Bioconjugates of siRNAs
RNA interference is an evolutionary conserved mechanism of specific gene silencing induced by double stranded RNA homologous to the target mRNA. Small interfering RNAs (siRNAs) are widely used for the control of gene expression in molecular biology and experimental pharmacology. Currently, siRNAs are successfully used for the validation of potent drug targets for anti-cancer therapy. However, application of siRNAs as therapeutics is limited by their sensitivity to ribonucleases, poor cellular uptake and rapid size-mediated renal clearance. These challenges must be overcome to develop a successful siRNA-based drug. Many of these limitations could be resolved with the use of chemical modifications improving the siRNA properties.
This review examines recent data regarding principals of the design of siRNA for the silencing of therapeutically relevant genes. A particular focus will be made on chemical modifications and their impact on siRNA potency, nuclease resistance and duration of the silencing effect. The types of chemical modifications, their location in siRNA structure influence siRNA properties in different modes: modulation of the interaction with RNAi proteins, the thermal stability and thermoasymmetry of the duplex and the sensitivity to the degradation by ribonucleases.
Special attention will be paid to the design of siRNA for the silencing of thermodynamically unfavorable targets: mutant and chimerical genes. In this case the utilization of computer algorithms for the selection of active siRNA cannot be applied. Modification of siRNA structure aimed at the correction of the thermoasymmetry by incorporation of nucleotide substitutions, blocking incorporation of the sense strand in the RISC complex by truncation of one overhang or inactivation of the sense strand can be successfully used. Mismatches in the central part of the duplex can facilitate the cleavage and dissociation of the “passenger” (sense) strand, whereas selective chemical modification protects the non-perfect duplex from accelerated degradation.
Peptides, steroids and other hydrophobic lipid groups can be attached to siRNA, extending the siRNA circulation time and enhancing direct cellular uptake. The potential of bioconjugation of siRNA with different biogenic molecules in altering the bioavailability and distribution of siRNAs following
2. Mechanism of RNAi
RNAi phenomenon was found during transfection of dsRNAs in
The selection of the "guide" strand is a key step determining the efficacy of RNAi induced by synthetic siRNAs. At the first step of RISC* assembly the intermediate complex RLC (RISC Loading Complex) consisting of Dicer, siRNA and R2D2 (
Dicer contains dsRNA-binding domain and PAZ-domain (PIWI/Argonaute/Zwille), that has an affinity to 3’-overhangs of siRNA [21, 22]. It was detected that R2D2 preferably binds with more thermodynamic stable flank of the siRNA duplex, whereas Dicer interacts with less stable siRNA flank [20, 23, 24, 25]. The orientation of siRNA relative to the protein complex Dicer-R2D2 determines the positioning of siRNA in the complex with Ago2 (a catalytic part of RISC*) (fig. 2 B). Ago2 is a protein of Argonaute family, containing PAZ and PIWI domains. The structure of PIWI domain similar to that of RNase H determines endoribonuclease activity of PIWI [26, 27]. Ago2 cleaves the one of the siRNA strand (“passenger” strand), another strand (“guide” strand) remains into the RISC* and guides the target RNA recognition and cleavage (fig. 2 C). The structure of the complex containing siRNA and Ago2 effects the selection of the strand (sense or antisense) that incorporates into RISC*, hence, determines the efficacy of mRNA-target cleavage. Evidently, the antisense strand that is homologous to the sequence of mRNA-target has to be “guide” strand in order to cleave mRNA. Ago2 replaces the Dicer-R2D2 dimer (fig. 2 B), interacting with Dicer via PAZ domain whereas the phosphate group at the 5’-end of the strand from the Dicer side (“guide” strand) interacts with PIWI domain that contains Mg2+ ion and basic amino acids.
The nucleotides located at 2 - 6 positions of the “guide” strand stabilizes the interaction between siRNA and Ago2 [29, 30, 31]. The presence of the phosphate at the 5’-end of the “guide” strand is essential for RISC* assembly [32, 33]. This orientation of siRNA with respect to catalytic PIWI-domain provides the cleavage of the complementary strand (“passenger” strand) between 9th and 10th nucleotides that facilitates the strand dissociation (fig. 2 C) [34, 35]. Ago2 and the “guide” strand are the main components of RISC* , however, other proteins could also interact with RISC* . Thus, the difference between thermodynamic stability of the duplex termini (thermodynamic asymmetry) determines the orientation of Dicer-R2D2 dimer and as a result, the structure of Ago2-siRNA complex. Therefore, thermodynamic properties of siRNA play a key role in the strand selection [20, 23, 24, 25] providing the preferential selection of the strand with low thermodynamic stability at the 5’-end of the duplex as the “guide” strand [37, 38].
It was shown, that the perfect match between 2 - 12 nt of the “guide” strand (corresponding to one turn of dsRNA helix) and the target mRNA is essential for effective recognition and binding of RISC* with mRNA target . The mechanism of RNA-target cleavage (occurs between 10th and 11th nt relative to the 5’-end of the “guide” strand ) and the following dissociation are similar to that of the “passenger” strand (described above). Cleaved target RNA and the “passenger” strand are degraded by ribonucleases after dissociation, whereas RISC* becomes available for acting in catalytic mode [7, 9, 17, 41]. Unlike antisense oligodeoxyribonucleotides (ODN), whose efficacy of action is determined by the efficacy of ODN hybridization with mRNA-target (fig. 3) , the formation of the duplex between the "guide" strand of siRNA and mRNA- target occurs due to the helicase activity of RISC* and virtually independent from the hybridization properties of the oligoribonucleotide . Silencing of gene expression by siRNAs is observed at the much lower ON concentrations than in the case of antisense ODN (IC50 for siRNAs is 100 – 1000 times lower, than IC50 for antisense ODNs) [43, 44].
