DOI: 10.1002/chem.201403430

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& Gene Regulation

Synthesis of Site-Specifically Phosphate-Caged siRNAs and Evaluation of Their RNAi Activity and Stability Li Wu,[a, b] Fen Pei,[a] Jinhao Zhang,[a] Junzhou Wu,[a] Mengke Feng,[a] Yuan Wang,[a] Hongwei Jin,[a] Liangren Zhang,[a] and Xinjing Tang*[a]

the internally caged siRNA was observed, irrespective of the presence of Mg2 + . Molecular dynamic simulations demonstrated that enhanced hydrolysis of the caging group on internally phosphate-caged siRNAs was due to easy fragmentation of the caging group upon formation of the pentavalent intermediate of the phosphotriester with attack by water. The caging group in the terminally phosphate-caged siRNA or single-stranded caged RNA prefers to form p–p stacks with nearby nucleobases. In addition to providing explanations for previous observations, this study sheds further light on the design of caged oligonucleotides and indicates the direction of future development of nucleic acid drugs with phosphate modifications.

Abstract: A complete set of new photolabile nucleoside phosphoramidites were synthesized, then site-specifically incorporated into sense or antisense strands of siRNA for phosphate caging. Single caging modification was made along siRNA strands and their photomodulation of gene silencing were examined by using the firefly luciferase reporter gene. Several key phosphate positions were then identified. Furthermore, multiple caging modifications at these key positions led to significantly enhanced photomodulation of gene silencing activity, suggesting a synergistic effect. The caging group on both the terminally phosphate-caged siRNA and the single-stranded caged RNA has comparatively high stability, whereas hydrolysis of the caged group from

Introduction

that can afford selective control over many biological processes). One strategy is to introduce a temporary chemical modification on a siRNA duplex to block gene silencing in its initial form. Light activation restores its RNAi silencing activity. This strategy has been successfully applied to photoregulate gene expression using caged antisense oligonucleotides,[3] caged small interfering RNAs,[4] caged mRNAs,[5] caged DNA decoys,[6] caged triplex-forming oligonucleotides,[7] and more recently caged miRNA antagomirs.[8] The most critical issue in developing photochemically modified siRNA is optimum modification on a siRNA duplex so as to maximize the effect of photoregulating its RNAi activity. Early on, statistical caging of the phosphate backbone was performed on a chemically stabilized RNAi effector by Shah et al.[4a] 1-(4,5-Dimethoxy-2-nitrophenyl)ethyl (DMNPE) was used for statistical labeling of the siRNA phosphate backbone with 3 % caging efficiency, thus rendering the siRNA molecule inactive. Light irradiation restored 80 % of the gene silencing activity. Subsequent work by the same authors indicated that the caging group was preferentially attached to the terminal phosphate moieties instead of the phosphate group in siRNA backbone as previously assumed.[9] Multiple caging modifications of siRNA duplexes have been made to totally inhibit their siRNA activity. However, complete recovery was not achieved. With the same caging strategy, more sterically demanding cyclododecyl DMNPE caging groups were introduced to the four termini of the double-stranded shRNA to prevent processing by Dicer, thus achieving efficient inhibition of silencing activity until UV exposure.[4c] Monroe et al. also incorporated the

RNA interference (RNAi) has been widely explored as a powerful tool with which to manipulate gene functions and constitutes a therapeutic strategy for many intractable diseases including cancer, neurodegenerative diseases, and viral infections. Short interfering RNAs (siRNAs) play a key role in controlling gene functions in biological processes. Recently, in vivo activity of nuclease-resistant siRNAs has also been reported,[1] shedding light on the mechanism of RNAi-mediated gene silencing, in conjunction with biochemical and genetic evidence.[2] In an effort to explore the utility of siRNAs in vivo, chemical modifications have been used to optimize RNA lipophilicity, increase duplex stability, and/or improve nuclease resistance. Moreover, a photolabile version of siRNA could be used to photoregulate a specific gene silencing by light with spatial and temporal resolution (considering that light is an excellent external trigger [a] Dr. L. Wu, F. Pei, J. Zhang, J. Wu, M. Feng, Y. Wang, Dr. H. Jin, Prof. L. Zhang, Prof. X. Tang State Key Laboratory of Natural and Biomimetic Drugs School of Pharmaceutical Sciences Peking University, Beijing 100191 (China) Fax: (+ 86) 10-8280-5635 E-mail: [email protected] [b] Dr. L. Wu College of Chemistry and Chemical Engineering University of Chinese Academy of Sciences Beijing 100049 (China) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201403430. Chem. Eur. J. 2014, 20, 1 – 10

