Current Topics in RNAi

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1 Current Topics in RNAi Essays on current, confusing, controversial, or otherwise interesting topics in the field of RNA interference Why Rational Pooling of sirnas is SMART Mike Straka, PhD, and Queta Boese, PhD There has been much discussion and some degree of confusion surrounding the use of sirna pools, as opposed to one or more individual sirnas, in gene silencing research. This article discusses the issue within a historical context and describes the theoretical and experimental basis supporting the advantages of pooling. RNAi in Nature RNA interference (RNAi) is an evolutionarily conserved mechanism observed in nearly every eukaryote studied thus far and represents a unique form of post-transcriptional gene silencing (reviewed in 1-6). In nature, RNAi is initiated by a long double-stranded RNA (dsrna) which can originate as transcripts from an invading virus, a mobilized transposon or other inappropriately transcribed endogenous sequence. Long dsrnas are processed by Dicer into a mixture of overlapping sirnas that interact with the RISC (Figure 1A). Populations of sirnas generated by this mechanism are efficient silencers and provide cells with a primitive immune system which is capable of combating viral invasion, controlling the expansion of endogenous transposons and monitoring transcription of endogenous genes. Figure 1. Pools of sirnas generated by nature and by rational design. A) In mammalian cells, Dicer digests long dsrna into a pool of duplexes that interact with RISC to induce silencing. B) Dharmacon's SMARTpool rationally designed sirnas mimic the pools generated in nature but are bioinformatically controlled for attributes associated with efficient and potent silencing. One obvious feature of RNAi in nature is that the sirnas generated by Dicer represent a set, or pool of sirnas that function together to silence gene expression. It was thus reasoned that by replicating the mechanism using pools of synthetic sirnas, individual genes could be silenced in a specific manner. In early attempts, conventionally designed sirnas targeting multiple sequences within the same mrna were used; however, gene silencing by pooled sirnas was often observed to be lower when compared to potent individual sirnas.

2 Figure 2. Silencing of stably transfected luciferase in HEK293 cells: rationally designed duplexes and pools are more potent silencers than those conventionally designed. Conventional Design, Green bars: 4 of 12 individual sirnas display ~90% knockdown; pools (P) are poor silencers. Rational Design, Blue bars: 12 of 12 of Dharmacon's SMARTselection rationally designed sirnas display 80-90% knockdown, and pooled duplexes (P) silence at >90%. Black bar: Non-specific Control sirna. The observed reduction in silencing by pools was attributed to saturation of the RISC with poor or non-functional sirnas that compete with the more functional duplexes for access to the RNAi apparatus. Figure 2 shows that only 4 of the 12 conventionally designed sirnas (4, 8, 10, 11) knock down stably transfected luciferase expression by 90% or more (F90). When these were pooled with the less effective duplexes (F65 or less), silencing by the pools was less effective compared to the potent sirnas, demonstrating that the effectiveness of pooled sirnas is dependent in part upon the potency of the constituent duplexes. Based on these types of results, it appeared that the inability to silence genes with pools of conventionally designed sirnas would be a barrier to gene silencing. However, Dharmacon scientists discovered that pools of rationally designed sirnas (Dharmacon SMARTpool sirnas) resulted in potent and consistent silencing. In order to interpret these results and to understand why certain sirna pools silence more effectively than others, it is useful to examine the differences between conventional sirna design and rational design. Conventional Design In their early work with cell lysates, Tuschl and colleagues developed a simple set of guidelines for the selection of sirnas (7,8). These initial studies focused on the structural attributes of sirnas (19-25mer duplexes with 3' dinucleotide overhangs) and resulted in a "conventional" design process for the generation of functional sirnas.

