We have evaluated the impact of 21 different types of chemical modifications on the activity and toxicity of a standard 21–22 bp siRNA targeting eGFP using all combinations of 48 ASs and 45 SSs, thereby generating a total of 2160 chemically different siRNA duplexes (see for AS and SS sequences). The modifications can be broadly categorized into 2′-substituted RNAs (OMe, F, DNA, AEM, APM, EA, AP, CE, GE), 4′-modified RNAs (HM), Locked RNAs (LNA, ALN, ADA, PYR, OX, AENA, CENA, CLNA) and RNAs with radical modifications of the ribose sugar ring (UNA, ANA and HNA; ; see ‘Material and Methods’ section for full names). In brief, HeLa cells stably expressing eGFP were transfected with siRNA duplexes (10 nM final concentration), and eGFP levels and cell viability were evaluated 72 h post-transfection.
Overview of chemically modified ASs and SSs
Modifications are well tolerated in the SS
We initially evaluated the impact of chemical modification on siRNA activity for all ASs and SSs in combination with an unmodified RNA strand (SS and AS termed W207 and W053, respectively). Modifications in the SS were well tolerated, and even heavily modified SSs supported knock down (KD) of eGFP levels to below 20% [e.g. HNA (GS2536), AEM (GS2543), APM (GS2548), HM (W044), OMe/F (JC5) and LNA (W037)] (a and ). Only in a few cases did introduction of multiple modifications result in reduced silencing potency, as seen for ANA (GS2366), HNA (GS2370), an additional UNA residue (W130) and full substitution using DNA (W007) and DNA/LNA (W008, W009). Bulky modifications were relatively well tolerated in the SS [e.g. ADA (W069), AEM (GS2542, GS2543) and APM (GS2547, GS2548)] as opposed to in the AS (see below). Interestingly, thermodynamic stabilization of the siRNA duplex by LNA could be achieved via SS modification, whereas a similar level of LNA modification in the AS abolished siRNA activity (compare SS W037 and AS W010; ). This illustrates that the SS can be functionalized by extensive modifications as long as the RNA duplex structure is not grossly distorted.
Figure 2. Silencing activity of chemically modified SSs and ASs. HeLa-eGFP were transfected with the indicated siRNAs (10 nM concentration) and eGFP levels were evaluated 72 h post-transfection. (a) Silencing activity of modified SSs in combination with the unmodified (more ...)
Position-dependent tolerance for AS modification
Modification of the AS had a strong impact on siRNA silencing activity with the most efficient ASs being only modestly modified (b and ). Accordingly, extensive modification reduced activity [e.g. HM (JW1187), LNA (W010), ANA (GS2373) and HNA (GS2540)] with the notable exception of the fully OMe/F-substituted AS (JC10). We generally found bulky modifications to be poorly tolerated in both the AS seed region [e.g. ADA (W068), PYR (W096), b; AEM and APM in position 3, data not shown] and the 3′-end [e.g. ADA (W095), PYR (W097), AEM (GS2549), APM (GS2544), OMe/EA (DO1002)], presumably due to helix distortion or altered strand selection.
The AS seed region (positions 2–8) guides the initial target recognition by RISC* (42
), and modifications in this region should therefore influence silencing activity. We found that single modifications in the seed region were generally well tolerated for non-bulky groups that do not strongly influence the thermodynamic stability [e.g. HNA (GS2538, GS2539), ANA (GS2378), OMe- (DHARM1), CENA (JC-F1, JC-F2, JC-F3) and HM (JW1186), b and Supplementary Figure 1
]. In contrast, modifications that stabilize the seed region, such as LNA (Supplementary Figure 1
) and AENA (JC-A1, JC-A2 and JC-A3; b) had a negative effect on silencing activity, presumably by altering strand selection or seed–target interactions. The strongly destabilizing UNA was better tolerated in the seed, but reduced silencing activity when introduced near the 5′-end where it could destabilize AS–target interactions (b, compare W124 and W123, and Supplementary Figure 1
). We also found some sensitivity to modification in the central positions of the AS, which must be capable of perfect base-pairing to the target to allow cleavage by Ago2 (7
); UNA (W126) and alternating OMe/F (JC10) were well tolerated, while HNA and ANA impaired silencing (GS2373 vs. GS2378 and GS2540, b). As expected, most modifications were well tolerated in the AS 3′-region [e.g. UNA (W125), HNA (GS2537), HM (W042), LNA (W075) F/OMe (JC10)] and 3′-overhang [e.g. ANA (GS2372), HNA (GS2537), ALN (W047), EA (JE1001), single HM (W059) and LNA (W006)], although UNA- (W127), double HM- (W054) and LNA modifications (W180) decreased silencing activity slightly (b).
