Inhibiting miRNA in Caenorhabditis elegans using a potent and selective antisense reagent
© Zheng et al; licensee BioMed Central Ltd. 2010
Received: 18 July 2009
Accepted: 1 April 2010
Published: 1 April 2010
Antisense reagents can serve as efficient and versatile tools for studying gene function by inhibiting nucleic acids in vivo. Antisense reagents have particular utility for the experimental manipulation of the activity of microRNAs (miRNAs), which are involved in the regulation of diverse developmental and physiological pathways in animals. Even in traditional genetic systems, such as the nematode Caenorhabditis elegans, antisense reagents can provide experimental strategies complementary to mutational approaches. Presently no antisense reagents are available for inhibiting miRNAs in the nematode C. elegans.
We have developed a new class of fluorescently labelled antisense reagents to inhibit miRNAs in developing worms. These reagents were synthesized by conjugating dextran with 2'-O-methyl oligoribonucleotide. The dextran-conjugated antisense reagents can be conveniently introduced into the germline of adult hermaphrodites and are transmitted to their progeny, where they efficiently and specifically inhibit a targeted miRNA in different tissues, including the hypodermis, the vulva and the nervous system. We show that these reagents can be used combinatorially to inhibit more than one miRNA in the same animal.
This class of antisense reagents represents a new addition to the toolkit for studying miRNA in C. elegans. Combined with numerous mutants or reporter stains available, these reagents should provide a convenient approach to examine genetic interactions that involve miRNA, and may facilitate studying functions of miRNAs, especially ones whose deletion strains are difficult to generate.
See related research article: http://jbiol.com/content/9/3/20
MicroRNAs (miRNAs) are single strand RNA molecules ~ 21-23 nucleotides long that play important roles in many biological processes through regulating gene expression . In animal cells, miRNAs act primarily by inhibiting mRNA translation and/or stability through a process involving partial complementary base-pairing with sequences at the 3'-untranslated region (3' UTR). Numerous miRNAs have been identified. To study their functions, antisense reagents against miRNAs have been developed as a reverse genetics tool. Synthetic oligonucleotide analogues, including 2'-O-methyl oligoribonucleotides , locked nucleic acids , 2'-O-methoxyethyl oligoribonucleotides , and morpholinos , have been tested. These antisense nucleotide analogues have been used to knock down miRNAs in cultured cells [2–4] and in live animals including zebrafish , D. melanogaster  and mice .
Caenorhabditis elegans has long been used as a model organism for studying the regulation and function of small non-coding RNA molecules, and yet no antisense reagents are available to reliably inhibit miRNAs in worms. Such a technique would be very useful for studying functions of miRNAs whose deletion strains are difficult to generate; for example, mutations causing lethality or sterility . In addition, to dissect functions of individual miRNAs that are clustered together, or to block intronic miRNAs [8, 9] without perturbing the function of the corresponding protein-coding genes, antisense reagents would offer a convenient approach to circumvent the limitation of using deletion strains.
Results and discussion
To apply dextran-(as-2'OMelin-4)8 to inhibit lin-4 in vivo, we injected the compound into gonads of adult hermaphrodites. Dextran-rhodamine (40 KDa) was coinjected as a fluorescent marker. About 16 h after injection, we collected rhodamine-labeled embryos (n = 50) under a fluorescence dissection scope. When these embryos reached adulthood, we scored for the egg laying defective (Egl) phenotype. In C. elegans, lin-4 is required during larval development to control the timing and pattern of cell division in the hypodermis of larva stage 1 (L1) and stage 2 (L2). lin-4 loss of function mutants (lin-4(lf)) display inappropriate reiterations of early fates at late developmental stages and show a retarded heterochronic phenotype in adults in the form of the absence of adult structures (such as vulva) and the failure of egg-laying [12, 13].
When using an injection with a concentration of 50 μM (all concentrations refer to the total concentration of 2'-O-methyl oligoribonucleotides in the sample as determined from the ultraviolet (UV) absorption at 260 nm) dextran-(as-2'OMelin-4)8 was effective in inhibiting lin-4 and caused Egl in about 70% of worms (Figure 1b-d). Raising the injection concentration to 100 μM or above increased Egl to over 90% in the labelled worms. In contrast, antisense 2'-O-methyl oligoribonucleotides that is not conjugated to dextran only had a small effect, even at 200 μM (Figure 1b). In order to examine the specificity of dextran-(as-2'OMelin-4)8 in inhibiting lin-4, we prepared two control dextran conjugates, dextran-(as-2'OMemiR-237)8 and dextran-(s-2'OMelin-4)8. Dextran-(as-2'OMemiR-237)8 contains 2'-O-methyl oligoribonucleotides complementary to miR-237, a miRNA of the lin-4 family with similar, but not identical, sequence as lin-4. Dextran-(s-2'OMelin-4)8 contains lin-4 sequence (sense). We did not observe Egl phenotypes, or other abnormalities, in worms labelled with either of these two control oligonucleotides (Figure 1b) which confirmed that dextran-(as-2'OMelin-4)8 inhibits lin-4 in a sequence specific manner, also suggesting that worms tolerate dextran conjugates of 2'-O-methyl oligoribonucleotides fairly well.
