Abstract
Small interfering RNAs (siRNAs) and microRNAs (miRNAs) are key regulators of posttranscriptional gene silencing, which is referred to as RNA interference (RNAi) or RNA silencing. In RNAi, siRNA loaded onto the RNA-induced silencing complex (RISC) downreugulates target gene expression by cleaving mRNA whose sequence is perfectly complementary to the siRNA guide strand. We previously showed that highly functional siRNAs possessed the following characteristics: A or U residues at nucleotide position 1 measured from the 5′ terminal, four to seven A/Us in positions 1–7, and G or C residues at position 19. This finding indicated that an RNA strand with a thermodynamically unstable 5′ terminal is easily retained in the RISC and functions as a guide strand. In addition, it is clear that unintended genes with complementarities only in the seed region (positions 2–8) are also downregulated by off-target effects. siRNA efficiency is mainly determined by the Watson–Crick base-pairing stability formed between the siRNA seed region and target mRNA. siRNAs with a low seed-target duplex melting temperature (Tm) have little or no seed-dependent off-target activity. Thus, important parts of the RNA silencing machinery may be regulated by nucleotide base-pairing thermodynamic stability. A mechanistic understanding of thermodynamic control may enable an efficient target gene-specific RNAi for functional genomics and safe therapeutic applications.
Introduction
Small RNA molecules, including small interfering RNAs (siRNAs) and microRNAs (miRNAs), are crucial regulators of posttranscriptional gene silencing referred to as RNA interference (RNAi) or RNA silencing. RNAi is an evolutionarily conserved pathway induced by siRNAs, 21–23-nt double-stranded RNAs (dsRNAs) with 2-nt 3′ overhangs (Figure 1). siRNAs incorporated into cells are transferred to an RNAi effector complex called the RNA-induced silencing complex (RISC; Hutvagner and Simard, 2008; Jinek and Doudna, 2009). The RISC assembles on one of the two strands of the siRNA duplex and is activated upon removal of the passenger strand (Martinez et al., 2002; Schwarz et al., 2002, 2003; Khvorova et al., 2003; Ui-Tei et al., 2004). We and others reported that asymmetrical features of both siRNA terminals are common to functional siRNAs (Amarzguioui and Prydz, 2004; Reynolds et al., 2004; Ui-Tei et al., 2004). An RNA strand with a thermodynamically unstable 5′ terminal is easily retained in the RISC. The activated RISC is a ribonucleoprotein complex that minimally consists of the core protein Argonaute (Ago) and a siRNA guide strand, which recognizes mRNAs with complementary sequences (Liu et al., 2004; Meister et al., 2004; Song et al., 2004). In siRNA-mediated RNAi, the Ago2 protein siRNA guide strand usually base-pairs with mRNA that is perfectly complementary and cleaves them (Figure 1). However, this can also lead to silencing of other genes with incompletely complementary sequences. This phenomenon is referred to as an off-target effect (Figure 1). The target recognition mechanism of the off-target effect is similar to that of miRNA-mediated gene silencing (Jackson et al., 2003, 2006a; Bartel, 2004; Scacheri et al., 2004; Lewis et al., 2005; Lim et al., 2005; Lin et al., 2005; Birmingham et al., 2006; Grimson et al., 2007). The transcripts with sequences complementary to the seed region (i.e., nucleotide positions 2–8 from the 5′ end of siRNA guide strand or miRNA loaded on Ago1–4 proteins) are mainly reduced. This is likely because seed nucleotides are present on the Ago surface in a quasi-helical form to serve as the entry or nucleation site for small RNAs in the RISCs (Ma et al., 2005; Yuan et al., 2005; Ui-Tei et al., 2008a). Thus, siRNA target recognition might be partially determined by structural features. However, the off-target effect silencing efficiency is mainly determined by the thermodynamic properties of nucleotide base-pairing between the siRNA guide strand seed region and their off-target mRNAs (Ui-Tei et al., 2008a). Understanding thermodynamic control of the siRNA off-target effect may make it possible to avoid the off-target effect for a target gene-specific RNAi.