Another mechanism of RISC activation - “bypass” route - employs the dissociation of the siRNA strands without preliminary cleavage of the sense strand by Ago2 . This type of RISC activation is inherent in miRNAs, containing unpaired bases in the central part, critical for Ago2 endoribonuclease activity (fig. 4). Moreover, the “bypass” rout was observed for siRNAs with chemically modified nucleotides surrounding the cleavage site (between 9th and 10th nt of the “passenger” strand ) impeding Ago2 action [34, 35, 46]. Silencing of gene expression proceeds via the arrest of mRNA translation, since RISC* binds to mRNA-target together with translation suppressor proteins (for example, helicase RCK/p54 from DEAD box family) . Complex of mRNA and proteins is deposited in “P-bodies” (processing bodies - distinct foci in the cytoplasm involved in mRNA turnover) . In “P-bodies” mRNA decapping followed by ribonuclease cleavage occurs. In the other cases, mRNA complexed with proteins can be deposited in “P-bodies” for a long time without degradation (fig. 4). Later, mRNAs can escape from “P-bodies” and be involved in translation machinery or can be degraded in a described above manner .
The “bypass” mechanism of RISC activation is less efficient than the “classical” one, since in this case RISC* does not act in a catalytic mode. The efficiency of RISC* assembly also is limited by the low rate of ATP-dependent dissociation of intact siRNA strands [35, 33, 39].
Thermodynamic stability siRNA duplex is an additional parameter determining the efficiency of RISC* assembly, hence, the efficiency of RNAi. Earlier developed algorithms for the selection of active siRNA sequences suggest selection of mRNA targets with approximately 50% GC-content. In the later studies, the percentage of G+C nucleotides in the structure of effective siRNAs varied from 30 to 50% [49, 50]. It was shown that low thermodynamic stability of the central part of the duplex (from 9th to 14th nt counting from 5’-end of the antisense strand) arising from AU-rich sequences or the presence of mismatches is a hallmark of active siRNAs . The average difference between Gibbs energy of central parts of active and inactive siRNAs was found to be about 1.6 kcal/mol. Unlike thermodynamic asymmetry, unstable center of duplex is not a sufficient criteria for selection of active siRNA .
Structural features of mRNA-target also effects the RNAi efficacy [51, 52, 53, 54]. The availability of the mRNA sequence for binding with siRNA (the absence of the hairpins in the secondary structure or overlapping with the binding regions of regulatory factors) as well as in the case of the ribozymes and the antisense ODN  can influence the silencing efficacy. In order to prove this, siRNAs targeted to mRNA sequences with different binding availability were synthesized . It was shown that siRNAs targeted to the region of the initiation of translation or the 3’-end of mRNA-target were inactive. Whereas siRNAs targeted to the regions forming the hairpins displayed low and average silencing activity. As expected high-performance siRNAs were those targeted to unstructured regions of mRNA [51, 52, 54].
Thus, the analysis of the secondary structure of mRNA-target and thermodynamic properties of siRNA duplex play important role in the selection of active inhibitors of gene expression.
3. Chemical modifications: The influence on RNAi efficacy
Currently, the development of RNAi-based drugs is an important aim in pharmacology. The optimization of siRNAs structure is required for biomedical applications; chemical modifications could be successfully used for this purpose. Different types of chemical modifications were widely used in antisense ODNs before the discovery of RNAi . The data collected in these experiments can be applied for optimization of the properties of siRNAs. There are two types of factors affecting both antisense ODNs and siRNAs efficacy: systemic factors that act on the level of the organism and intracellular factors defining the activity of the therapeutics in the cells. The first type of factors includes: i) fast (approx. 5 min) siRNA elimination from organism resulted from its hydrophobicity and size of the molecule (approx. 14 kDa) [56, 57], ii) high nuclease sensitivity of siRNA [58, 59, 60, 61, 62] and iii) inefficient or/and non-specific delivery into target cells . Intracellular factors are related to RISC assembly and the efficiency of binding to and cleaving the target.
3.1. Chemical modifications of the nucleotides
3.1.1. Modifications of ribose (furanose) ring
All ribose modifications can be divided in two main groups: the modifications related to the replacement of hydrogen atoms in furanose by different groups and structural modifications of the furanose cycle.
220.127.116.11. Substituents in furanose
The 2’-OH group of the ribose is the main target for modification, since this group is involved in the phosphodiester bonds cleavage by endoribonucleases via trans-etherification mechanism . Thus, the modification of 2’-OH defends siRNA against ribonucleases . The size of the substituent is an important characteristic determining its tolerance by RNAi machinery. The small groups (2’-O-methyl (2’-O-Me), 2’-fluoro (2’-F) etc.) (fig. 5 А) virtually do not disturb the conformation of siRNA duplex and are better tolerated than bulky groups: 2’-O-methoxyethyl (2’-O-MOE), 2’-O-allyl (2’-O-allyl) etc. The replacement of 2’-OH by electrophilic groups also stabilizes C3’-endo conformation of the ribose (fig. 6) which corresponds to A-helix geometry of the duplex obligatory for effective RNAi . This sugar conformation arrange for the axial location of the substituents reducing total energy of the system and increasing the affinity to the complementary RNA target. As a result the duplex melting temperature (ΔTm) increases approximately 1 0С per modification .
