**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 [59] , 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 [59], 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) [162]. 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\*[162]. 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 nt overhang instead of 2) at the 3' end in some cases did not affect significantly the silencing activity [40]. 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 [40], probably due to inefficient interaction of this siRNAs with PAZ-domen of Dicer [29]. siRNAs, containing overhangs at the 5'-ends of the duplex were inactive in RNAi [40], and induced side effects related to the PKR-mediated activation of the innate immunity [169].

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 *C.elegans* [170]. Later, it was assumed that the minor fraction of dsRNA in the preparations of ssRNA obtained by *in vitro* transcription was the agent that silence the expression of the target genes 1. However, the ability of antisense, but not sense ssRNA (22 – 40 nt) to induce gene silencing was experimentally verified in *C.elegans* [171]. It was demonstrated that mRNA degradation induced by ssRNA proceeds via RISC\* assembling (fig. 9) [131,122]. The evaluation of the silencing activity of anti-*luciferase* sssiRNA in HeLa cells demonstrated, that the required concentration of ss-siRNA was 8-fold higher, than the concentration of classical siRNA to reach the similar level of gene silencing. In the other report, it was shown, that the concentration of ss-siRNA essential for the RISC assembling was 10 – 100 higher than the concentration of double stranded siRNA analogous [122]. High sensitivity of single stranded RNA to ribonucleases [62], and less efficient interaction with the RNAi machinery proteins [131,122] were supposed to be the possible reasons of the lower efficiency of RNAi induction by ssRNA.

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**

210 Practical Applications in Biomedical Engineering

**silencing** 

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

**4.2. The influence of the siRNA overhangs structure on the efficiency of gene** 

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 [59] , 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 [59], 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) [162]. 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\*[162]. 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

siRNA cannot be predicted and should be determined experimentally.

number of studies conducted on HeLa cells (Table 4, 2nd type of siRNA).

activation of the innate immunity [169].

in siRNA design for increasing the efficiency of RNAi.

**4.3. Single stranded analogous of siRNA (ss-siRNA)** 

It was found the 1 nt shortening of the overhang (1 nt overhang instead of 2) at the 3' end in some cases did not affect significantly the silencing activity [40]. 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 [40], probably due to inefficient interaction of this siRNAs with PAZ-domen of Dicer [29]. siRNAs, containing overhangs at the 5'-ends of the duplex were inactive in RNAi [40], and induced side effects related to the PKR-mediated

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

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 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 *EGFP* were used [174]. The interfering activity of dsRNA was evaluated at concentration ranging from 50 pMol to 50 nMol using HEK293 cells transfected with the plasmid encoded EGFP. It was shown that at subnanomolar concentrations (50 *pM* – 200 *pM*) DsiRNAs were significantly more active than siRNA. Among duplexes tested the DsiRNA 27 bp in length with blunt ends displays the highest interfering activity: this DsiRNA (1 nM) inhibited expression of the target gene by 95% (Table 5) while corresponding siRNA was almost inactive [174]. It turned out, that this DsiRNA (27 bp, blunt ends) is processed by Dicer yielding pull of different siRNAs 21bp in length. Seven siRNAs (21 bp with 3'-dinucleotide overhangs) generated by single-nucleotide shift along the *EGFP* mRNA sequence homologous to DsiRNA were synthesized to answer the question can the synthetic siRNA be as active as this particular DsiRNA. It was shown that at the concentrations 50 pM and 200 pM neither each synthetic siRNA nor pull of synthetic siRNAs silence expression of EGFP as efficiently as corresponding DsiRNA. Interestingly, in the case if DsiRNA was processed by Dicer *in vitro* and then transfected into the cells the silencing efficiency of this processed DsiRNA was similar to the activity of synthetic siRNAs [174]. It has been suggested, than after DsiRNA cleavage Dicer did not dissociated from the complex with DsiRNA and thus governed the mode of interaction of R2D2 (*D*.*melanogaster*) or TRBP (*H.sapiens*) and, as a consequence, the unequivocal orientation of siRNA within the RISC. This provides for incorporation into the RISC the antisense strand of the duplex.

However, if an equivocal binding of Dicer with dsRNA takes place which results in a formation of a set of siRNA cleavage products, the possibility of formation of incompetent RISC\*, containing sense strand of siRNA as a leading strand is increased. Thus, optimization of a structure of DsiRNA is a current task.

Structure - Functions Relations in Small Interfering RNAs 213

**Level of target gene silencing, %** 

> 95 94

> 30 82

sense antisense

sense antisense 23 23

24 24

26 26

strand (L), bp

27 25

25 27 **Ref** 

172

**DsiRNA structure peculiarities** 

> 27/25D 25D/27

> 25D/27

1 25D/27 95 173

10 27/25D

1) The structures of dsRNA – substrates of Dicer: L+2 – duplex of L bp with two nucleotide overhangs at the 3'- ends, L–2 – duplex of L bp with two nucleotides overhangs at the 5' ends, L+0 – duplex of L bp with blunt ends where L

of DsiRNA Scheme of DsiRNA Length of the strand (L), bp

2) Asymmetric DsiRNA (25 bp), containing at the 3'-end of the sense (antisense) strand 2 nt overhang, the opposite end

of DsiRNA Scheme of DsiRNA Strand Length of the

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 [174].

