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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 59:B771-B783 (2004)
© 2004 The Gerontological Society of America

Emerging Technologies

Trendy RNA Tools for Aging Research

Custer C. Deocaris1,2, Sunil C. Kaul1, Kazunari Taira1,2 and Renu Wadhwa1,

1 Gene Function Research Center, National Institute of Advanced Industrial Science & Technology (AIST), Tsukuba Science City, Japan.
2 Department of Chemistry and Biotechnology, School of Engineering, University of Tokyo, Japan.

Address correspondence to Dr. Renu Wadhwa, Gene Function Research Center, National Institute of Advanced Industrial Science & Technology (AIST), 1-1-1 Higashi, Tsukuba Science City 305-8562, Japan. E-mail: renu-wadhwa{at}aist.go.jp


   Abstract
Top
 Abstract
The RNA Toolbox
 RNA-Based Proteome Probes
 Gene Silencing by RNA...
 References
 
Aging is an inevitable biological phenomenon. Attempts to understand its mechanisms and, consequently, to therapeutically decelerate or even reverse the process are limited by its daunting complexity. Rapid and robust functional genomic tools suited to a wide array of experimental model systems are needed to dissect the interplay of individual genes during aging. In this article, we review principles that transcend the view of RNA, from a molecule merely mediating the flow of genetic information, into a unique molecular tool. In the form of catalytic molecular scissors (ribozymes), antibody-like antagonists (aptamers) and gene silencers (interfering RNAs, RNAi) can be effectively used to dissect biofunctions conserved throughout the evolution. In this review, application of recent RNA tools in aging research is discussed.

AGING, most commonly defined as functional decline with age, is the most fundamental feature of life expressed at all levels of living organisms. Our understanding of this phenomenon is limited by its sheer vastness, ambiguity, and complexity, affecting the entire physiology of an organism. In the past 50 years, numerous age-related changes at biochemical, cellular, and organismal levels have been catalogued along with their evolutionary, social, and demographic dimensions. Attempts to interpret these colossal data has generated different theories on aging, more aptly termed "senescence" (1). These fall into two major categories: (i) genetic basis of functional decline as an inevitable outcome of the evolutionary conserved biofunctions; and (ii) functional crisis due to inbuilt limits of living systems to repair genetic and stochastic damage (accumulated mutations, error catastrophes, posttranslational defects, oxidative stressors, and so forth) (2–5). Owing to the recent advances in molecular biology, gerontologists in this decade have extended concepts of aging from a "fatalistic" conundrum—we age because our bodies follow an evolutionarily selected, genetically–programmed, and tightly regulated continuum of development, maturation, and finally, demise—to adopting a "reductionist" approach in identifying bases of functional decline at molecular and cellular levels. Elucidation of gene expression profiles, their regulatory pathways, and genotype–phenotype correlations have defined in toto our current conceptual paradigms of aging as an extremely intricate interplay of genes. Some key players eventually leading to long- lived or short-lived organisms (worms, flies, and mice) have been identified (6–8). Future attempts using different model systems and molecular approaches shall comprehensively aim to extend the healthy years of human life span and delay the onset of old age diseases.

The fact that each species has a typical range of life span attests to the existence of genetic determinants in aging. Humans, for example, have an average life span 15–20-fold greater than mice even after normalizing for some biodemographic contributions of the major killers: cancer, diabetes, and cardiovascular diseases (9). Studies on zygotic twins showed that genes contribute to 25% of the variation in life span, whereas others propose a larger genetic contribution of 52% (10). Perhaps the best evidence of its genetic basis are the heritable mutations that accelerate aging phenotypes, for example, Hutchinson-Gilford and Werner's syndromes, caused by mutational defects in the lamin A/C (11) and in the DNA helicase (12), respectively. This rubric of human aging diseases suggests that molecular glitches in the mechanism for genomic integrity underlie at least some of the known aging phenotypes. Early fusion experiments strengthen the contention of the genetic basis of human senescence. Hybrids obtained from fusing normal with immortal human cells invariably exhibited limited division potential. It appeared that the finite proliferative capacity of normal human cells was dominant and that immortal cells had acquired recessive changes in their genetic program, which allowed them to escape senescence. Several of these whole cell and microcell fusion studies have lent support to the hypotheses that senescence indeed results from active, genetic mechanisms (13). Consistently, more recent exciting research endeavors on cloning and somatic cell nuclear transfer attest to such a genetic behavior of aging systems and have, in principle, placed into proper perspective our blissful imaginative view of the "brave new clonal world." While not dampening expectations of many science fiction writers, cloning does not necessarily restore the telomere clock. Surprisingly, this is quite unlike ordinary conception wherein our children always start to age at zero level and not at the level of the telomeric clocks of the parents during conception. More so, rederived cell lines from cloned fetuses obtained by nuclear transfer experiments had the same proliferative capacity and rate of telomere shortening as the donor cell lines, underscoring the truism of aging being innate and genetically determined (14,15).

