• miRNA target site identification and validation
  • Screening for miRNAs that regulate target gene expression
  • Screening for miRNAs that affect a cellular process

MicroRNAs (miRNAs) are small, regulatory RNAs that are expressed in animals and plants and affect the translation or stability of target mRNAs. The 17-24 nt, single-stranded (ss) miRNAs are derived from longer, primary transcripts termed "pri-miRNAs" [1]. The pri-miRNAs, which can be more than 1000 nt in length, contain an RNA hairpin in which one of the two strands includes the mature miRNA [1]. The hairpin, which typically comprises 60-120 nt, is cleaved from the pri-miRNA in the nucleus by the double-strand-specific ribonuclease, Drosha [1]. The resulting precursor miRNA, or "pre-miRNA," is transported to the cytoplasm via a process that involves Exportin-5 [2-4]. The pre-miRNA is further cleaved by Dicer [5] to generate a short, partially double-stranded (ds) RNA in which one strand is the mature miRNA. The mature miRNA is taken up by a protein complex that is similar, if not identical, to the RNA Induced Silencing Complex (RISC) that supports RNA interference (RNAi) [6], and miRNA-bound complex functions to regulate translation.

miRNA functional analyses can be carried out using tools that are similar to those used to analyze protein encoding genes. Up-regulation of the miRNAs can help to identify gain-of-function phenotypes while down-regulation or inhibition studies can help to identify loss-of-function phenotypes. The combination of up- and down-regulation can be used to identify not only genes that are regulated by miRNAs but also cellular processes that are affected by specific miRNAs. Two recent publications have described antisense molecules that can inhibit the activities of miRNAs [7, 8].

Pre-miR miRNA Precursor Molecules

As described below, Ambion has recently used the Pre-miR™ design criteria to design synthetic miRNAs for miRNAs listed in the Sanger miRNA Registry. These synthetic miRNAs are known as Pre-miR miRNA Precursor Molecules (patent pending). A reciprocal set of miRNA inhibitors, called Anti-miR™ miRNA Inhibitors (patent pending), is also available. This miRNA database provides a valuable resource for researchers and includes miRNA sequence information, references, and links directly to raw data from the Sanger miRNA Registry--the world's foremost repository of miRNA information. Pre-miR miRNA Precursor Molecules and Anti-miR miRNA Inhibitors can be obtained one at a time to facilitate in-depth studies of single miRNAs or in groups to facilitate screening studies.

Advantages of Synthetic miRNAs

Up-regulation of miRNAs in cells can be accomplished by transfecting cells with either synthetic miRNAs [9] or plasmids that express miRNAs [10]. The use of synthetic miRNAs has several advantages:

  • The transfection efficiency of synthetic miRNAs can approach 100% for immortalized cells. These high transfection efficiencies are particularly beneficial for high-throughput applications like miRNA functional screening.
  • Synthetic miRNAs can be readily electroporated into primary cells without inducing significant cell death.
  • Synthetic miRNAs can be transfected at different concentrations, facilitating dose response studies.
  • The rules for miRNA maturation and activation from pri-miRNA and pre-miRNA precursors are not fully understood, making it difficult to ensure uptake and activation of the intended miRNA molecule from plasmid-based expression systems. In contrast, the sequences of the synthetic miRNAs that are altering translation are known.

Strand Specificity of Small RNAs

For a synthetic miRNA to be useful, it must be active, robust, and most importantly, strand specific. Small RNAs like miRNAs and siRNAs exhibit strand specificity, where one of the two complementary RNA strands ("active strand") in the RNA molecule is preferentially incorporated into the miRNA (or siRNA) pathway [11] (the other strand is referred to as the "off-strand"). The sequence compositions and duplex stabilities of the small RNA molecules dictate the strand that will be active in the cell. Transfected miRNAs should be similarly strand specific to ensure efficient uptake of the active miRNA strand and exclusion of the incorrect, complementary (or partially complementary) off-strand.