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 [174]. The silencing effect caused by DsiRNA was longer in comparison with conventional siRNA: DsiRNAs efficiently silence *EGFP* expression in NIH3T3 cells for 10 days while corresponding siRNA

**Target gene / model system Optimal or the only** 

L+2 5'– NNNNNNNNNNNNNNNNNNNNNNN-3' 3'– NNNNNNNNNNNNNNNNNNNNNNN-5'

L-2 5'-NNNNNNNNNNNNNNNNNNNNNNNN-3'

L+0 5'-NNNNNNNNNNNNNNNNNNNNNNNNNN-3'

3'-NNNNNNNNNNNNNNNNNNNNNNNN-5'

3'-NNNNNNNNNNNNNNNNNNNNNNNNNN-5'

was blocked by replacement of two ribonucleotides with deoxyribonucleotides – **N**.

27/25D 5'-NNNNNNNNNNNNNNNNNNNNNNNNNNN-3' 3'-**NN**NNNNNNNNNNNNNNNNNNNNNNN-5'

25D/27 5'-NNNNNNNNNNNNNNNNNNNNNNN**NN**-3'

3) The numbers correspond to different sequences within the target gene *F.luciferase.* **Table 5.** The influence of DsiRNA structure in its silencing activity

displays silence activity not longer than 4-7 days [174].

3'-NNNNNNNNNNNNNNNNNNNNNNNNNNN-5'

*F.luciferase* **4**3) Plasmid DNA transfected in НEK 293 cells

> **La antigene in** НЕК 293 cells

*STAT1,* endogenous gene in HeLa cells

varied from 19 to 45 bp. The structure

The structure

**studied concentration of DsiRNA, nM** 

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 [172]. 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 [161]. 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\*.



1) The structures of dsRNA – substrates of Dicer: L+2 – duplex of L bp with two nucleotide overhangs at the 3'- ends, L–2 – duplex of L bp with two nucleotides overhangs at the 5' ends, L+0 – duplex of L bp with blunt ends where L varied from 19 to 45 bp.


2) Asymmetric DsiRNA (25 bp), containing at the 3'-end of the sense (antisense) strand 2 nt overhang, the opposite end was blocked by replacement of two ribonucleotides with deoxyribonucleotides – **N**.


3) The numbers correspond to different sequences within the target gene *F.luciferase.*

**Table 5.** The influence of DsiRNA structure in its silencing activity

212 Practical Applications in Biomedical Engineering

binding of the duplex with Dicer.

antisense incorporation into RISC\*.

*EGFP***,**  Plasmid DNA encoded EGFP transfected in НEK 293 cells

*EGFP***,**  Endogenous gene in NIH3T3 cells

*EGFP***,**  Plasmid DNA encoded EGFP transfected in НEK 293 cells

*F.luciferase* **1**3)**,** Plasmid DNA transfected in НEK 293 cells

*F.luciferase* **2**3) Plasmid DNA transfected in НEK 293 cells

*F.luciferase* **3**3) Plasmid DNA transfected in НEK 293 cells

**Target gene / model system Optimal or the only** 

**studied concentration of DsiRNA, nM** 

of a structure of DsiRNA is a current task.

However, if an equivocal binding of Dicer with dsRNA takes place which results in a formation of a set of siRNA cleavage products, the possibility of formation of incompetent RISC\*, containing sense strand of siRNA as a leading strand is increased. Thus, optimization

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 [172]. 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

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 [161]. 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

50 21+2 1)

1 27+0

1 19+2

10 27/25D

**DsiRNA structure peculiarities** 

23-2 23+2 24-2 24+2 25-2; 25+2; 26+0; 27+0; 27+2; 27-2

> 30+0 35+0 40+0 45+0

27+0 27/25D 2) 25D/27

25D/27

27/25D 25D/27

27/25D 25D/27 **Level of target gene silencing, %** 

> > 80 97

> > 97 96

> > 23 78

**Ref** 

174

161

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 [174].

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 [174]. The silencing effect caused by DsiRNA was longer in comparison with conventional siRNA: DsiRNAs efficiently silence *EGFP* expression in NIH3T3 cells for 10 days while corresponding siRNA displays silence activity not longer than 4-7 days [174].

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 [173]. 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).

Structure - Functions Relations in Small Interfering RNAs 215

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

Recently another approach to enhance RNAi efficiency based on modification of siRNA structure was proposed [99]. 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 [99]. Thus, sisiRNA have an advantage over conventional siRNA with unfavorable duplex thermoasymmetry. In addition unlike siRNAs with the classic structure the extensive chemical modification of antisense strand of sisiRNA has only little effect on its silencing activity. As an example, the silencing activity of anti-*EGFP* sisiRNA with antisense strand having 6 LNA-nucleotides or alternative 2'-F / 2'-O-Me-nucleotides and internally segmented sense strand with 6 LNA-nucleotides in each part was significantly higher than that of conventional siRNA bearing the same modifications [99]. Besides, both mentioned sisiRNA display high nuclease resistance. Thus, sisiRNAs as sequence specific inhibitors of gene expression are very promising, because they provide

efficient RNAi even at the unfavorable thermodynamic parameters RNA duplex.