2’-O-methyl modification is one of the widely used 2’- modifications for the enhancement of nuclease resistance of siRNA [59, 62] and impeding the induction of the interferon response in eukaryotic cells [64, 66, 67, 68, 69, 70]. Obviously, the number and location of the modified nucleotides in the duplex are crucial for the silencing. The increase of the number of modifications was shown to decrease the silencing activity of siRNAs; totally modified siRNAs frequently had no activity [58, 59, 71]. However, in a number of experiments the activity of siRNA with the totally modified sense strand was compatible with that of unmodified analog[72, 73]. This can be related to functional unequivalence of siRNA strands [74, 75]. The introduction of 2 - 4 2’-О-Me modifications in the both strands of siRNA is well tolerated. Moreover, in the case of selective modification of the nuclease-sensitive sites within siRNA the increase of the duplex nuclease resistance and prolonged silencing were detected .
On the contrary, the replacement of ribonucleotides of siRNAs with deoxyribonucleotides characterized by C2’-
Introduction of the bulky groups in the 2’-position of the furanose causes the conformational disturbance of the siRNA duplexes resulting in the decrease of siRNA silencing activity . However, the modification of duplex termini and overhangs with 2’-О-methoxyethyl (2’-O-MOE) or 2’-O-allyl groups (fig. 5 A) was tolerant [46, 83, 84]. The random replacement of 2’-OH in the both strands of the siRNA (up to 70 %) by 2,4-dinitrophenyl ethers (2’-O-DNP) (fig. 5 A) causes the increase of the silencing activity. Since the thermodynamic stability of the modified duplex was comparable with that of unmodified duplex  (that indicates the similar hybridization properties ), the observed effect may be related to the increase of the nuclease resistance and cellular uptake of the modified siRNA [85, 86, 87]. In order to increase the nuclease resistance of the siRNA duplexes, other chemical modifications - 2’-aminoethoxymethyl (2’-AEM), 2’-aminopropoxymethyl (2’-APM), 2’-aminoethyl (2’-EA), 2’-cyanoethyl (2’-СE), 2’-guanidinoethyl (2’-GE) etc.- may be used (fig. 5 А). It was shown that totally modified siRNAs were inactive, whereas the silencing activity of the partially modified siRNAs depends on the location of the modifications in the duplex structure .
Introduction of the modifications at the 4’-position of the furanose results in the slight increase of the RNA-duplex termostability (ΔTm 1 0С per modification) [89, 90]. Usually the 4’-thio (4’-S) ribonucleosides (fig. 5 А) are used for modification of siRNA, especially, at the termini [91, 92, 93]. The replacement of the several ribonucleotides at the 5’-end of the antisense strand  or four ribonucleotides at the sense strand termini and at the 3’-end of the antisense strand  by 4’-S- analogs only slightly decrease the silencing activity of siRNAs, whereas the modification of the central part of the duplex significant inhibits RNAi [91, 92]. The introduction of the 4’-S-modifications in siRNA structure increases nuclease resistance of the duplex and improves the pharmacokinetics of siRNA. Unfortunately, this modification simultaneously increases the binding of siRNAs with blood serum components and increases their cytotoxicity .
18.104.22.168. Structural modifications of the furanose ring
This type of modifications includes bicyclic derivatives of the nucleotides (LNA, ENA, CLNA, CENA, AENA et al.), acyclic nucleotides (UNA, PNA) or nucleotides containing pyranose ring (ANA, HNA) instead of ribose (fig. 5 B). LNA and ENA (2'-О,4'-С-methylene- and 2'-О,4'-С-ethylene bicyclic nucleotide analogs are the frequently used bicyclic derivatives which provide the significant increase of the duplex thermostability (ΔTm up to 10 0С per modification) [94, 95]. The ribose in LNA and ENA is fixed in C3’-
Experimental data on the activity of LNA- and ENA-modified siRNAs are contradictory. In some cases modifications of the 5’- and/or 3’-ends of the both siRNA strands were well tolerant [65, 96, 97, 98], whereas in the other studies ENA-analogs at the 3’-end of the sense strand of siRNAs reduced or abolished the silencing activity . The difference in activity may be connected with the difference in the thermodynamic asymmetry of the modified siRNA [37, 38]. The replacement of the several ribonucleotides with LNA in the antisense strand was shown to be well tolerant [71, 96], whereas extensive modification was tolerant only in the case of sisiRNA with segmented sense strand  (see section 1.3.5).
The thermodynamic stability and nuclease resistance of the siRNA could be increased by introduction of HNA and ANA nucleotides with pyranose ring (hexitol nucleic acid and altritol nucleic acid, respectively (fig. 5 B)) instead of ribose [100, 101]. The modified duplex contaning these analogs adopts the A-helix geometry , however, the location of the ANA- or HNA-analogs in siRNA structure is critical for silencing activity. Remarkably, the introduction of the ANA- or HNA-analogs at the 3’-termini of one or both strands maintain or increase the silencing activity [102, 103]. The presence of ANA-analogs at the 5’-end of the antisense strand substantially decreased the siRNA activity. It was suggested that modifications at the 5’-end of the antisense strand impede the action of cellular kinases due to steric obstacles . The decrease of the phosphorylation efficacy of the 5’-terminal nucleotide of the antisense strand is critical for efficient interaction of siRNA with Ago2 PIWI domain followed by mRNA-target cleavage [32, 33]. siRNAs with HNA or ANA-analogs of nucleotides in the central part of the sense strand display the effective gene silencing, whereas, similar modifications of the antisense strand substantialy decreased the siRNA activity [88, 103].