3 The criteria for conventional design as described by Tuschl are: 1. Identify a region that begins bases downstream of the start codon to avoid nucleotide sequences that may be occupied by translational or regulatory proteins. 2. In the remaining coding sequence, identify regions with the motif AA(N19)TT, where N equals any nucleotide. If no suitable sequences are found, the search can be relaxed to identify sequences with the motif NA(N21). 3. Evaluate the GC content of potential sirna sequences; ideally the GC content should be near 50%, or between 30-70%. 4. Compare the candidate sirna with known expressed sequence tags (ESTs) in publicly available databases to ensure specificity; e.g. BLASTx The above parameters have been incorporated into a variety of publicly available sirna design programs. Such computational tools facilitate the screening for potential sirna sequences, and are sponsored on the Web by various public and private institutions and commercial sirna suppliers including Dharmacon, Cold Spring Harbor Laboratories, and the Whitehead Institute. Among these, Dharmacon's sidesign Center is unique in that the conventional criteria are enhanced by the incorporation of 8 additional selection criteria representing a subset of the advanced rational design algorithm, discussed in the following section. Rational Design As a field of research advances, technological innovations enable increasingly larger gains in knowledge. Such is the case with the technologies pioneered by Dharmacon scientists. In contrast to the simplicity of conventional design, the sophistication of Dharmacon's SMARTselection technology represents the state of the art in rational sirna design; it is an in silico process based on a weighted algorithm which incorporates numerous sequence-specific and thermodynamic parameters as well as bioinformatic analysis to identify unique functional sirnas. The basis for Dharmacon's rational design algorithm has been previously described by Reynolds et al. (9). The distinguishing features of Dharmacon's rational design algorithm are: 1. Evaluating targets based on attributes associated with functional sirnas 2. Performing systematic sequence comparisons and extensive bioinformatic analysis 3. Effectively eliminating non-functional sirnas from the candidate pool The algorithm utilizes the fundamental principles described by Reynolds and colleagues along with further refinements; the refinements include but are not limited to GC content, sirna internal stability profiles, potential competing internal or foldback structures, and base preferences at key positions (9). For example, functionality is predicted for sirnas that have, in the sense strand, an A at position 3; a U at position 10; a base other than G at position 13; and/or an A (but not a C or G) at position 19. Individually, these parameters each convey only a limited selection advantage, yet when combined in a weighted manner, highly functional sirnas can be sorted from a mixed population of sequences (9). Figure 3 shows the silencing of an endogenous housekeeping gene, GAPDH, by conventionally designed sirnas vs rationally designed sirnas. All four of the rationally designed duplexes silenced GAPDH expression more than 90%, while only two of the four conventionally designed sirnas were effective at that level. These data, along with the data in Figure 2, show that conventional sirna design is inconsistent and may require the investigator to test multiple duplexes to obtain effective silencing. In contrast, rational design consistently generates highly functional sirnas. When such highly functional sirnas are pooled (Figure 2, blue bars), silencing is consistently very effective. In fact, Dharmacon SMARTpool sirnas are guaranteed to knock down gene expression by at least 75%; in many cases the level of silencing is significantly higher, in the range of 90-95%.

4 Figure 3. Comparison of GAPDH silencing by rationally vs conventionally designed sirnas. Rational selection of sirnas results in consistent and effective gene silencing. Pooling Reduces Off-Target Activity We have shown that Dharmacon's SMARTselection technology enables consistent and potent silencing by individual sirnas; an extension of the same SMARTselection algorithm is used to create SMARTpool reagents which consist of four SMARTselection designed duplexes, targeting different sequences of the same mrna, combined into one reagent. Pooling multiple sirnas into one silencing reagent conveys several distinct advantages over the use of one or more individual duplexes, and here we discuss in detail the experimental evidence in support of this claim. The evidence demonstrates that Dharmacon's science is superior to that of other vendors, and why SMARTselection technology delivers the most consistent and reliable silencing in the industry. One of the undesirable observations in gene silencing experiments is the off-target effect, which is the silencing of an unintended target (or targets). This can occur through sequence identity of either strand of the duplex with an mrna that is not the intended target. An excessively high concentration of sirna, 100 nm or higher, can also cause nonspecific off-target activity. In either case, a gene (or genes) which is not the intended target is silenced and can lead to incorrect or confusing experimental results and interpretations. How is off-target activity evaluated and interpreted? Global off-target effects are evaluated by gene expression profiling. Using microarray analysis, the profile, or signature, of gene regulation by an sirna can be displayed in terms of a gene being either up- or down-regulated by some standard magnitude, generally 2-fold. By convention, down-regulated genes are displayed as green and upregulated genes as red. A magnified portion of such an analysis is shown in Figure 4. Numerous observations of off-target activity, including the data shown in Figure 4, indicate that individual sirnas likely have unique signatures; that is, each duplex will silence its own specific set of off-target genes. Figure 4 shows the gene expression profile of four rationally designed sirnas targeting human Cyclophilin B; these were used at 100 nm in duplicate experiments. The data show that the off-target signatures of the four duplexes differ from each other and also between the two experiments, representing variation within biological replicates. The data in Figure 4 also demonstrate that, at the same concentration as the component duplexes, to a large extent pooled sirnas have less off-target activity. The explanation lies in the effective concentration of the duplexes in the pool: at a total sirna concentration of 100 nm, the individual duplexes comprising the pool are at a much lower concentration, approximately 25 nm. Thus the off-target activity of each duplex is reduced by a concentrationdependent mechanism, as described below.

