**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 [42]. 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 [63]. 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.

#### *3.1.1.1. Substituents in furanose*

192 Practical Applications in Biomedical Engineering

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.

**Figure 4.** Scheme of RNAi with the "bypass" rout of RISC\* assembly (miRNA-like action). RISC\* binds

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 [42]. 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 [63]. Intracellular factors are

with mRNA-target (n is equal 1, if the antisense strand is fully complementary to mRNA) [10].

related to RISC assembly and the efficiency of binding to and cleaving the target.

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

**3.1. Chemical modifications of the nucleotides** 

*3.1.1. Modifications of ribose (furanose) ring* 

of the furanose cycle.

**3. Chemical modifications: The influence on RNAi efficacy** 

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 [64]. Thus, the modification of 2'-OH defends siRNA against ribonucleases [62]. 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 [58]. 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 [65].

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 nucleasesensitive sites within siRNA the increase of the duplex nuclease resistance and prolonged silencing were detected [59].

The C3'-*endo* conformation could be stabilized by introduction of 2'-F-modified nucleotides in siRNA (fig. 5 A). Since fluorine atom is more electronegative than oxygen atom, the increase of the binding affinity between siRNA strands and target RNA (ΔTm 2 – 4 0С per modification) is observed [65]. Remarkably, the silencing activity of 2'-F-modified siRNAs virtually does not depend on the number of modifications in contrast to 2'-O-Me-modified siRNAs. The silencing activity of siRNAs with 2'-F-modifications in the sense and the antisense strands was compatible with that of unmodified analogs [76]. The replacement of pyrimidine nucleotides in the duplex with 2'-F-analogs was well tolerated by RNAi machinery both *in vitro* [46, 77, 73] and *in vivo* [78]. The silencing activity of siRNA containing 2'-F modifications located in the site of Ago2 cleavage and unmodified analog was similar [79], as well as long-term silencing effects *in vivo* of modified siRNAs and unmodified analogs [78]. It should be noted that epimers of 2'-F-nucleotides (2'-fluoro-β-Darabinonucleotides, FANA (fig. 5 A)) with DNA-like C2'-*endo*-conformation of furanose cycle [80] (fig. 7) introduced in siRNA structure also improve the nuclease resistance of the duplex. It was found that partial modification of both strands with FANA or total modification with FANA of the sense strand of siRNAs ensure the A-geometry helix [81] and effective RNAi. These modifications were shown to alter the duplex thermodynamic stability insignificantly [76].

Structure - Functions Relations in Small Interfering RNAs 195

On the contrary, the replacement of ribonucleotides of siRNAs with deoxyribonucleotides characterized by C2'-*endo*-conformation of the furanose cycle (fig. 5 A) reduces their affinity to mRNA-target (ΔTm -0.5 0С per modification) (fig. 7) [65]. Mainly, deoxyribonucleotides are used for the defense of siRNA overhangs from exonucleases [13, 59]. However, the modification of the other duplex regions does not impede effective RNAi [40, 82]. The introduction of deoxyribonucleotides in the duplex region responsible for the recognition of the target mRNA, so called "seed" region (about 8 nt. from the 5' end of the antisense strand [29]) results in the increase of siRNA specificity since lesser stability of DNA/RNA hybrids as compared with RNA duplex [82]. siRNAs with the totally modified antisense or sense strands had no activity regardless of preferable for

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 [76]. 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 [85] (that indicates the similar hybridization properties [86]), 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

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 [91] or four ribonucleotides at the sense strand termini and at the 3'-end of the antisense strand [93] 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

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-

depends on the location of the modifications in the duplex structure [88].

RNAi A-helix geometry of the duplexes [32].

and increases their cytotoxicity [65].

*3.1.1.2. Structural modifications of the furanose ring* 

**Figure 5.** Analogs of nucleotides and nucleosides used for siRNA modification. **A.** Substituents in furanose. **B.** Structural modifications of furanose cycle. **C.** Backbone modifications. "B" – base (**А** – **C**). **D.** Nucleobase modifications. "R" – ribose residue.