Strand Specificity of Small RNAs

To create synthetic miRNAs that function like natural miRNAs, Ambion scientists tested a variety of RNA designs that were produced by Ambion's Custom RNA Synthesis Group. To test for strand activation, a series of reporter plasmids were produced using the pMIR-REPORT™ miRNA Reporter Expression Vector (Cat# 5795), which contains the coding sequence for luciferase. A sequence complementary to the mature miRNA sequence or complementary to the off-strand of the precursor miRNA being evaluated was inserted into the 3'-UTR of the luciferase gene. The levels of luciferase gene expression in cells transfected with a combination of reporter vector and six different variants containing the let-7b or miR-33 miRNA sequences are shown in Figure 1. While the siRNA-like synthetic miRNAs provided efficient reduction of the plasmid reporter, the off-strand of the dsRNA molecule provided similar activity as well (Figure 1). The lack of strand specificity of the siRNA-like design makes it impossible to ensure that any target gene regulation or cellular phenotype observed is the result of activity of the mature miRNA and not the complementary strand of the siRNA. Other variants showed a similar lack of strand specificity or alternatively, very low activity. One design permutation, however, provided very efficient reduction of the miRNA-specific reporter and essentially no reduction of the off-strand reporter (Figure 1). Interestingly, the activities of the let-7b and miR-33 molecules produced using this design were higher than any of the other design variants, further indicating that the miRNA pathway was preferentially taking up the correct miRNA molecule into the transfected cells.


Figure 1. Comparison of Different miRNA Inhibitor Designs. Multiple different synthetic miRNA permutations for miR-33 and let-7b were tested for the activities of the miRNA strand and the off-strand or complementary strand of the dsRNA molecules (representative data shown). The strand assay included a series of luciferase reporter vectors that were specific to the two strands of the various miR-33 and let-7b synthetic miRNAs. In the 3'UTR of the luciferase gene, we inserted a sequence complementary to the mature miR-33 or let-7b miRNA sequence or complementary to the off-strand of the various synthetic miRNA designs that were evaluated. Shown in Figure 1 are the levels of luciferase expression in cells transfected with a combination of reporter vector and six different variants containing the let-7b or miR-33 miRNA sequence. In each pair, the bars represent experiments performed using 10nM (left) and 3 nM (right) synthetic miRNA. The siRNA-like synthetic miRNAs provide efficient reduction of the plasmid reporter, but the off-strand of the dsRNA molecule provides similar activity. The lack of strandedness of the siRNA-like design makes it impossible to ensure that any target gene regulation or cellular phenotype is the result of activity of the mature miRNA and not the complementary strand of the siRNA. Other variants showed a similar lack of strandedness or very low activity. In contrast, the Pre-miR design provided very efficient reduction of the miRNA-specific reporter and essentially no reduction of the off-strand reporter (shaded bars). Error bars represent standard deviations for triplicate transfections.

Validation of Synthetic Pre-miR Design

The most effective miR-33 sequence was tested for strand specificity in several different cell lines to ensure that the observed effect was not cell type specific. The let-7b miRNA was not used because all but HepG2 cells express high levels of let-7b that would mask the effects of the synthetic miRNA. The miR-33 off-strand had essentially no activity in any of the cell types that were used (Figure 2). Additionally, the reduction in the miRNA-specific reporters by the miR-33 molecule mirrored the transfection efficiencies of the cell types tested.


Figure 2. Correct Strand Activation of Pre-miR™ miRNA Precursor Molecules. HeLa, MCF7, SK-N-As, and HepG2 cells were co-transfected with 10 nM of miR-33 or Pre-miR Negative Control #1 (NC#1) and 200 ng of mature miR-33 luciferase reporter plasmid (pMIR-REPORT™ vector; Ambion) or off-strand miR-33 luciferase reporter plasmid (Off-strand reporter). Twenty-four hours post-transfection, the expression of luciferase was measured. The expression of luciferase in cells transfected with the Pre-miR Precursor miR-33 was divided by the expression of luciferase in cells transfected with Pre-miR Precursor Negative Control #1. Error bars represent standard deviations for triplicate transfections.


To confirm that the best design could be successfully applied to other miRNAs, siRNA and enhanced design synthetic miRNAs (the Pre-miR design) were prepared for miR-1, miR-10, and miR-124. HeLa cells were transfected with the synthetic miRNAs at a final concentration of 3 and 10 nM, respectively. The expression of co-transfected reporters for each of the miRNAs was monitored 24 hours post-transfection. As was observed for miR-33 and let-7b, the Pre-miR design miRNAs, referred to as Pre-miR miRNA Precursor Molecules, were significantly more active than the corresponding siRNA-like synthetic miRNAs (Figure 3).


