**1. Introduction**

186 Practical Applications in Biomedical Engineering

ActaAnaesthesiolScand, 43, 308-315.

Electronic Engineers Sensors Journal, 1211-1214.

[22] Karason S., Karlsen K.L., Lundin S. et al. (1999). "A simplified method for separate measurements of lung and chest wall mechanics in ventilator-treated patients". In:

[23] Kwan K.Y., Kaler K.V.I.S., Mintchev M. P. (2002). "High-pressure balloon catheter for real-time pressure monitoring in the esophagus". In: Institute of Electrical and

> RNA interference is an evolutionary conserved mechanism of specific gene silencing induced by double stranded RNA homologous to the target mRNA. Small interfering RNAs (siRNAs) are widely used for the control of gene expression in molecular biology and experimental pharmacology. Currently, siRNAs are successfully used for the validation of potent drug targets for anti-cancer therapy. However, application of siRNAs as therapeutics is limited by their sensitivity to ribonucleases, poor cellular uptake and rapid size-mediated renal clearance. These challenges must be overcome to develop a successful siRNA-based drug. Many of these limitations could be resolved with the use of chemical modifications improving the siRNA properties.

> This review examines recent data regarding principals of the design of siRNA for the silencing of therapeutically relevant genes. A particular focus will be made on chemical modifications and their impact on siRNA potency, nuclease resistance and duration of the silencing effect. The types of chemical modifications, their location in siRNA structure influence siRNA properties in different modes: modulation of the interaction with RNAi proteins, the thermal stability and thermoasymmetry of the duplex and the sensitivity to the degradation by ribonucleases.

> Special attention will be paid to the design of siRNA for the silencing of thermodynamically unfavorable targets: mutant and chimerical genes. In this case the utilization of computer algorithms for the selection of active siRNA cannot be applied. Modification of siRNA structure aimed at the correction of the thermoasymmetry by incorporation of nucleotide substitutions, blocking incorporation of the sense strand in the RISC complex by truncation of one overhang or inactivation of the sense strand can be successfully used. Mismatches in the central part of the duplex can facilitate the cleavage and dissociation of the "passenger" (sense) strand, whereas selective chemical modification protects the non-perfect duplex from accelerated degradation.

© 2012 Chernolovskaya et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Chernolovskaya et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Peptides, steroids and other hydrophobic lipid groups can be attached to siRNA, extending the siRNA circulation time and enhancing direct cellular uptake. The potential of bioconjugation of siRNA with different biogenic molecules in altering the bioavailability and distribution of siRNAs following *in vivo* delivery will be discussed. The combination of these approaches can lead to the development of siRNAs with therapeutic value.

Structure - Functions Relations in Small Interfering RNAs 189

protein kinase PKR that inhibits translation. The expression of numerous genes, including genes encoding interferons and cytokines, alters [11, 12]. Thus, sequence-specific decrease of target mRNA level is not observed. It was found that RNAi in mammalian cells can be induced by chemically or enzymatically (*in vitro*) synthesized siRNAs or endogenously expressed siRNAs 19-21 bp in length. These siRNAs mimic the products of long dsRNAs processing by Dicer and can be involved directly into the effector phase of RNAi mechanism

The selection of the "guide" strand is a key step determining the efficacy of RNAi induced by synthetic siRNAs. At the first step of RISC\* assembly the intermediate complex RLC (RISC Loading Complex) consisting of Dicer, siRNA and R2D2 (*D.melanogaster*) or TRBP [18, 19, 20] (Homo sapiens) forms (fig. 2 A). R2D2 protein along with its human analog contains

**Figure 2.** RISC\* assembly. **A.** RLC-complex, consisting of Dicer, dsRNA-binding protein, R2D2 and

Dicer contains dsRNA-binding domain and PAZ-domain (PIWI/Argonaute/Zwille), that has an affinity to 3'-overhangs of siRNA [21, 22]. It was detected that R2D2 preferably binds with more thermodynamic stable flank of the siRNA duplex, whereas Dicer interacts with less stable siRNA flank [20, 23, 24, 25]. The orientation of siRNA relative to the protein complex Dicer-R2D2 determines the positioning of siRNA in the complex with Ago2 (a catalytic part of RISC\*) (fig. 2 B). Ago2 is a protein of Argonaute family, containing PAZ and PIWI domains. The structure of PIWI domain similar to that of RNase H determines endoribonuclease activity

passing the phase of initiation (fig. 1) [13, 14, 15, 16, 17].

two dsRNA-binding domains and a Dicer binding domain.

siRNA. **B.** Interaction of Ago2 with RLC. **C.** RISC\* formation.