In conclusion, modification of the AS must take the functional regions into account and should preferably be restricted to the peripheral regions.
Identification of highly efficient siRNAs
We next investigated what chemical modifications could enhance siRNA activity beyond that of an unmodified siRNA duplex. We found two ASs (W006, JC-F1) and six SSs (DO003, GS2369, JC1, W60, GS2542 and GS2534) that performed significantly better than unmodified RNA when paired to an unmodified opposing strand (b). However, the efficiency of a SS in combination with an unmodified ASs (W053) does not necessarily allow the prediction of its behaviour in combination with other ASs. Our data from 2160 siRNAs provide valuable insight into how chemically different ASs and SSs can be combined to generate highly modified and functional siRNAs. We identified a total of 134 highly efficient (HE) siRNAs exhibiting enhanced silencing activity compared with unmodified siRNA ( and Supplementary Table 1
). Interestingly, the most efficient siRNA (JW1186-W043) maintained stronger silencing than unmodified siRNAs (W053-W207) even at a lower concentration (3 nM, Supplementary Figure 2
), demonstrating a higher potency that may prove important for in vivo
applications. Hereby our data show that several types of chemical modifications can indeed enhance siRNA activity beyond that of an unmodified siRNA even in short-term cell-culture experiments.
Rescue of AS activity by optimal SSs
We found only modestly modified ASs among the HE siRNAs (b; and ), yet high levels of siRNA modification would be preferable for many in vivo
applications. Interestingly, the activity of most ASs in our screen could be improved by combining the ASs with specific ‘optimal’ SSs [, compare ‘optimal SS’ (red line) and unmodified SS (W207, black line)]; the most prominent effect was seen for extensively modified ASs [e.g. OMe (W106), LNA (W209) and ANA (GS2373)]. The activity of certain ASs was improved by several optimal SSs (e.g. W097, GS2549, JC-A2 and GS2544) whereas other ASs were only rescued by one or few SSs (e.g. W209 by GS2542, JC-A3 by W194 and W106 by GS3543, and Supplementary Table 1
). Conversely, certain ASs allowed the use of heavily modified SSs that failed to support RNAi when combined with an unmodified AS; the fully LNA/DNA substituted SS W009, produced a potent ~80% KD in combination with the UNA-modified AS W123 but gave only a 30% KD in combination with unmodified AS W053 (Supplementary Table 1
). These observations imply that the reduced activity often observed upon extensive siRNA modification can be compensated by careful matching of specifically modified SSs and ASs. However, our data also identifies SSs that act as more ‘general optimal SSs’ to improve the performance of many ASs and are therefore broadly applicable in siRNA design. In particular, SSs modified in the 3′-overhang by UNA and HM (W131 and W043, respectively) or destabilized in the 3′-end by EA and OX (DO003 and JC1, respectively) produced a strong KD in combination with many ASs ( and ).
Figure 3. Optimal SSs enhance the activity of ASs. Relative eGFP expression of HeLa-eGFP cells transfected with all investigated ASs (X-axis, ASs name given in bold) in combination with all 45 SSs (represented by grey dots) or with selected SSs (coloured triangles/lines). (more ...)