Figure 1
Functional siRNA Sequences
RNA interference efficiency in mammalian cells varies considerably depending on the siRNA sequence (Holen et al., 2002; Harborth et al., 2003). We showed that there are three siRNA classes based on their RNAi gene silencing activity (Ui-Tei et al., 2004). Class I siRNAs, which are highly functional in mammalian RNAi, have A or U residues at nucleotide position 1, four to seven A/Us in nucleotide positions 1–7 (AU ≥ 57%) and G/C at position 19, with the nucleotide position measured from the 5′ end of the guide strand (Figure 1). In addition, a GC stretch of no more than nine nucleotides occurs in class I siRNA sequences. Class III siRNAs have opposite features with respect to the first three conditions and cause the least RNAi-silencing effects. The remaining siRNAs belong to class II and are a mixture of functional and non-functional siRNAs.
We and others demonstrated that functional siRNA with an unstable RNA strand 5′ terminal in the siRNA duplex is functional as a guide strand (Amarzguioui and Prydz, 2004; Reynolds et al., 2004; Ui-Tei et al., 2004); A or U residues at the 5′ end of the guide strand are especially important. In RNAi, thermodynamic asymmetry is not essential for target gene silencing because the passenger strand of most double-stranded siRNAs loaded onto RISC are cleaved by catalytic activity of the Ago2 protein and degraded (Figure 1; Kawamata et al., 2009; Yoda et al., 2010). Thus, in this case, A/U nucleotide itself at 5′ terminal might be strongly contributed to the RNAi activity, as nucleotide monophosphates, AMP, and UMP, bind to Ago2 with up to 30-fold higher affinity than either CMP or GMP (Frank et al., 2010). However, when the siRNA duplex is loaded into other Ago proteins without slicer activity, siRNAs might be unwound into a single-strand from the thermodynamically unstable 5′ terminal as shown in miRNA-mediated gene silencing (Figure 1; Matranga et al., 2005; Miyoshi et al., 2005; Leuschner et al., 2006; Kawamata et al., 2009; Yoda et al., 2010). As off-target gene silencing is performed using both mechanisms for eliminating the passenger strand, siRNA thermodynamic asymmetry in addition to A/U nucleotide itself at the 5′ terminal might be involved in seed-dependent off-target effects.
Seed-Dependent Off-Target Effect Efficiency Varies Depending on Seed Sequence
Accumulated evidence from large-scale knockdown experiments (Jackson et al., 2003, 2006a; Scacheri et al., 2004; Lin et al., 2005; Birmingham et al., 2006) suggests that siRNA can generate off-target effects through a mechanism similar to that of miRNA target silencing (Lewis et al., 2005; Lim et al., 2005; Grimson et al., 2007). The 3′UTRs of off-target transcripts or miRNA targets are complementary to the guide strand seed region (i.e., nucleotide positions 2–8; Figure 2; Lim et al., 2005; Lin et al., 2005; Birmingham et al., 2006; Jackson et al., 2006a). We determined the relationship between class I siRNA seed sequences and off-target effect using the expression reporter plasmid, psiCHECK, which encodes the Renilla luciferase gene. Three tandem repeats of seed-matched target sequences (Figure 3C) complementary to the entire seed-containing region (positions 1–8), but not to the remaining non-seed region (positions 9–21), were introduced into the region corresponding to the 3′UTR of the luciferase mRNA to generate psiCHECK-sm and used to determine the efficiency of the seed-dependent unintended off-target effect (see Figure 4A; Ui-Tei et al., 2008a). Although all siRNAs examined exhibited high activity for intended gene silencing at 50 nM, the off-target gene silencing calculated using psiCHECK-sm was much less effective and more susceptible to changes in siRNA concentration (Ui-Tei et al., 2008a). These findings indicated that variations in the efficiency of unintended off-target gene silencing were due to a difference in the interactions between the guide strand RNA entrapped in the RISC and mRNA.
Figure 2
Figure 3
Figure 4
Seed-Dependent Off-Target Effect Efficiency Varies Depending on Seed Region GC Content
Class I siRNA seed region GC content used in our previous study (Ui-Tei et al., 2008a) ranged from 0 to 57%. To further determine the relationship between seed region GC content exceeding 57% and off-targeting efficiency of the corresponding siRNA, six functional class II siRNAs with high GC content in the seed region were arbitrarily selected (Figure 3A), and their capability to exert off-target effects was examined using luciferase reporter assays (Figure 4; Ui-Tei et al., 2009). Note that two of the six class II siRNAs (siLuc-1063 and siLuc-1430) possessed a 100% GC content in the seed region (Figure 3A). In contrast to class I siRNAs, which have little or no off-target effects, class II siRNAs were frequently associated with a considerable level of off-target gene silencing on the seed-matched targets (Figure 4). This apparent difference in the off-target effect may be due to differences in the GC content in the seed region between functional class I and II siRNAs.