RNAs are a major factors providing for efficient gene silencing.

According to literary data the structural and thermodynamic peculiarities of interfering

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 [37]. 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

Small interfering RNAs are not optimal to be used as therapeutics *in vivo*, therefore, development of the approached aimed to improve or/and optimize the properties of siРНК is of significance nowadays. One of the approaches widely used is chemical modification of siRNAs. At the beginning this approach was proposed as a way to increase nuclease resistance of siRNA in the presence of serum, in the cells and bloodstream [61, 62]. The enhancement of nuclease resistance of siRNA by chemical modification not resulting in the drop of its silencing activity represent a complex task, since silencing activity of siRNA is

strand.

**4.5. Small segmented RNA** 

conventional siRNA diplex.

**5. Conclusion** 

**Figure 11.** Design of chemically modified DsiRNA. By bold letter **N** deoxyribonucleotides are shown, short arrows – cleavage site of DsiRNA by Dicer.

It was shown, that chemical modification of DsiRNA affects its silencing activity in respect to *STAT1* mRNA only at the lowest concentration used (100 pM), while at higher concentration of DsiRNA (1 and 10 nM) the drop of silencing activity was detected only for extensively modified molecules, containing combination of several 2'-F- and 2'-O-Me nucleotides in a row. DsiRNA containing 11 2'-O-Me nucleotides in the antisense strand was very nuclease resistant: the fraction of intact DsiRNA was found in reaction mixture after 24 h of incubation in the presence of serum. However, such an extensive modification affects pattern of DsiRNA cleavage by Dicer and lead to the formation of two products of 21 and 22 bp instead of one product of 21 bp formed with unmodified DsiRNA. Replacement of one or two 2'-O-Me nucleotides in the vicinity of Dicer cleavage sites by the natural ones facilitated formation of 21 bp cleavage product. The introduction from 1 to 4 mismatches into central part of each strand of anti-*EGFP* DsiRNA significantly reduced its silencing activity [174].

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 [64]), 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 [173].

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 [173]. 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**

214 Practical Applications in Biomedical Engineering

short arrows – cleavage site of DsiRNA by Dicer.

sensitive motives (Fig. 11).

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 [173]. When choosing sites of modifications authors try to avoid the region needed for Dicer to execute dsRNA cleavage, despite presence there of nuclease

**Figure 11.** Design of chemically modified DsiRNA. By bold letter **N** deoxyribonucleotides are shown,

It was shown, that chemical modification of DsiRNA affects its silencing activity in respect to *STAT1* mRNA only at the lowest concentration used (100 pM), while at higher concentration of DsiRNA (1 and 10 nM) the drop of silencing activity was detected only for extensively modified molecules, containing combination of several 2'-F- and 2'-O-Me nucleotides in a row. DsiRNA containing 11 2'-O-Me nucleotides in the antisense strand was very nuclease resistant: the fraction of intact DsiRNA was found in reaction mixture after 24 h of incubation in the presence of serum. However, such an extensive modification affects pattern of DsiRNA cleavage by Dicer and lead to the formation of two products of 21 and 22 bp instead of one product of 21 bp formed with unmodified DsiRNA. Replacement of one or two 2'-O-Me nucleotides in the vicinity of Dicer cleavage sites by the natural ones facilitated formation of 21 bp cleavage product. The introduction from 1 to 4 mismatches into central part of each strand of anti-*EGFP* DsiRNA significantly reduced its silencing activity [174].

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 [64]), blocked IFN-α secretion by human PBMC and did not induce immune response in RIG-I-dependent T98G cells

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 [173]. 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

opposite to unmodified DsiRNA and 2'-F-modified disРНК, respectively [173].

Recently another approach to enhance RNAi efficiency based on modification of siRNA structure was proposed [99]. 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 [99]. Thus, sisiRNA have an advantage over conventional siRNA with unfavorable duplex thermoasymmetry. In addition unlike siRNAs with the classic structure the extensive chemical modification of antisense strand of sisiRNA has only little effect on its silencing activity. As an example, the silencing activity of anti-*EGFP* sisiRNA with antisense strand having 6 LNA-nucleotides or alternative 2'-F / 2'-O-Me-nucleotides and internally segmented sense strand with 6 LNA-nucleotides in each part was significantly higher than that of conventional siRNA bearing the same modifications [99]. Besides, both mentioned sisiRNA display high nuclease resistance. Thus, sisiRNAs as sequence specific inhibitors of gene expression are very promising, because they provide efficient RNAi even at the unfavorable thermodynamic parameters RNA duplex.

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 [37]. 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.