In contrast to siRNA duplex stabilizing modifications, the presence of acyclic nucleotide analogs (UNA) (fig. 5 B) in the siRNA increase the conformational flexibility of the duplex and decrease its thermodynamic stability (ΔTm -5 to-8 °С per modification). The location of the UNA-analogs in siRNA was shown to play a key role, since one modification can result in the significant reduction or augmentation of the silencing activity of siRNA [99, 104]. It was suggested that the replacement of ribonucleotides at the 3’-end of the sense strand increases the duplex thermodynamic asymmetry and acquires antisense strand incorporation into RISC* [104, 105]. It was found experimentally that the introduction of 1 - 3 UNA nucleotides at the 3’-end of the sense strand, including the 3’-overhangs, increase to some extent the siRNA activity [106, 107]. Quite the opposite, the modification of the first and the second positions of the “guide” strand abolishes the 5’-end phosphorylation and reduce siRNA silencing activity.
Peptide nucleic acids analogs (PNA) containing N-(2-aminoethyl)-glycin polyamide backbone could be used for modification of siRNA. It was shown that in the antiparallel PNA/RNA duplex RNA adopts the right-handed helix characterized by anticonformation at the N-glycosidic bond and C3’-
Thus, virtually all ribose modifications provide the increase of the nuclease resistance of siRNA. However, the optimization of their number and location in the duplex structure (depending on the type of modification) is required to make them tolerable in the process of RNAi.
3.1.2. Backbone modifications
This type of modifications includes the replacement of phosphate group (PO) with phosphorothioate (PS) or boranophosphonate (PB) groups, the replacement of the 3’,5’-phosphodiester bond with 2',5'-bond or the amide bond instead of the ester bond (fig. 5 C). All of these modifications increase the nuclease resistance of siRNA, however their impact on the efficacy of RNAi is varied . PS-modification is known to reduce the melting temperature of the duplex and oligoribonucleotide binding affinity . However, the experimental data related to the activity of PS-modified siRNAs are discrepant. The comparable silencing activity of the PS-modified siRNAs and their unmodified analogs was shown in the studies [83,111], whereas in the other studies the silencing activity of the PS-modified siRNAs was lower, than that of their unmodified analogs [58, 73, 112]. The modification of the central part of the duplex decreases siRNA activity . Remarkably, siRNAs with blunt 3’-ends containing totally PS-modified one or both strands were active . However, high binding affinity of the phosphorothioate analogs to serum albumin, IgG, IgM, lactoferrin and the cellular membrane receptors is disadvantageous . It results in the development of toxic effects both
Thus, the most promising modification of the siRNA backbone is the boranophosphonate modification, since it provides the increase of the nuclease resistance of the duplex and its silencing activity and is not accompanied by the development of toxic effects. However, the price of PB-analogs limits their usage.
3.1.3. The chemical modifications of the nucleobases
In contrast to the other types of modifications, the modifications of heterocyclic bases (nucleobases) have no influence on the nuclease resistance of RNAi. Earlier it was found that the presence of modified nucleobases in the antisense ODN altered their hybridization properties . It is known that the replacement of the uridine by 5-bromo- or 5-iodouridine (fig. 5 D) improves their ability to interact with adenine owing to the increase of the acidity of the imine and results in the stabilization of the base pair . The replacement of the adenine by 2,6-diaminopurine results in the formation of the additional H-bond, hence, in the stabilization of the nucleotide base pair . At the start, it was suggested that the introduction of these analogs in the antisense strand of siRNA will enhances its binding affinity to mRNA and increases the RNAi efficacy. Unexpectedly, the decrease of silencing activity of modified siRNA in comparison with unmodified ones was observed. This effect is possibly related with the decrease of the dissociation rate of siRNA duplex on the step of RISC* formation . The replacement of uridine by 2-thiouridine (s2U) or pseudouridine (ψ) (fig. 5 D) provides the increase of the duplex thermodynamic stability due to the stabilization of the ribose in the C3’-
3.2. Chemical modification of siRNA termini
This type of modifications includes the modification of the nucleotides at the 3’-overhangs and the 5’-ends of the duplex. These modifications could be used for the solution of different problems: i) to increase the nuclease resistance of siRNA towards exoribonucleases (inverted 3’-3’ or 5’-5’ deoxyriboses [61, 121](fig. 8), dideoxycytosine , ENA-analog of the thymidine etc.), ii) to facilitate asymmetric RISC* assembly (the replacement of the 5’-OH by 5’-OMe group in the terminal nucleoside ) and ii) to provide the efficient accumulation of siRNA in cells (attachment of lipophilic molecules [57, 124, 125], folic acid , peptides [14, 127, 128], aptamers ). The fluorescent residues  and biotin  are widely used as a termini modification for siRNA detection.