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Full Paper completely ablate gene silencing activity, which could be fully DMNPE group nonspecifically throughout fully modified 2’restored upon light activation. fluoro-siRNA to achieve optochemical control over siRNA activiHerein, for the first time, we report the synthesis of a comty in vivo.[10] However, the DMNPE-caged 2’-fluoro-siRNAs did plete set of four new 1-(2-nitrophenyl)ethyl (NPE) protected not fully inhibit siRNA function, and siRNA activity could not nucleoside phosphoramidites (dA0, dG0, dC0, dT0) and their be completely restored through UV irradiation. Although this methodology provides a means to regulate siRNA function by site-specific incorporation in siRNA strands, as shown in light, one main limitation of these light-activated RNAi reFigure 1. 2-Cyanoethyl-1-(2-nitrophenyl)ethyl-N,N’-diisopropylagents is the statistical incorporation of the caging group, phosphoramidite (N0) was also synthesized for terminal caging which may account for the “leakiness” of the caged siRNAs. In modification. Systematic screening of caged siRNA activity was addition, the positions of caged phosphate moieties were carried out in HEK293A by using a dual luciferase assay, and likely variable with the nonspecific labeling technique. No positheir inactivation and light-induced recovery of RNAi-mediated tion or structure–function correlation has been obtained from gene silencing are presented. In this work, several key caging statistical placement of the caging groups in the siRNA duplex. positions with effective photomodulation of siRNA activity However, inhibition of RNAi with caging groups may depend were identified. Further site-specific installation of multiple highly on the caging position. Several potentially critical posicaging groups at these key positions greatly suppressed siRNA tions for the disruption of RNAi have been previously reported activity, and light irradiation fully restored their functions. Furthrough siRNA modification and structural evaluation of RNAther investigation indicated that the caging group was found induced silencing complex (RISC).[8b] to be easily hydrolyzed only when the caged RNA formed a duplex with its complementary strand. Furthermore, hydrolyRecently, Heckel et al. incorporated a 2-(2-nitrophenyl)propyl sis of the caging group was independent of the 2’-hydroxyl (NPP)-caged deoxyguanosine nucleotide into the antisense group, the complementary DNA or RNA oligonucleotide, and strand of an siRNA.[4b] By NPP-caging one of the nucleotides at the presence of Mg2 + . Molecular dynamics simulations sugposition 9–11, siRNA activity was reduced to 10 %. However, ingested that the enhanced hydrolysis of caging groups on interstability of the caging group was detected, because an innally phosphate-caged siRNA duplexes may be due to the easy crease in RNAi activity was observed after 28 h in cells that were kept in the dark. Deiters et al. installed 6-nitropiperonyloxymethyl (NPOM)-caged uridine and guanosine nucleotides within the seed region and the central argonaute-cleavage region of a siRNA molecule.[11] Although the caging group was known to be able to effectively suppress recognition of target mRNA while maintaining stability, only a few positions with caged uridine and guanosine nucleotides were investigated. Therefore, site-specific introduction of caging groups in siRNA duplexes would be beneficial to understand the detailed mechanism of the photomodulation of RNAi activity. However, a systematic study of the positional effect of caging modifications in the siRNA phosphate backbone on gene silencing activity has not yet been reported. Such a study is necessary to understand the electrostatic interaction, steric effects, and photocontrolled gene expression with respect to the caging position and the effect on the activation of the RNAi pathway. Ideally, a single cage Figure 1. moiety in a siRNA duplex could a) Phosphate-caged phosphoramidite monomers and b) their site-specific incorporation into single-stranded RNA. &