5 Figure 4. Microarray analysis of off-target effects by individual and SMARTpool sirnas targeting human Cyclophilin B. Four individual SMARTselection designed duplexes (C1-C4) were used to target human Cyclophilin B in two experiments (A.C1-A.C4 and B.C1-B.C4). The duplexes were also pooled and tested in duplicate (A.pool and B.pool). All sirnas were transfected at 100 nm. Figure 5. Microarray analysis and concentration curve of off-target effects by conventionallydesigned and SMARTselection designed sirnas. Off-target activity is concentrationdependent, and SMARTselection designed sirnas have less off-target effects than conventionally designed sirnas. In addition to unique signatures, the magnitude of off-target activity of sirnas is concentration-dependent. Figure 5 shows a single sirna targeting human GAPDH used at concentrations ranging from 100 nm to 0.1 nm, as indicated across the top of the figure. The top row of green bars shows down-regulation of the target, and the green bars under the target band display offtarget activities at the indicated concentrations. The data show that the rationally designed sirna displays a strikingly lower level of off-target activity, particularly at concentrations above 1nM. Furthermore, the rationally designed sirna silences GAPDH at 0.5 nm while the conventionally designed duplex requires at least 10 nm for comparable

6 silencing. At 1 nm, the concentration at which off-target activity is fairly well reduced, the conventionally designed duplex also loses efficacy against the target. The data in Figures 4 and 5 demonstrate that rational design and pooling reduce off-target activity without loss of silencing potency. Thus the investigator can achieve silencing at a lower effective concentration of sirna. Pooling Reduces the Rate of False-Positive and False-Negative Results Another class of undesirable observations in RNAi are false-positive and false-negative results. Let's first consider false-positives. In a screen using a phenotypic assay such as cell proliferation, in which inhibition of cell growth is a positive response, it is easy to see how single sirnas, even if rationally designed, can indicate a falsepositive and lead the investigator to consume precious time and resources tracking down a false hit. An example of this type of situation is shown in Figure 6. In Figure 6, 64 rationally designed sirnas were used to screen 16 genes purportedly involved in cell growth (four individual duplexes per target). Designating 60% survival as the cutoff (i.e. less than 60% survival indicates target involvement and thus is a potential positive), positive "hits" are indicated in the red box. These "hits" are then subject to secondary screening, shown in Figure 7. Figure 6. False positive results in a proliferation screen. In this phenotypic assay, the criteria for a "hit" is any value below 60% cell survival; hits are then subject to further analysis. Unverified Positives Following up on the initial screen, the investigator goes on to perform a secondary screen of the positives obtained in Figure 6. Two of the genes indicated as "hits" are then targeted with all four of the individual sirnas and the corresponding pool of the four. Figure 7 shows the result of the secondary screen. In Figure 7, black bars indicate target silencing: all duplexes and pools are effective silencers. Survivability, the screening phenotype, is indicated by grey and red bars. Red bars (A4 and B3) show survivability using two of the "positive" duplexes from the initial screen, and again these duplexes inhibit cell growth. The grey bars show survivability using the other three duplexes targeting the corresponding genes; these duplexes give the true phenotype (ie., no effect on cell growth). Thus, the two duplexes which inhibit growth (red bars) are revealed as false-positives. The blue bars show survivability using a SMARTpool consisting of the same four duplexes: the pooled sirnas give the true phenotype - no effect on cell growth. Thus, if the investigator had performed the initial screen using pooled sirnas, the false-positive would have been avoided.