The type of the applicable terminal modification has to be selected experimentally. In many cases the modification of the 5’- and 3’-ends of the sense (“passenger”) strand is well tolerated [59, 131, 111, 130], however the data on modification of the antisense (“guide”) strand are discrepant. It is known that the phosphorylation of 5’-OH groups of the antisense strands of synthetic siRNAs by the kinases [33, 122] is essential for interaction with PIWI-domain of Ago2 and for correct selection of the “guide” strand [29, 30, 31]. Therefore the replacement of the 5’-OH group by the 5’-OMe in the sense strand blocks its phosphorylation and the incorporation into RISC* as a “guide” strand . On the other hand, the same modification of the antisense strand resulted in the decrease of siRNA silencing activity [59, 131, 122]. However, the attachment of the fluorescein residue to the 5’-phosphate of the antisense strand via hexamethylene linker is well tolerated , suggesting that this modification does not prevent the interactions of the 5’-terminal phosphate of siRNA with PIWI-domain. The 3’-end of the antisense strand recognized by PAZ-domains of Dicer and Ago2  is less sensitive to modification [14, 33, 59]. It was shown that the attachment of puromycin or biotin to the 3’-end of the antisense strand  or the replacement of the 3’-terminal ribonucleotide by ddC, or the attachment of the aminopropyl linker via phosphodiester bond  virtually does not change the silencing activity. However, the introduction of the 2-hydroxyethylphosphate, ENA-analog of thymidine  or fluorescent dyes  at the 3’-end of the antisense strand abolished the silencing. The replacement of the terminal nucleotides of siRNA in both strands by 5’-5’- or 3’-3’-dioxyribose (fig. 8) results in the increase of the nuclease resistance and does not reduce the silencing activity [59, 61].
Anionic siRNA cannot effectively pass through the electrostatic and hydrophobic barriers of the cellular membrane to enter the cytoplasm and to induce RNAi. Conjugation of siRNAs with the lipophilic molecules (cholesterol, derivatives of the oleic, lithocholic and lauric acids), peptides, antibodies, aptamers and other compounds is an effective tool to overcome this problem [132, 133]. The cholesterol was suggested as the first candidate for conjugation, since the natural mechanisms of cholesterol transport exist in mammalian organisms. Apolipoprotein B (ApoB) expressed in the liver and intestine cells is involved in the assembly and secretion of the lipid-protein particles known as very-low-density lipoproteins (VLDL) and low-density lipoproteins (LDL) and in the transport and metabolism of the cholesterol. Since ApoB located on the cellular surface is specific to LDL-receptors, responsible for the delivery of the lipoproteins into cell [132, 134], it was suggested that LDL-receptors could deliver the cholesterol-conjugated siRNAs inside the cells via receptor-mediated endocytosis. It was proved experimentally that LDL-receptors bind with the LDL-particles preliminary associated with cholesterol-conjugated siRNA or the conjugate of siRNA and the oleic or lithocholic acids, however the mechanism of their penetration into cell is not well-defined  (Table 1). Moreover, the involvement of the transmembrane protein SID-1 in the transport of the lipophilic conjugates was also shown . It was suggested that SID-1 facilitates the dsRNA penetration into cells forming the channels for diffusion or mediating the interactions with other proteins [125, 135]. The successful use of the cholesterol and the derivatives of lithocholic, oleic and lauric acids for siRNA modification was demonstrated
The conjugation of siRNAs with the peptides could also improve their cellular accumulation (Table 1). 11 amino acid cationic peptide derived from cell-permeable Tat protein and responsible for its nuclear localization was used in a number of studies [14, 127]. It was found that the attachment of this peptide (TAT-peptide) with additional cysteine to the 3’-end of the antisense strand of siRNA results in its effective accumulation in HeLa cells and effective silencing of the target genes
The addition of the cell penetrating peptides (transportan and penetrantin) to the 5’-end of the sense strand of siRNA improves the penetration ability and the silencing activity of anti-
Another approach to the enhancement of siRNA cellular accumulation is the delivery of siRNA conjugated with antibodies or aptamers . The aptamers are structured synthetic nucleic acids with size less than 15 kDa (Table 1), they can be chemically modified for the defense from the nucleases . The penetration of these conjugates occurs via specific interactions with receptors on the surface of the target cells, providing the effective cellular accumulation of siRNAs . The conjugates of anti-
The attachment of the folic acid to siRNA is a promising approach to increasing cellular uptake of siRNA. These conjugates can penetrate into virtually all human cancer cells, since expression of the expression of the folate receptors on the surface of the cancer cells is substantially higher than in normal cells . The efficient accumulation of fluorescein-labeled siRNA with folate attached to the 5’- or 3’-end of the sense strand via disulfide bond was observed in solid tumor in mice  (Table 1). In the other studies [144, 145]the delivery of siRNA into cells was performed with folate-containing structures. These experiments (
The introduction of biomolecules in siRNA is one of the promising approaches of non-viral delivery. In contrast to other non-viral methods (cationic lipids and polymers, high-pressure injections) the advantages of conjugation include the cell-specificity and the absence of toxic effect [63, 146, 147, 148, 149]. The employment of cholesterol- and folate-contaning siRNAs
3.3. Combination of chemical modifications
The application of different modifications simultaneously is a widely-used approach for the optimization of siRNA properties. Two main strategies are considered in order to increase nuclease resistance of siRNA. The first one is based on the extensive modification of siRNA. In this case optimal design of the duplex (i.e. selection of modifications) could be created experimentally by the analysis of the data on nuclease resistance and biological activity. For example, different types of modifications were used in totally modified siRNA, displaying high activity in mice infected with Hepatitis B virus. The sense strand of this siRNA contains deoxyribonucleotides instead of the purine ribonucleotides, 2’-F-modifications and 5’-5’- and 3’-3’-terminal deoxyribonucleotides instead of the pyrimidine bases; the antisense strand contains alternating 2’-F / 2’-O-Me groups; one PO-bond between 3’-terminal nucleotides was replaced by PS-bond  (Table 2). Other examples of siRNA containing different combinations of chemical modifications are presented in the table 2. However, the extensive modification of siRNA often is not well-tolerated by RNAi machinery and rather often leads to the reduction or blocking of the silencing activity [40, 59, 99]. Therefore, minimization of the number of modifications in siRNA structure is required. It was shown, that duplexes with limited number of LNA-modifications could be active and as resistant to nucleases as the totally modified 2’-O-Me / 2’-F-siRNAs, which were inactive [40, 59, 99, 99] (Table 2). Since siRNAs are degraded mainly by endoribonucleases , it was suggested that the increase of the thermostability will increase the nuclease resistance . On the other hand, it is known that the pattern of siRNA degradation in the presence of serum is similar to the cleavage by RNase A . Therefore, the selective modification of the nuclease sensitive sites of siRNAs, mapped in the presence of serum, represents an alternative approach to the extensive modification of siRNA. This rational approach of siRNA modification allows to minimize the number of modified nucleotides in the duplex and as a consequence, to maintain its silencing activity . The optimization of siRNA design also includes the defense of the 3’-terminal nucleotides against exonuclease cleavage (see section 1.2.2).