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Full Paper fragmentation of a pentavalent intermediate of phosphotriester upon attack by water, whereas the caging group on the terminally phosphate-caged siRNA duplexes or single-stranded caged RNA prefers to form p-p stacks with nearby nucleotides, which inhibits fragmentation of the caging group. This study also provides new evidence for previous observations, such as the preferential caging of the termi- Figure 2. Synthesis of phosphate-caged phosphoramidite monomers. nal phosphate of siRNA duplexes, the intrinsic instability of statistically caged siRNA duplexes, and incomplete recovery of ficiency of photolysis under 365 nm light irradiation. This kind siRNA activity of heavily caged siRNAs. of photolabile derivative has been widely used in caging nucleic acids for biological applications.[3e–i, 4b] Starting from 1-(2-nitrophenyl)ethanol, a synthetic intermediate, N,N,N’,N’-tetraisopropyl-[1-(2-nitrophenyl)ethyl]phosphorodiamidite (P0) was first Results and Discussion synthesized and then used in the preparation of four nucleoRational design of phosphate-caged siRNAs side phosphoramidite monomers (dA0, dG0, dC0, dT0) from the Gene silencing through the RNAi pathway includes loading of corresponding protected nucleosides (Figure 2). The reaction a siRNA into RISC, guidance of the antisense strand in the RISC mixtures were purified by silica gel column flash chromatograto target mRNA, and cleavage of the target mRNA for further phy to give 50–60 % yields. We also evaluated another synthetgene silencing. We envisaged that chemical modifications of ic route to dA0, dG0, dC0, dT0 that involved coupling of bis(diithe siRNA duplex at different positions would influence RISC sopropylamino)deoxynucleosid-3’-yl-O-phosphine with 1-(2-niloading and subsequent processing. Recently, it was shown trophenyl)ethanol, but the yields were relatively low (data not that different regions of the siRNAs play distinct roles in target shown). 2-Cyanoethyl-1-(2-nitrophenyl)ethyl-N,N’-diisopropylrecognition, cleavage, and product release.[12] A detailed study phosphoramidite (N0) was also synthesized for 5’-terminal on the positional effect has been conducted to evaluate the phosphate labeling. 31P NMR spectra of these nucleoside phosdegradation of passenger RNAs with 2’-MeORNA or misphoramidites showed the expected multiple singlets between matched modifications.[13] However, no systematic investiga145 and 150 ppm for several diastereoisomers due to the chiral moieties of 1-(2-nitrophenyl)ethyl and phosphine (see tion on the positional effect of caged siRNA is available with Figure S11 in the Supporting Information). respect to both suppression and light restoration of their RNAi In a previous study, Chiu and Rana found that, as long as activity. To achieve site-specific caging phosphate of siRNA duthe A form structure of the duplexes was not disrupted, siRNA plexes, we synthesized 1-(2-nitrophenyl) ethanol phosphoramiactivity was compatible with 2’-modifications, including the indite and four novel phosphate-caged phosphoramidites. These dividual substitution with deoxynucleotides.[14] Heckel further phosphoramidites were then site-specifically incorporated at confirmed that the introduction of single deoxynucleotides did any phosphate position in the sense and antisense strands of not affect siRNA activity.[4b] In our case, we synthesized a series a siRNA duplex for targeting by firefly luciferase gene of caged siRNAs containing single deoxynucleotide modifica(Figure 1). By screening of these phosphate-caged siRNAs, the tion with phosphoramidite monomers (dA0, dG0, dC0, dT0) in positional effect of phosphate-caged modifications on siRNA different positions of siRNA sequence. Two concerns were concould be evaluated in terms of gene silencing and light restosidered: 1) The choice of caged deoxynucleotides instead of riration, and key positions may be determined to maximize the bonucleotides will eliminate the possible hydrolysis of the photoregulation of gene expression. We expect that the sitecaging group mediated by the 2’-hydroxyl group in ribonuspecific caging method will be helpful to further understand cleotides; 2) Synthesis of 1-(2-nitrophenyl) ethyl protected the mechanistic interactions of caged siRNAs in future studies, phosphoramidites ribonucleoside monomers were much more and provide further information about the structure–function challenging due to the strong steric conflict between the relationships observed in previous studies.[4a, 9, 10] caging group and 2’-tert-butyldimethylsilane. The siRNA sequence (antisense strand (ASc): 5’-GCG AAG Synthesis of phosphate-caged phosphoramidite monomers AAG GAG AAU AGG GTT-3’ and sense strand (Sc): 5’-CCC UAU and phosphate-caged siRNAs UCU CCU UCU UCG CTT-3’) was chosen for targeting firefly luciferase mRNA because it was previously identified as a stable The 1-(2-nitrophenyl)ethyl photolabile group was chosen for native siRNA duplex by screening a series of siRNA conthese studies because of its easy accessibility, high stability in structs.[15] All caged oligonucleotides were synthesized by siteRNA solid-phase synthesis, and because of its relatively high efChem. Eur. J. 2014, 20, 1 – 10

