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CHALLENGES
forthe Analytical Chemist I
n the world of science, many discoveries are the result of unexplained, accidental findings. These findings are not viewed as legitimate until significant evidence supports the phenomenon, opening the door to research that ventures into new territory within a particular scientific discipline. One such example is the discovery of microRNA (miRNA), an 18–24-nucleotide noncoding (i.e., does not code for protein) RNA molecule in the genes of humans, plants, and animals. Within the past few years, miRNA research programs have flourished because of results that were stumbled upon in Ambros’s laboratory, where the developmental gene pathway of the soil nematode C. elegans was being studied (box on p 4760). Researchers discovered that the gene lin-14 was negatively regulated by the gene lin-4, which encoded two small noncoding strands of RNA, one containing 22 nucleotides and the other 61 (1). Both lin-14 and lin-4 control larval development and have recently been shown to regulate the life span of C. elegans (2). It was predicted that the 61-nucleotide strand would fold into a stem–loop structure, believed to be the precursor of the smaller, 22-nucleotide strand. At that time, this discovery
SAPNA K. DEO
With the emergence of microRNA as a key player in gene regulation, rapid, sensitive, and quantitative microRNA detection methods are imperative.
Kyle A. Cissell Suresh Shrestha Sapna K. Deo Indiana University Purdue University Indianapolis © 2007 AMERICAN CHEMICAL SOCIETY
was viewed as an anomaly, unlikely to occur frequently in nature. Less than a decade later, however, another noncoding, small RNA molecule was discovered, again in C. elegans (3). Ruvkun and colleagues discovered that this small, noncoding RNA molecule encoded by the gene let-7 regulated the transcription of lin-14. The discovery of let-7 sparked the recent boom in miRNA research. Soon, >100 new genes for miRNAs were discovered in humans, worms, and Drosophila (4–6). Gene regulation by miRNAs plays a role in cell proliferation, cell death, tumorigenesis, and mammalian cell development (7, 8). Thousands of published miRNA sequences are located in the database miRBase (9, 10). Many of these miRNA sequences overlap from species to species, but numerous unique sequences exist. The importance of miRNA and its role in gene interference cannot be stressed enough, as evidenced by the 2006 Nobel Prize in Medicine, awarded to Fire and Mello for their work in RNA interference. miRNA raises questions as to the actual function of genes, in particular, the purpose of noncoding strands of RNA encoded from genes. It has long been thought that most RNA codes for the translation of proteins. Could there possibly be more to RNA than previously speculated? In fact, a significant amount of RNA in a cell does not code for the translation of proteins— rather, it regulates gene expression. For example, of the 62% of the mouse genome that is transcribed, ~50% of the RNA has been found to be noncoding (11, 12). The biogenesis of miRNA is rather complex, comprising many steps (Figure 1). Before mature miRNA is formed, primary miRNA (pri-miRNA), a long strand of RNA containing stem–loop structures (up to 1 kb in length), is initially transcribed by RNA polymerase II from the gene (13) and further excised by the endonuclease Drosha (14). After excision, the pri-miRNA, now called pre-miRNA, is exported to the cytoplasm via Exportin-5 (Exp-5; 15–18 ). This pre-miRNA consists of a long J U LY 1 , 2 0 0 7 / A N A LY T I C A L C H E M I S T R Y
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RNA polymerase
Pri-miRNA
mRNA target Mature miRNA
Drosha processing Pre-miRNA
RISC
Dicer processing
Exportin-5-mediated transport into cytoplasm
Mature miRNA
FIGURE 1. miRNA biogenesis. RNA polymerase II generates pri-miRNA. After cleavage by the endonuclease Drosha, pre-miRNA is formed and transported into the cytoplasm. The premiRNA is recognized by the Dicer, which cleaves it into a biologically active, mature miRNA. The single-stranded miRNA associates with the RNA-induced silencing complex (RISC) and binds to the 3´ untranslated region of its target mRNA, either to excise mRNA directly or to regulate protein translation indirectly through gene silencing.
stem of ~25–30 nucleotides along with a small loop structure. After the pre-miRNA is released from the Exp-5, it is cleaved by an RNase III enzyme known as Dicer, to generate mature miRNA. The mature miRNA, in conjunction with the RNA-induced silencing complex, anneals to the 3´ untranslated region of its complementary target mRNA. Once bound to the target mRNA, the miRNA can induce cleavage of the mRNA, regulating protein translation machinery directly, or regulate translation indirectly through gene silencing. Therefore, if we are to fully understand the roles of miRNA in the cell, detecting these small molecules with high sensitivity and specificity becomes of paramount importance.
