Inhibiting miRNA in Caenorhabditis elegans using a potent and selective antisense reagent

Background 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. Results 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. Conclusion 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

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 [8]. 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. 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'OMe lin- 4) 8 was effective in inhibiting lin-4 and caused Egl in about 70% of worms (Figure 1b-d).
In dextran-( as-2'OMe lin-4) 8 , each dextran molecule is conjugated to eight copies of antisense 2'-O-methyl oligoribonucleotides. Having a high density of oligonucleotides on the surface of dextran molecule might increase the steric hindrance and compromise the hybridization efficiency of the antisense oligonucleotide to its target miRNA. In order to test whether we could improve the potency of these dextran conjugated antisense reagents by varying the coupling stoichiometry, we decreased the amount of 2'-O-methyl oligoribonucleotides used for conjugation. In addition, we also linked a fluorescent label (rhodamine B isothiocyanate) with dextran so that we could visualize the distribution of these antisense reagents directly. We prepared two rhodamine-dextran ( Rh dextran) conjugates of 2'-O-methyl oligoribonucleotides, Rh dextran-( as-2'OMe lin-4) 4 and Rh dextran-( as-2'OMe lin-4) 1 , by varying the equivalents of 2'-O-methyl oligoribonucleotides added to the conjugation reaction (Figure 2). Each Rh dextran-( as-2'OMe lin-4) 4 or Rh dextran-( as-2'OMe lin-4) 1 on average contains four or one copy of lin-4 antisense 2'-O-methyl oligoribonucleotide, respectively (Figures 2 and 3a). These two dextran conjugates were comparably efficient in inhibiting lin-4, yet both were much more potent than dextran-( as-2'OMe lin-4) 8 . At 20 μM or above, both Rh dextran-( as-2'OMe lin-4) 4 and Rh dextran-( as- 2'OMe lin-4) 1 caused Egl in nearly 100% of labelled worms (Figure 3b). In contrast, dextran-( as-2'OMe lin-4) 8 was completely ineffective at 20 μM. In addition, fluorescence imaging of Rh dextran-( as-2'OMe lin-4) 1 confirmed that the conjugate was localized fairly evenly in the cytosol after being taken up by cells.
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 Rh dextran-( as-2'OMe lin-4) 1 : (1) the formation of vulval structure; (2) adult specific alae formation and col-19 expression; and (3) the stagespecific seam cell division programmes.
During larval development, the lateral hypodermal seam cells follow a characteristic division pattern. After each division, one of the daughter cells fuses with the hypodermis (hyp7) and the other daughter cells divides again at a later stage until it terminally differentiates at the L4 molt ( Figure 6a) [16]. Seam cells of lin-4(e912) animals repeat the L1 fates in hypodermal cell lineages and are unable to exit the cell cycle at the L4 molt. In order to determine how seam cell development is altered in lin-4 knockdown animals, we followed the seam cell division pattern by observing GFP-labelled seam cells (wIs51, strain JR667). Both L1 and L2 divisions in lin-4 knockdown appeared to be normal, with each V cell (including V1 -V4 and V6) divided once in L1 (100%, n = 10, data not shown) and twice in L2 (100%, n = 10; Figure 6b, c). However, in early L3, V cells in lin-4 knockdown repeated the L2 division pattern, so that each V cell gave rise to twice as many daughter cells as the corresponding seam cells of the control animals (100%, n = 6; Figure 6d, e). This pattern of reiterated L2 seam cell division is consistent with a reduced, but not absent, activity of lin-4, possibly resulting in an intermediate level of lin-14 over expression [17]. Together, these data showed that Rh dextran-( as-2'OMe lin-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 Rh dextran-( as-2'OMe lsy-6) 1 and Rh dextran-( as-2'OMe let-7) 1 using the same procedure as for making Rh dextran-( as-2'OMe lin-4) 1 . These two dextran conjugates were designed to block lsy-6 and let-7, respectively, two miRNAs of known functions in C. elegans.  The lsy-6 microRNA regulates left-right asymmetry of ASE neurons, a pair of chemosensory neurons that share many bilaterally symmetrical features, yet differ in their ability to discriminate different ions by expressing distinct sets of chemoreceptors of the gcy gene family [18]. lsy-6 is only present in ASEL (left ASE) of adult worms. It promotes gcy-7 expression through repression of cog-1, a transcription factor that negatively regulates gcy-7 expression [18]. In order to score the inhibition of lsy-6 activity, we used two reporter strains expressing GFP in either ASEL (gcy-7 prom ::gfp, strain OH3191) or ASER (right ASE; gcy-5 prom ::gfp, strain OH3192). Rh dextran-( as-2'OMe lsy-6) 1 was highly effective in inhibiting lsy-6. At the injection concentration of only 3 μM, the compound inhibited lsy-6 with nearly 100% penetrance, assayed by the repression of gcy-7 prom ::gfp expression in ASEL (Figure 7a), and by the induction of ectopic gcy-5 prom ::gfp expression in ASEL (Figure 7b).