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Full Paper tions of 0.05, 0.1, 0.2, and 0.5 nm (Figure S1). In addition, an irradiation time course for blank cells (Figure S2) and transfected cells (Figure S3) was performed, and no difference in gene silencing was observed within 3 min UV irradiation under the photolysis conditions (UV-LED light, 365 nm, 7 mW cm 2). We then evaluated gene silencing activity with the set of caged antisense guide strand RNAs (AS00–AS19) paired with the unmodified sense strand (Sc). Firefly luciferase activity with only vectors was set as 100 % with renilla luciferase activity as internal control (Figure S4A). A scrambled siRNA was used as a negative control (NC) and, as expected, showed no inFigure 3. Sequences of natural and caged firefly luciferase siRNAs strands used for this study, AS: antisense hibition of firefly luciferase exstrand; S: sense strand; f: 2’-F-modification; P: phosphate; ASc: control native antisense strand; Sc: control native pression. Cells treated with posisense strand. tive control siRNA (ASc/Sc) targeting firefly luciferase displayed a 40 % level of gene expression. Compared with the positive specific incorporation of phosphate-caged phosphoramidites control siRNA duplex, caged modification at the 5’-end of the (Figure 3). 2’-O-TBDMS and 2’-F phosphoramidites with exocyantisense strand resulted in 76 % firefly luciferase activity clic amino groups protected with benzoyl (Bz for A and C) or before irradiation, indicating that installation of the photolabile isobutyryl (ibu for G) groups were used for the synthesis of the unit at this position partially masked the phosphate moiety. corresponding RNA oligonucleotides. The coupling time for Light irradiation (2 min) removed the photolabile group and phosphate-caged phosphoramidites was extended to 25 min activated siRNA activity, resulting in an enhancement of gene to ensure maximum coupling efficiency, and the step coupling silencing up to 41 % of control level, similar to that of positive efficiencies of phosphate-caged phosphoramidites were similar control (ASc/Sc). These results are consistent with previous reto those of normal RNA nucleotide phosphoramidites. ports.[4a, 9, 17] It is well known that the interaction of RISC with mRNA is highly dependent on seed region nucleotides 2nd– Photoregulation of phosphate-caged siRNA activity 8th of the guide strand.[18] In our case, a slight inhibition of The dual luciferase assay was performed to evaluate RNAi acsiRNA activity in comparison to that of the positive control tivity, using a previously established siQuant vector.[15] The siRNA was observed proceeding along caging positions 2nd– siRNA sequence is designed to target firefly luciferase mRNA 8th of the antisense strand, except for positions 4 and 8, and for gene silencing, in which renilla luciferase is included as an the silencing activity of these siRNAs was restored to a level internal control. Reporter vectors with the corresponding that was similar to that of the positive control after light expocaged or noncaged siRNA duplexes were cotransfected into sure. It has also been reported that base pair mismatches and HEK293A cells, and luciferase activities were measured after chemical modification at nucleotide positions 10th–11th of an 20 h. The fluorescence signals generated by firefly luciferase siRNA antisense strand interrupt RNAi activity.[13, 19] To our sur[16] were normalized to those by renilla luciferase. prise, the results presented in this study showed that caged phosphate residues around the cleavage site (i.e., positions To evaluate the positional effect of phosphate-caged modifi9th–11th of the antisense strand) do not have an effect on cations on RNAi activity, we first introduced the phosphatesiRNA activity. caged residues one by one along the antisense guide strand Caged sense RNA strands S00–S19 (Figure 3) were also syn(AS00–AS19, as shown in Figure 3 and Table S1). These caged thesized. These caged sense strands were then hybridized to antisense strand RNAs were then mixed with their complementhe control antisense guide strand (ASc) to form caged siRNAs tary unmodified sense strand (Sc) to form caged siRNA duplexthat were further evaluated for gene silencing activity. All es. A preliminary study of firefly luciferase gene silencing using these caged siRNA duplexes exhibited similar activity to the the positive control duplex and terminal phosphate-caged corresponding positive controls (Sc/ASc) even without light acsiRNA duplexes (AS00/Sc) was conducted in HEK293A cell line tivation (Figure S4B). A high tolerance towards the caging and their dose dependence was also evaluated in concentra&

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Full Paper group installed at the 5’-end of the sense strand of siRNA was also observed, suggesting that capping of the 5’-end of the sense strand has no effect on silencing activity of the whole siRNA duplex. To verify whether caged sense and antisense guide strands of siRNA duplexes have a synergistic effect on the photomodulation of their gene silencing activity, we further tested the complete set of caged antisense guide strand RNAs (AS00– AS19) matched with the corresponding caged sense stands (S00–S19), as shown in Figure 4 (see also Figure S5). These Figure 5. Photocontrolled firefly luciferase expression of siRNA duplexes with natural sense strand and modified antisense strand with multiple phosphate-caged modifications. The concentration of siRNA duplex was 0.5 nm. AS20 (caging group at 20th phosphate), AS21 (caging group at 5’-end and 20th phosphate), AS22 (caging group at 5’-end, 16th and 20th phosphate), AS23 (caging group at 5’-end, 6th and 20th phosphate), AS24 (caging group at 5’-end, 6th, 16th, and 20th phosphate). Each set measured in more than triplicate.

of AS21/Sc completely restored RNAi activity to the same level as that of the positive control siRNA duplex (ASc/Sc). Significantly, photoactivation of caged siRNA duplex (AS23/Sc) bearing three caging modifications at positions 6th, 20th and the 5’-end resulted in nearly 3.3-fold decrease in luciferase activity. In contrast, introduction of three caging modifications at positions 16th, 20th and the 5’-end (AS22) rendered only a moderate decrease in caged siRNA activity. Modification of four caging groups at positions 6th, 16th, 20th and the 5’-end (AS24) also led to inactivation of siRNA, with 73 % of luciferase activity remaining. After brief UV irradiation, the caging groups were fully cleaved and the RNAi activity was restored completely. These results further suggested that caging modifications at different phosphate positions of siRNA (i.e., position 6th and the 5’-end of the antisense strand) showed a synergistic effect on the photoregulation of RNAi activity, and multiple caged phosphates at the key positions greatly increased the photomodulation efficiencies.