Strategies for enhancing siRNA activity
The HE siRNA duplexes were found to harbour modifications either in the 3′-overhang (e.g. W006, JE1001, GS2372, W043, W047, W060, W131), within the siRNA body (e.g. JC-F1, DO003, JC-S1, JC1, GS2369) or in both regions (e.g. GS2383, JW1189, GS2542, GS2534) ( and ), and we therefore speculated that two mechanisms were responsible for enhancing siRNA activity: (i) Modification of the siRNA body to introduce thermodynamic asymmetry to favour AS incorporation into RISC. (ii) Modification of SS and AS 3′-overhangs to enhance serum stability or affect strand selection by RISC.
Destabilization of SS 3′-ends enhances silencing efficiency
Given that strand selection during RISC loading, and thereby AS activity, is dependent on the thermodynamic profile of the siRNA duplex we investigated the impact of modifications on thermodynamic asymmetry. The chemical modifications used in this study have both stabilizing (OMe, F/OMe, HNA, ANA, ALN, LNA) and destabilizing (DNA, AEM, APM, OXE, EA, AP, CE, UNA) properties. We found 15 of the 20 most efficient siRNA duplexes to have chemical modifications favouring AS incorporation by altering the thermodynamic profile. W123 contained a 5′-end destabilizing UNA, whereas JC-F1 and JC-S1 contained CENA and CLNA resulting in stabilization of the AS 3′-end (see and and Supplementary Table 1
). Nearly a third of the HE siRNAs (38/134) contained these three ASs suggesting that altering the thermodynamic asymmetry through AS modification is a major determinant of siRNA activity.
A similar trend was found among the HE SSs; the 3′-end OX-destabilized JC-1 and the 3′-end EA destabilized DO003 improved the performance of most ASs (, orange and green, respectively, and a, columns 1, 2, 13,14) and resulted in an average 25% improvement in activity for 22 and 23 ASs, respectively (). Furthermore, JC1 and DO003 were highly represented among the 134 HE siRNAs and among the top three SSs for each AS (). Conversely, we found both SS 3′-end stabilization (e.g. GS2369 and GS2383; a, columns 11, 23 and 12, 24) and SS 5′-end destabilization (e.g. JC2 and W129; a, columns 9, 21 and 10, 22) to negatively influence the activity of many ASs. The stabilization of the SS 5′-end should improve siRNA performance, and moderate 5′-end stabilization (e.g. GS2368; a, columns 4, 16) did improve the performance of most ASs; however more stabilizing modifications (e.g. multiple HNAs (GS2534; a, columns 5, 17), single and extensive LNA modifications [(W011 and W013; a, columns 6, 18 and 7, 19)] impaired silencing. This suggests that, although favouring AS selection, extensive thermodynamic stabilization of an siRNA duplex is detrimental to silencing activity. In accordance, the activity of stabilized ASs (e.g. W106, W209 and GS2373) was enhanced both by the 3′-end destabilized SSs (DO003 and JC1) and by the 5′-end destabilized JC-2 although this SS should disfavour AS incorporation into RISC based on thermodynamic asymmetry ( and Supplementary Table 1
). This highlights that siRNA thermodynamic stability should fall within a range compatible with the components of the RNAi machinery, and that both siRNA thermodynamic asymmetry and stability are critical parameters to consider during siRNA design. Based on our screen, relative destabilization of the SS 3′-end (using DO003 and JC1) represented the more reliable strategy to improve siRNA performance unless high stability of the duplex is specifically required.
Figure 4. Improvement of siRNA performance by introduction of additional thermodynamic asymmetry and modification of 3′-overhangs. (a) The performance of ASs (exemplified by the representative ASs DO1001 and JC-S3) can be modified by altering the overall (more ...)
Modification of 3′-overhangs influences siRNA activity
The observation that several highly efficient siRNAs (e.g. W006-W060, W006-W043 and W006-W131) are modified in the 3′-overhangs only, highlights the importance of overhangs for silencing activity (, and ). In fact, ASs modified exclusively in the 3′-overhang by LNA (W006), EA (JE1001), ALN (W047) and ANA (GS2372) were found in half (66/134) of the HE siRNAs. The impact of 3′-overhang modification was highly chemistry dependent; the UNA- (W127), LNA- (W180) and HM-modified (W054) ASs showed a significantly reduced silencing activity whereas ASs modified by ANA (GS2372), EA (JE1001) and ALN (W047) were equally efficient to unmodified RNA (W053) (b). Interestingly, the LNA-modified overhang in W006 (5′-LNA-LNA-RNA-3′) containing an additional 3′-RNA residue resulted in significantly improved silencing (b; see the Discussion section below).