Seed-Dependent Off-Target Effect is Determined by Thermodynamic Stability in the Duplex Formed Between the siRNA Guide Strand Seed Region and Target mRNA
The results shown above indicated that siRNAs with high GC content in the seed sequence have strong seed-dependent off-target effects. Thus, one possible efficiency regulator of the seed-dependent off-target effect might be the thermodynamic stability of the nucleotide duplex. The melting temperature (Tm) and standard free energy change (ΔG) of the seed–target duplex formation are good measures of the thermodynamic stability of the protein-free seed–target duplex. In a previous experiment using class I siRNAs (Ui-Tei et al., 2008a), we verified a close linear relationship between ΔG and Tm in seed region positions 2–8; a strong positive correlation between luciferase activity and ΔG (r = 0.69), and a strong negative correlation between Tm and luciferase activity (r = −0.72) was found. By replacing class I siRNAs with a mixture of class I and II siRNAs, the ΔG range expanded from −13 and −7 to between −19 and −7 kcal/mol (Figures 5A,B), while the Tm range expanded from −8 and 28°C to −8 and 55°C (Figures 5A,C). Correlation coefficients between luciferase activity and ΔG or Tm were 0.72 or −0.76, respectively, indicating a close relationship between the seed-dependent off-target effect and the seed duplex ΔG and Tm. The linear relationships among these parameters were almost invariant (Figure 5A). The dissociation constant (Kd) of the 17 siRNAs calculated using the formula ΔG = −RTln(1/Kd) indicated that the highest Kd was more than 108 times greater than the lowest one (Table 1). Therefore, it may follow that in both functional class I and II siRNA-mediated gene silencing, the degree of off-target effects is governed primarily by the thermodynamic stability of the seed-target duplex formed between the seed region of the siRNA guide strand and its mRNA counterpart. In Figure 6A, all possible 7-nt seed sequences (47 = 16,384) were ordered as a function of GC content and Tm values of their double-stranded counterparts, and the siRNAs were plotted against the absence or presence of off-target effects. The data suggest that Tm values between 21 and 25°C serve as a Tm boundary, which may discriminate off-target-free seed sequences from off-target-positive ones. Approximately 22% of 7-nt sequences had Tm values under 21°C, indicating that limited seed sequences are available for selecting siRNAs with reduced off-target effects.
Table 1
| Luciferase activity (% at 50 nM) | Seed region GC number | Tm 2–8 (°C) | ΔG 2–8 (kcal/mol) | Kd (M) | |
|---|---|---|---|---|---|
| CLASS I | |||||
| siLuc-309 | 76 | 0 | −8.1 | −7.2 | 5.3 × 10−6 |
| siVIM-812 | 95 | 1 | 8.8 | −9.3 | 1.5 × 10−7 |
| siGRK4-934 | 102 | 1 | 6.7 | −9.6 | 9.2 × 10−8 |
| siOct-821 | 64 | 2 | 12.2 | −10.3 | 2.8 × 10−8 |
| siLuc-774 | 75 | 2 | 14.6 | −10 | 4.7 × 10−8 |
| siVIM-1128 | 79 | 3 | 21.2 | −12.8 | 4.1 × 10−10 |
| siLuc2-153 | 52 | 3 | 21.0 | −11.7 | 2.6 × 10−9 |
| siVIM-596 | 49 | 3 | 26.4 | −11.6 | 3.1 × 10−9 |
| siOct-797 | 29 | 3 | 25.7 | −12.4 | 8.1 × 10−10 |
| siVIM-270 | 25 | 3 | 26.2 | −12.2 | 1.1 × 10−9 |
| siLuc-36 | 49 | 4 | 28.4 | −13 | 3.0 × 10−10 |
| CLASS II | |||||
| siGRK4-189 | 9 | 4 | 40.1 | −15.5 | 4.3 × 10−12 |
| siLuc-1120 | 44 | 5 | 42.3 | −16.7 | 5.7 × 10−13 |
| siLuc-49 | 6 | 6 | 46.3 | −17.6 | 1.3 × 10−13 |
| siLuc-1048 | 30 | 6 | 49.7 | −17.8 | 8.9 × 10−14 |
| siLuc-1063 | 18 | 7 | 54.5 | −18.6 | 2.3 × 10−14 |
| siLuc-1430 | 20 | 7 | 54.5 | −18.6 | 2.3 × 10−14 |
Relative luciferase activities and Tms, ΔGs, and Kds at seed regions of class I and II siRNAs.