Aging in a Culture Dish
When normal cells are raised in a culture dish, they recapitulate the basic aging process of the organism. This phenomenon, termed "replicative senescence," was first documented by Hayflick and Moorehead in 1961 (16), and their discovery became the pillar for the biology of cellular aging. When cells are explanted from the body, they divide vigorously for a specific (depending on the source and age) number of population doubling (or the Hayflick limit), and "tire-down" until, finally, they completely cease to proliferate. In addition, various treatments (gamma irradiation, oxidative stress, activated Ha-ras oncogene, cancer chemotherapeutic agents, cross-linked DNA, ceramide, phosphatidylinositol-3-kinase inhibitor, DNA demethylating agents, histone deacetylase inhibitors, and inhibition of cyclic guanine monophosphate [cGMP] production) can result in rapid onset of permanent growth arrest and senescence-like morphological and biochemical alterations. This is referred to as "premature senescence" or "stasis," with the terms "senescence" or "replicative senescence" being kept for cells that have reached the Hayflick limit (16–18). Numerous studies in the recent past have established that cellular senescence is engendered by the activation and intricate interactions between tumor suppressor genes, Rb and/or p53, and their regulators, such as p16INK4a, p21WAF1, and adenosine diphosphate ribosylation factor (ARF) (19,20). What triggers this activation is not fully understood. DNA damage that occurs spontaneously with each cell division in the form of attrition of the telomeres (21–23) is implicated as one of the activating signals.

Telomeres are the ends of chromosomes, which consist of highly repetitive and conserved DNA (tandem repeats of TTAGGG, 4–15 kilobases [kb] in length in humans), that protect chromosome ends from fusion and [organize chromosomes?] organization of chromosomes during cell division. Due to the end-replication problem, a tiny end of the telomere is lost during each cell division and hence telomeres serve as "mitotic clocks or replicometers" that keep track of how many times their predecessors have divided (24,25). Telomere-driven human cell senescence is also modified by the presence of oxidative stress due to a telomere-specific damage repair deficiency (26) and by not-so-well-understood pathways involving tumor suppressor genes and oncogenes (27). In corollary, species with shorter life spans tend to lose more telomeric repeats with age than species with longer life spans (28). Yet, it seems to oppose the observation that telomeres of shorter-lived mice are longer than that of humans (29). Telomere lengths, in addition, do not show clear-cut correlation with tissue renewal times in vivo (30).

Confronted with this seemingly "madding" crowd of aging pathways, would it then be possible to define its genetic circuitry, understand it in terms of how the program unfolds, and appreciate how multitudes of these genes interact with each other and with the environment? To this goal, more powerful and direct genetic tools are needed. Fortunately, the recent completion of the human genome project has opened vistas in investigating gene functions and regulatory pathways. In the past, traditional RNA tools such as the cDNA library and RNA antisense probes have been assets for these studies. Recent unconventional and innovative approaches that exploit the very architecture and function of the RNA molecule have extended its applications in basic genomics, biotechnology, and biomedicine. Use of the modern RNA technology in aging research is discussed here.


   THE RNA TOOLBOX
Top
 Abstract
 The RNA Toolbox
RNA-Based Proteome Probes
 Gene Silencing by RNA...
 References
 
Anyone interested in understanding the influence of a known gene in aging processes, or who is planning on a hunting adventure for some hitherto unknown candidate aging gene, has a wide array of experimental RNA tools available. Each class of functional RNAs would have its attractive offers along with some inherent limitations.

Catalytic RNA Scissors
In the 1989 Nobel Prize-winning discovery in chemistry, the RNA molecule was portrayed as an enzyme (31,32). Ribozymes are distributed quite ubiquitously in nature. Accepted to be incarnates of the (hypothetical) hydrothermal RNA world (33), the biochemistry of RNA catalysis, as opposed to protein enzymatic action, provides an attractively plausible map of the origin of present-day metabolism and of life in general (34).