Why detect miRNA? These tiny miRNA molecules can be found in plant, human, and animal tissues and clearly play many functional roles in cells as they regulate gene activity. Croce and colleagues reported that the miRNAs mir-125b, mir-145, mir-21, and mir-155 are significantly deregulated in human cancerous tissues compared with normal tissues; this finding links miRNA transcription regulation to increased malignancy (19). An analysis of primary tumors showed down-regulation of mature miRNAs compared with the levels of miRNA primary transcripts; this indicates posttranscriptional regulation of miRNA function (20). Many miRNAs are present in human embryonic stem cells; their presence is probably tied to development (21–23). Also, miRNA has been found to be involved in maintaining the infectious state of latent herpes simplex virus cells (24).
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In the landmark publication by Ambros and his colleagues, miRNA in C. elegans was shown to play roles in larval development (1). miRNA also has been found to play a role in apoptosis in insects and in the development of plants (25–30). Many other accounts of miRNA research indicate that they act as regulators and play key roles in inhibition or promotion of protein translation. Although miRNA performs gene regulation through translational repression or mRNA cleavage, the specific mechanism by which this occurs and the role of miRNA expression levels are not fully understood. To understand the functions of miRNA, it is necessary to understand how and where it is produced as well as the global changes within an organism associated with variations in miRNA expression level. The expression profiles may serve as molecularlevel diagnostic tests for diseases as well as new targets in drug discovery. Selective and highly sensitive detection methods will pave the way for extended understanding of miRNA function and further validate its purposes within organisms (31). Detection of these molecules, however, introduces many demands.
Challenges Before miRNA detection is described, it is important to point out that the main problem is the small size of miRNA. Currently, the vast majority of miRNA detection methods rely on hybridization, in which a target miRNA molecule hybridizes with a complementary labeled oligonucleotide probe. Because miRNAs are small, designing these probes is difficult. The signal transducers must be devised such that they do not interfere with hybridization. The small size of miRNA also makes selective pairing of miRNAs difficult. With any small hybridization probe, the oligonucleotide annealing temperature is low, thereby lessening the stringency of hybridization and greatly increasing the risk of cross-hybridization. If a method is not selective enough, a mismatch in one base may still produce a signal, giving a false positive reading. Another problem is the sensitivity of the assay. Cellular miRNA concentration can be as low as 1000 molecules per cell (32). If the assay calls for isolation of miRNA, its sensitivity will be limited by the efficiency of the isolation method; this step also increases the overall assay time. To isolate miRNA from cells, enrichment and amplification must be performed. The majority of the work in this area is performed by large companies. This is an open field of research to which chemists can contribute by developing isolation methods that can work on small samples, discriminate between pre-miRNA and mature miRNA, and allow high miRNA enrichment. If a highly sensitive method of miRNA detection is developed, it may be possible to avoid the amplification step and decrease total assay time. Furthermore, high sensitivity is important to determine small changes in the expression levels of miRNA, which have functional importance. Another challenge is in situ detection. When a sample that is a mixture of pre- and mature miRNA is studied, the oligonucleotide probe can anneal nonspecifically to pre-miRNA. This can cause false positive readings for expression levels of mature
d se es ld oc go Pr ith w
S en ha ilve r nc em en t
Pr wi oce th sse QD d
miRNA, because precursor miRNA levels are not indica(a) Biotin–X-hydrazide O Base tive of their corresponding O O Base Base RNA RNA NalO4 RNA mature miRNA levels. A major N O O HO OH challenge for in situ detection Biotin is the method by which the hybridization probe is delivered (b) to the cell. For example, probes may interfere with the normal actions of the cell by Oligonucleotide probes inducing mRNA cleavage or Biotin-labeled sparking a cell signaling pathmiRNA way. In addition, washing steps cannot be used—therefore, Hybridization homogeneous assays are needed. Furthermore, the solubility of oligonucleotide–label conjugates plays a major role with in situ detection. Ultimately, miRNA detection methods should be rapid, sensitive, and FIGURE 2. Overcoming low sensitivity by using