### **2. Mechanism of RNAi**

RNAi phenomenon was found during transfection of dsRNAs in *C.elegans* [1] and is inherent in different organisms (flies, vertebrates, higher plants [2, 3, 4]). RNAi mechanism was initially examined in details in *Drosophila melanogaster*, but later it was found that the mechanism is highly conservative between the organisms. There are two stages of RNAi (fig. 1): at the first stage (phase of initiation) specific ribonuclease Dicer binds to and cleaves long dsRNAs yielding short (21-23 nt) duplexes with 2-overhanged nucleotides at the 3' ends (or siRNAs); at the second stage (effector phase) siRNAs molecules incorporate into multiprotein complex (RISC – RNA-induced silencing complex). One of siRNA strands ("passanger") undergo cleavage and dissociation from the complex upon RISC activation, the other strand ("guide") remains in the complex. Activated complex RISC\* specifically binds to RNA target and cleaves it (fig. 1) [5, 6, 7, 8, 9, 10] . Long dsRNAs (> 30 bp) activate the innate immune response in the mammalian cells (except for non-differentiated or low differentiated cells) resulting in non-specific RNA degradation by RNase L, activation of

**Figure 1.** Scheme of RNAi. A. Initiation phase B. Effector phase.

protein kinase PKR that inhibits translation. The expression of numerous genes, including genes encoding interferons and cytokines, alters [11, 12]. Thus, sequence-specific decrease of target mRNA level is not observed. It was found that RNAi in mammalian cells can be induced by chemically or enzymatically (*in vitro*) synthesized siRNAs or endogenously expressed siRNAs 19-21 bp in length. These siRNAs mimic the products of long dsRNAs processing by Dicer and can be involved directly into the effector phase of RNAi mechanism passing the phase of initiation (fig. 1) [13, 14, 15, 16, 17].

188 Practical Applications in Biomedical Engineering

**2. Mechanism of RNAi** 

Peptides, steroids and other hydrophobic lipid groups can be attached to siRNA, extending the siRNA circulation time and enhancing direct cellular uptake. The potential of bioconjugation of siRNA with different biogenic molecules in altering the bioavailability and distribution of siRNAs following *in vivo* delivery will be discussed. The combination of

RNAi phenomenon was found during transfection of dsRNAs in *C.elegans* [1] and is inherent in different organisms (flies, vertebrates, higher plants [2, 3, 4]). RNAi mechanism was initially examined in details in *Drosophila melanogaster*, but later it was found that the mechanism is highly conservative between the organisms. There are two stages of RNAi (fig. 1): at the first stage (phase of initiation) specific ribonuclease Dicer binds to and cleaves long dsRNAs yielding short (21-23 nt) duplexes with 2-overhanged nucleotides at the 3' ends (or siRNAs); at the second stage (effector phase) siRNAs molecules incorporate into multiprotein complex (RISC – RNA-induced silencing complex). One of siRNA strands ("passanger") undergo cleavage and dissociation from the complex upon RISC activation, the other strand ("guide") remains in the complex. Activated complex RISC\* specifically binds to RNA target and cleaves it (fig. 1) [5, 6, 7, 8, 9, 10] . Long dsRNAs (> 30 bp) activate the innate immune response in the mammalian cells (except for non-differentiated or low differentiated cells) resulting in non-specific RNA degradation by RNase L, activation of

these approaches can lead to the development of siRNAs with therapeutic value.

**Figure 1.** Scheme of RNAi. A. Initiation phase B. Effector phase.

The selection of the "guide" strand is a key step determining the efficacy of RNAi induced by synthetic siRNAs. At the first step of RISC\* assembly the intermediate complex RLC (RISC Loading Complex) consisting of Dicer, siRNA and R2D2 (*D.melanogaster*) or TRBP [18, 19, 20] (Homo sapiens) forms (fig. 2 A). R2D2 protein along with its human analog contains two dsRNA-binding domains and a Dicer binding domain.

**Figure 2.** RISC\* assembly. **A.** RLC-complex, consisting of Dicer, dsRNA-binding protein, R2D2 and siRNA. **B.** Interaction of Ago2 with RLC. **C.** RISC\* formation.