The combinations of chemical modifications are used for optimization of other siRNA properties. In order to achieve
The increase of the RNAi efficacy was also observed after stabilization of the 3’-end of the antisense strand of siRNA by s2U or ψ analogs and destabilization of the 5’-end by introduction of D at the 3’-end of the sense strand . It should be noted that the decrease of the thermostability of the duplex at the 3’-end of the sense strand is more preferable in comparison with the stabilization of the duplex at the 5’-end of the sense strand if the duplex stabilization is not needed .
Thus, in order to optimize siRNA design by chemical modifications the following principles should be considered: modifications should provide 1) the RNA-like A-helix geometry of the duplex; 2) the access of the terminal 5’-OH group of the antisense strand for phosphorylation; 3) low thermostability of the 5’-end of the antisense strand, hence, the modification has to increase the favorable thermoasymmetry or has no effect on the duplex thermostability.
The information on the influence of chemical modifications on siRNA properties is summarized in the table 3.
4. The impact of the siRNA structure on the efficiency of RNAi
The investigation of the structures of natural siRNAs formed by dsRNA processing with Dicer and endogenous miRNA revealed the common siRNA structures. "Classical" small interfering RNA resembles duplexes 18 - 23 bp in length [18, 22, 156, 157] with 2 nt overhangs at 3'-ends [40, 45] (fig. 9).
However, it was clearly demonstrated, that the silencing activity of structurally similar duplexes with different sequences varies significantly. As it was mentioned above thermodynamic asymmetry of the duplex and to a lesser extend the structure of mRNA target determines the siRNA activity [44, 45, 57, 58, 60]. Possibility to optimize the duplex structure was demonstrated in a number of publications. Introduction of nucleotide substitutions in siRNA, resulting in a formation of non-canonical base pairs or mismatches [38, 158, 159] is a perspective approach for the optimization of the thermodynamic properties of the duplex. The other approach utilize the inactivation of the sense strand of siRNA (for example, segmentation) to guarantee the incorporation of the antisense strand into activated RISC* independent from the thermodynamic properties of the duplex . The silencing activity of siRNA can be increased by the lengthening of the duplex and converting siRNA into Dicer substrate DsiRNA [160, 161] or by the modifications of the overhands affecting duplex thermodynamic asymmetry . Single stranded analogs of siRNA can be also used as inducers of RNAi, in this case the problem of the strand selection does not exist [40, 77]. All mentioned approaches will be described in details below.
4.1. The influence of nucleotide substitutions on the silencing activity of siRNA
Thermodynamic properties of siRNA play a key role on the stages of RISC activation and mRNA target cleavage, determining the efficiency of strand dissociation, strand selection and mRNA cleavage. Since that, nucleotide substitutions affecting thermodynamic stability of the duplex could change the silencing activity of siRNA. The nature of the changes depends on the type of substitutions and the location of the substitutions in siRNA structure. The data on the impact of nucleotide substitutions in the silencing activities of siRNAs reported in the literature are summarized in Table 4.
The kinetics of mRNA cleavage directed by anti-
Nucleotide substitution at the 3’-end of the sense strand of anti-
The elevation of the thermodynamic asymmetry by single nucleotide substitution at the 1th position of the antisense strand was reported to increase or have no influence on the silencing activity of different siRNAs, targeted to
Introduction of the nucleotide substitutions at the 3’-end of the sense strand of siRNA duplex resulted in the formation of the mismatches, has led to the creation of the structurally new class of small interfering RNAs – fork-like siRNAs (fsiRNAs) . More effective silencing of the expression of
The increase of the thermodynamic asymmetry of the duplex by the introduction of the mismatches at the ends of the strands cannot guarantee the positive influence on the activity. It is known, that the region of the “guide” strand spanning from 2 to 6 position determines the efficiency of the interaction of siRNA with Ago2 [21, 30, 31]; this fact limits the introduction of substitutions in this region. Nevertheless, С→U substitution in 2d position of the antisense strand of siRNA targeted
Introduction of nucleotide substitutions in the central part of siRNA duplex could be used for the destabilization the central region and the subsequent elevation of the silencing activity. Nevertheless, this type of structure modification could switch the mode of siRNA action to miRNA-like mechanism  (fig. 4). It was demonstrated in experiments on HeLa cells, that substitutions in 9th and 10th positions of the sense strand of siRNA leads to the 2 - 4.5-fold increase of IC50 as compared with IC50 for siRNA with classical duplex 36. Substitutions in the sense strand or in both strands of siRNA  reduced the efficiency of RNAi (Table 4).