Figure 5
Figure 6
Thermodynamic Control of RNA Strand Incorporation into the Risc by Chemical Modifications
RNA strand incorporation into the RISC is determined by siRNA duplex thermodynamics. The RNA strand with lowest binding stability in the 5′ end of the guide strand is preferentially incorporated into the RISC. Thus, rational chemical modifications can be used to improve selective guide strand loading into the RISC (Table 2). High-affinity modifications [e.g., locked nucleic acid (LNA)] at the 5′ end of the passenger strand increase selective loading of the guide strand (Elmén et al., 2005). In addition, base modifications of a high-affinity 2-thiouracil base at the 3′ end of the guide strand and a low-affinity dihydrouracil base at the 3′ end of the passenger strand can be used to the same effect (Sipa et al., 2007). Furthermore, a moderately active siRNA duplex is significantly improved by modifying the high-affinity 4′-thioribonucleoside (Hoshika et al., 2007). Similarly, other modifications, such as high-affinity 5-methyluracil and 5-methylcytosine modifications (Terrazas and Kool, 2009), or low-affinity 2,4-difluorotoluene and 5-nitroindole modifications (Addepalli et al., 2010), may also control the efficiency of RISC loading.
Table 2
| Chemical modification | Nucleotide position | Modified base-pairing stability | Functional modification | Reference |
|---|---|---|---|---|
| LNA | The 5′ end of the passenger strand | Increase the stability at 5′ end of the passenger strand | Elmén et al. (2005) | |
| 4′-Thioribonucleoside | Four residues on both ends of the passenger strand and 3′ end of the guide strand | Increase the stability at 3′ end of the guide strand | Enhancement of selective RISC loading of the guide strand | Hoshika et al. (2007) |
| 2-Thiouracil | The 3′ end of the guide strand | Increase the stability at 3′ end of the guide strand | Sipa et al. (2007) | |
| Dihydrouracil | The 3′ end of the passenger strand | Decrease the stability at 3′ end of the passenger strand | ||
| 2′-O-methyl | Position 2 of the guide strand and positions 1 + 2 of the passenger strand | The conformational alteration of RISC by the guide strand modification reduces the rate of RISC formation to dissociate off-target transcripts with weaker binding to the guide strands | Reduction of the seed-dependent off-target effect | Jackson et al. (2006b) |
| 2′-Deoxy (DNA) | Positions 1–8 of the guide strand and positions 12–21 of the passenger strand | Decrease the stability in the seed region of the guide strand | Ui-Tei et al. (2008b) |
Thermodynamic siRNA modification.
Elimination of Seed-Dependent off-Target Effect by Chemical Modifications
The seed-dependent off-target effect is also eliminated by chemical modifications (Table 2). 2′-O-methyl modification of the guide strand seed region alters the RISC conformation and reduces seed-dependent off-target effects by dissociating off-target transcripts with weak binding to the guide strand (Jackson et al., 2006b). Low-affinity dihydrouracil base, 2,4-difluorotoluene, or 5-nitroindole modifications in the seed region may also reduce the seed-dependent off-target effects. Furthermore, we revealed that 2′-deoxy modification (DNA replacement) of nucleotides 1–8 in the guide strand and 12–21 in the passenger strand (DNA:RNA chimeric siRNA, chiRNA; Figure 3B) reduces thermodynamic stability in the seed-target duplex, and almost completely eliminates off-target effects with little or no loss of target gene silencing activity (Ui-Tei et al., 2008b). In contrast, replacing the 3′-proximal RNA sequence of the guide strand with its DNA counterpart resulted in almost complete loss of gene silencing activity of the passenger strand. As shown in Figure 7, most functional class II siRNAs could not effectively eliminate the off-target effects by DNA replacement in the seed region (Ui-Tei et al., 2009). We examined the relationship