We speculated that the influence of 3′-overhang modifications could result from differences in siRNA serum stability; however, no clear correlation between stability and silencing activity was observed for overhang-modified siRNAs ( and and data not shown). Instead modification of the 3′-overhang was found to influence strand selection, as modifications disfavoured in the AS 3′-overhang [e.g. HM (W054), UNA (W127), LNA (W180); b and 4b] were found to favour AS selection when incorporated into SS 3′-overhangs [HM (W043), LNA (W194) and UNA (W131); b]. In detail, the silencing activity of the LNA-modified AS W180 (b, column 9) was enhanced to a level indistinguishable from unmodified siRNA (b, column 5) when paired to a SS with disfavoured overhangs, such as LNA (W194), UNA (W131) and HM (W043) (b, columns 10–12). Similar SS 3′-overhang effects were seen for the UNA-modified AS W127 (b, columns 13–16) and HM-modified AS W054 (b, columns 17–20). The HM modification was particularly strongly disfavoured as the HM-modified W054 was a significantly poor AS (b, column 17), while the HM-modified SS W043 had the most potent rescue effect on ASs with disfavoured overhangs (b, columns 8, 12, 16, 29). This suggests that HM-modified overhangs may be more strongly disfavoured than UNA- (W131) and LNA-modified (W194) SS and can therefore be broadly used in SS design to favour AS-strand selection.
Figure 5. Enhancing serum stability of siRNAs with minor loss of activity. (a) The biostability of modified siRNAs was evaluated by incubation in 80% FBS. While a low level of chemical modification results in only modest increase in stability (left panel), more (more ...)
Modified overhangs alter strand selection by RISC
To further investigate the impact of 3′-overhang modifications on altered strand selection, we evaluated the activity of both strands in the siRNA duplex using reporters containing a perfect target site for either the SS or AS downstream of a luciferase open reading frame (d). We expected disfavoured modifications in the AS 3′-overhang [LNA (W180), UNA (W127), HM (W054)] to increase incorporation of the unmodified SS (W207) into RISC*, thereby leading to higher SS silencing activity. Indeed, the modest silencing effect of the SS (W207) in an unmodified duplex (5 nM concentration) was significantly increased when combined with an AS with a disfavoured overhang (c, compare column 2 with 4, 6, 8). We therefore suggest that disfavoured modifications in 3′-overhangs can be used to favour the incorporation of opposing strands, irrespective of the thermodynamic asymmetry of the siRNA duplex.
Intriguingly, we found the AS W006 to be significantly more efficient than an unmodified AS (W053) when paired to W207 suggesting the 3-nt 5′-LNA-LNA-RNA-3′ overhang motif to be favoured for RISC loading even over the natural RNA–RNA 3′-overhang (b and 4b). In agreement, a SS (JW1104) with the 5′-LNA-LNA-RNA-3′ overhang motif exhibited significantly enhanced activity compared with the unmodified SS (W207) (c, compare columns 2 and 10) and a similar effect was obtained when combining JW1104 with the disfavoured ASs W180 (LNA), W127 (UNA) and W054 (HM) (c, compare columns 4, 6, 8 and 12, 14, 16). We found other highly efficient ASs modified exclusively in the 3′-overhang by EA (JE1001), ANA (GS2372) and ALN (W047) () to be favoured during RISC loading as they lowered the activity of the opposing SS (W207) as compared with the unmodified AS W053 (Supplementary Figure 3
). Consequently, favoured overhangs such as the 5′-LNA-LNA-RNA-3′ motif and others can be broadly utilized to favour AS incorporation into RISC*, thereby enhancing siRNA potency.