Figure 3. Pre-miR™ Precursor miRNA Molecules are Hyperactive. siRNA- and Pre-miR-designed Precursor miRNAs for miR-10, miR-1, and miR-124 were used to co-transfect HeLa cells along with 200 ng of luciferase reporter vectors (pMIR-REPORT™ vector; Ambion) with miR-10, miR-1, and miR-124 target sites. Luciferase expression in the transfected cells was monitored 24 hours post-transfection. The luciferase expression in the miRNA-transfected cells was divided by the luciferase expression Pre-miR Negative Control #1 or Silencer® Negative Control siRNA #1. Error bars represent standard deviations for triplicate transfections.


Ultimately, the synthetic miRNAs must function in cells in a manner that is consistent with naturally occurring miRNAs. Cells were transfected with four different Pre-miR miRNAs and the expression of genes that are known or predicted to be regulated by the four miRNAs was measured. The Pre-miR miRNAs reduced the expression of the natural target genes by 50-80% (Figure 4).


Figure 4. Pre-miR™ miRNA Precursor Molecules Regulate Natural Targets of Endogenous miRNAs. Cell assays, RT-PCR, and protein assays were set up to monitor the expression of genes targeted by miR-1, miR-20, miR-196, and let-7. HeLa cells were transfected with 10 nM Pre-miR Negative Control #1 (NC Pre-miR), miR-1, miR-20, and miR-196 Pre-miR Precursor miRNAs. HepG2 cells were transfected with 10 nM Pre-miR Negative Control #1 and let-7 Pre-miR Precursor miRNAs. Cells were monitored for target protein activity or expression or mRNA expression two days post-transfection. Relative reduction in target gene expression was measured relative to untransfected cells. Error bars represent standard deviations for triplicate transfections.

Applications

Key applications for the Pre-miR miRNA Precursor Molecules and Anti-miR miRNA Inhibitors include:

  • miRNA target site validation--Pre-miR miRNA Precursor Molecules and Anti-miR miRNA Inhibitors are transfected into cells, and the expression of an endogenous gene in the transfected cell population is measured. Reduced expression of a target protein in Pre-miR miRNA Precursor Molecule transfected cells relative to negative control transfected cells provides evidence that the gene is indeed regulated by the miRNA. Increased expression of the target protein in Anti-miR miRNA Inhibitor transfected cells provides additional support for the miRNA/target gene interaction.
  • miRNA target site identification--A single Pre-miR miRNA Precursor Molecule or Anti-miR miRNA Inhibitor is co-transfected with a reporter plasmid (e.g. pMIR-REPORT miRNA Reporter Expression Vector) containing a region of a gene that is thought to interact with the miRNA. Down-regulation of the reporter in Pre-miR miRNA Precursor Molecule co-transfections suggests that the gene sequence responds to miRNA regulation. Up-regulation of the reporter in Anti-miR miRNA Inhibitor co-transfections is further evidence that the miRNA regulates gene expression through the sequence in the reporter vector.
  • Screening for miRNAs that regulate target gene expression--An assay that measures the expression of a protein or a reporter construct (e.g. pMIR-REPORT miRNA Reporter Expression Vector) containing the 3'-UTR of a gene is used to identify Pre-miR miRNA Precursor Molecules or Anti-miR miRNA Inhibitors that influence the expression of the protein or the reporter. Transfecting a series of cells with individual Pre-miR miRNA Precursor Molecules or Anti-miR miRNA Inhibitors can be used to identify important regulatory miRNAs. Those Pre-miR miRNA Precursor Molecules and Anti-miR miRNA Inhibitors that affect the expression of the target gene represent miRNAs that directly or indirectly regulate the expression of that gene.
  • Screening for miRNAs that affect a cellular process--Cells are sequentially transfected with a collection of Pre-miR miRNA Precursor Molecules or Anti-miR miRNA Inhibitors and then assayed for a phenotype like cell cycle arrest or differentiation. Those Pre-miR miRNA Precursor Molecules or Anti-miR miRNA Inhibitors that induce the desired phenotype represent miRNAs that directly or indirectly participate in the cellular process being studied.

Scientific Contributors
David Brown, Rich Jarvis, Mike Byrom, Angie Cheng, Vince Pallotta, Dmitriy Ovcharenko, Lance Ford • Ambion, Inc.