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

204 Practical Applications in Biomedical Engineering

2) "-" data is not available.

Thermostability

Thermoasymmetry

Nuclease resistance

Duration of circulation in the bloodstream

Cellular uptake

Target cellular uptake

Specificity of action

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

1) Bold letters – deoxyribonucleotides, underlined letters – 2'-O-Me, italics – 2'-F-analogs, B\_ – 5'-5'-, \_B – 3'-3'-inverted

**siRNA property The role of chemical modification Examples of modifications** 

2'-О-Ме, 2'-F, 2'-F-ANA, 2'- O-MOE, 2'-O-DNP, 4'-S, LNA, ENA et al.

UNA, LNA, nucleobase modifications

Virtually all modifications

Chol, 2'-O-DNP, folate

> Peptides, antibodies, aptamers

1) see above; 2) UNA, 2'-O-Me, deoxyribonucleotides 3) 2'-O-Me 4) 2'-O-Me

To increase the thermostability of siRNA by providing the optimal conformation of nucleotides for base pairing65, 99, 120.

To increase the thermoasymmetry of the duplex by the decrease of thermostability at the 3'-flank of the sense strand or/and the increase of the thermostability at the 5'-flank of the sense strand of the duplex 99, 105, 107, 120.

To increase the duplex nuclease resistance by introduction of the chemically modified analogs of nucleotides in its structure153,76.

To increase siRNA affinity to serum proteins and the

To provide the effective mechanisms of siRNA penetration: receptor-mediating endocytosis, diffusion via cellular membrane or other ways (if the mechanism is unknown)153, 86, 126.

To increase the specificity of uptake by the attachment of the molecules with high binding affinity to surface of the cells of particular organs and tissues154.

To increase the specificity of the silencing by: 1) the increase of duplex thermoasymmetry99, 105, 107, 120; 2) alteration of binding affinity of the antisense strand of siRNA to mRNA-target155, 99, 82; 3) the absence of receptors providing the activation of immune response 64; 4) blocking the phosphorylation of the 5'- OH group of the sense strand of siRNA 123.

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

time of circulation 57, 65. PS, 4'-S, Сhol

deoxyribose, underlined bold letters – LNA-analogs, s – phosphorothioate bond between nucleotides.

The investigation of the structures of natural siRNAs formed by dsRNA processing with Dicer and endogenous miRNA revealed the common siRNA structures. "Classical" small interfering RNA resembles duplexes 18 - 23 bp in length [18, 22, 156, 157] with 2 nt overhangs at 3'-ends [40, 45] (fig. 9).

However, it was clearly demonstrated, that the silencing activity of structurally similar duplexes with different sequences varies significantly. As it was mentioned above thermodynamic asymmetry of the duplex and to a lesser extend the structure of mRNA target determines the siRNA activity [44, 45, 57, 58, 60]. Possibility to optimize the duplex structure was demonstrated in a number of publications. Introduction of nucleotide substitutions in siRNA, resulting in a formation of non-canonical base pairs or mismatches [38, 158, 159] is a perspective approach for the optimization of the thermodynamic properties of the duplex. The other approach utilize the inactivation of the sense strand of siRNA (for example, segmentation) to guarantee the incorporation of the antisense strand into activated RISC\* independent from the thermodynamic properties of the duplex [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.