**Figure 6.** C3'-*endo* conformation of the ribose with the 2'-substituent. R – electron-acceptor group.

**Figure 7.** C2'-*endo* (DNA-like) conformation of ribose with the substituent (R) in 2'-position.

On the contrary, the replacement of ribonucleotides of siRNAs with deoxyribonucleotides characterized by C2'-*endo*-conformation of the furanose cycle (fig. 5 A) reduces their affinity to mRNA-target (ΔTm -0.5 0С per modification) (fig. 7) [65]. Mainly, deoxyribonucleotides are used for the defense of siRNA overhangs from exonucleases [13, 59]. However, the modification of the other duplex regions does not impede effective RNAi [40, 82]. The introduction of deoxyribonucleotides in the duplex region responsible for the recognition of the target mRNA, so called "seed" region (about 8 nt. from the 5' end of the antisense strand [29]) results in the increase of siRNA specificity since lesser stability of DNA/RNA hybrids as compared with RNA duplex [82]. siRNAs with the totally modified antisense or sense strands had no activity regardless of preferable for RNAi A-helix geometry of the duplexes [32].

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 [76]. 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 [85] (that indicates the similar hybridization properties [86]), 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 [88].

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 [91] or four ribonucleotides at the sense strand termini and at the 3'-end of the antisense strand [93] 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 [65].

#### *3.1.1.2. Structural modifications of the furanose ring*

194 Practical Applications in Biomedical Engineering

**Figure 5.** Analogs of nucleotides and nucleosides used for siRNA modification. **A.** Substituents in furanose. **B.** Structural modifications of furanose cycle. **C.** Backbone modifications. "B" – base (**А** – **C**).

**Figure 6.** C3'-*endo* conformation of the ribose with the 2'-substituent. R – electron-acceptor group.

**Figure 7.** C2'-*endo* (DNA-like) conformation of ribose with the substituent (R) in 2'-position.

**D.** Nucleobase modifications. "R" – ribose residue.

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'-*endo*conformation (fig. 6) due to the methylene or ethylene "bridge" between 2'- and 4' positions, respectively.

Structure - Functions Relations in Small Interfering RNAs 197

PNA/RNA duplex RNA adopts the right-handed helix characterized by anticonformation at the N-glycosidic bond and C3'-*endo*-conformation of the ribose [108], with the similar to the RNA-like A-helix geometry [58]. PNAs are stable in the human blood serum as well as in the cellular extract; they are resistant to protein kinase A and several types of peptidases [109]. It was found that the introduction of PNA-analogs in the sense and / or in the antisense strands of the duplex increase nuclease resistance of siRNA. The increase of the siRNA silencing activity was observed when modifications were introduced in the sense strand. In the other cases the activity of PNA-containing siRNA was comparable with activity of the unmodified siRNA [110]. Due to the functional inequivalence of siRNA strands the modifications in the sense strand a better tolerated than modifications in the

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

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 [76]. PS-modification is known to reduce the melting temperature of the duplex and oligoribonucleotide binding affinity [71]. 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 PSmodified siRNAs was lower, than that of their unmodified analogs [58, 73, 112]. The modification of the central part of the duplex decreases siRNA activity [113]. Remarkably, siRNAs with blunt 3'-ends containing totally PS-modified one or both strands were active [73]. However, high binding affinity of the phosphorothioate analogs to serum albumin, IgG, IgM, lactoferrin and the cellular membrane receptors is disadvantageous [114]. It results in the development of toxic effects both *in vitro* and *in vivo*, even siRNAs containing alternate PS-analogs were toxic in cell cultures [83, 111]. Introduction of boranophosphonate analogs (fig. 5 C) increases nuclease resistance of the duplex as compared with PScontaining and unmodified siRNA [65] and increases RNAi efficacy. It was found that an optimal position for PB-modifications is the terminal region of the sense strand of siRNA, whereas the modification of the central part of the duplex resulted in the significant decrease of silencing activity [65]. This effect is likely connected with the inhibition of the sense strand cleavage by Ago2 during RISC\* formation. The replacement of the 3',5'- by 2',5'-phosphodiester bond or 3',5'-amide bond (fig. 5 C) increases the nuclease resistance of the duplex and is tolerant when located in the sense strand [115] or the 3'-overhangs of

antisense strand.