Enhancing siRNA serum stability by chemical modification
The identification of highly nuclease-resistant siRNAs is a key concern for in vivo
applications and great efforts have been applied to enhance stability by chemical modification (14
). Like other studies, we find siRNA serum stability to be positively correlated with the level of RNA modification for most chemistries. While substitution of 3′-overhangs led to a modest increase in serum stability (e.g. UNA, ALN, HM, LNA, ANA; a, left panel), partial or full modification of the siRNA body led to efficient stabilization (OMe/F, OMe, DNA/LNA LNA; a, right panel). Although a large number of substitutions within an siRNA does indeed enhance stability, highly modified siRNAs generally displayed poor silencing. Fully OMe/F-substituted siRNAs have previously been reported to be both highly stable and more potent than standard siRNAs (30
); however, in our hands a similar siRNA (JC5-JC10) exhibited high stability, but produced a very modest 26% KD (a and Supplementary Table 1
). This highlights the importance of identifying other strategies for siRNA stabilization that support high silencing activity. Interestingly, we found that LNA-mediated stabilization of selected positions within the siRNA duplex led to similar or even enhanced stability as compared with fully substituted duplexes (a, compare W010-W037, W010-W004-W179, JC10-W037 with JC5-JC10 and W106-W009). In fact, the partially LNA-modified W006-W037 and W010-W004-W179 exhibited higher stability than the fully OMe/F-substituted JC5-JC10, while retaining a potent 87% and 66% KD activity, respectively ( and a). Moreover, we found that the sisiRNA design (41
) that utilizes an LNA-stabilized segmented SS (W004 + W179) may be broadly applied to introduce LNA-stabilization with minor impact on activity. The sisiRNA constructs W010-W004-W179 and JC10-W004-W179 retained both high silencing activity and serum stability when modified extensively with LNAs, whereas an intact LNA-modified SS (W037) was detrimental to siRNA activity (W010-W037 and JC10-W037, ). In fact, the sisiRNA design allowed the LNA-stabilization of several differentially modified ASs without major loss of silencing activity (b). This implies that siRNA stabilization is preferentially achieved by introducing few LNA modifications, preferentially using the sisiRNA design, rather than generating fully substituted duplexes. Furthermore, selected modifications allow further functionalization of the siRNA duplex through modification of the remaining nucleotide positions.
Reducing siRNA toxicity through chemical modification
Chemical modifications have previously been shown to affect the toxicity of siRNAs (44
) and we therefore evaluated the viability of the siRNA transfected cells after 72 h (as number of nuclei per well relative to mock transfections). The majority of modified SSs resulted in a similar high degree of viability in combination with unmodified RNA AS whereas the viability varied more dramatically among the modified ASs in combination with the RNA SS (). We found cell viability to correlate with siRNA potency for the majority of ASs (Supplementary Figure 4
) with only a few non-functional siRNAs resulting in low cell viability (). This suggests that the observed siRNA toxicity arises from efficient ASs interfering with the endogenous RNAi pathway. Indeed, plotting the relative eGFP levels versus relative viability allowed us to identify distinct subgroups of ASs; the unmodified W053, and the LNA-modified W006 displayed high silencing potency and moderate cell viability (, yellow and purple triangles, respectively), the OMe- (W106) and heavily LNA- (W209) modified ASs had poor activity and high viability (, light blue and brown triangles, respectively), while the HNA-modified GS2538 had high activity and very low viability (, red triangles). Interestingly, silencing activity and viability of particularly the UNA- (W123) but also the HM- (JW1186) modified ASs were both high in combination with most SSs (, green and dark blue triangles, respectively). Hereby our data shows that although most potent siRNAs cause reduced viability, it is possible to generate highly potent and non-toxic siRNAs by selecting particular chemical modifications.
Figure 6. Identification of highly efficient siRNAs with low toxicity. Scatter plot showing target cell viability versus eGFP expression (siRNA activity) for all tested siRNAs (grey dots) and for selected ASs in combination with all 45 SS (coloured dots). Silencing (more ...)