer's guide to potent multi-target RNAi

Abstract

Artificial microRNA (amiRNA) sequences embedded in natural microRNA (miRNA) backbones have proven to be useful tools for RNA interference (RNAi). amiRNAs have reduced off-target and toxic effects compared to other RNAi-based methods such as short-hairpin RNAs (shRNA). amiRNAs are often less effective for knockdown, however, compared to their shRNA counterparts. We screened a large empirically-designed amiRNA set in the synthetic inhibitory BIC/miR-155 RNA (SIBR) scaffold and show common structural and sequence-specific features associated with effective amiRNAs. We then introduced exogenous motifs into the basal stem region which increase amiRNA biogenesis and knockdown potency. We call this modified backbone the enhanced SIBR (eSIBR) scaffold. Using chained amiRNAs for multi-gene knockdown, we show that concatenation of miRNAs targeting different genes is itself sufficient for increased knockdown efficacy. Further, we show that eSIBR outperforms wild-type SIBR (wtSIBR) when amiRNAs are chained. Finally, we use a lentiviral expression system in cultured neurons, where we again find that eSIBR amiRNAs are more potent for multi-target knockdown of endogenous genes. eSIBR will be a valuable tool for RNAi approaches, especially for studies where knockdown of multiple targets is desired.

Figure 1.

Overview of the wtSIBR cassette and amiRNA screening. (A) Nucleotide sequence of the 150-bp wtSIBR cassette from mouse and predicted miRNA secondary structure. The guide and passenger strands are labeled green and yellow, respectively. Microprocessor cleavage sites are marked with closed arrows and the +1 and –1 nucleotides relative to cleavage are indicated. Dicer cleavage sites are marked with open arrows. (B) Schematic representation of amiRNA screening. COS7 cells were co-transfected with an HA-tagged reporter construct and an amiRNA cloned into the guide strand position of the wtSIBR backbone located in an exon following the GFP open reading frame (GFP-SIBR). amiRNAs are liberated by the microprocessor, exported from the nucleus where they are further processed and loaded into the RNA-induced silencing complex (RISC). Effective sequences lead to reduced reporter levels due to mRNA degradation or translational repression. (C) Representative quantitative western blot using an HA antibody to measure reporter expression and (D) calculated reporter knockdown efficiency of cadm1 amiRNAs. Knockdown percentage was calculated relative to reporter co-transfection with a vector containing an empty wtSIBR cassette (empty). Actin was used as a loading control.

Figure 2.

Distinct structural features are associated with effective and ineffective amiRNA sequences. (A) Example miR-155 nucleotide sequences from different organisms and predicted secondary structures of distinct guide/passenger strand mismatches. (B) Frequency bias of different mismatch structures found in effective and ineffective amiRNA sequences. Effective amiRNA n = 50, ineffective amiRNA n = 84, **P < 0.01, Pearson's χ²-test. (C) G/C nucleotide content of effective and ineffective amiRNA sequences. Effective amiRNA n = 50, ineffective amiRNA n = 79, ***P < 0.001 Student's two-tailed t-test. Error bars represent SEM. (D) Nucleotide frequency bias of effective and ineffective amiRNA sequences and (E) Normalized U/A or G/C nucleotide frequency bias of effective amiRNA sequences. Nucleotide positions are relative to the 5′ microprocessor cleavage site. Effective amiRNA n = 183, ineffective amiRNA n = 79, **P < 0.01, ***P < 0.001 Pearson's χ²-test with Šidák correction for multiple comparisons.

Figure 3.

The eSIBR backbone enhances knockdown potency. (A) Nucleotide substitutions on and near the miR-155 basal stem which added the indicated UG and CNNC motifs to create the eSIBR backbone. Black circles indicate the wild-type sequence; blue circles are the modified sequence. Nucleotide numbers are relative to microprocessor cleavage sites. (B) Representative western blots and (C) quantification of reporter knockdown efficiency from data in Supplementary Figure S1 in COS7 cells co-transfected with the indicated amiRNAs in wild-type or modified SIBR backbones. Knockdown percentage was calculated relative to reporter co-transfections with a control vector containing an empty wtSIBR cassette (empty, not shown). Actin was used as a loading control. cadm1.715 and nrxn1.1271 n = 4 independent experiments, all others n = 3 independent experiments. (D) Reporter knockdown efficiency as in (C) from single experiments with additional amiRNAs in the wtSIBR or eSIBR backbone. (E) Comparison of potency-of-knockdown between constructs containing amiRNAs in modified backbones (plotted points) relative to their counterparts in the wtSIBR backbone (dotted line). If more than one experiment was conducted for an amiRNA, the average value is plotted. n = 16 amiRNAs, ***P < 0.001, n.s. = not significant, one-way ANOVA with Tukey's post-hoc comparison to the wtSIBR backbone group. For all graphs error bars represent SEM.

Figure 4.