Thus, the introduction of nucleotide substitutions could be successfully used for the design of siRNA characterized by favorable thermodynamic asymmetry and low thermostability of the central part of the duplex. This approach can be applied for the creation active siRNA targeted to any desired sequence for silencing of mutated or chimerical genes. However, there is not enough data publish up to date for the formulation of the universal rules for the design of fork-like siRNA and siRNA with destabilized center, the activity of resulted siRNA cannot be predicted and should be determined experimentally.
4.2. The influence of the siRNA overhangs structure on the efficiency of gene silencing
2 - 3 nt overhangs at the 3’-ends of siRNA are a distinguishing feature of both natural siRNA generated by Dicer and synthetic siRNA [40, 45, 162]. The overhangs are important for the interaction of siRNA with PAZ-domens of Dicer and Ago2 [21, 27, 168]. However, the data on the activity of siRNA with blunt ends (with no overhangs) are rather contradictory: in mammalian cells the activity of this type of siRNA did not vary significantly from the activity of siRNA with 2-nt overhangs targeted to the same sequence , controversially, in extracts from drosophila cells blunt ends siRNA were less active [40, 45]. The assumption that observed difference could be accounted for the difference in the mechanism of action of heterodimer Dicer-R2D2 in drosophila and its analogue Dicer-TRBP in humans , did not find the experimental support, since it was reported recently, that in HeLa S3 the activity of the blunt ended siRNA was similarly lower (60% silencing) than the activity of classical siRNA (80 % silencing) . Moreover, the presence of 2 nt overhang at the 3’-end of only one of the strands leads to the selection of this strand as a guide strand due to the preferential loading into RISC*. The presence of the mismatches at the 5’-end of the antisense strand resulting in the formation of thermodynamically asymmetrical duplex increases the efficiency of the incorporation of antisense strand, containing 2 nt overhang, into RISC*. Efficient silencing of the target gene expression by fork-like structurally asymmetrical siRNA was achieved in a number of studies conducted on HeLa cells (Table 4, 2nd type of siRNA).
It was found the 1 nt shortening of the overhang (1 ntoverhang instead of 2) at the 3’ end in some cases did not affect significantly the silencing activity . The sequential lengthening of the 3’-end overhang in the sense or the antisense strand up to 7 nt induced the substantial reduction of the silencing activity of siRNA , probably due to inefficient interaction of this siRNAs with PAZ-domen of Dicer . siRNAs, containing overhangs at the 5’-ends of the duplex were inactive in RNAi , and induced side effects related to the PKR-mediated activation of the innate immunity .
Therefore, exploitation of the structurally asymmetrical siRNAs, containing 2 nt overhang only at the 3’-end of the antisense strand together with the reduction of the thermodynamic stability of the duplex at the 5’-end region of the antisense strand could be successfully used in siRNA design for increasing the efficiency of RNAi.
4.3. Single stranded analogous of siRNA (ss-siRNA)
Before the discovery of RNAi, it was demonstrated that long (several hundred bases in length) single stranded RNA are capable of sequence specific silencing of the expression of homologous genes in
Thus, the application of non-modified ss-siRNA as inhibitors of gene expression is not beneficial in comparison with double stranded siRNA.
4.4. dsRNA (27-30-mers) as Dicer substrates
During the search of the effective inductors of RNAi, able to silence target gene at low (nanomolar and lower) concentrations, it was found that dsRNAs 25-30 bp in length which are substrates of Dicer (here and after DsiRNA – Dicer-substrate siРНК), are significantly more active in comparison with “classic” or “conventional” siRNAs [161, 172, 173, 174]. To compare interfering activity of DsiRNA and conventional siRNA the duplexes of different length (21-30 bp) and structure (presence or absence of 3’ or 5’ dinucleotide overhangs) targeting
It is known, that one of the functions of Dicer within the cell is a processing of pre-miRNA via binding with their 3’-end, containing two hanging nucleotides followed by cleavage of the duplex at a distance of 21-22 nucleotides from the 3’-end . Taking into account that Dicer has a low affinity to DNA, a set of asymmetric dsRNA was synthesized, containing at the 3’-end of the sense or antisense strand two nucleotides overhang, while the opposite end was blocked for Dicer binding by the replacement of two ribonucleotides in the complementary strand by deoxyribonucleotides [161, 172] (Table 5, structures 25D/27 and 27/25D). It was anticipated that such a structure of DsiRNAs provide for an unequivocal binding of the duplex with Dicer.
According to mass-spectrometry the cleavage of these DsiRNA by Dicer (Table 5, structures 25D/27 and 27/25D) yields the same product in both cases . However, the silencing activity of siRNAs derived from these DsiRNAs was different; which possibly is explained by the peculiarities of DsiRNA interactions with Dicer, determined the efficiency of sense or antisense incorporation into RISC*.
It was shown, that in HEK293 and HeLa cells the DsiRNAs with antisense strand having dinucleotide overhang at the 3’-end were more active than those with dinucleotide overhang at the 3’-end of the sense strand (Table 5) [161, 172]. Thus, it is possible to predict the structure of forming siRNAs upon processing of DsiRNA by Dicer [161, 172], however, the activity of these siRNAs will depend on the thermodynamic parameters of their duplexes. It worth mentioning that in the case of unfavorable context of the target sequence within mRNA DsiRNAs have a significant advantage over conventional siRNA .