In order to confirm that these antisense reagents act specifically by inhibiting lin-4, we examined several molecular and cellular markers to characterize the development of animals labelled with Rhdextran-(as-2'OMelin-4)1: (1) the formation of vulval structure; (2) adult specific alae formation and col-19 expression; and (3) the stage-specific seam cell division programmes.
Finally, since lin-4 functions through lin-14, mutations in lin-14 should suppress the phenotype of lin-4 knockdown. Indeed, at 20°C, inhibition of lin-4 in lin-14(n179), a lin-14 nonnull mutant, with Rhdextran-(as-2'OMelin-4)1 (50 μM injection concentration) only caused Egl in 2.6% of labelled worms (n = 190) and all the examined young adult worms displayed normal alae (n = 30).
Together, these data showed that Rhdextran-(as-2'OMelin-4)1 caused developmental retardation consistent with lin-4 knockdown, confirming its efficacy and specificity in inhibiting lin-4 during development.
In order to test whether these conjugated antisense agents can be used to inhibit other miRNAs in worms, we prepared Rhdextran-(as-2'OMelsy-6)1 and Rhdextran-(as-2'OMelet-7)1 using the same procedure as for making Rhdextran-(as-2'OMelin-4)1. These two dextran conjugates were designed to block lsy-6 and let-7, respectively, two miRNAs of known functions in C. elegans.
We have developed a new class of antisense reagents that potently and selectively inhibit miRNAs in C. elegans. This offers an experimental approach complementary to mutational strategy for the study of the functions of miRNA in vivo.
2'-O-methyl oligoribonucleotides were either purchased from the Integrated DNA Technologies (IDT, Iowa, USA) or synthesized in-house by the standard solid phase phosphoramidite chemistry using an ABI 394 DNA/RNA Synthesizer (Applied Biosystems, California, USA). Sequences of 2'-O-methyl oligoribonucleotides used in this study are:
s-2'OMelin-4 (sense): 5' - UCCCUGAGACCUCAAGUGUGA - 3'
as-2'OMelin-4 (antisense): 5' - UCACACUUGAGGUCUCAGGGA - 3'
as-2'OMemiR-237: 5' - AGCUGUUCGAGAAUUCUCAGGGA - 3'
as-2'OMelet-7: 5' - AACUAUACAACCUACUACCUCA - 3'
as-2'OMelsy-6: 5' - CGAAAUGCGUCUCAUACAAAA - 3'
as-2'OMemiR-84: 5' - UACAACAUUACAUACUACCUCA - 3'
as-2'OMemiR-42: 5' - UCUGUAGAUGUUAACCCGGUGA - 3'
For bioconjugation, an n-hexyl linker containing a disulfide bond (Thio-Modifier C6 S-S, Glen Research, Virginia, USA) was attached to the 5'-end of 2'-O-methyl oligoribonucleotides. A,C,G,U-2'-OMe-RNA CE phosphoramidite monomers and A,C,G,U-2'-OMe RNA synthesis supports were from AZCO Biotech (California, USA). MAL-dPEG4 ™-NHS ester was from Quanta BioDesign Ltd (Ohio, USA). Dextran amine (40 KD) was purchased from Molecular Probes (Oregon, USA). Other reagents and solvents were from Aldrich (Missouri, USA). The UV and visible absorption spectra were recorded on a Shimadzu 2401 PC spectrometer.
Conjugating 2'-O-methyl oligonucleotides with dextran
To prepare Rhdextran-(as-2'OMelin-4)1, for example, dextran amine (40 KD, ~ 8 amines/dextran, 10 mg) was first reacted with Rhodamine B isothiocyanate (RBITC, 0.4 mg, 0.75 μmol) in 0.1 mL anhydrous DMSO at 37°C for 8 h. MAL-dPEG4-NHS ester (3 mg, 5.84 μmol) was then added and the reaction was continued at room temperature overnight. The reaction mixture was dialyzed against water through a regenerated cellulose membrane (Float A Lyzer®, molecular wight cut off [MWCO] = 3500, Spectrum Laboratories, Inc. California, USA) to remove excess reagents. After freeze drying, the solid product was dissolved in water (0.25 mL) to make a 1 mM rhodamine-dextran stock solution.