#### **4.1. The influence of nucleotide substitutions on the silencing activity of siRNA**

Structure - Functions Relations in Small Interfering RNAs 207

**Reference** 

158

159

74

166

165

**%** 

98 94.5 99.5 99.8 99.8 99.6 99

67.5 79.5

> 79 92

77.5 84.5

82.5 89.5

> 86 72 95

> 92 89 20

> 0 50 0

**Substitutions 2,3) Silencing efficiency,**

**Target, model system Type of** 

*P.luciferase*, **(+2/+ 23)**,

Hela cells

*P.luciferase*, **(+21/+ 43),**

Hela cells

*Lamin A/C* **, (+829/+ 851),**  Hela cells

*Lamin A/C***, (+608/+ 630),**  Hela cells

*Dnm T1***, (+70/+ 89),** Hela cells

*Dnm T1***, (+185/+ 203),** Hela cells

Hela cells

Hela cells

*F.luciferase***,** 

NIH3T3 cells

*MGC29643,*  **(+576/+596)**  Неla cells

*GPR39, (MGC29643*  **(+576/+596)а)** Неla cells

*Jagged-1,*  **(+3382/+3402)** 

plasmid, transfected into НЕК 293Т cells

*R.luciferase/P.luciferase* **<sup>с</sup> ,**

plasmid, transfected into

*R.luciferase/ P.luciferase* **a,** plasmid, transfected into

plasmid, transfected into

plasmid, transfected into

plasmid, transfected into

**siRNA 1)**

1









*- G9 → C9 G6,9 → C6,9 G6,9,13 → C6,9,13 G3,6,9,13,17 → C3,6,9,13,17* 

*C8 → G8*  A12 → U12 *C8,* A12 → *G8,* U12

C12 → G12 *A8 → U8 A8,* C12 → *U8,* G12

*C18 → U18*

*C5 A12C15 → U5G12U15* 

*C18A→ A18U C1G → U1U*

A18C → U18U, A12→U12

C18A → U18U, U12→A12

G18A → U18U, G12→U12

U18U → A18A, G12→U12

2

1

2

1 2

1 2

1 2

1 2

1

2

1

2

1

1 -

1 -

1 -

 **4**)

Thermodynamic properties of siRNA play a key role on the stages of RISC activation and mRNA target cleavage, determining the efficiency of strand dissociation, strand selection and mRNA cleavage. Since that, nucleotide substitutions affecting thermodynamic stability of the duplex could change the silencing activity of siRNA. The nature of the changes depends on the type of substitutions and the location of the substitutions in siRNA structure. The data on the impact of nucleotide substitutions in the silencing activities of siRNAs reported in the literature are summarized in Table 4.

The kinetics of mRNA cleavage directed by anti-*F.luciferase and anti*-*sod1* siRNA, containing nucleotide substitutions in one of the strands of the duplex were exanimated in details in [38]. It was shown, that the substitution of cytosine with uracil at the 5'-end of the sense strand of anti-*F.luciferase* siRNA, resulting in U:G pair formation (stabilized by two hydrogen bonds)[163]), reduced the target mRNA cleavage rate. At the same time, the rate of the antisense RNA (complementary to the first target) cleavage increased substantially. The analysis of the binding of siRNA strands with RISC\* revealed, that single nucleotide substitution at the position 1 of the sense strand reduce the incorporation of the antisense strand in the complex, whereas the efficiency of the sense strand incorporation into RISC\* increased (fig. 10 A).

Nucleotide substitution at the 3'-end of the sense strand of anti-*sod1* siRNA resulting in the formation of А:G mismatch (fig. 10 B), caused the opposite effect: the rate of mRNA cleavage increased, while the rate of antisense mRNA cleavage decreased. This result indicates that the efficiency of the sense strand incorporation into RISC\* decreases. The observed effect could be caused both by preferential incorporation of the antisense strand into RISC complex, and by non-perfect complementarity between the sense strand of siRNA and its target (antisense mRNA), but the results of the additional experiments with the substitution of G by C at the 5'-end of the antisense strand (fig. 10 B) verified the key role of preferential RISC loading [38]. Thus, it was clearly demonstrated, that single nucleotide substitutions at the flank regions of siRNA could change the thermodynamic asymmetry and to determine the preferential incorporation of one strand into RISC\*: destabilization of the duplex at the 3'-end of the sense strand increase the activity of siRNA , the destabilization of the opposite end of the duplex, reduce the silencing activity [38].

**Figure 10.** The scheme of the experiment described in 38. **А.** Substitution at the 5'-end of the sense strand of anti-*F.luciferase* siРНК. **B.** Substitution at the 3'-end of the sense strand (left) or at the 5'-end of the antisense strand (right) of anti-*sod1* siRNA.


increased (fig. 10 A).

siRNAs reported in the literature are summarized in Table 4.