between the relative luciferase activity and the ΔG or Tm of the DNA:RNA seed duplex in 11 class I and six class II chiRNAs (Figure 5D–F). We verified a close linear relationship between ΔG and Tm in the seed region (Figure 5D), a strong positive correlation between luciferase activity and ΔG (r = 0.71) and a strong negative correlation between Tm and luciferase activity (r = −0.79), irrespective of the presence or absence of DNA replacement in the seed region (Figure 5E,F). However, DNA replacement increased ΔG and reduced both the seed-target duplex Tm and luciferase activity considerably. Tm was reduced to less than 20°C in all the class I chiRNAs, while the relative luciferase activity at 50 nM exceeded 60%, the minimum relative luciferase activity necessary for a practical off-target effect. In contrast, the relative luciferase activity at 50 nM was 30% or less in three of six cases treated with functional class II chiRNAs, even though the seed-target Tm was reduced; this demonstrates a strong negative correlation with ΔG for seed-target duplex formation (Figure 5D). Therefore, it appears that the reduced off-target effect in chiRNA-dependent gene silencing is generally attributable to a reduction in the thermodynamic stability of the DNA:RNA hybrid in the seed–target duplex. According to Tables 1 and 3, DNA replacement throughout the guide strand seed region is roughly equivalent to a 13°C reduction in Tm, a 5 kcal/mol increment in ΔG, and about two to three G/C → A/U changes in the seed duplex. In Figure 6B, Tm values of all possible 7-nt DNA:RNA hybrids are ordered and plotted against chiRNAs with or without off-target silencing activity. For DNA:RNA hybrids, 28–41°C might be a boundary line that discriminates off-target-free from off-target-positive seed sequences. However, this boundary had higher Tm values, as compared to those of RNA duplexes shown in Figure 6A. This might be partially due to different parameters used in calculating Tm values of RNA duplexes and DNA:RNA hybrids. The proportion of 7-nt DNA:RNA hybrids with Tm values under 28°C was about 88%, indicating that most 7-nt sequences are available for off-target effect-reduced RNA silencing by replacing RNA with DNA in the seed region.
Table 3
| Luciferase activity (% at 50 nM) | Seed region GC number | Tm 2–8 (°C) | ΔG 2–8 (kcal/mol) | Kd (M) | |
|---|---|---|---|---|---|
| CLASS I | |||||
| chiLuc-309 | 103 | 0 | −12.2 | −4.9 | 2.6 × 10−4 |
| chiVIM-812 | 94 | 1 | −0.5 | −5.5 | 9.3 × 10−5 |
| chiGRK4-934 | 95 | 1 | 0.2 | −5.6 | 7.3 × 10−5 |
| chiOct-821 | 95 | 2 | 4.7 | −7.1 | 6.2 × 10−6 |
| chiLuc-774 | 65 | 2 | −4.8 | −6.8 | 1.0 × 10−5 |
| chiVIM-1128 | 79 | 3 | 8.8 | −7.9 | 1.6 × 10−6 |
| chiLuc2-153 | 91 | 3 | 5.4 | −7.8 | 1.9 × 10−6 |
| chiVIM-596 | 63 | 3 | 8.8 | −7.8 | 1.9 × 10−6 |
| chiOct-797 | 84 | 3 | 1.3 | −7.8 | 1.9 × 10−6 |
| chiVIM-270 | 78 | 3 | 3.1 | −7.6 | 2.7 × 10−6 |
| chiLuc-36 | 68 | 4 | 19 | −8.0 | 1.4 × 10−6 |
| CLASS II | |||||
| chiGRK4-189 | 12 | 4 | 28.1 | −9.6 | 9.2 × 10−8 |
| chiLuc-1120 | 69 | 5 | 31.1 | −9.8 | 6.5 × 10−8 |
| chiLuc-49 | 31 | 6 | 30.4 | −11.0 | 8.6 × 10−9 |
| chiLuc-1048 | 16 | 6 | 45.2 | −9.7 | 7.7 × 10−8 |
| chiLuc-1063 | 58 | 7 | 41.4 | −12.0 | 1.6 × 10−9 |
| chiLuc-1430 | 53 | 7 | 35 | −12.0 | 1.6 × 10−9 |
Relative luciferase activities and Tms, ΔGs, and Kds at seed regions of class I and II chiRNAs.
Figure 7
Genome-Wide Analysis Revealed that siRNA with Low Stability in the Seed-Target Duplex is Capable of Inducing Target Gene-Specific Silencing
The hypothesis that off-target gene silencing is determined primarily by seed-target duplex stability was apparent in genome-wide expression profiling using class I siRNA with high or low seed Tm value (Figure 8). The reporter assay described above predicted that siRNA with high seed Tm value would be good inducer, while that with low seed Tm value would be poor inducer of the off-target effect.