*3.1.2. Backbone modifications* 

RNAi.

siRNA [116].

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 [74]. 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 [99] (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 [100], 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 [103]. 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 ANAanalogs 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'-*endo*-conformation of the ribose [108], with the similar to the RNA-like A-helix geometry [58]. PNAs are stable in the human blood serum as well as in the cellular extract; they are resistant to protein kinase A and several types of peptidases [109]. It was found that the introduction of PNA-analogs in the sense and / or in the antisense strands of the duplex increase nuclease resistance of siRNA. The increase of the siRNA silencing activity was observed when modifications were introduced in the sense strand. In the other cases the activity of PNA-containing siRNA was comparable with activity of the unmodified siRNA [110]. Due to the functional inequivalence of siRNA strands the modifications in the sense strand a better tolerated than modifications in the antisense strand.

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*

196 Practical Applications in Biomedical Engineering

positions, respectively.

siRNA activity [88, 103].

reduce siRNA silencing activity.

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'-*endo*conformation (fig. 6) due to the methylene or ethylene "bridge" between 2'- and 4'-

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 [74]. 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

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 [100], 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 [103]. 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 ANAanalogs 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

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

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

only in the case of sisiRNA with segmented sense strand [99] (see section 1.3.5).

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 [76]. PS-modification is known to reduce the melting temperature of the duplex and oligoribonucleotide binding affinity [71]. 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 PSmodified siRNAs was lower, than that of their unmodified analogs [58, 73, 112]. The modification of the central part of the duplex decreases siRNA activity [113]. Remarkably, siRNAs with blunt 3'-ends containing totally PS-modified one or both strands were active [73]. However, high binding affinity of the phosphorothioate analogs to serum albumin, IgG, IgM, lactoferrin and the cellular membrane receptors is disadvantageous [114]. It results in the development of toxic effects both *in vitro* and *in vivo*, even siRNAs containing alternate PS-analogs were toxic in cell cultures [83, 111]. Introduction of boranophosphonate analogs (fig. 5 C) increases nuclease resistance of the duplex as compared with PScontaining and unmodified siRNA [65] and increases RNAi efficacy. It was found that an optimal position for PB-modifications is the terminal region of the sense strand of siRNA, whereas the modification of the central part of the duplex resulted in the significant decrease of silencing activity [65]. This effect is likely connected with the inhibition of the sense strand cleavage by Ago2 during RISC\* formation. The replacement of the 3',5'- by 2',5'-phosphodiester bond or 3',5'-amide bond (fig. 5 C) increases the nuclease resistance of the duplex and is tolerant when located in the sense strand [115] or the 3'-overhangs of siRNA [116].

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.

Structure - Functions Relations in Small Interfering RNAs 199

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 PIWIdomain 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 [123]. 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 [111], 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 [21] 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 [130] or the replacement of the 3'-terminal ribonucleotide by ddC, or the attachment of the aminopropyl linker via phosphodiester bond [122] virtually does not change the silencing activity. However, the introduction of the 2-hydroxyethylphosphate, ENA-analog of thymidine [74] or fluorescent dyes [111] 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

**Figure 8.** Inverted deoxyribose applied for the protection of 5'- or 3'-termini of siRNA.

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

reduce the silencing activity [59, 61].

*3.2.1. Bioconjugates* 

#### *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 [42]. 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 [117]. 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 [58]. 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 [58]. 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' *endo*-conformation (fig. 6) [118, 119]. Dihydrouridine (D) contains non-aromatic ring that does not participate in staking interactions (fig. 7) which resulted in the destabilization of siRNA [120], but one substitution with D at the 3'-end of the sense or the antisense strand does not change siRNA silencing activity. The replacement of U by Ψ and s2U at the 3'-end of the sense strand reduces the silencing activity, whereas introduction of the same modification at the 3' end of the antisense strand creates favorable thermodynamic asymmetry and provides siRNAs with higher activities than the parent siRNA. Thus, these modifications could be applied for the design of thermoasymmetric siRNAs [120] (see section 3.3).