**4.1. The influence of nucleotide substitutions on the silencing activity of siRNA** 

Thermodynamic properties of siRNA play a key role on the stages of RISC activation and mRNA target cleavage, determining the efficiency of strand dissociation, strand selection and mRNA cleavage. Since that, nucleotide substitutions affecting thermodynamic stability of the duplex could change the silencing activity of siRNA. The nature of the changes depends on the type of substitutions and the location of the substitutions in siRNA structure. The data on the impact of nucleotide substitutions in the silencing activities of

The kinetics of mRNA cleavage directed by anti-*F.luciferase and anti*-*sod1* siRNA, containing nucleotide substitutions in one of the strands of the duplex were exanimated in details in [38]. It was shown, that the substitution of cytosine with uracil at the 5'-end of the sense strand of anti-*F.luciferase* siRNA, resulting in U:G pair formation (stabilized by two hydrogen bonds)[163]), reduced the target mRNA cleavage rate. At the same time, the rate of the antisense RNA (complementary to the first target) cleavage increased substantially. The analysis of the binding of siRNA strands with RISC\* revealed, that single nucleotide substitution at the position 1 of the sense strand reduce the incorporation of the antisense strand in the complex, whereas the efficiency of the sense strand incorporation into RISC\*

Nucleotide substitution at the 3'-end of the sense strand of anti-*sod1* siRNA resulting in the formation of А:G mismatch (fig. 10 B), caused the opposite effect: the rate of mRNA cleavage increased, while the rate of antisense mRNA cleavage decreased. This result indicates that the efficiency of the sense strand incorporation into RISC\* decreases. The observed effect could be caused both by preferential incorporation of the antisense strand into RISC complex, and by non-perfect complementarity between the sense strand of siRNA and its target (antisense mRNA), but the results of the additional experiments with the substitution of G by C at the 5'-end of the antisense strand (fig. 10 B) verified the key role of preferential RISC loading [38]. Thus, it was clearly demonstrated, that single nucleotide substitutions at the flank regions of siRNA could change the thermodynamic asymmetry and to determine the preferential incorporation of one strand into RISC\*: destabilization of the duplex at the 3'-end of the sense strand increase the activity of siRNA , the

destabilization of the opposite end of the duplex, reduce the silencing activity [38].

**Figure 10.** The scheme of the experiment described in 38. **А.** Substitution at the 5'-end of the sense strand of anti-*F.luciferase* siРНК. **B.** Substitution at the 3'-end of the sense strand (left) or at the 5'-end of

the antisense strand (right) of anti-*sod1* siRNA.


Structure - Functions Relations in Small Interfering RNAs 209

The elevation of the thermodynamic asymmetry by single nucleotide substitution at the 1th position of the antisense strand was reported to increase or have no influence on the silencing activity of different siRNAs, targeted to *sod1 / F.luciferase* mRNA or the complementary sequence (antisense mRNA) [164] (Table 4). In turn, the equal efficiency of the cleavage of *htt* mRNA and *htt* antisense mRNA by thermodynamically symmetrical siRNA suggests the

Introduction of the nucleotide substitutions at the 3'-end of the sense strand of siRNA duplex resulted in the formation of the mismatches, has led to the creation of the structurally new class of small interfering RNAs – fork-like siRNAs (fsiRNAs) [158]. More effective silencing of the expression of *R.luciferase/P.luciferase* gene by fsiRNAs then by classical siRNA targeted to the same sequences was demonstrated in Hela cells (Table 4). The most pronounced augmentation of the activity was observed for siRNA with initially moderate activity [158]. Fork-like siRNA with two nucleotide substitutions at the 3'-end of the sense strand was found to be the most active [11, 158, 159] (Table 4). Introduction of from 2 to 4 nucleotide substitutions at the 3'-end of the sense strand of siRNA targeted *sod1* 

The increase of the thermodynamic asymmetry of the duplex by the introduction of the mismatches at the ends of the strands cannot guarantee the positive influence on the activity. It is known, that the region of the "guide" strand spanning from 2 to 6 position determines the efficiency of the interaction of siRNA with Ago2 [21, 30, 31]; this fact limits the introduction of substitutions in this region. Nevertheless, С→U substitution in 2d position of the antisense strand of siRNA targeted *Jagged-1* mRNA increased the silencing effect from 0 to 50% in НЕК 293Т cells transfected with vector, containing cDNA fragment of the target gene [165] (Table 4). This data suggests, that the substitutions in the antisense strand resulted in the formation of non-canonical base pairs is more tolerable than the substitutions resulted in the formation of mismatches. In the other study, single nucleotide substitutions at 19th position of the sense strand or at the 1st position of the antisense strand resulting in the formation of the mismatches as well as non-canonical base-pairs reduced the activity of anti-*EGFP* siRNA in H1299 cells. It was assumed that the reduction of the efficiency of the interaction of TRBP (but not the Dicer) with siRNA was the possible reason