probes. selective; require minimal (a) The target miRNA is biotinylated by converting the 3´ hydroxyls on the ribose ring into a dialdehyde, which further reamounts of sample; and be ca- acts with biotin–X-hydrazide. (b) Immobilized oligonucleotide probes complementary to the target miRNA are reacted with the biotinylated miRNA, followed by addition of a streptavidin–quantum-dot (QD) probe. The biotinylated miRNA pable of in situ application. miRNA expression levels may also be processed with gold in a colorimetric procedure that results in a buildup of metallic silver on the gold surface. (Adapted with permission from Ref. 42.) are gaining popularity as signatures for clinical diagnostics. Existing detection methods are mainly used for research purpos- membrane (3, 5, 34, 35). This method is time-consuming, often es and are not ideal for diagnostics. To be clinically useful, these taking days for completion; however, it is considered the gold methods should be able to detect several miRNAs simultaneous- standard for miRNA detection and validation. A complication is ly, to yield an expression profile. Validation criteria also need to that blotting methods are not sensitive and therefore require large amounts of sample (~10–30 µg) and have detection limits be established before these methods can be used practically. in the nanomole range. This sensitivity problem has been miniExisting detection methods mized with the addition of locked nucleic acids (LNAs) to the The detection of miRNA is based on hybridization, which can be probe sequence, increasing the detection sensitivity 10-fold combroken down into the categories of PCR, blotting, and microar- pared with normal RNA or DNA oligonucleotides (36). LNAs ray-based methods. No matter what the method, each assay must are RNA analogs in which the furanose ring in the sugar-phosproduce a signal upon hybridization—that is, a transducer must phate backbone is locked in place by a 2´-O, 4´-C methylene be present in order to translate the hybridization event into a bridge. LNAs exhibit increased thermal stability compared with measurable signal. their DNA and RNA oligonucleotide counterparts. Although PCR and blotting methods, although biological in nature, are PCR and northern blotting are important tools for miRNA dementioned because they have played a significant role in miRNA tection, simpler, faster, and more sensitive methods should be exdiscovery and are useful for validating new detection assays. PCR plored. Microarray technology is being used more and more by scimethods use the annealing and extension events of complementary primer oligonucleotides to amplify miRNA in the sample. entists seeking ways to decrease sample volume, test multiple PCR is helpful when only minute amounts of miRNA are ex- samples simultaneously, and decrease assay time. The miRNA tracted from cells. Because of the small length of the miRNA microarray chips can contain immobilized oligonucleotide oligonucleotides, the primer oligonucleotide must also be short. probes that are complementary to a miRNA target sequence. This short primer length ultimately demands a very low melting These oligonucleotide probes contain a signal transducer, such as temperature, affecting the efficiency of the PCR. This problem Cy 3, Cy 5, or biotin, to characterize the hybridization event. In can be combated by detecting miRNA precursors rather than some cases, the signal transducer may be directly conjugated to mature miRNA (33); however, pre-miRNA levels may not be the miRNA in the sample. The miRNA is then added to each well, which is washed to remove unhybridized molecules. Each homologous with the mature miRNA levels in the cell. Perhaps the most used miRNA detection method is northern well produces a particular color upon staining or yields fluoresblotting, in which a complementary labeled oligonucleotide cence signals, which are then measured to determine the amount probe binds to a target miRNA captured on a nitrocellulose of hybridized miRNA in a sample well. J U LY 1 , 2 0 0 7 / A N A LY T I C A L C H E M I S T R Y
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In this method, isolated miRNA is oxidized to conO vert the 3´ terminal hydroxN yls on the ribose ring into a HO N OH O + HN dialdehyde, which then reO O acts with a biotin–X-hydraH2N–N O O N H zide via condensation to biotinylate the target miRNA Oligonucleotide Target (Figure 2a). The biotinylatprobes miRNA ed miRNA is then added to the immobilized capture probe. After a wash, hybridNanoparticle Hydrazine Hybridization ization is detected by using a Time streptavidin–quantum-dot conjugate, as shown in FigFIGURE 3. Electrocatalytic detection. ure 2b. When the hybridized probes are excited with Complementary oligonucleotide probes are immobilized on a glass slide and hybridized with miRNA. OsO2 nanolight, the quantum dots particles are fused to the hybridized miRNA target–probe. Upon addition of