The eSIBR backbone increases cleavage by the microprocessor. (A) Schematic of microprocessor activity assay. GFP-SIBR mRNA is either exported from the nucleus to be translated or the amiRNA is cleaved by the microprocessor and the mRNA is degraded. (B) Representative western blots showing GFP levels in COS7 cells transfected with GFP-SIBR constructs containing the indicated amiRNAs in wild-type or modified SIBR backbones. Actin was used as a loading control. (C) Comparison of GFP levels measured by quantitative western blot of COS7 cells transfected with GFP-SIBR constructs carrying amiRNAs in modified backbones (bars) relative to their corresponding wtSIBR counterparts (dotted line). (D) Representative SIBR pri-miRNA and gapdh loading control qRT-PCR amplification plots using cDNAs synthesized from COS7 cell total RNA following transfection of hairpin nlgn3.1900 in wtSIBR or modified backbones. Insets are of the threshold cycle region of the amplification curves. (E) Comparison of SIBR pri-miRNA levels measured by qRT-PCR of COS7 cells transfected with GFP-SIBR constructs carrying amiRNAs in modified backbones (bars) relative to their corresponding wtSIBR counterparts (dotted line). If more than one experiment was conducted for an amiRNA sequence, the average value was used. (C) n = 16 amiRNAs, (E) n = 18 amiRNAs, **P < 0.01, ***P < 0.001, n.s. = not significant, one-way ANOVA with Tukey's post-hoc comparison to the wtSIBR group. Error bars represent SEM.

Figure 5.

Chaining amiRNAs targeting distinct genes increases knockdown potency. (A) Schematic of GFP-SIBR constructs expressing a single hairpin or triple hairpins used in this figure. Single hairpin constructs contain an amiRNA targeting a single gene. Triple hairpin constructs contain three unique amiRNA sequences targeting different genes of the same family (e.g. cadm1, 2 and 3 or nlgn1, 2 and 3) or contain three unique scrambled sequences (scrambled1, 2 and 3). (B) Representative western blots showing knockdown fidelity for all combinations of HA-reporter constructs co-transfected with single wtSIBR amiRNAs and a control vector containing an empty wtSIBR cassette (empty) in COS7 cells. Cross-targeting is marked by an asterisk. (C) Representative western blots and (D) quantification of reporter knockdown efficiency in COS7 cells co-transfected with GFP-SIBR constructs carrying indicated single or triple amiRNA sequences in either wtSIBR or eSIBR backbones. Knockdown percentage was calculated relative to reporter co-transfections with a control vector containing an empty wtSIBR cassette (not shown). Data for single eSIBR amiRNAs are not shown. Actin was used as a loading control. Values represent the average of 3 independent experiments. (E) Comparison of potency-of-knockdown for triple-amiRNA expressing constructs (bars) relative to their counterparts with single-amiRNAs in the wtSIBR backbone (dotted line) and (F) comparison of potency-of-knockdown for triple eSIBR amiRNA expressing constructs (bar) relative to counterparts single amiRNAs in the eSIBR backbone from experiments in (D). Values represent the average of all values for 3 independent experiments of the 5 conditions as in (D) (total n = 18 per group). **P < 0.01, ***P < 0.001, student's two-tailed t-tests against the relative control group (dotted line). (G) Comparison of potency-of-knockdown for triple-amiRNA constructs in eSIBR backbones (blue bars) relative to their counterparts expressing triple-amiRNAs in the wtSIBR backbone (black bars) from data in (D). n = 3 independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001, Student's two-tailed t-test. (H) Comparison of pri-miRNA levels measured by qRT-PCR in COS7 cells following transfection of single and triple cadm1.1358- and nlgn2.1283-containing SIBR constructs in either the wtSIBR or eSIBR backbones. pri-miRNA levels are relative to the single wtSIBR amiRNA condition. n = 3 independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001, Student's two-tailed t-test against the single wtSIBR amiRNA condition. For all graphs, error bars represent SEM.

Figure 6.

eSIBR enhances multi-target knockdown potency in primary neuron cultures. (A) Schematic of lentiviral vectors used for multiple endogenous gene knockdown. The cytomegalovirus promoter (CMV, yellow) promotes antisense-strand expression (relative to viral RNA) of amiRNAs located in an intron preceding a nuclear-localized GFP (nlsGFP, green) coding sequence. amiRNA triplets target cadm1, 2 and 3 or contain three unique scrambled sequences (scrambled1, 2 and 3). Orange boxes represent viral-specific sequences. LTR, long-terminal repeat, RRE, Rev-response element, cPPT, central polypurine tract, WPRE, woodchuck hepatitis virus posttranscriptional regulatory element, SA, splice acceptor, SD, splice donor. (B) Representative western blots and (C) quantification of endogenous Cadm family knockdown efficiency in 14DIV cultured rat hippocampal neurons transduced with viral constructs carrying the indicated amiRNA sequences in wtSIBR or eSIBR backbones. Antibodies were against Cadm1, Cadm3 or Cadm1–3 (pleio-Cadm). Knockdown percentage was calculated relative to uninfected control cultures (no virus). Actin was used as a loading control. (D) Comparison of endogenous Cadm potency-of-knockdown for viral vectors containing amiRNAs in eSIBR backbones (blue bars) relative to their counterparts in the wtSIBR backbone (black bars). (C) and (D) n = 4 independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001, Student's two-tailed t-test. Error bars represent SEM.