Ribozymes may be classified into three major groups: self-splicing introns, RNAse P, and the small catalytic RNAs. A multitude of self-splicing introns (groups I and II) totaling more than 1700 different types, are generally found in the nucleus and mitochondria of lower eukaryotes, prokaryotes, plants, and some bacteriophages. Groups I and II introns differ fundamentally from the typical introns in that they display self-splicing activities independent of the spliceosome. Furthermore, these introns have been shown to catalyze many trans reactions such as endonuclease, ligase, nucleotidyl tranferase, and phosphatase reactions with RNA as well as DNA substrates (35). A subclass of group II introns, for example, the Ll. LtrB group II intron from Lactococcus lactis bacterium, even has the unique ability to retrohome, that is, to insert itself into cognate intronless alleles or ectopic sites via a reverse splicing mode (36). These self-splicing introns are tapped to study gene function and to edit disease-relevant gene targets, for example, ßs-globin mRNA in sickle cell anemia-derived erythroid precursor cells (37).

RNase P ribozymes are a key enzyme in the biosynthesis of tRNAs. Its target recognition is not based on RNA sequence context but rather on the target's structural features, that is, the RNA target must bear a resemblance to the acceptor stem and T-stem structure of its natural precursor tRNA substrate. Recent modifications, such as the addition of guide sequences, have empowered it for gene-specific targeting as well (38). The M1GS ribozyme, which is engineered from the Escherichia coli catalytic RNA subunit M1 ribozyme, has been demonstrated to cleave mRNAs of herpes simplex virus 1, human cytomegalovirus, and cancer-causing BCR-ABL proteins in vitro (39–41). While, in recent publications, the catalytic activities of these myriad ribozymes are starting to be extensively explored in biotechnology, we focus this review on their diminutive catalytic siblings: small, yet potent, and which have been widely used and are more tractable and less expensive to synthesize in the laboratory.

Small Catalytic RNAs
Members of the small catalytic RNA family are the hammerhead, hairpin, hepatitis D virus, and the Varkud satellite (VS) ribozymes. Each is distinguished by its respective secondary structures, as shown in Figure 1.



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  		Figure 1. Ribozyme tools as derived from nature's RNA species. A, Hammerhead ribozyme. Using the standard nomenclature proposed by Hertel and colleagues (187), the conserved bases are shown in bold letters and the cleavage site is indicated by an arrow. In order that it cleaves in trans, the hammerhead ribozyme structure has two separate strands that function as the enzyme (E), composed of the catalytic core or stem-loop II (boxed), and substrate-binding region (S), stems I and III. The catalytic core captures Mg2+ ions to form "scissors" and cleaves adjacent to a site of a gene target with the triplet sequence NUX (N is any base and X is A, C, or U) (188). B, Hairpin ribozyme. The ribozyme-substrate complex comprises 2 structural domains, Domain A (with 2 intermolecular helices, H1 and H2, and a loop LA) and Domain B (with helices 3 and 4, and LB). To trans-cleave its target, Domain A docks upon Domain B, and the nucleotides of both loops (LA and LB) must interact to mediate a general acid–base catalysis. This type of cleavage is metal ion independent and requires a more constrained mRNA target sequence: BNGUC, where B = any nucleotide except adenosine (189). Other promising ribozyme tools include (C); a hepatitis D virus ribozyme motif (genomic and antigenomic) has four double-stranded regions (P1–P4). P1:P2 and P3:P4 form pseudoknot and stem-loop motifs, respectively. In the model, P1 defines the cleavage site; D, Varkud satellite ribozyme with structural domains I–VI arranged like the letter "H." Its cleavage site is indicated by an arrow. [Figures adapted from Puerta-Fernandez (190), with permission.]

 
The smallest of these ribozymes is the hammerhead. Originally derived from species of economically devastating plant pathogens, the viroids, it functions to mediate the self-cleavage of nascent viroid oligomeric RNAs in the rolling circle style of replication (42). Recently, active hammerhead domains are isolated from satellite DNAs of newts (43), the parasite Schistosoma (44), and the cave cricket Dolichopoda (45). A detailed mechanism of how this enzyme cleaves its substrate has been reviewed recently by Kato and Taira (46). Mediated by binding of a divalent metal ion, the hydroxide ion bound to a divalent metal deprotonates the cleavage site 2'-OH, followed by the nucleophilic attack of a 2'-OH group on the 3'-phosphate finally generating 5'-OH and 2',3'-cyclic phosphate termini. A second divalent metal ion may stabilize the transition state (47). Scott and colleagues have demonstrated that some monovalent cations (Li+ and ) at high concentrations can also activate a ribozyme (48).