The silencing activity of a longer dsRNAs with blunt ends (27 – 45 bp) decreases with the increase of dsRNA length. It turns out that this drop of silencing activity correlate well with the drop of efficiency of cleavage of longer dsRNAs by Dicer . The silencing effect caused by DsiRNA was longer in comparison with conventional siRNA: DsiRNAs efficiently silence
Despite the promising results on silence activity of Dicer substrates the optimization of the properties of DsiRNAs by chemical modification is relevant. The influence of 2’-F- and 2’-O-Mе modifications within DsiRNA type 25D/27 on the silencing activity and nuclease resistance was studied . When choosing sites of modifications authors try to avoid the region needed for Dicer to execute dsRNA cleavage, despite presence there of nuclease sensitive motives (Fig. 11).
It was shown, that chemical modification of DsiRNA affects its silencing activity in respect to
On the other hand, chemical modification of DsiRNA can block induction of the unspecific immune response in mammalian cells, linked with the presence of triphosphate groups at the 5’ ends of DsiRNA or/and immunostimulating motives in the duplex [43,175, 176, 177, 178, 179,180]. So, DsiRNA, which contains in the antisense strand 10 -11 2’-O-Me nucleotides (including 2’-O-Me-cytidine and 2’-O-Me-uridine nucleotides known as the most effective blockers of interferon response activation via endosomal TLR ), blocked IFN-α secretion by human PBMC and did not induce immune response in RIG-I-dependent T98G cells opposite to unmodified DsiRNA and 2’-F-modified disРНК, respectively .
Full replacement of natural nucleotides by the analogs (2’-O-Me, 2’-F, etc.) is unacceptable as these DsiRNA will not be processed by Dicer . Therefore, upon the introduction of chemical modifications in the structure of DsiRNA one should avoid modifying the nucleotides from 1 to 8 from 5’ end of the sense strand and complementary region of the sense strand (Fig. 11). Moreover, two overhanging nucleotides at the 3’-end of the antisense strand as well as two deoxyribonucleotides at the 3’-end of the sense strand facilitate the correct orientation of Dicer and selection of the antisense strand of DsiRNA as a leading strand.
4.5. Small segmented RNA
Recently another approach to enhance RNAi efficiency based on modification of siRNA structure was proposed . It was shown that siRNA in which the sense strand is subdivided in two parts of 10-12 nucleotides long, are more active in comparison with conventional siRNA duplexes. The efficiency of this internally segmented siRNA or sisiRNA (small internally segmented interfering RNA) (Fig. 9) was provided by unequivocal incorporation of the antisense strand into RISC*, since the nick in the central part of the sense strand blocked its usage as a guiding strand and facilitate the dissociation of the sense strand from the duplex . Thus, sisiRNA have an advantage over conventional siRNA with unfavorable duplex thermoasymmetry. In addition unlike siRNAs with the classic structurethe extensive chemical modification of antisense strand of sisiRNA has only little effect on its silencing activity. As an example, the silencing activity of anti-
According to literary data the structural and thermodynamic peculiarities of interfering RNAs are a major factors providing for efficient gene silencing.
Prediction of silencing activity of any siRNA based on its thermodynamic profile is an efficient method of selection of active molecules. This approach is used in a number of software and algorithms for search and selection of such siRNA . An alternative approach to the problem of creating efficient sequence-specific inhibitors of gene expression is the use of structure-modified siRNAs: fork-like siRNAs, DsiRNAs, or sisRNAs which silencing activity is less dependent on their thermodynamic parameters as compared with conventional siRNA diplex.
Small interfering RNAs are not optimal to be used as therapeutics
Chemical modification of siRNA can be also used to develop delivery vehicles for siRNA. It is well known that naked or free siRNA is not able to penetrate through cellular membrane and accumulate in the cells. In the case of in vivo conditions, the small size of siRNA molecule promotes its rapid clearance . In other words, the systems for in vivo delivery of siRNAs should provide for efficiency of cellular accumulation, specificity of delivery (if applicable), duration of silencing as well as safety and possibility of systemic administration. Today no one delivery approach encounters these criteria.
On the other hand, the low efficiency of RNAi can be a result of its unfavorable sequence determining thermodynamic properties and/or by stable secondary structure at the target site within mRNA [37, 38, 51, 52, 54]. There are several algorithms to select siRNAs of high-performance [182, 183, 184, 185, 186], however it is impossible to predict precisely silencing activity of any particular siRNA in vivo. In addition, the design of siRNA targeting chimeric or mutant genes represent of complex task since the limitations of target site selection does not allow to use these algorithms for choosing the sequence siRNA. This is why structure modifications of siRNA improving their thermodynamic properties are of interest. The fork-like siRNA [158, 159], able to silence target gene more efficiently that conventional siRNA is a promising approach for creating high-performance siRNA targeted any sequence of the gene. Despite this, the number of works in this field is negligible so further research of properties of siRNA with modified structure are relevant.
To obtain high-performance siRNA often optimization of multiple parameters is required, so using modification of the siRNA structure together with its chemical modification is a promising approach. Thus, advanced research of chemically and/or structurally modified siRNA, as well as development and optimization of the algorithms for modification are important.
This research was supported by the Russian Academy ofScience under the programs Molecular and Cell Biology(grant no. 22-1) and Science to Medicine (grant no. 37), the Russian Foundationfor Basic Research (grant nos. 11-04-01017-a and 11-04-12095-ofi-m-2011) Ministry of Science and Education of the Russian Federation (grant no. 14.740.11.1058)and the SiberianBranch of Russian Academy of Sciences (grant no. 85).