hydrazine, the OsO2 nanoparticles act as catalysts for the oxidation of hydrazine, which produces an electrochemical signal. (Adapted from Ref. 43.) fluoresce, and this is detected with a laser confocal scanner. This method produces a Many cited studies have used microarrays to detect and pro- lower detection limit of ~0.4 fmol, much lower than that of file miRNA. For example, Croce and colleagues designed a mi- northern blotting methods. croarray for profiling 245 miRNAs from humans, mice, and As an alternative to quantum-dot probes, gold nanoparticles Arabidopsis (37 ). They modified target miRNA by reacting it conjugated to streptavidin were used (Figure 2b). Once the tarwith primers containing biotin. The biotin-containing targets get miRNA is labeled with the nanoparticle–streptavidin comwere allowed to hybridize with solid-phase oligonucleotides rep- plex and hybridized with the complementary oligonucleotide resenting the 245 different miRNA sequences. After wash steps probe, silver is added. During silver enhancement, colloidal to remove unbound miRNA, the hybridized probes were react- gold, in the presence of a reducing agent and silver ions, cated with streptavidin–Alexa647 conjugate. The affinity of the bi- alyzes the reduction of silver ions to metallic silver, which forms otin–streptavidin conjugate allows the Alexa647 dye to serve as on the surface of the gold nanoparticle. This buildup of metallic the signal transducer that detects hybridization. Fluorescence silver can be detected via a CCD camera mounted on a micromeasurements determined the intensity of each well in order to scope. This colorimetric method of miRNA detection has a characterize the expression profile of the target miRNA. This lower detection limit of only 0.1 fmol. The hybridization methmethod showed that only 2.5 µg of total RNA sample is needed ods based on quantum dots and gold nanoparticles are both to detect miRNA, compared with ~10 µg for northern blotting. highly sensitive and are viable alternatives to the time-consumPerhaps the greatest advantage of the microarray platform ing northern blotting method. over other detection methods is its high-throughput-screening Recently, a novel method of miRNA detection was introduced capability. Because of this advantage, microarrays are widely used by Gao and Yang. It is based on electrocatalytic OsO2 nanopartito characterize miRNA expression profiles (38–41). Although cle tags and produces a detection limit in the femtomole range the cost of microarray technology is front-loaded with the over- (43; Figure 3). In this method, complementary oligonucleotide head costs of the robotics needed for fabrication, in the long run, capture probes are immobilized on a glass slide coated with indimicroarrays are cost-effective in large laboratories where thou- um tin oxide. This is followed by washing with periodate-treated sands of samples are screened daily. However, this method is not target miRNA and hybridization. At this point, OsO2 nanopartisuitable for small research laboratories. Microarrays have other cles are added to the reaction chamber. A condensation reaction drawbacks, including low sensitivity due to minimal sample vol- occurs between the drug isoniazid and the 3´ terminal dialdeume and the possibility of cross-hybridization, which may occur hydes of the miRNA molecules. Once the capped nanoparticles when samples differ by only one base. Also, no specific standard are fused to the miRNA, the hybridization event can be characexists for data analysis or validation. The only true validation terized via the electrochemical current produced by the nanoparmethods available are blotting methods, which have been stan- ticle-catalyzed oxidation of hydrazine. If a miRNA molecule does dardized. not hybridize with the probe, then the nanoparticles cannot conjugate to the miRNA and an electrochemical signal will not result. In this approach, the signal transducer is chemically bound Emerging detection methods To overcome the low sensitivity observed with organic fluo- to the target rather than to the oligonucleotide capture probe; rophores in hybridization assays, Ruan and colleagues have eval- this increases the sensitivity and selectivity of the assay. Modificauated probes such as quantum dots and gold nanoparticles (42). tions of the Gao and Yang method use Ru(PD)2Cl2 and Os4758