The hairpin ribozyme was first identified in the negative strand of the satellite RNA associated with the tobacco ringspot virus (49). The hairpin motif is the first ribozyme approved for human clinical trials for the treatment of HIV (50). It catalyzes trans-cleavage reactions and also the reversible cleavage for a second RNA molecule via similar transesterification chemistry. Unlike the hammerhead, the hairpin ribozyme has more pronounced RNA ligase activity (10 times faster than its cleavage activity) and does not have a strict requirement for metal ions to serve as its Lewis acid. A hammerhead ribozyme, in contrast, has 100-times-faster cleavage compared to its ligation activity (51,52). The hairpin motif has the advantage of improved substrate recognition and binding because it obviates the need for a hard-to-achieve highly stable association and high sequence specificity for an RNA substrate demanded by the hammerhead motif (53).

Another type is the infectious human delta virus (HDV) ribozyme. To activate its killer instinct in the human population, it corroborates with the hepatitis B virion, and, consequently, its pathobiology assigns HDV a satellite RNA "personality" (54). HDV contains two cis-acting ribozymes (genomic and antigenomic) that are both required for its replication (55). To be trained to cleave in trans, either of the two minimal delta RNAs of HDV ribozyme is cloned, with its 5'-strand of the P1 helix replaced by a second RNA molecule that will act as a substrate. Sequence specificity is defined by the 3'-P1 sequence. With an estimated half-life of more than 100 hours, HDV ribozymes are highly stable ex vivo and are well suited for use in the human cell environment. Despite that fact, relatively few ex vivo gene inactivation work is being done on HDV ribozyme (56–58).

The VS ribozyme from the mitochondria of Neurospora (59) is dubbed as the largest nucleolytic ribozyme. The nuclear magnetic resonance structure of the active conformation of the VS ribozyme cleavage site has just recently been published (60). With an elegant damage selection experiment, Beattie and Collins were able to deduce its mechanism of action and locate the cleavage site in the VS catalytic core (61). Since cleavage by VS ribozyme requires a "kissing" interaction between its hairpin loops, stem-loops I and V (62), this stem-loop interaction might be appropriate to target RNA substrates with complex structures for gene inactivation.

Ramifications in Natural Ribozyme Motifs
New design features have been added to the natural ribozymes to improve their in vitro, in vivo, as well as therapeutic and marketability values. These classical models of trans-acting RNA catalysts, though they have proven useful in analyzing functions of genes in the eukaryotic cell, have some inherent technical problems. Inside the cell, the activity of ribozymes is affected by its level and site of expression, cleavage specificity, stability, and mRNA target accessibility (63). Since mRNA folds in highly complex superstructures, not all of its sequences that are theoretically cleavable by ribozymes work. In ruling out nontargets, there are available freewares, such as MFOLD (http://bioinfor.math.rpi.edu/~mfold/rna/), that have better predictive power over in vitro accessibility assays (64). However, its utility is most often limited by the size of the RNA to be analyzed, and, more often, selection of targets must still be determined experimentally.

Allosteric ribozymes are a type of catalytic RNAs that empowers a researcher to manipulate the RNA cleavage activity by means of an effector molecule that binds to a ribozyme's allosteric site (or noncatalytic binding region). These effectors include, but are not limited to, small organic compounds, metal ions, proteins, and oligonucleotides. By far, only oligonucleotide-regulated ribozymes have been useful in functional genomics research. Other nonconventional allosteric ribozymes demonstrate novel biotechnological utility such as serving as a nanocomponent in biosensors (65) to indicate specific analytes, for example, cyclic adenosine monophosphate (camp) (66,67), adenosine triphosphate (68), and the bronchodilator theophylline (69), from a complex ad mixture. Possibilities for target ligands and "ribozymic" switches are virtually limitless. A recent gene-suppressive allosteric ribozyme, called the "maxizyme," was heralded as having cleavage activity that can be switched–on or off by the binding of the ribozyme sensor arm of a target sequence. This discerning maxizyme, composed of two dimerizing truncated hammerheads (minizymes), offers promising clinical use in targeting HIV-1 tat mRNA (70), repairing mRNA of abl in bcr:abl translocations in Philadelphia syndrome (71), and in the restoration of mutant p53 function in tumors (72).