#### **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 [122], ENA-analog of the thymidine [74] etc.), ii) to facilitate asymmetric RISC\* assembly (the replacement of the 5'-OH by 5'-OMe group in the terminal nucleoside [123]) and ii) to provide the efficient accumulation of siRNA in cells (attachment of lipophilic molecules [57, 124, 125], folic acid [126], peptides [14, 127, 128], aptamers [129]). The fluorescent residues [111] and biotin [130] 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 PIWIdomain 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 [123]. 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 [111], 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 [21] 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 [130] or the replacement of the 3'-terminal ribonucleotide by ddC, or the attachment of the aminopropyl linker via phosphodiester bond [122] virtually does not change the silencing activity. However, the introduction of the 2-hydroxyethylphosphate, ENA-analog of thymidine [74] or fluorescent dyes [111] 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].

**Figure 8.** Inverted deoxyribose applied for the protection of 5'- or 3'-termini of siRNA.

#### *3.2.1. Bioconjugates*

198 Practical Applications in Biomedical Engineering

price of PB-analogs limits their usage.

*3.1.3. The chemical modifications of the nucleobases* 

the design of thermoasymmetric siRNAs [120] (see section 3.3).

are widely used as a termini modification for siRNA detection.

**3.2. Chemical modification of siRNA termini** 

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

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 [42]. 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 [117]. 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 [58]. 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 [58]. 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' *endo*-conformation (fig. 6) [118, 119]. Dihydrouridine (D) contains non-aromatic ring that does not participate in staking interactions (fig. 7) which resulted in the destabilization of siRNA [120], but one substitution with D at the 3'-end of the sense or the antisense strand does not change siRNA silencing activity. The replacement of U by Ψ and s2U at the 3'-end of the sense strand reduces the silencing activity, whereas introduction of the same modification at the 3' end of the antisense strand creates favorable thermodynamic asymmetry and provides siRNAs with higher activities than the parent siRNA. Thus, these modifications could be applied for

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 [122], ENA-analog of the thymidine [74] etc.), ii) to facilitate asymmetric RISC\* assembly (the replacement of the 5'-OH by 5'-OMe group in the terminal nucleoside [123]) and ii) to provide the efficient accumulation of siRNA in cells (attachment of lipophilic molecules [57, 124, 125], folic acid [126], peptides [14, 127, 128], aptamers [129]). The fluorescent residues [111] and biotin [130]

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 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 receptormediated endocytosis. It was proved experimentally that LDL-receptors bind with the LDLparticles 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 [125] (Table 1). Moreover, the involvement of the transmembrane Structure - Functions Relations in Small Interfering RNAs 201

COS-7 cells

LNCaP and PC-3 cells

HEK 293

Brain of

cells *F.luciferase* 

*F.lucifera*

*Bcl-2* 

mouse 69 – 81 142

*se* 50 128

*PLK1* 80 129

> 90 141

N-terminal Cys-S-(CH2)6 at the 5'-end of the sense strand

at the 5'-end of the sense strand 4)

biotin-tetraethylene glycol at the 3'-end of the sense strand

biotin-tetraethylene glycol at the 3'- or 5'-end of the sense strand

protein SID-1 in the transport of the lipophilic conjugates was also shown [135]. 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 *in vitro* [124] and *in vivo* [57] (Table 1). The conjugate of siRNA and cholesterol, attached to the 5'-end of the sense strand, is able to penetrate into the human liver cells in the absence of the transfection agents. siRNA bearing cholesterol in the sense strand inhibits *ß-Gal* gene expression more effectively than siRNAs with the cholesterol in the antisense strand or in the both strands. In the presence of the transfection agent the activity of the unmodified siRNA was comparable with that of the conjugates [124]. The accumulation of siRNA-Chol was detected in liver, heart, kidneys, fatty and pulmonary tissues after intravenous injection of the radioactively labeled conjugates (containing cholesterol at the 3'-end of the sense strand) in mice. It should be noted that cells of these organs express LDL-receptors at high level. Other examples of cholesterol-modified