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(dmpy)2(IN)Cl+ instead of OsO2 nanoparticles (a) (44, 45). These electrochemical methods are simple, sensitive, and rapid. Another advantage of using electrochemistry is the low cost of the assay. Molecular beacons (MBs) are another tool for miRNA detection. MBs are stem–loop Hybridization resulting in structures consisting of a double-stranded stem loop opening and generation of signal ~4 or 5 bp in length, containing a donor and quencher fluorophore at the 5´ and 3´ ends, along with a single-stranded loop complemenAddition of tary to the target DNA or RNA base sequence miRNA (46; Figure 4a). In the absence of a target, the Quenching of fluorescence fluorescence of the donor is quenched; in the presence of a target, the annealing of the MB (b) leads to separation of the donor and acceptor Q F fluorophores, resulting in fluorescence emission upon excitation. 3´ 5´ + F Q MBs are very selective, differentiating tar5´ C 5´ gets with a mismatch of as little as a single base. 3´ 5´ A 5´ One of the problems of using MBs to detect 3´ 5´ 3´ miRNA is that the small size of miRNA means 3´ 3´ that the loop of the MB must be small in order to hybridize with the target miRNA. Because of the small size of the loop, the stems may be B B unable to dissociate from one another, resulting in low fluorescence. Another disadvantage is the lack of signal amplification, which limits sensitivity. To improve this situation, multiple fluorophores have been attached to the stems FIGURE 4. MB hybridization. of MBs. Famulok and colleagues accomplished di- (a) MBs consist of a stem–loop structure with a fluorophore and quencher conjugated to opposite rect detection through hybridization of mi- ends of the stem, resulting in a quenching of fluorescence. Once the MB is hybridized with miRNA, RNA to an MB or through signal-amplifying the stem is separated, producing a fluorescence signal. (b) iHP-let-7 ribozyme (B) is hybridized with ribozymes (47 ). In the ribozyme approach, a the miRNA let-7 at C, which allows the hairpin to bind to A. Hybridization of the hairpin probe results in its cleavage (crooked arrow) and a measurable signal from the fluorophore. (Adapted from Ref. 47.) hairpin probe labeled with donor and quencher fluorophores is used as a signal transducer (Figure 4b). The basis of this assay is that the ribozyme iHP-let-7 will these probes react and hybridize with 50–100 ng total tissue catalyze the cleavage of the hairpin probe upon hybridization of miRNA, unbound probes are hybridized with fluorescence the miRNA let-7 with its target. Once cleavage results, a fluores- quenchers to minimize background. The resulting probe–micence signal is obtained. A detection limit of 50 fmol miRNA RNA hybridized molecule is passed through a capillary where was observed—an improvement over northern blotting tech- the probes are excited via a series of lasers, and the fluorescence niques that yield detection limits in the nanomole range. For de- emission is recorded on a CCD camera. A defining attribute of this method is its ability to differentiate tection that uses the MB without docking to a ribozyme, results were an order of magnitude lower in sensitivity (Figure 4a). The between single-base mismatches, as shown through the studies of ribozyme method has drawbacks, however, when applied in situ. the let-7 gene family members. The genes let-7a and let-7c differ Ribozymes are subject to digestion through ribonuclease activi- by only one base yet produce a threefold difference in the numty. To be applied in situ, the ribozymes must be engineered such ber of coincident events counted by the CCD camera upon hythat they are stable in the presence of ribonucleases. bridization of the let-7a-labeled probe to let-7a and let-7c. AnA miRNA detection method that uses fluorescence correla- other positive result from this method lies in the ability to detect tion spectroscopy has been developed (48). In this method, miRNAs in tissues where they were previously not detected via oligonucleotide probes composed of DNA and LNA comple- microarrays or northern blotting because of their low sensitivity. Fang and colleagues use nanoparticle-amplified surface plasmentary to the 5´ and 3´ ends of target miRNA were labeled with Oyster 556 and Oyster 656 fluorescent probes, which each mon resonance (SPR) imaging to detect polyadenylated synthetproduce spectrally distinct signals upon laser excitation. Once ic miRNA as well as miRNA from total RNA samples (49). This
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A short history of miRNA. ing, such as various ligands and proteins. In addition, symmetric binding and 1993 – 2003 Northern blotting PCR detection in the nanomole range. strong interaction at the 5´ end is preferred for miRNA–mRNA binding. Re2000 Ruvkun lab discovers let-7 in C. elegans (3 ). search efforts need to be targeted toward 2001 miRNA officially named (4 –6 ). the development of computational methods that allow binding to be predicted 2002 Croce lab links miRNA levels to cancer (33 ). and then experimentally validated. These 2004 – present Microarray detection in the femtomole to attomole range. computational studies would prove highly beneficial in the design of novel thera2007 and beyond Standardization of rapid, sensitive, and selective detection methods; pies that involve binding interactions. is subattomole detection possible? Miniaturization and microfluidics have played a significant role in