Protein chaperones are coerced to "hitch a ride" on ribozymes to improve these RNA catalysts' stability and cleavage activities. To date, several of these proteins were used in enabling rapid and faithful RNA–RNA annealing, strand transfer and exchange, and RNA ribozyme-mediated cleavage under physiological conditions. Examples of these useful chaperones are p7 nucleocapsid of HIV-1 (73), glyceraldehyde 3-phosphate dehydrogenase (74), and A1 (75). Another interesting turn in its development was the concept of RNA helicase-associated ribozymes (or sliding ribozymes). The idea is that ribozymes can be instructed to sequester intracellular helicases to clear-steer its mRNA target sequence of unwanted secondary and tertiary structures. Warashina and colleagues (76) attached a cytoplasmic RNA transport signal derived from the simian type-D retroviral RNA to a basic hammerhead ribozyme. Alternatively, a poly(A) motif was used in recruiting helicase eIF4AI via intermolecular associations with poly(A)-binding protein and the poly(A)-binding protein-interacting protein-1 to accomplish the same trick (77). This batch of sliding ribozymes were effective at knocking-down mortalin, an hsp70 family member involved in the control of cell proliferation, in contrast to the conventional ribozyme tools that were futile (78,79).

Ribozymes may be harnessed as agents that may be useful in manipulation of components in the aging process. Already, this has been realized by researchers focused at reversing immortality as a universal treatment against neoplasms. A prime example is the volume of work on ribozymes directed against telomerase, an enzyme absent in most normal somatic cells but present in immortal cells. Hammerhead motifs aimed at hTERT, the RNA moiety of telomerase, successfully attenuated telomerase activity, interfered with cell growth, and induced cell death by apoptosis in human hepatocellular carcinoma (80), endometrial carcinoma (81), melanoma (82), breast carcinoma (83), ovarian carcinoma (84), and nasopharyngeal carcinoma (85). In nontumor systems, these finely regulated RNA tools will help to offer new perspectives on the expanding role of telomerase in many aging-associated diseases such as osteoarthritis (86), macular degeneration (87), and cardiomyopathy (88).

Randomized Ribozyme Libraries
In the gene discovery marathon, much of the excitement in the ribozyme applications stems from its application in elucidating the role of different genes and nontranslated sequences with the development of ribozyme libraries (89). Such a library is composed of ribozymes with substrate recognition sequences that have been randomized. When such a library is introduced into a cell line, any gene involved in a specific cellular pathway under scrutiny will be suppressed by a specific active ribozyme species. Depending on the selection strategy, cells displaying a desired phenotype will be selected (rescued), and genomic DNA encoding the ribozyme will be subcloned and eventually sequenced. The identification of the gene target will be deduced by BLASTing the complementary sequence of the ribozyme substrate arm to the human genome databank. Although this "classic" ribozyme strategy has contributed to some successful gene target identification pursuits (89–94), the approach is vulnerable to the aforementioned factors that plagued early ribozyme scientists, such as the steric hindrance of mRNA problem, among many others.

RNA helicase-associated ribozymes was later recruited in the combinatorial scenario as these hybrids exhibit more robust gene suppressive activity than previous randomized ribozymes (95). In the span of 1 year, a gamut of genes involved in different biological phenotypes of cells has been identified. These include the apoptotic machinery mediated by Fas (96) and the tumor necrosis factor-{alpha} (TNF{alpha}) (97), retinoic acid-induced cell differentiation pathway of neuronal cells (98), migration and invasive properties of tumor cells (99,100), and Alzheimer's disease (101).