To conjugate 2'-O-methyl oligoribonucleotides containing a 5'-disulfide (5' S-S) group with rhodamine-dextran, we first reduced the 5'-disulfide group to a free thiol using tris(2-carboxyethyl) phosphine (TCEP), a water soluble reducing reagent. as-2'OMelin-4 (5' S-S, 30 nmol) was dissolved in 100 μl of deaerated sodium phosphate buffer (100 mM, pH = 7.0). An excess amount of TCEP was added to the solution under the protection of Argon. One hour later, cold ethanol (0.5 mL) was added to precipitate the oligonucleotide. The supernatant was removed after centrifugation (14000 rpm for 10 min) and the precipitated oligonucleotide was redissolved in a sodium phosphate buffer (100 mM, pH 7.0, 70 μL). The oligonucleotide solution was then mixed with the rhodamine-dextran stock solution (30 μL) prepared above. The mixture was stirred under argon overnight. Excess mercaptoethanol was added to cap the remaining unreacted maleimide group. The reaction mixture was dialyzed against water using a cellulose membrane (MWCO = 10,000) and lyophilized to yield the final product. The dried product was re-dissolved in water to prepare a stock solution. The concentration of stock solution was typically in the range of 1 mM which was determined by measuring the UV absorption of 2'-O-methyl oligoribonucleotide at 260 nm. The UV absorption was converted to the oligonucleotide concentration using the OligoAnalyzer program accessible online http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/. Rhodamine absorption at 260 nm was corrected according to its peak absorption at 559 nm. The conjugation yields were typically around 50%. The average stoichiometry of conjugation was calculated from the total amount of oligoribonucleotide in the final product divided by the amount of dextran added to the reaction.
RhDextran-(as-2'OMelin-4)4, dextran-(as-2'OMelin-4)8 and other dextran conjugates containing different sequences of 2'-O-methyl oligonucleotides were synthesized similarly. When preparing RhDextran-(as-2'OMelin-4)4, four equivalents of 2'-O-methyl oligonucleotides containing a 5'-disulfide group were used to react with the dextran-linked maleimide group. No Rhodamine B isothiocyanate was used when synthesizing dextran-(as-2'OMelin-4)8.
Conjugation products purified by dialysis still contained a small amount of unconjugated oligonucleotides as analyzed by the polyacrylamide gel electrophoresis (PAGE), and were used for most of the experiments except for the ones shown in Figure 8 and Figure 10. To completely remove unconjugated oligonucleotides from dextran-conjugates, the reaction mixture was first concentrated under vacuum to a small volume (≤30 μL) and then mixed with 0.27 mL formamide (>99%, Ambion, Texas, USA). The mixture was boiled briefly and loaded into 5% preparative denaturing polyacrylamide gel. After running the gel at 400 V for 10 min, we confirmed the separation of free 2'-O-methyl oligoribonucleotides from dextran conjugates by viewing the gel over a fluorophore-coated thin-layer chromatography plate (Silica Gel 60, F254, Merck, Germany) under the UV illumination (265 nm). Dextran-conjugated products that remained near the origin of the gel showing red fluorescence were cut out and transferred into a dialysis membrane (MWCO = 1000) containing 2 mL of 0.5× TBE buffer (Bio-Rad, CA, USA). The dialysis membrane was sealed and the product in the gel was recovered by electrophoresis (300 V for 20 min). The TBE buffer in the dialysis membrane containing dextran conjugates was transferred to another cellulose dialysis membrane (MWCO = 10000) and dialyzed against water 3 times over 16 hrs to remove salts and urea. The final products were obtained as a powder after lyophilization.
Worm injection and assay of miRNA inhibition in vivo
Dextran conjugates of 2'-O-methyl oligoribonucleotides were injected into both gonads of young adult worms of either wild-type (N2) or transgenics expressing GFP in an ASE neuron (OH3191 or OH 3192). Rhodamine dextran (40 KD, 8 mg/mL final concentration) was included in the injection solution if the injected reagents contained no fluorescent label, for example, dextran-(as-2'OMelin-4)8 or unconjugated 2'-O-methyl oligoribonucleotides. For each experiment, we routinely injected a sample into ~ eight worms. About 16 h later, we collected rhodamine labelled embryos (n = 50) laid by injected worms under a fluorescence dissection scope (SteREO Discovery.V12, Carl Zeiss, Göttingen, Germany), and scored their phenotypes when they reached appropriate larval or adult stages. Staging of animal development was based on gonad size and morphology.
We also attempted delivering antisense reagents using a standard soaking method for RNAi (. However, this method turned out to be ineffective. After incubating L1 larvae with Rhdextran-(as-2'OMelin-4)1 (300 μM) or Rhdextran-(as-2'OMelet-7)1 (300 μM) for 48 h in the soaking solution (M9 solution (0.25 ×, without Mg2+) with 3 mM spermidine and 0.05% gelatin), we recovered L1 larvae on NGM plates. All the worms developed normally without showing any observable phenotype expected from lin-4 or let-7 knockdown.
egg laying defective
green fluorescence protein
molecular weight cut off
We thank the Caenorhabditis Genetics Center for providing worm strains and Dr X Wang for providing the microinjection apparatus. We are also grateful to Dr J Liu for help with the microinjection. This project is supported by the grants from the Welch Foundation (I-1510) and the National Institute of Health.
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