Introduction of nucleotide substitutions in the central part of siRNA duplex could be used for the destabilization the central region and the subsequent elevation of the silencing activity. Nevertheless, this type of structure modification could switch the mode of siRNA action to miRNA-like mechanism [167] (fig. 4). It was demonstrated in experiments on HeLa cells, that substitutions in 9th and 10th positions of the sense strand of siRNA leads to the 2 - 4.5-fold increase of IC50 as compared with IC50 for siRNA with classical duplex 36. Substitutions in the sense strand or in both strands of siRNA [167] reduced the efficiency of RNAi (Table 4).

Thus, the introduction of nucleotide substitutions could be successfully used for the design of siRNA characterized by favorable thermodynamic asymmetry and low thermostability of the central part of the duplex. This approach can be applied for the creation active siRNA

equal probability of sense or antisense strand incorporation into RISC\* [38].

and *htt* mRNAa correspondingly increased the efficiency of silencing [38].

of the observed effect [24] (Table 4).

1)


2) Numeration of the nucleotides from the 5'-end.

3) Substitutions in the sense strand are presented in normal letters; substitutions in the antisense strand are presented in *italic*.

4) *P.luciferase s* (а) – a gene or a fragment of gene encoded sense (s ) or antisense (a) RNA.

5) *АPPL(S)/luciferase –* recombinant gene, containing mutant London (L) or Swedish (S) type of *APP* allele.

b) Silencing activity of structurally modified siRNA is presented as the ration of IC50 (primary siRNA) / IC50 (modified siRNA), where IC50 is the concentration of siRNA, inducing –50 % silencing of the target gene.

**Table 4.** The influence of the nucleotide substitutions in siRNA on the efficiency of RNAi

The elevation of the thermodynamic asymmetry by single nucleotide substitution at the 1th position of the antisense strand was reported to increase or have no influence on the silencing activity of different siRNAs, targeted to *sod1 / F.luciferase* mRNA or the complementary sequence (antisense mRNA) [164] (Table 4). In turn, the equal efficiency of the cleavage of *htt* mRNA and *htt* antisense mRNA by thermodynamically symmetrical siRNA suggests the equal probability of sense or antisense strand incorporation into RISC\* [38].

208 Practical Applications in Biomedical Engineering

**siRNA 1)**

1 -

1 -

2 -

2 -

1 -

1 -

1 -

<sup>1</sup>5'-NNNNNNNNNNNNNNNNNNNNN-3' 3'-NNNNNNNNNNNNNNNNNNNNN-5'

– a gene or a fragment of gene encoded sense (s

<sup>2</sup>5'-NNNNNNNNNNNNNNNNNNN-3' 3'-NNNNNNNNNNNNNNNNNNNNN-5'

2) Numeration of the nucleotides from the 5'-end.

G1 → U1 C19 → A19 *G1 → U1 C19 → A19*

G1 → U1 C19 → A19 *G1 → U1 C19 → A19*

C18C → U18U

G18C → U18A

G19 → C19

G19 → A19 G19 → C19 G19 → U19 *C19 → A19 C19 → U19 C19 → G19* 

C1 → A1 A19 → C19 A19 → G19 A19 → U19 G9 → A9 G9 → C9 G9 → U9 A10 → C10

other substitutions in the sense strand (2 – 8, 12 – 18 positions)

3) Substitutions in the sense strand are presented in normal letters; substitutions in the antisense strand are presented in

b) Silencing activity of structurally modified siRNA is presented as the ration of IC50 (primary siRNA) / IC50 (modified

5) *АPPL(S)/luciferase –* recombinant gene, containing mutant London (L) or Swedish (S) type of *APP* allele.

siRNA), where IC50 is the concentration of siRNA, inducing –50 % silencing of the target gene. **Table 4.** The influence of the nucleotide substitutions in siRNA on the efficiency of RNAi

Structure of siRNA Strand Length of the

) or antisense (a) RNA.