Another miRNA, let-7, controls the larval-to-adult transition by repressing the translation of lin-41 and hbl-1. Mutations in let-7 lead to a retarded terminal differentiation of seam cells, which results in the elimination of alae and a bursting of the animal at the vulva as the animal undergoes the fourth molt [19]. We observed a dosedependent bursting or no-alae phenotype, consistent with inhibition of let-7 activity in the progeny of hermaphrodites injected with Rh dextran-( as-2'OMe let-7) 1 (Figure 8). When Rh dextran-( as-2'OMe let-7) 1 was injected at 20 μM, it caused bursting vulva in about 40% of labelled worms and eliminated the formation of alae in nearly 80% of worms. Raising the concentration of Rh dextran-( as-2'OMe let-7) 1 to 50 μM or above increased the penetrance, and none of the labelled animals showed alae when they reached young adults. In contrast, an injection of Rh dextran-( as-2'OMe mir-84) 1 directed against another let-7 family microRNA caused no observable phenotype. Since mir-84 mutations do not cause visible phenotypes [20], this result is consistent with these antisense reagents inhibiting the targeted miRNA with high specificity. Further, in a lin-41 nonnull mutant, lin-41(ma104), Rh dextran-( as-2'OMe let-7) 1 (50 μM) failed to produce the bursting  . This again suggested that these antisense reagents acted specifically by blocking their corresponding microRNAs.
In order to confirm that this class of antisense reagents is also effective in inhibiting microRNAs during embryo development, we prepared Rh dextran-( as-2'OMe mir-42) 1 against mir-42, a member of the mir-35 family which consists of eight microRNA genes of similar sequences. This family of microRNAs is expressed only during embryogenesis [21] and functions redundantly to control embryonic development [8,22]. Deletion of the seven microRNAs (mir35 -41, strain MT14119) leads to a temperature-sensitive late embryonic or L1 lethal phenotype. At 15°C, about 10% of the embryos displayed this phenotype, while the remaining 90% developed normally (Figure 9). In this mir35-41 null background, inhibition of mir-42 with Rh dextran-( as-2'OMe mir-42) 1 dramatically increased embryonic or L1 lethality: at the injection concentration of 3 μM, 77% of labelled worms were embryonic or L1 lethal. In contrast, inhibition of mir-42 had little effect in N2 strain (Figure 9).
Among four miRNAs tested (lin-4, lsy-6, let-7 and mir-42), the dose of antisense reagents required for the effective inhibition of individual miRNA varied from as low as three micromolar (lsy-6 and mir-42) to as high as tens of micromolar (let-7). Two factors may account for the difference in the apparent potency of these antisense reagents. First, the cellular expression level of different miRNAs varies over a wide range [23], so a higher concentration of antisense reagents is needed to block more abundant miRNAs. Second, to inhibit a miRNA that is expressed late in larval development, it would require a higher concentration of reagent in the zygote to compensate for the dilution effect of cell division and larval growth. let-7, for example, is not expressed until the third larval stage and functions in the fourth stage [19]. By that time, the antisense reagent would be further diluted as animals grow and expand in size. In principle, a higher concentration of antisense reagents can be used to overcome the dilution effect. However, it should be cautioned that, as we raised the dose, we also noticed that an increasing number of embryos failed to hatch at concentrations above 50 μM. The extent to which these antisense reagents affect embryo development seemed to vary. Among the reagents tested, antisense reagents (antimirs) against lin-4 and mir-237 were best tolerated, with nearly 100% of the embryos hatched normally at 100 μM. By comparison, the antimir against let-7 was least tolerated, with close to 40% of embryos hatched at 100 μM ( Figure 10). We have not yet explored the causes for this differential effect, although we anticipate that the future development of other bioconjugates employing different classes of oligonucleotides, for example locked nucleic acids or 2'-O-methoxyethyl oligoribonucleotides, may offer an improvement on the potency with concomitant reduction of the perturbation on embryo development.
In order to test whether these antisense reagents can be used combinatorially to inhibit more than one miRNAs at a time, we coinjected Rh dextran-( as-2'OMe lsy-6) 1 and Rh dextran-( as-2'OMe lin-4) 1 into gonads of OH3192 strain (gcy-5 prom ::gfp). Together, these two reagents caused Egl in all labelled worms and they induced ectopic gcy-5 prom ::gfp expression in ASEL (Figure 11). In contrast, Rh dextran-( as-2'OMe lin-4) 1 alone did not alter the expression pattern of gcy-5 prom ::gfp, and Rh dextran-( as-2'OMe lsy-6) 1 by itself failed to cause Egl ( Figure 11). The results again confirmed the specificity of these dextran-conjugated antisense reagents and suggested that multiple antisense reagents can be used in combination to block more than one miRNAs in  C. elegans. As numerous deletion strains of single miRNA genes appear to be grossly normal, it has been suggested that redundancy might mask their functions [8]. Mixing these antisense reagents would allow the study of the combinatorial effects of multiple miRNAs on gene expression and should facilitate screening genetic interactions using mutants or reporter strains.

Conclusions
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.
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'OMe lin-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.
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 fluorophorecoated 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'OMe lin-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.