Figure 8
As anticipated, both siRNAs effectively reduced the amount of completely matched target mRNA to less than 20% as a result of intended RNAi (red arrows in Figure 8). In contrast, a high level of off-target effects was evident in the case of transfection with siRNA with high seed Tm value. Conversely, transfection with siRNA with low seed Tm value exhibited little off-target effects. Thus, it was concluded that the level of off-target gene silencing is determined by the thermodynamic stability of the seed duplex formed between the siRNA guide strand and the target mRNA.
Conclusion
In this review, we demonstrated that siRNA seed-dependent off-target effect efficiency is controlled by thermodynamic properties of the nucleotide duplex. This conclusion was drawn from the following: (1) The functional siRNA duplex is asymmetric in its terminal nucleotide base-pairing. An RNA strand with an unstable 5′ terminal is effective as a guide strand, probably because it is easily retained in the RISC. (2) The siRNA off-target effect efficacy can be determined by seed region nucleotide duplex thermodynamic properties. The seed-dependent off-target effect efficiency is positively and negatively correlated with ΔG and Tm in seed region positions 2–8. Thus, small RNA-mediated gene silencing is partly regulated by nucleotide base-pairing thermodynamic stability.
Statements
Acknowledgments
We thank Eigo Shimizu for excellent assistance in preparing Figure 6. This work was partially supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT), and the Cell Innovation Project (MEXT) to Kumiko Ui-Tei.
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
References
1
AddepalliH.MeenaPeng, C. G.WangG.FanY.CharisseK.JayaprakashK. N.RajeevK. G.PandeyR. K.LavineG.ZhangL.Jahn-HofmannK.HadwigerP.ManoharanM.MaierM. A. (2010). Modulation of thermal stability can enhance the potency of siRNA. Nucleic Acids Res.38, 7320–7331.10.1093/nar/gkq568
2
AmarzguiouiM.PrydzH. (2004). An algorithm for selection of functional siRNA sequences. Biochem. Biophys. Res. Commun.316, 1050–1058.10.1016/j.bbrc.2004.02.157
3
BartelD. P. (2004). MicroRNAs: genomics, biogenesis, mechanism, and function. Cell116, 281–297.10.1016/S0092-8674(04)00045-5
4
BirminghamA.AndersonE. M.ReynoldsA.Ilsley-TyreeD.LeakeD.FedorovY.BaskervilleS.MaksimovaE.RobinsonK.KarpilowJ.MarshallW. S.KhvorovaA. (2006). 3′ UTR seed matches, but not overall identity, are associated with RNAi off-targets. Nat. Methods3, 199–204.10.1038/nmeth0606-487a
5
ElménJ.ThonbergH.LjungbergK.FriedenM.WestergaardM.XuY.WahrenB.LiangZ.ØrumH.KochT.WahlestedtC. (2005). Locked nucleic acid (LNA) mediated improvements in siRNA stability and functionality. Nucleic Acids Res.33, 439–447.10.1093/nar/gki193
6
FrankF.SonenbergN.NagarB. (2010). Structural basis for 5′-nucleotide base-specific recognition of guide RNA by human AGO2. Nature465, 818–822.10.1038/nature09039
7
GrimsonA.FarhK. K.JohnstonW. K.Garrett-EngeleP.LimL. P.BartelD. P. (2007). MicroRNA targeting specificity in mammals: determinants beyond seed pairing. Mol. Cell27, 91–105.10.1016/j.molcel.2007.06.017
8
HarborthJ.ElbashirS. M.VandenburghK.ManningaH.ScaringeS. A.WeberK.TuschlT. (2003). Sequence, chemical and structural variation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing. Antisense Nucleic Acid Drug Dev.13, 83–105.10.1089/108729003321629638
9
HolenT.AmrzguiouiM.WiigerM. T.BabaieE.PrydzH. (2002). Positional effects of short interfering RNAs targeting the human coagulation trigger tissue factor. Nucleic Acids Res.30, 1757–1766.10.1093/nar/30.8.1757
10