Figure 4. Photomodulation of firefly luciferase expression with caged siRNA duplexes consisting of the caged antisense RNAs and the corresponding caged sense RNA strand. The concentration of siRNA duplexes was 0.2 nm. Each set measured in more than triplicate.

caged siRNA duplexes, for example the antisense strand AS01 with sense stand S18 (duplex AS01/S18), displayed a profile of photomodulating siRNA activity that was similar to that of siRNAs containing caging groups only in the antisense strand (Figure S4A). Compared with other positions, the caging group at positions 1st, 2nd, and 5th–7th in the seed region of the antisense guide strand led to more inhibition of siRNA activity to different extents, and light irradiation fully restored their RNAi activity. Interestingly, caging modification at position 6 of the antisense strand, showed more synergistic inhibition of RNAi activity compared with modification of the other positions in the seed region. The enhanced inhibition of gene silencing activity of caged siRNAs with caging modification on both the antisense and sense strands may suggest that the caging group in the sense strand may partially block loading of the siRNA duplex into RISC or inhibit the active conformation of RISC. According to the screening result of single phosphate-caged siRNAs, several key phosphate positions were identified and selected for multiple caging modifications in the antisense guide strand (Figure 5). The introduction of a second caging group in the phosphate of dangling TT position near to 3’-end of AS00, caused more inactivation of the RNAi effect, because 68 % of firefly luciferase activity for AS21/Sc (caging group at 5’-end and 20th phosphate) was observed, whereas only a single caged phosphate at the dangling TT position (AS20/ Sc) had no effect on RNAi activity. Furthermore, light irradiation Chem. Eur. J. 2014, 20, 1 – 10

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Chemical and enzymatic stability of phosphate-caged siRNA duplexes Unlike the demonstration of the crystal structure of RISC and previous reports of caged siRNAs,[18] the presence of a single caging group positioned at the cleavage site or the seed region did not lead to enhanced inhibition of siRNA activity for single phosphate-caged siRNAs in this study. We suspect that the stability of the caged siRNA may compromise the blocking of gene silencing. 2’-Fluoro-ribonucleoside modifications seem to be the best-tolerated alteration for maintaining RNAi activity and such derivatives have been widely used to increase the resistance to hydrolytic and enzymatic degradation of RNA.[1, 20] Thus, to achieve better thermodynamic or chemical stability, we replaced all the ribonucleotides with 2’-fluoro-ribonucleoside (FNA). Here, fully 2’-fluoro-modified antisense guide strand of caged siRNAs (FAS06, FAS10, FAS16, and FAS00), their 5’-end prephosphorylated caged oligonucleotide (FAS06P, FAS10P, FAS16P) with single phosphate-caged deoxynucleotide at 6th, 5

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Full Paper 10th, 16th position and the 5’-end prephosphorylated antisense strand (FAScP) were also synthesized (Figure 3). RNAi activities of their duplexes with natural sense strand (Sc) were further investigated according to different siRNA interaction regions with Ago2 (Figure S6). For the 5’-terminus caged siRNA duplex (FAS00/Sc), a clear inactivation of siRNA activity was observed, which was identical to that of the negative control siRNA. After UV irradiation (2 min), the silencing activity of FAS00/Sc was restored to the level of that of the positive control siRNA (ASc/Sc), leading to an approximate 2.4-fold decrease. However, duplex FAS06/Sc (bearing a caging group in the seed region), duplex FAS10/Sc (bearing a caging group at the cleavage site), and duplex FAS16/Sc (bearing a caging group in dissociation region) exhibited similar levels of firefly luciferase activity either with or without light irradiation. Further evaluation of phosphorylated siRNAs (FAS06P, FAS10P, and FAS16P) paired with the corresponding natural sense strand also showed that no improvement of their RNAi activities was observable (Figure S6). These results indicated that the increase in siRNA stability with 2’-fluoro-modification had no effect on the photomodulation of phosphate-caged siRNA gene silencing activity. Improving the nuclease resistance of siRNA is another important issue for therapeutic applications of synthetic siRNAs.[21] The native siRNA sequence we used has previous been shown to be highly stable towards enzyme-catalyzed degradation.[15] To establish whether phosphate-caged modification of siRNAs can decrease the nuclease resistance compared with native siRNA, the susceptibility of siRNAs to snake venom phosphodiesterase (SVPD), was first examined (Figure 6). Unmodified ASc/Sc and modified caged siRNA duplexes (AS00/Sc, AS21/Sc, AS22/Sc, AS23/Sc, AS24/Sc) with different numbers of caging groups, were incubated at 37 8C in the presence of SVPD. After 1 h incubation, the hydrolyzed band for the unmodified siRNA duplex was observed, however, the hydrolyzed band for the modified siRNA duplexes was not clearly visible. Thus, modified siRNA duplexes are generally more stable than native siRNA duplexes in the presence of SVPD. In addition, the tolerance of the siRNAs to Ribonuclea-