**Substitutions 2,3) Silencing efficiency,**

**%** 

0 45

0 80

55 50

1 b**)** 0.3 1.15 1.2 1.25 4.5 3.3 2.15 2.3 0.2 – 1.3

sense antisense

sense antisense **Reference** 

164

159

159

24

36

strand, nt

21 21

19 21

**Target, model system Type of** 

**,**

*sod1/F.luciferase* **<sup>c</sup>**

*sod1/F.luciferase* **a,** plasmid, transfected into

*АPP(L)/luciferase* **5),** plasmid, transfected into

НЕК 293 cells

НЕК 293 cells

НЕК 293 cells

НЕК 293 cells

*EGFP, (+306/+324)* 

H1299

*EGFP, (+396/+414)*

H1299

1)

*italic*. 4) *P.luciferase s* (а)

*F.luciferase*,

Неla SS6 cells

siRNA type

*АPP(S)/luciferase***,** plasmid, transfected into

human lung carcinoma

human lung carcinoma

plasmid, transfected into

plasmid, transfected into

Introduction of the nucleotide substitutions at the 3'-end of the sense strand of siRNA duplex resulted in the formation of the mismatches, has led to the creation of the structurally new class of small interfering RNAs – fork-like siRNAs (fsiRNAs) [158]. More effective silencing of the expression of *R.luciferase/P.luciferase* gene by fsiRNAs then by classical siRNA targeted to the same sequences was demonstrated in Hela cells (Table 4). The most pronounced augmentation of the activity was observed for siRNA with initially moderate activity [158]. Fork-like siRNA with two nucleotide substitutions at the 3'-end of the sense strand was found to be the most active [11, 158, 159] (Table 4). Introduction of from 2 to 4 nucleotide substitutions at the 3'-end of the sense strand of siRNA targeted *sod1*  and *htt* mRNAa correspondingly increased the efficiency of silencing [38].

The increase of the thermodynamic asymmetry of the duplex by the introduction of the mismatches at the ends of the strands cannot guarantee the positive influence on the activity. It is known, that the region of the "guide" strand spanning from 2 to 6 position determines the efficiency of the interaction of siRNA with Ago2 [21, 30, 31]; this fact limits the introduction of substitutions in this region. Nevertheless, С→U substitution in 2d position of the antisense strand of siRNA targeted *Jagged-1* mRNA increased the silencing effect from 0 to 50% in НЕК 293Т cells transfected with vector, containing cDNA fragment of the target gene [165] (Table 4). This data suggests, that the substitutions in the antisense strand resulted in the formation of non-canonical base pairs is more tolerable than the substitutions resulted in the formation of mismatches. In the other study, single nucleotide substitutions at 19th position of the sense strand or at the 1st position of the antisense strand resulting in the formation of the mismatches as well as non-canonical base-pairs reduced the activity of anti-*EGFP* siRNA in H1299 cells. It was assumed that the reduction of the efficiency of the interaction of TRBP (but not the Dicer) with siRNA was the possible reason of the observed effect [24] (Table 4).

Introduction of nucleotide substitutions in the central part of siRNA duplex could be used for the destabilization the central region and the subsequent elevation of the silencing activity. Nevertheless, this type of structure modification could switch the mode of siRNA action to miRNA-like mechanism [167] (fig. 4). It was demonstrated in experiments on HeLa cells, that substitutions in 9th and 10th positions of the sense strand of siRNA leads to the 2 - 4.5-fold increase of IC50 as compared with IC50 for siRNA with classical duplex 36. Substitutions in the sense strand or in both strands of siRNA [167] reduced the efficiency of RNAi (Table 4).

Thus, the introduction of nucleotide substitutions could be successfully used for the design of siRNA characterized by favorable thermodynamic asymmetry and low thermostability of the central part of the duplex. This approach can be applied for the creation active siRNA targeted to any desired sequence for silencing of mutated or chimerical genes. However, there is not enough data publish up to date for the formulation of the universal rules for the design of fork-like siRNA and siRNA with destabilized center, the activity of resulted siRNA cannot be predicted and should be determined experimentally.

Structure - Functions Relations in Small Interfering RNAs 211

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

Thus, the application of non-modified ss-siRNA as inhibitors of gene expression is not

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.

reasons of the lower efficiency of RNAi induction by ssRNA.

beneficial in comparison with double stranded siRNA.

**4.4. dsRNA (27-30-mers) as Dicer substrates** 