HoshikaS.MinakawaN.ShinonoyaA.ImadaK.OgawaN.MatsudaA. (2007). Study of modification pattern-RNAi activity relationships by using siRNAs modified with 4′-thioribonucleosides. Chembiochem8, 2133–2138.10.1002/cbic.200700342
11
HutvagnerG.SimardM. J. (2008). Argonaute proteins: key players in RNA silencing. Nat. Rev. Mol. Cell Biol.9, 22–32.10.1038/nrm2321
12
JacksonA. L.BartzS. R.SchelterJ.KobayashiS. V.BurchardJ.MaoM.LiB.CavetG.LinsleyP. S. (2003). Expression profiling reveals off-target gene regulation by RNAi. Nat. Biotechol.21, 635–637.10.1038/nbt831
13
JacksonA. L.BurchardJ.SchelterJ.ChauB. N.ClearyM.LimL.LinsleyP. S. (2006a). Widespread siRNA “off-target” transcript silencing mediated by seed region sequence complementarity. RNA12, 1179–1187.10.1261/rna.184206
14
JacksonA. L, Burchard, J.LeakeD.ReynoldsA.SchelterJ.GuoJ.JohnsonJ. M.LimL.KarpilowJ.NicholsK.MarshallW.KhvorovaA.LinsleyP. S. (2006b). Position-specific chemical modification of siRNAs reduces “off-target” transcript silencing. RNA12, 1197–1205.10.1261/rna.184206
15
JinekM.DoudnaJ. A. (2009). A three-dimensional view of the molecular machinery of RNA interference. Nature457, 405–412.10.1038/nature07755
16
KawamataT.SeitzH.TomariY. (2009). Structural determinants of miRNAs for RISC loading and slicer-independent unwinding. Nat. Struct. Mol. Biol.16, 953–960.10.1038/nsmb.1630
17
KhvorovaA.ReynoldsA.JayasenaS. D. (2003). Functional siRNAs and miRNAs exhibit strand bias. Cell115, 209–216.10.1016/S0092-8674(03)00801-8
18
LeuschnerP. J.AmeresS. L.KuengS.MartinezJ. (2006). Cleavage of the siRNA passenger strand during RISC assembly in human cells. EMBO Rep.7, 314–320.10.1038/sj.embor.7400637
19
LewisB. P.BurgeC. B.BartelD. P. (2005). Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell120, 15–20.10.1016/j.cell.2004.12.035
20
LimL. P.LauN. C.Garrett-EngeleP.GrimsonA.SchelterJ. M.CastleJ.BartelD. P.LinsleyP. S.JohnsonJ. M. (2005). Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature433, 769–773.10.1038/nature03315
21
LinX.RuanX.AndersonM. G.McDowellJ. A.KroegerP. E.ResikS. W.ShenY. (2005). siRNA-mediated off-target gene silencing triggered by a 7 nt complementation. Nucleic Acids Res.33, 4527–4525.10.1093/nar/gki623
22
LiuJ.CarmellM. A.RivasF. V.MarsdenC. G.ThomsonJ. M.SongJ. J.HammondS. M.Joshua-TorL.HannonG. J. (2004). Argonaute2 is the catalytic engine of mammalian RNAi. Science305, 1437–1441.10.1126/science.1102513
23
MaJ.-B.YuanY. R.MeisterG.PeiY.TuschlT.PatelD. J. (2005). Structural basis for 5′-end-specific recognition of guide RNA by the A. fulgidus piwi protein. Nature434, 666–670.10.1038/nature03514
24
MartinezJ.PatkaniowskaA.UrlaubH.LührmannR.TuschlT. (2002). Single-stranded antisense siRNAs guide target RNA cleavage in RNAi. Cell110, 563–574.10.1016/S0092-8674(02)00908-X
25
MatrangaC.TomoariY.ShinC.BartelD. P.ZamoreP. S. (2005). Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes. Cell123, 607–620.10.1016/j.cell.2005.08.044
26
MeisterG.LandthalerM.PatkaniowskaA.DorsettY.TengG.TuschlT. (2004). Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs. Mol. Cell15, 185–197.10.1016/j.molcel.2004.07.007
27
MiyoshiK.TsukumoH.NagamiT.SiomiH.SiomiM. C. (2005). Slicer function of Drosophila Argonautes and its involvement in RISC formation. Genes Dev.19, 2837–2848.10.1101/gad.1370605
28
ReynoldsA.LeakeD.BoeseQ.ScaringeS.MarshallW. S.KhvorovaA. (2004). Rational siRNA design for RNA interference. Nat. Biotechnol.22, 326–330.10.1038/nbt936
29
ScacheriP. C.Rozenblatt-RosenO.CaplenN. J.WolfsbergT. G.UmayamL.LeeJ. C.HughesC. V.ShanmugamK. S.BhattacharjeeA.MeyersonM.CollinsF. S. (2004). Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells. Proc. Natl. Acad. Sci. U.S.A.101, 1892–1897.10.1073/pnas.0308698100
30