se A (RNase A) was also examined (Figure S7). After 0.5 h incubation, unmodified siRNA duplex was significantly hydrolyzed, whereas AS23/Sc and AS24/Sc, corresponding to three and four photolabile modifications at the antisense strand, suffered much less degradation within 2 h, suggesting that siRNA duplexes carrying multiple caging groups were significantly more resistant to RNase A compared with the native siRNA duplex. Finally, the stability of siRNAs in PBS buffer containing fetal bovine serum was investigated. Modified siRNA duplexes, similar to the native siRNA (ASc/Sc), suffered only low levels of hydrolysis after 1 h incubation, and the corresponding hydrolyzed bands increased to a similar level when the incubation time was extended to 7 h (Figure S8). This indicated that caging modification did not decrease the stability of siRNA duplexes in the presence of fetal bovine serum. Hydrolysis of the caging group in phosphate-caged siRNAs and molecular dynamics simulation The work described above shows that a single caged phosphate at the seed region and cleavage site is not sufficient to block RNAi activity. Moreover, their failure to block siRNA activity is not due to the chemical or enzymatic instability of modified siRNAs themselves, as evidenced by experiments described above (Figure 6 and Figure S6–8). The groups of Friedman and Monroe previously speculated that the caging group on statistically labeled siRNA may be unstable in cellular environments.[9, 10] In our case, we introduced single caged phosphate group site-specifically in a RNA oligonucleotide and obtained the pure caged antisense or sense RNA strand instead of previous statistically distributed phosphate caged siRNA, which facilitated a further investigation of detailed hydrolysis mechanisms. When the caged antisense RNA was mixed with the control sense strand in standard PBS buffer at 37 8C, the siRNA duplex (AS00/Sc and AS06/Sc) was formed. An identical experiment was also carried out for only these caged antisense stands (AS00 and AS06). Aliquots of the latter RNA solutions were removed at recorded time points (0, 6, 14, and 24 h) and then subjected to HPLC analysis. As shown in Figure 7, no caging group was hydrolyzed for 5’-terminally phosphate-caged siRNA duplex (AS00/Sc) even with long incubation time (24 h). However, hydrolyzed product, which was confirmed to be the corresponding siRNA duplex without the caging group (ASc/Sc), was detected for siRNA duplex AS06/Sc even without light irradiation. The degree of caging group hydrolysis was different, depending on the modified position in the RNA strand. Moreover, caging groups on the internal phosphate were more easily hydrolyzed than caging groups close to the termini of the siRNAs (Figure S9). However, no hydrolyzed product was detected for any of the single-stranded RNAs, even after 24 h incubation under the same conditions, even though they have the same phosphotriester moiety. These results suggest that hydrolysis of the caging group was not dependent on the cellular environment, and that the internally phosphate-caged siRNAs, but not the terminally phosphate-caged siRNA or caged single-stranded RNAs, were more susceptible to hydroly-

Figure 6. Native PAGE of modified siRNA duplexes and control siRNA duplex hydrolyzed by SVPD. The experimental conditions are as described in the Experimental Section.

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Full Paper minally phosphate-caged RNA/ DNA duplexes (AS10/ODN or AS00/ODN) was observed (Figure S10). To evaluate the possibility of Mg2 + -mediated hydrolysis of caged siRNA duplexes, we added different concentrations of ethylenediaminetetraacetic acid (EDTA) to chelate the trace amount of Mg2 + that was possibly present in the buffer (even though no Mg2 + salt was used during the processing or workup of siRNA duplexes). If Mg2 + has an effect on the hydrolysis of internally phosphate-caged siRNAs, its chelation with EDTA will eliminate or greatly inhibit Mg2 + -mediated hydrolysis of the caging group. Our results indicated that the relative extent of hydrolysis of the caging group in caged siRNA duplexes was Figure 7. HPLC analysis of the caged siRNAs at 37 8C with increasing incubation time (0, 6, 14, and 24 h). Caged not affected by the amount of siRNAs: a) AS00, b) AS00/Sc, c) AS06, and d) AS06/Sc. Peaks at 13.2 min correspond to the excess natural sense EDTA added, which suggested strand (Sc), peaks at 15.5 min correspond to the deprotected product (ASc/Sc), and the remaining peak corresponds to the phosphate-caged siRNAs. that Mg2 + did not contribute to the hydrolysis of internally caged phosphate moiety in siRNA duplexes (Figure S11). sis. These results also shed some light on previous reported Chemical modifications of oligonucleotides might induce observations.[4a, 9, 10] In statistically distributed caging of the conformational variations as well as other effects such as an insiRNA phosphate moiety, terminal phosphates were preferenteraction between the group at the modified position and tially caged compared with internal phosphates, which is probneighboring nucleotides.[24] Thus, a structural understanding of ably due to the rapid hydrolysis of internal caging groups in the siRNA duplex. Furthermore, heavily caged siRNA duplexes chemical modifications is helpful to explain the physical and greatly enhanced the inhibition of their RNAi activity. However, biological properties of the modified siRNA duplexes. We full recovery of RNAi was not achieved with light irradiatiotherefore investigated the effect of molecular structure in n.[4a, 9, 10] According to our studies, caging groups in phosphates a water environment on the hydrolysis of the caged moiety in phosphate-caged siRNAs by using molecular dynamics simulashould be easily photoremoved even with multiple site-specific tions. Starting from two initial forms with the caging moiety lolabeling of phosphate moieties (Figure 5). More inhibition and cated either out of the nucleobases or between two nucleoless recovery of siRNA activity for heavily caged siRNAs was bases, the structures of the single-stranded antisense RNA more likely because of the caging of nucleobases instead of (AS10) were simulated for 20.0 ns, and two respective conforthe phosphate group under statistical caging conditions, as mations generated during the last 5.0 ns period of simulation evidenced by the fact that caged nitrogen atoms of nucleobaswere also obtained. The total energy (ETotal) of these two cones are difficult to uncage.[22] Further studies were carried out to establish an underlying formations was 2311.5 and 2523.9 kcal mol 1, respectively. mechanism that could account for the difference between hyAs shown in Figure 8, the caging group in the backbone of the drolysis of the internally caged phosphate of siRNA duplex and caged, single-stranded RNA could rotate freely to maintain of single-stranded RNA. It is clear that the 2’-hydroxyl group in a comparatively stable conformation (ETotal = 2523.9 kcal the RNA nucleotide may mediate hydrolysis of the phosphomol 1), in which p–p stacking interaction between the caging [23] triesters, however, we designed these caged siRNAs with rigroup (o-nitrobenzyl) and neighboring nucleobases was observed. We also analyzed the structures of siRNA duplexes bonucleotides substituted by the corresponding deoxynucleoAS00/Sc (caging modification at 5’-end) and AS10/Sc (caging tides, which prevents direct hydrolysis by the attack of 2’-hymodification at position 10) starting from two similar initial droxyl group from the same nucleotide. To exclude the effect forms as single-stranded AS10. Under the same molecular dyof possible helix conformations (A form or B form) with the namics stimulation, only one conformation was obtained for complementary strand, we replaced the complementary RNA each siRNA duplex with ETotal of 5205.5 kcal mol 1 (AS00/Sc) strand with oligodeoxynucleotide (ODN). However, no inhibition of the caging group hydrolysis for either internally or terand 4595.9 kcal mol 1 (AS10/Sc), respectively. Similar to Chem. Eur. J. 2014, 20, 1 – 10