This combinatorial ribozyme library technology has pervaded the gene discovery activities in some aging research laboratories. One of the first papers describes the findings on dual function of telomerase in senescence and carcinogenesis by Li and colleagues (94). In the study, mouse fibroblasts were transduced with a ribozyme library, and foci were selected to identify putative target genes for the transformation. Telomerase-targeted ribozymes was serendipitously identified in the foci, and this suggested that the commonly accepted role of telomerase in maintaining cellular immortalization is more complicated than what had been previously thought. Immusol, an American biotech company, focused on expanding its palette of gene targets for drug development in aging-associated neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases, using their hairpin-based ribozyme library. In their drug discovery platform, a ribozyme library was introduced into the SK-N-MC neuroblastoma cell line to identify rescued cells after C2-ceramide and TNF{alpha}-induced apoptotic signals. Upon several rounds of selection and ribozyme rescue, three neuroprotective ribozymes and their respective cellular targets were validated and further studied with microarray analysis and yeast two-hybrid screens (102). These studies underscore the diverse application of this novel functional genomics strategy, enabling the targeted discovery of genes, whether previously known or not, that are involved during the aging process.

The study on the molecular mechanism underlying senescence as a whole has shed light on the principles of carcinogenesis and organismal aging. To be able to regulate induction in senescence (premature senescence or stress-induced senescence) in cells independent of telomere status is equally an important approach as with the unleashing of the suicide program. Interestingly, strategies to quell malignancy by forcing cells to senesce without primarily killing the tumor target are less popular despite their equivocal clinicopathologic impact. Ribozyme libraries may then be tapped to uncover a plethora of these genes, which we can manipulate in the effector programs of induced senescence. High-throughput technologies such as microarray screening of the aging transcriptome (103–109), and various functional assays, that is, yeast two-hybrid screens (110–113), have revealed quite an interesting collection of genes and pathways for senescent phenotypes in various tissues and organisms. In turn, combinatorial ribozyme technology has been proven invaluable for fast-tracking gene identification, target validation, and drug discovery simultaneously. The method of silencing by ribozymes may be particularly important in forward genetic strategies that need to select for genes in key regulatory steps of a pathway that can be selected even with a modest degree of knockdown. Complete silencing (e.g., with knockouts using antisense oligos and with knockdowns with RNAi) can be lethal to cells especially when the gene of study is essential for life (114).


   RNA-BASED PROTEOME PROBES
Top
 Abstract
 The RNA Toolbox
 RNA-Based Proteome Probes
Gene Silencing by RNA...
 References
 
In theory, the three-dimensional configuration of an RNA molecule, which has a structure complementary to a protein, can bind to a target protein with great specificity and affinity thus mimicking the action of antibodies without the hassle of raising BALB/c mice or rabbits.

SELEX (Systematic Evolution of Ligands by EXponential enrichment) technology, a technique in combinatorial chemistry, is applied to generate these RNA aptamers and intramers (intracellularly expressed aptamers). The basic strategy is to prepare the starting library by synthesizing a random stretch of 22 to 100 nt to create an enormous diversity of possible sequences that span an array of different conformations with different binding properties. Then, the library is incubated with the target molecule under conditions favorable for binding. The subset of nucleic acid–protein complexes are then selectively partitioned and amplified for enrichment and subsequent rechallenge. Finally, the RNA molecule is reverse-transcribed and sequenced. By SELEX technology, aptamers can be generated to bind in vitro or in vivo to virtually any protein of interest. There are aptamers reported to be specific against intact cells or tissues such as platelets (115), lymphocytes and leukocytes (116), lung tumor cell lines (117), and glioblastoma cells (118). Excellent reviews are available elsewhere on SELEX (119–121).

Protein targets that do not bind nucleic acids, in nature, give very high specificity and dissociation constants in the nanomolar to picomolar range after SELEX. This has been shown for aptamers raised against fibroblast growth factor (122) and vascular endothelial growth factor (123). Even at very low concentrations, RNA aptamers have been demonstrated to ablate protein function in the reaction tube; however, they lose potency when brought in the intracellular milieu. Outside the cell, aptamers are subjected to electrophoresis, optimal buffer systems, and are isolated from other molecules, while inside the cells they experience differences in macromolecular interactions and varying pH of the cytoplasm. However, there are few that overcame this major stumbling block of aptamer technology. Intracellularly active aptamers (intramers) have found a straightforward use in defining cellular proteomes (124). There are still quite a few reports on intramers that were used successfully to dissect the proteome, as a greater number of postgenomic researchers all have eyes for the transcriptome. Mayer and coworkers (125) demonstrated manipulation of the regulatory pathway of the ARF in vitro by aptamers by targeting the guanine–nucleotide exchange factor of the Sec7 domain, a regulatory protein. Armed with only the fungal metabolite brefeldin A, a guanosine triphosphatase inhibitor, it would not have been possible for ARF enthusiasts to dissect the role of other regulatory guanosine triphosphatases.