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 *EGFP* and *CDK9* [14]. The endocytosis was suggested

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-*EGFP* and anti-*GL2* siRNAs in COS-7, C166-GFP, EOMA-GFP and CHO-AA8-Luc Tet-Off

Transportan-

AptamersiRNA

AntibodysiRNA

siRNA CLIKKALAALAKLNIKLLYGASNLTWG

Antibodies to insulin receptor

Antibodies to transferrin receptor

3) In this investigationthe biological activity of the conjugate was not measured (n.d.). 4) The conjugate of siRNA andaptamer synthesized by *in vivo* transcription (without linker).

siRNAs with high penetration ability are presented in the Table 1.

to be the mechanism of the conjugates penetration into cells [136, 137].

1) Lipo – lipophilic residues, Chol – cholesterol residue.

2) siRNA with linker are denoted «R».

**Table 1.** Bioconjugates of siRNAs



1) Lipo – lipophilic residues, Chol – cholesterol residue.

2) siRNA with linker are denoted «R».

200 Practical Applications in Biomedical Engineering

**conjugate 1) Modification 2) Linker,** 

**siRNA** 

Lipo-siRNA

Сhol-siRNA

FolatesiRNA

Penetratin-

ТАТ-siRNA YGRKKRRQRRR

siRNA CRQIKIWFQNRRMKWKK

LDL-receptors could deliver the cholesterol-conjugated siRNAs inside the cells via receptormediated endocytosis. It was proved experimentally that LDL-receptors bind with the LDLparticles 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 [125] (Table 1). Moreover, the involvement of the transmembrane

**site of attachment to siRNA**

trans-4-hydroxy proline linker at the 3'-end of the sense strand

aminocaproic acidpyrrolidine linker at the 3' end of the sense strand


6-aminohexyl at the 5'-end of the sense strand

at the 5'-end of the sense strand

N-terminal Cys-HN-(CH2)3 at the 3'-end of the antisense strand

C-terminal Cys –S-(CH2)6 at the 5'-end of the sense strand

N-terminal Cys-S-(CH2)6 at the 5'-end of the sense strand

C-terminal Cys –S-(CH2)xat the 5'-end of the sense strand

**Cell or target organ** 

mouse

mouse

mouse lungs

β-Gal/Huh -7 cells

HeLa

HeLa cells

Lungs of mouse

> COS-7 cells

Lungs of mouse

**Target gene** 

liver *Apo-B-1* 55-25 125

liver *Apo-B-1* 50 57

cells n.d. **3)** n.d. 126

*GFP* 

*P38 MAP kinase* 

*F.lucifera*

*P38 MAP kinase*  40 127

*β-Gal* 55 124

*CDK9* 60 – 70 14

*se* 60 128

40 127

20 127

*P38 MAP kinase* 

**Silencing efficacy, %** 

**Ref.** 

3) In this investigationthe biological activity of the conjugate was not measured (n.d.).

4) The conjugate of siRNA andaptamer synthesized by *in vivo* transcription (without linker).

#### **Table 1.** Bioconjugates of siRNAs

protein SID-1 in the transport of the lipophilic conjugates was also shown [135]. 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 *in vitro* [124] and *in vivo* [57] (Table 1). The conjugate of siRNA and cholesterol, attached to the 5'-end of the sense strand, is able to penetrate into the human liver cells in the absence of the transfection agents. siRNA bearing cholesterol in the sense strand inhibits *ß-Gal* gene expression more effectively than siRNAs with the cholesterol in the antisense strand or in the both strands. In the presence of the transfection agent the activity of the unmodified siRNA was comparable with that of the conjugates [124]. The accumulation of siRNA-Chol was detected in liver, heart, kidneys, fatty and pulmonary tissues after intravenous injection of the radioactively labeled conjugates (containing cholesterol at the 3'-end of the sense strand) in mice. It should be noted that cells of these organs express LDL-receptors at high level. Other examples of cholesterol-modified siRNAs with high penetration ability are presented in the Table 1.