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Full Paper Conclusion A complete set of four novel phosphate-caged nucleoside phosphoramidites were synthesized and incorporated site-specifically along a siRNA sequence. By scanning each caged phosphate position along the siRNA strands, two regions of a siRNA sequence have been identified as being more sensitive to the introduced caging group based on a systematic evaluation of the binary correlation between the modified positions in siRNAs and inhibition/light-restoration of siRNA activity. Caging phosphate at the 5’-end of the antisense strand and caging phosphate in the seed region has an effect on photomodulation of RNAi activity to different extents. In addition, simultaneous introduction of 3–4 caging modifications at certain identified key phosphate positions rendered a substantial decrease of caged siRNA activity until UV exposure fully restored their RNAi activity. Detailed analysis indicated that the inefficient photomodulation of their RNAi activity was not due to less effective blocking of siRNA loading and function. Instead, the caging group was actually hydrolyzed from the phosphate backbone of caged RNAs. Further experiments confirmed that 2’-hydroxyl- or Mg2 + -mediated hydrolysis were not responsible for hydrolysis of the caging group, but that duplex formation of internally phosphate-caged siRNA led to its hydrolysis. By molecular dynamics stimulations, it was revealed that the caging group on the phosphate of single-stranded RNA was found to hide between RNA nucleobases through p-p interaction with a nearby nucleobase due to its flexible rotation, which renders the fragmentation of a pentavalent intermediate difficult, even with attack by water. Upon hybridization of caged single-stranded RNA with its complementary oligonucleotide, a duplex is formed with a structure that is more rigid. The caging group was forced to protrude out of the siRNA duplex, which facilitated fragmentation of the pentavalent intermediate upon nucleophilic attack by water molecules (not OH ), as indicated by readily hydrolysis of caged internal phosphate of the caged siRNA duplex. This observation indicated a new duplex-formation-induced hydrolysis of the phosphotriester, which may provide positive guidance for the design of caged siRNAs and for the chemical modification of siRNAs for use as scientific tools and nucleic acid drugs.

Figure 8. Views of the calculated average RNA structures of caged regions with minimum total energy. Caging group is marked with a shaded area. a) Two conformations of AS10, b) AS00/Sc, and c) AS10/Sc. Starting models of studied AS10, AS00/Sc, and AS10/Sc were built from the A canonical structures by using Discovery Studio 2.5 software. Molecular dynamics simulations were carried out by using the SANDER module of AMBER 11.