the diagnostic method has been designed in a microarray format, thus enabling field. Many papers have been published on microfluidic detecthe detection of multiple miRNAs in parallel. To detect miRNA, tion of DNA and RNA on microchips. For example, Baeumner 3´-thiol-modified LNA oligonucleotides, which are complemen- and colleagues developed a microchip that could detect pathotary to target miRNA, are immobilized onto a solid surface gens on the basis of target mRNA sequences with fluorescence through a heterobifunctional linker. After immobilization, mi- (54 ). Detection methods for miRNA could be adapted easily to RNA from the sample is hybridized with the complementary microchip platforms. The small sample volume and minimal LNA oligonucleotides. The hybridized miRNA is then polyade- assay time make the use of microfluidics desirable for miRNAnylated with poly (A) polymerase. Once polyadenylated, the based diagnostics. Currently, miRNAs are studied only in terms of their biologmiRNA is then exposed to thymine-modified gold nanoparticles that hybridize with the poly (A) tail. The bound gold nanoparti- ical role. Studies targeting their structure and mechanism of cles are detected via SPR imaging. An advantage of this method binding may shed light on their activity. In addition, the effects is the signal enhancement provided by the gold nanoparticles. of small ligands as well as macromolecules on miRNA activity The lower detection limit is 5 amol, which is one of the lowest have not been studied. These types of fundamental evaluation can be performed through analytical techniques. We have reported for miRNA. reached a point where hundreds of different miRNAs have been Future developments discovered. It is well accepted that these tiny molecules are synMore and more, researchers are looking into the roles of miRNA thesized in cells for a purpose; however, to decipher the role of in different cellular processes; however, progress in the develop- these miRNAs in human health and in plant and animal biology, ment of sensitive, accurate, and easy-to-use detection methods the development of miRNA detection methods must continue. has not been as rapid. Cellular regulations are dynamic processes that can be affect- Cissell would like to thank Indiana University for support through the Indiana ed by simple perturbations. Therefore, an ideal method for University Research Support Grant Award. We would also like to thank David studying gene regulation by miRNA would provide for single- Harvey, Frank Schultz, and Sylvia Daunert for review of the manuscript and cell direct monitoring of miRNA with high spatial and temporal Emre Dikici for drawing Figure 1. resolution. Advances in microscopy techniques and the availability of reporters with high quantum yield should make such de- Kyle A. Cissell is a graduate student at Indiana University Purdue Unitection possible. Researchers have been working in the field of versity Indianapolis (IUPUI). Suresh Shrestha is currently employed direct labeling of miRNA in the living system (50). Another by Eli Lilly and Co. Sapna K. Deo is an assistant professor at IUPUI. problem that needs attention is the ability to detect changes in The research interests of her group include development of novel biothe level of miRNA on top of the existing background level of analytical methods for the detection of nucleic acids, small molecules, and proteins. The research involves the use of fluorescent and miRNA in a single cell. The development of computational programs to determine luminescent proteins and molecules. Address correspondence about binding between miRNA and their respective mRNA targets is this article to Deo at
[email protected]. another open area of research. One example of such a program is MicroInspector (51). With this program, miRNA binding sites References are found from a database of known miRNA–mRNA binding (1) Lee, R. C.; Feinbaum, R. L.; Ambros, V. Cell 1993, 75, 843–854. sites, binding strength of hybridization, examination of second- (2) Boehm, M.; Slack, F. Science 2005, 310, 1954–1957. ary structures, and the organism the miRNA originated from. (3) Reinhart, B. J.; et al. Nature 2000, 403, 901–906. Other bioinformatics methods that use different demarcation (4) Lau, N. C.; et al. Science 2001, 294, 858–862. criteria have been developed as well (52, 53). (5) Lee, R. C.; Ambros, V. Science 2001, 294, 862–864. The binding between miRNA and its target mRNA occurs (6) Lagos-Quintana, M.; et al. Science 2001, 294, 853–858. through base-pairing interactions; however, in mammals, it is not (7) Hwang, H. W.; et al. Br. J. Cancer 2006, 94, 776–780. completely complementary. Other factors may assist in this bind- (8) Grishok, A.; et al. Cell 2001, 106, 23–34. 1993
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Ambros lab discovers the first miRNA, lin-4, in C. elegans (1).
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