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 *EGFP* and *CDK9* [14]. The endocytosis was suggested to be the mechanism of the conjugates penetration into cells [136, 137].

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-*EGFP* and anti-*GL2* siRNAs in COS-7, C166-GFP, EOMA-GFP and CHO-AA8-Luc Tet-Off

cells [128] (Table 1). It was suggested that penetration of the conjugates does not occur via pino- or endocytosis, but is mediated by diffusion through plasma membrane [128]. Besides the increase of transfection efficacy, the attachment of the peptides to siRNAs may enhance their specificity. Bioconjugates of siRNAs with peptides inhibiting the RISC assembly and containing specific sequence cleavable by cell-specific peptidases are promising agents for cell-specific gene silencing [138]. The efficient inhibition of the exogenous *GFP* expression was observed in choriocarcinoma Jeg-3 cells after electroporation of the conjugate of siRNA and a peptide with the "LEVD" sequence recognized by caspase-4, whereas in caspase-4 deficient HEK 293 cells the conjugate was inactive [138].

Structure - Functions Relations in Small Interfering RNAs 203

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 [61] (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 [59], it was suggested that the increase of the thermostability will increase the nuclease resistance [99]. 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 [62]. 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 [76]. The optimization of siRNA design also includes the defense of the 3'-terminal

The combinations of chemical modifications are used for optimization of other siRNA properties. In order to achieve *in vivo* both the efficient cellular uptake and their high nuclease resistance, the 2'-O-Me-modified or/and PS-modified siRNAs conjugated with cholesterol were used [57, 151]. In order to increase the thermoasymmetry of the duplex, the nucleotide analogs with opposite effect on the siRNA thermostability can be used simultaneously. For instance, LNA- and UNA-analogs introduced in siRNA simultaneously was shown to enhance the silencing activity of the siRNA due to the increase of thermoasymmetry of the duplex: duplex stabilization of the duplex by LNA-analogs at the

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 [120]. 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

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

5'-end and destabilization by UNA-analogs at the 3'-end of the sense strand [99].

nucleotides against exonuclease cleavage (see section 1.2.2).

duplex stabilization is not needed [99].

Another approach to the enhancement of siRNA cellular accumulation is the delivery of siRNA conjugated with antibodies or aptamers [132]. 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 [139]. 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 [132]. The conjugates of anti-*BCL2* or anti-*PLK1* (polo-like kinase 1) siRNA and PSMA-receptor specific aptamer, effectively penetrate into prostate cancer LNCaP cells and induce effective gene silencing [129]. Injection of these conjugates into tumor expressing PSMA-receptors results in tumor regression [140]. It was shown also that the conjugates of siRNA and anti-transferrin or anti-insulin monoclonal antibodies attached to siRNA via streptavidin-biotin linker effectively reduces the exogenous *F.luciferase*  expression in HEK 293 cells and rat glial cells implanted in the brain [141, 142] (Table 1).

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 [143]. The efficient accumulation of fluoresceinlabeled siRNA with folate attached to the 5'- or 3'-end of the sense strand via disulfide bond was observed in solid tumor in mice [126] (Table 1). In the other studies [144, 145] the delivery of siRNA into cells was performed with folate-containing structures. These experiments (*in vivo* and *in vitro*) verified the advantage of siRNA modification by folate: specific delivery into cancer cells and high silencing effect of these siRNAs was observed.

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 *in vivo* is less specific, since LDL- and folate-receptors are expressed by different cells [125, 150], however, this approach is could be usefully applied when strict cell or tissue selectivity is not required.