single-stranded AS10, p–p stacking interaction of the nitrobenzyl moiety with the terminal nucleobase was found in the structure of AS00/Sc. However, for AS10/Sc, the caging group protruded out of the siRNA duplex and formed a flexible branch. This conformation rendered it accessible for the nucleophilic attack on the phosphotriester by water molecules (not OH , due to their extremely low concentration under neutral buffer conditions) to form a pentavalent intermediate, leading to facile fragmentation and elimination of the caging group instead of cleavage of the RNA phosphate backbone. In contrast, the strong p–p stacking interaction between the caging group and the neighboring nucleobases makes it difficult to fragment even with the formation of the pentavalent intermediate, thus enhancing the stability of the caging groups in single-stranded RNA or terminally caged siRNA duplex. HPLC studies and molecular dynamics simulations provided a solid theoretical basis for the observations that the caging group is more easily hydrolyzed as internally phosphate-caged siRNA duplexes rather than 5’-end phosphate-caged siRNA duplexes and phosphate-caged single-stranded RNA. Furthermore the study clarifies why caging group hydrolysis is not sensitive to the cellular environment, but is dependent on the conformation of the caging group in siRNA duplexes. The data also provides insight into some previously reported phenomena, including preferential caging at terminal phosphates of siRNA duplexes, instability of caged siRNAs in gene silencing activity, and incomplete recovery of heavily caged siRNA, which is due to the difficult photocleavage of multiple caging groups on nucleobases instead of on the phosphate backbone.[4a, 9, 10] &

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Experimental Section General procedures All the reagents for organic synthesis were analytically pure and used as received. Solvents for synthesis and purification were distilled over CaH2. Silica gel column chromatography was carried out on Merck silica gel C-300. NMR spectra were recorded at 400 MHz (1H) with a Bruker Avance III 400 spectrometer. 1H and 31P NMR spectra were referenced using internal standard (CH3)4Si and external standard 85 % H3PO4, respectively. All single-stranded oligonucleotides were custom-synthesized with 1 mmole standard CPG with an ABI 394 DNA/RNA synthesizer. Deprotected oligonucleotides were purified with a Waters HPLC system by using Waters reverse-phase adsorption of mixed type anion exchange solid-phase extraction column (5 mm bead, 9.6 mm  150 mm), eluting at

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Full Paper 1.0 mL min 1 with a gradient of acetonitrile/50 mm triethylammonium bicarbonate buffer (TEAB, pH 8.5). Buffer A (0.05 m TEAB), buffer B (acetonitrile); B, 0–30 % in 20 min, 30–100 % in 25 min, 100 % in 30 min, 100–0 % in 33 min, 0 % in 35 min; running temperature, 60 8C. Mass spectra were recorded with a Waters Xevo G2 Q-TOF by ESI-MS under negative mode. Analytical PAGE was carried out with a BioRad Mini-protein tetra system and imaged by using ChemiDoc XRS. Photoirradiation in vitro experiments with oligonucleotide samples and photoirradiation of HEK293A cells were conducted with a UV-LED lamp (365 nm, 7 mW cm 2) and luciferase activity experiments were conducted with a Fluoroskan Ascent FL luminometer (Thermo Scientific). Details of the experimental materials used are listed in the Supporting Information.

[4]

[5]

Synthesis of deoxynucleosid-3’-yl-O-[1-(2-nitrophenyl)ethyl]N,N’-diisopropyl phosphoramidites (dA0, dG0, dC0, dT0)

[6]

A solution of N,N,N’,N’-tetraisopropyl-[1-(2-nitrophenyl)ethyl] phosphorodiamidite (P0, 0.93 g, 2.1 mmol) and tetrazole (140 mg, 2 mmol) in anhydrous CH2Cl2 (3 mL) was added slowly to a solution of four corresponding DMT protected nucleosides (A, C, G, T, 2 mmol), respectively, in anhydrous CH2Cl2 (2 mL), while stirring at RT for 4 h. The desired products were purified by silica gel column chromatography with 50–60 % yield as white foam solids. The compounds had more than 95 % purity judging from 31P NMR spectra and were characterized by 1H, 13C NMR, and ESI-MS analyses. Details of the characterization of these compounds are provided in the Supporting Information.

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[9] [10] [11] [12] [13]

Acknowledgements [14]

We thank Profs. Quan Du and Suwei Dong (Peking University) for reading this manuscript and for their helpful suggestions. The work was funded by the National Natural Science Foundation of China (Grant No. 21302008 and Grant No. 21372018), the 973 Program (Grant No. 2012CB720600), and the Program for New Century Excellent Talents in University (Grant No. NCET-10-0203). Dr Li Wu was also supported by the Chinese Postdoctoral Science Foundation (Grant No. 2012M510290).

[15] [16] [17] [18] [19] [20]

Keywords: caged siRNA · nucleosides · photoactivation · regioselectivity · RNA hydrolysis

[21] [22]

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Received: May 7, 2014 Published online on && &&, 0000

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FULL PAPER & Gene Regulation L. Wu, F. Pei, J. Zhang, J. Wu, M. Feng, Y. Wang, H. Jin, L. Zhang, X. Tang* && – && Synthesis of Site-Specifically Phosphate-Caged siRNAs and Evaluation of Their RNAi Activity and Stability

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Custom-made cages: A complete set of new photolabile nucleoside phosphoramidites were synthesized and incorporated site-specifically into any phosphate position (see scheme). These phosphate-caged siRNAs were then systematically evaluated for their photo-

modulation of RNAi activity, and key phosphate positions were identified. In addition, duplex-forming-induced hydrolysis of the caging group in internally phosphate-caged siRNA was also suggested as an explanation for previous observations.

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