7 These data demonstrate that pooled duplexes reduce the rate of false-positives, again validating the use of rationally designed sirnas and SMARTpool technology. Figure 7. Secondary screen of two "positives" from Figure 6 (A and B). Pooling helps avoid false-positives. Black Bars: target knockdown Red Bars: survivability using the individual duplex giving "positive" for two genes from the red box in Figure 6 Grey Bars: survivability using remaining three sirnas targeting the corresponding genes; these give the true phenotype, which is target knockdown with no effect on survivability Blue Bars: Pools give the true phenotype - gene knockdown but no effect on cell survival Ctl: Controls With regard to false-negatives, the rationale for pooling is more theoretical than experimental. One of the causes of a false-negative result is the existence of SNPs (single nucleotide polymorphisms). If an individual sirna happens to target a sequence containing a SNP, silencing will be ineffective and the target will be registered as a negative, in this case a false-negative. SMARTpool sirnas, however, because they consist of four duplexes targeting different regions of the mrna, have a higher probability of avoiding the SNP region by at least one of the duplexes. Thus even if a target contains a SNP, a SMARTpool reagent has a much greater likelihood of silencing it when compared to an individual duplex or non-rationally designed pools. Other causes of false-negative results include previously unidentified isoforms or polymorphisms, and changes or updates in the curated sequence. The same rationale supporting the use of pooled sirnas can be applied: rational design and SMARTpool technology generates sirnas that have a higher probability of avoiding these kinds of sequence mismatches than conventional design or pooling.

8 SUMMARY - Benefits of SMARTpool sirnas In addition to the scientific reasons outlined here, SMARTpool sirna reagents are an economical strategy for both new and seasoned investigators alike. Whether used as an entry point into RNAi studies or as screening tools, investigators will realize savings in time, cost, and resources required to conduct a series of silencing experiments. For example, to screen 1000 targets using just two individual sirnas, one would need to conduct approximately 2200 to 2300 experiments including confirmation of hits. In contrast, only about 1400 experiments are needed to screen and confirm hits using SMARTpool reagents consisting of four duplexes. Furthermore, SMARTpool reagents carry a guarantee that mrna levels will be reduced by 75% (under standard cell culture conditions) or will be replaced; this assures the investigator that precious time, effort and money will not be wasted, as may happen with the use of poorlydesigned sirnas. Synthetic sirna-mediated gene silencing exploits a naturally occurring endogenous cellular pathway. The potency and specificity of silencing are highly dependent upon the particular sirna sequence used, which in turn is the direct result of the technology used in its design. Dharmacon s design algorithm relies on and emulates the natural process which selects for the most stable and potent silencers. It has been asserted that pooled sirnas are ineffective (10); however, the data shown in this article demonstrate that the pools were comprised of poorly-designed and inconsistent sirnas. The sirnas as designed in this study clearly fall well below acceptable standards of functionality and would not be considered appropriate for use. The extensive experimental evidence presented here in part demonstrates that the functionality of individual duplexes and pools is directly related to the strength of the design algorithm the more sophisticated and refined the algorithm, the better the performance. If an sirna pool consists of poorly-designed duplexes which are inconsistent silencers, it should not be surprising that the pool and its associated phenotype or knockdown is not consistent with the duplexes. In sharp contrast, rational design generates potent duplexes, and pools of highly functional duplexes demonstrate effective silencing: Dharmacon s SMARTselection and SMARTpool technologies represent the most advanced, potent and consistent gene silencing reagents available in the industry. Additional benefits of SMARTpool technology are decreased off-target activity resulting in lower rates of false-positive and false-negative results. We have presented evidence that the Dharmacon rational design algorithm is superior to any other design available, providing investigators the broadest range of potent, consistent and reliable silencing reagents, whether the need is for individual duplexes or pooled sirnas. SMARTpool reagents enable rapid and reliable screening of potential targets with high confidence that the rate of off-target activity and false results will be low. Interesting hits obtained via screening can then be examined in greater detail through the use of individual duplexes in conjunction with sequence information. In conclusion, pooling sirnas is only as SMART as the technology used in their design. References 1. Zamore PD, RNA interference:listening to the sound of silence. Nat. Struct. Biol., (9): Tuschl T, RNA interference and small interfering RNAs. Chembiochem., (4): Sharp PA, RNA interference Genes Dev., (5): McManus MT and Sharp PA, Gene silencing in mammals by small interfering RNAs. Nature Rev. Genet., (10): Hannon GJ, RNA interference. Nature, (6894): Hutvagner G and Zamore PD, RNAi: Nature abhors a double strand. Curr Opin. Genetics Dev., (2): Elbashir SM, Lendeckel W, and Tuschl T, RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev., (2): Elbashir SM, et al, Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods, (2): Reynolds A, et al, Rational sirna design for RNA interference. Nature Biotechnol., (3): Brown, D, Byrom, M, Krebs, J, Kelnar, K, Jarvis, R, Campbell, A, Ford, L. Ambion TechNotes 12(1), February 2005 (