#### **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 [61] (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 [59], it was suggested that the increase of the thermostability will increase the nuclease resistance [99]. 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 [62]. 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 [76]. The optimization of siRNA design also includes the defense of the 3'-terminal nucleotides against exonuclease cleavage (see section 1.2.2).

202 Practical Applications in Biomedical Engineering

is not required.

**3.3. Combination of chemical modifications** 

deficient HEK 293 cells the conjugate was inactive [138].

cells [128] (Table 1). It was suggested that penetration of the conjugates does not occur via pino- or endocytosis, but is mediated by diffusion through plasma membrane [128]. Besides the increase of transfection efficacy, the attachment of the peptides to siRNAs may enhance their specificity. Bioconjugates of siRNAs with peptides inhibiting the RISC assembly and containing specific sequence cleavable by cell-specific peptidases are promising agents for cell-specific gene silencing [138]. The efficient inhibition of the exogenous *GFP* expression was observed in choriocarcinoma Jeg-3 cells after electroporation of the conjugate of siRNA and a peptide with the "LEVD" sequence recognized by caspase-4, whereas in caspase-4

Another approach to the enhancement of siRNA cellular accumulation is the delivery of siRNA conjugated with antibodies or aptamers [132]. 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 [139]. 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 [132]. The conjugates of anti-*BCL2* or anti-*PLK1* (polo-like kinase 1) siRNA and PSMA-receptor specific aptamer, effectively penetrate into prostate cancer LNCaP cells and induce effective gene silencing [129]. Injection of these conjugates into tumor expressing PSMA-receptors results in tumor regression [140]. It was shown also that the conjugates of siRNA and anti-transferrin or anti-insulin monoclonal antibodies attached to siRNA via streptavidin-biotin linker effectively reduces the exogenous *F.luciferase*  expression in HEK 293 cells and rat glial cells implanted in the brain [141, 142] (Table 1).

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 [143]. The efficient accumulation of fluoresceinlabeled siRNA with folate attached to the 5'- or 3'-end of the sense strand via disulfide bond was observed in solid tumor in mice [126] (Table 1). In the other studies [144, 145] the delivery of siRNA into cells was performed with folate-containing structures. These experiments (*in vivo* and *in vitro*) verified the advantage of siRNA modification by folate: specific delivery into cancer cells and high silencing effect of these siRNAs was observed.

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 *in vivo* is less specific, since LDL- and folate-receptors are expressed by different cells [125, 150], however, this approach is could be usefully applied when strict cell or tissue selectivity

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 The combinations of chemical modifications are used for optimization of other siRNA properties. In order to achieve *in vivo* both the efficient cellular uptake and their high nuclease resistance, the 2'-O-Me-modified or/and PS-modified siRNAs conjugated with cholesterol were used [57, 151]. In order to increase the thermoasymmetry of the duplex, the nucleotide analogs with opposite effect on the siRNA thermostability can be used simultaneously. For instance, LNA- and UNA-analogs introduced in siRNA simultaneously was shown to enhance the silencing activity of the siRNA due to the increase of thermoasymmetry of the duplex: duplex stabilization of the duplex by LNA-analogs at the 5'-end and destabilization by UNA-analogs at the 3'-end of the sense strand [99].

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 [120]. 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 [99].

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


Structure - Functions Relations in Small Interfering RNAs 205

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

The information on the influence of chemical modifications on siRNA properties is

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

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 [99]. 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 [162]. 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.

**Figure 9.** Structural repertoire of siRNA. The triangle indicates single stranded nick.

**4. The impact of the siRNA structure on the efficiency of RNAi** 

thermostability.

summarized in the table 3.

overhangs at 3'-ends [40, 45] (fig. 9).

1) Bold letters – deoxyribonucleotides, underlined letters – 2'-O-Me, italics – 2'-F-analogs, B\_ – 5'-5'-, \_B – 3'-3'-inverted deoxyribose, underlined bold letters – LNA-analogs, s – phosphorothioate bond between nucleotides.

2) "-" data is not available.

**Table 2.** Extensive and selective modification of siRNA


**Table 3.** Influence of the chemical modifications on siRNA properties

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.