SchwarzD. S.HutvágnerG.DuT.XuZ.AroninN.ZamoreP. D. (2003). Asymmetry in the assembly of the RNAi enzyme complex. Cell115, 199–208.10.1016/S0092-8674(03)00759-1
31
SchwarzD. S.HutvágnerG.HaleyB.ZamoreP. D. (2002). Evidence that siRNAs function as guides, not primers, in the Drosophila and human RNAi pathways. Mol. Cell10, 57–548.10.1016/S1097-2765(02)00651-2
32
SipaK.SochackaE.Kazmierczak-BaranskaJ.MaszewskaM.JanickaM.NowakG.NawrotB. (2007). Effect of base modifications on structure, thermodynamic stability, and gene silencing activity of short interfering RNA. RNA13, 1301–1316.10.1261/rna.538907
33
SongJ. J.SmithS. K.HannonG. J.Joshua-TorL. (2004). Crystal structure of Argonaute and its implications for RISC slicer activity. Science305, 1434–1437.10.1126/science.305.5686.944
34
TerrazasM.KoolE. T. (2009). RNA major groove modifications improve siRNA stability and biological activity. Nucleic Acids Res.37, 346–353.10.1093/nar/gkn958
35
Ui-TeiK.NaitoY.NishiK.JuniA.SaigoK. (2008a). Thermodynamic stability and Watson-Crick base pairing in the seed duplex are major determinants of the efficiency of the siRNA-based off-target effect. Nucleic Acids Res.36, 7100–7109.10.1093/nar/gkn042
36
Ui-TeiK.NaitoY.ZennoS.NishiK.YamatoK.TakahashiF.JuniA.SaigoK. (2008b). Functional dissection of siRNA sequence by systematic DNA substitution: modified siRNA with a DNA seed arm is a powerful tool for mammalian gene silencing with significantly reduced off-target effect. Nucleic Acids Res.36, 2136–2151.10.1093/nar/gkn042
37
Ui-TeiK.NaitoY.TakahashiF.HaraguchiT.Ohki-HamazakiH.JuniA.UedaR.SaigoK. (2004). Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference. Nucleic Acids Res.32, 936–948.10.1093/nar/gkh247
38
Ui-TeiK.NishiK.NaitoY.ZennoS.JuniA.SaigoK. (2009). “Reduced base-base interactions between the DNA seed and RNA target are the major determinants of a significant reduction in the off-target effect due to DNA-seed-containing siRNA,” in Proceedings of the 2009 Micro-NanoMechatronics and Human Science (MHS2009), Nagoya, 298–304.10.1109/MHS.2009.5351913
39
YodaM.KawamataT.ParooZ.YeX.IwasakiS.LiuQ.TomariY. (2010). ATP-dependent human RISC assembly pathways. Nat. Struct. Mol. Biol.17, 117–123.10.1038/nsmb.1742
40
YuanY.-R.PeiY.MaJ. B.KuryavyiV.ZhadinaM.MeisterG.ChenH. Y.DauterZ.TuschlT.PatelD. J. (2005). Crystal structure of A. aeolicus Argonaute, a site-specific DNA-guided endoribonuclease, provides insights into RISC-mediated mRNA cleavage. Mol. Cell19, 405–419.10.1016/j.molcel.2005.05.023
Summary
Keywords
siRNA, RNAi, off-target effect, thermodynamic stability, seed region
Citation
Ui-Tei K, Nishi K, Takahashi T and Nagasawa T (2012) Thermodynamic Control of Small RNA-Mediated Gene Silencing. Front. Gene. 3:101. doi: 10.3389/fgene.2012.00101
Received
24 April 2012
Accepted
18 May 2012
Published
04 June 2012
Volume
3 - 2012
Edited by
Michael Rossbach, Genome Institute of Singapore, Singapore
Reviewed by
Tohru Yoshihisa, Nagoya University, Japan; Timothy Bowen, Cardiff University School of Medicine, UK
Copyright
© 2012 Ui-Tei, Nishi, Takahashi and Nagasawa.
This is an open-access article distributed under the terms of the Creative Commons Attribution Non Commercial License, which permits non-commercial use, distribution, and reproduction in other forums, provided the original authors and source are credited.
*Correspondence: Kumiko Ui-Tei, Department of Biophysics and Biochemistry, Genome Information Biology, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. e-mail: ktei@bi.s.u-tokyo.ac.jp
This article was submitted to Frontiers in Non-Coding RNA, a specialty of Frontiers in Genetics.
Disclaimer
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