Aptamers, like the ribozymes, are an interesting class of reporter tools to study RNA-protein interaction. Monitoring proteins in real time and in nonhomogeneous solution has always been a difficult task. Fang and colleagues (126) configured a fluorophore-labeled aptamer for the ultrasensitive detection of platelet-derived growth factor. By monitoring fluorescence anisotropy (an optical property that uses polarized light), real-time monitoring of the binding between the aptamer and the protein was detected. This work on RNA optics was further elaborated with the development of "structure-switching signaling aptamers" (127) and with immobilized RNA aptamers that rapidly measure picomolar levels of cancer-associated proteins (inosine monophosphate dehydrogenase II, vascular endothelial factor, basic fibroblast growth factor) in human serum and cellular extracts (128). We extrapolate based on these developments that aptamer technology could help facilitate our understanding of the proteomics of aging cells by supplementing protein analytical techniques such as western blotting, high-pressure liquid chromatography, and enzyme-linked immunosorbent assay in the detection of the expression and modification of proteins closely correlated with the phenomenon.


   GENE SILENCING BY RNA INTEREFERENCE (RNAI)
Top
 Abstract
 The RNA Toolbox
 RNA-Based Proteome Probes
 Gene Silencing by RNA...
References
 
The most popular newcomer in the arena of RNA technology is RNA interference (RNAi). In 1998, Fire and colleagues (129) discovered that when they injected double-stranded RNAs (dsRNAs) into Caenorhabditis elegans, the worm mounted a sequence-specific gene-silencing phenomenon. With Drosophila embryo extracts, Zamore and colleagues (130) and Elbashir and colleagues (131) observed that long dsRNA substrates were fragmented into short interfering RNAs (siRNAs) of ~22 nt (130), and subsequent introduction of chemically synthesized siRNAs similarly initiated RNAi (131). This new type of specific gene-silencing phenomenon seems to associate with two ancient genetic processes, cosuppression in plants and quelling in Neurospora crassa. RNAi is tasked to counteract parasitic genomic invaders, such as viruses, and regulate endogenous genetic processes such as transposon silencing and chromosomal rearrangement (132–134). Since its discovery, papers on RNAi have exploded over the last few years, and it is dubbed as today's fastest growing tool for molecular biologists, as polymerase chain reaction was dubbed a decade ago.

RNAi in Functional Genomics
What makes RNAi a tool to reckon with? In contrast to the conventional gene identification protocols involving homologous recombination and mutagenesis screens, RNAi technology offers an advantage to switch off expression of any gene specifically, rapidly, and effectively. Already, several key genes related to aging (examples are shown in Table 1) have been fished from C. elegans simply by feeding the worms with dsRNA or with bacteria-expressing dsRNAs (135) or soaking them up in a solution containing the dsRNA library (136). For example, Asencio and coworkers at Spain's Universidad Pablo de Olavide have rapidly dissected the role of 8 candidate aging genes, including clk-1, in the ubiquinone biosynthesis pathway, which showed an extension in life span in RNAi-silenced worms (137). Ashrafi and colleagues (138) used a library of 16,757 cloned dsRNA and reported 417 genes implicated in fat storage regulation; among its long list of genes are included components of the insulin-like signaling pathways linked with longevity from worms (139) to mice (140).


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  		Table 1. Functional Genomic Studies on Genes Relevant to Aging Using Ribozymes (Rz) and RNAi.

 
While experiments with RNAi seem to be easy with worms and fruit flies, to achieve RNAi with mammalian systems, on one end, appeared almost impossible in the early days. With the exception of embryonic mammalian cells (mouse oocytes and embryos) (141,142), long dsRNAs (>30 nt) are known to activate the interferon pathway, dsRNA-dependent protein kinase (PKR), and 2',5'-oligoA synthase, leading to a global translation inhibition and RNA degradation (143). This limitation was later overcome by several groups who introduced chemically synthesized siRNAs (~20–21 nt) against luciferase and lamin A/C genes, and showed that these siRNAs resulted in the specific loss in target gene activity sans the nonspecific effects (144,145). In mice, gene suppression in vivo was successful after directly injecting siRNAs via intramuscular (146), intravenous (147), and intraocular (148) routes. These results have led to widespread use for direct study of the functions of several human genes and have bright prospects for RNAi-based gene therapy.

Because of the greater silencing capability and target specificity of RNAi as compared with ribozymes, scientists are racing to find a genome-wide functional annotation and comprehensive screening of the human genome using of the concept of RNAi libaries (149–153). Most recently, Berns and colleagues successfully constructed a human RNAi library of 7914 genes (using a total of 23,742 shRNA vectors targeting 3 regions per mRNA). In addition to what is already known about p53 pathways (from 27 years of research), their group revealed five new modulators of p53-dependent growth arrest and described a novel siRNA bar-code strategy to facilitate the swift identification of individual shRNA vectors associated with a specific phenotype within a large population of vectors (151).

Mechanism of Gene Suppression by RNAi
During the initial steps of RNAi, Dicer (a RNAse III enzyme) reduces long dsRNA and complex hairpin RNAs (hsRNA) into two intermediaries: siRNAs that degrade mRNA and microRNAs that halt translation. Recently, it has been shown that siRNAs with partially complementary binding sites in its 3' UTR can also function as endogenous microRNAs (miRNAs) (154). Cleavage is mediated by RNA-induced silencing complex (RISC), a process that is restricted to the cytoplasm (155), which finally decimates the target mRNA species. On the other hand, miRNAs consist of single-stranded RNA of ~20 bases processed from ~70 nt hairpin precursors (156) and are incorporated into a ribonucleoprotein that includes two members of the RISC family. To be processed by RISC, the siRNAs have to be phosphorylated at the 5'-end, otherwise they will be acted upon by an endogenous kinase (157).

Some Technical Notes
Despite their reputable knockdown potency, gene-suppressive activities of siRNAs still give variable results. The optimal design of siRNAs would still be a hit-or-miss thing. Analysis of the average internal stability profile of siRNA duplexes is warranted to avoid using duplexes with poor silencing activity (158). Reynolds and colleagues (159) identified eight characteristics associated with siRNA functionality, namely, a low G/C content, a bias towards low internal stability at the sense strand 3'-terminus, a lack of inverted repeats, and sense strand base preferences (positions 3, 10, 13, and 19). Programs to design siRNA are now available at the Whitehead Institute's biocomputing home page (www.jura.wi.mit.edu/bio) and at manufacturers' web sites (http://www.igene-therapeutics.co.jp, www.dharmacon.com, and www.qiagen.com). One may also need to consider the following: whether to use synthetic siRNA or vector-based shRNA and miRNA, the sequence target site of a particular mRNA target, background knowledge on the abundance and regulation of expression of the target gene, the type of delivery system (viruses, liposome and cojugates, electroporation, cholesterol, poly-L-lysine, chitosan nanoparticles, and nanomagetites). For vector-based system, a choice of polII- and polIII-based promoters is available, with the latter (e.g., U6 and H1 promoters) being more convenient because it does not trigger significant interferon responses (160,161). Several helpful details on these technical issues are exhaustively discussed in other articles (159,162–164).

Concluding Remarks
As new gene functions are discovered at the transcriptosomal and proteosomal levels, the universal tendency of any complex genetic system is to unleash a brand new set of interactomes operative in a perturbed system in order to gain maximal entropy. There are yet many mysteries that will unfold and bring us closer (but paradoxically further) to answering the basic questions on how and why we age. One thing is more certain: These new RNA tools have sparked a paradigm shift in our appreciation of our own genome and those of other species, of how a tritiered balancing of molecular architecture, information, and catalysis can be achieved as seen with the ribozymes and aptamers, and of how ancient genetic pathways can be resurrected as revolutionary tools in functional genomics as with RNAi technology. Then, if one imagines that tools as powerful as these have been handed over to biotech companies, we would likely now be amidst the breaking floodgates of RNA-based drugs designed to switch off disease genes, such as in cancer and AIDS. We are, by all indication, in the early days of finding the "holy grail" of rationally designed drugs that can manipulate longevity and cellular aging.



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  		Figure 2. Schematic diagram of the molecular pathways involved in RNAi. Short double-stranded RNA, formed endogenously by the activity of Dicer or introduced exogenously, complexes with RNA-induced silencing complex, which traps the antisense strand, hooks it to the target mRNA, and cleaves it

 

   Acknowledgments
 
We thank Masayuki Sano and Yoshio Kato for helpful discussions.


   Footnotes
 
Decision Editor: James R. Smith, PhD

Received May 8, 2004

Accepted May 26, 2004


   References
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