Dicer contains dsRNA-binding domain and PAZ-domain (PIWI/Argonaute/Zwille), that has an affinity to 3'-overhangs of siRNA [21, 22]. It was detected that R2D2 preferably binds with more thermodynamic stable flank of the siRNA duplex, whereas Dicer interacts with less stable siRNA flank [20, 23, 24, 25]. The orientation of siRNA relative to the protein complex Dicer-R2D2 determines the positioning of siRNA in the complex with Ago2 (a catalytic part of RISC\*) (fig. 2 B). Ago2 is a protein of Argonaute family, containing PAZ and PIWI domains. The structure of PIWI domain similar to that of RNase H determines endoribonuclease activity of PIWI [26, 27]. Ago2 cleaves the one of the siRNA strand ("passenger" strand), another strand ("guide" strand) remains into the RISC\* and guides the target RNA recognition and cleavage (fig. 2 C). The structure of the complex containing siRNA and Ago2 effects the selection of the strand (sense or antisense) that incorporates into RISC\*, hence, determines the efficacy of mRNA-target cleavage. Evidently, the antisense strand that is homologous to the sequence of mRNA-target has to be "guide" strand in order to cleave mRNA. Ago2 replaces the Dicer-R2D2 dimer (fig. 2 B), interacting with Dicer via PAZ domain [28] whereas the phosphate group at the 5'-end of the strand from the Dicer side ("guide" strand) interacts with PIWI domain that contains Mg2+ ion and basic amino acids.

Structure - Functions Relations in Small Interfering RNAs 191

target together with translation suppressor proteins (for example, helicase RCK/p54 from DEAD box family) [47]. Complex of mRNA and proteins is deposited in "P-bodies" (processing bodies - distinct foci in the cytoplasm involved in mRNA turnover) [48]. In "Pbodies" mRNA decapping followed by ribonuclease cleavage occurs. In the other cases, mRNA complexed with proteins can be deposited in "P-bodies" for a long time without degradation (fig. 4). Later, mRNAs can escape from "P-bodies" and be involved in

translation machinery or can be degraded in a described above manner [10].

**Figure 3.** Silencing of gene expression by antisense ODNs: **A.** The degradation of the duplexes: ODN / mRNA or ODN / pre-mRNA by RNase H. **B.** Inhibition of mRNA translation or pre-mRNA

The "bypass" mechanism of RISC activation is less efficient than the "classical" one, since in this case RISC\* does not act in a catalytic mode. The efficiency of RISC\* assembly also is limited by the low rate of ATP-dependent dissociation of intact siRNA strands [35, 33, 39].

Thermodynamic stability siRNA duplex is an additional parameter determining the efficiency of RISC\* assembly, hence, the efficiency of RNAi. Earlier developed algorithms for the selection of active siRNA sequences suggest selection of mRNA targets with approximately 50% GC-content. In the later studies, the percentage of G+C nucleotides in the structure of effective siRNAs varied from 30 to 50% [49, 50]. It was shown that low thermodynamic stability of the central part of the duplex (from 9th to 14th nt counting from 5'-end of the antisense strand) arising from AU-rich sequences or the presence of mismatches is a hallmark of active siRNAs [37]. The average difference between Gibbs energy of central parts of active and inactive siRNAs was found to be about 1.6 kcal/mol. Unlike thermodynamic asymmetry,

unstable center of duplex is not a sufficient criteria for selection of active siRNA [37].

Structural features of mRNA-target also effects the RNAi efficacy [51, 52, 53, 54]. The availability of the mRNA sequence for binding with siRNA (the absence of the hairpins in the secondary structure or overlapping with the binding regions of regulatory factors) as well as in the case of the ribozymes and the antisense ODN [55] can influence the silencing efficacy. In order to prove this, siRNAs targeted to mRNA sequences with different binding availability were synthesized [53]. It was shown that siRNAs targeted to the region of the initiation of translation or the 3'-end of mRNA-target were inactive. Whereas siRNAs targeted to the regions forming the hairpins displayed low and average silencing activity. As expected highperformance siRNAs were those targeted to unstructured regions of mRNA [51, 52, 54].

splicing by ODNs those are not RNase H substrates [43].

The nucleotides located at 2 - 6 positions of the "guide" strand stabilizes the interaction between siRNA and Ago2 [29, 30, 31]. The presence of the phosphate at the 5'-end of the "guide" strand is essential for RISC\* assembly [32, 33]. This orientation of siRNA with respect to catalytic PIWI-domain provides the cleavage of the complementary strand ("passenger" strand) between 9th and 10th nucleotides that facilitates the strand dissociation (fig. 2 C) [34, 35]. Ago2 and the "guide" strand are the main components of RISC\* [36], however, other proteins could also interact with RISC\* [10]. Thus, the difference between thermodynamic stability of the duplex termini (thermodynamic asymmetry) determines the orientation of Dicer-R2D2 dimer and as a result, the structure of Ago2 siRNA complex. Therefore, thermodynamic properties of siRNA play a key role in the strand selection [20, 23, 24, 25] providing the preferential selection of the strand with low thermodynamic stability at the 5'-end of the duplex as the "guide" strand [37, 38].

It was shown, that the perfect match between 2 - 12 nt of the "guide" strand (corresponding to one turn of dsRNA helix) and the target mRNA is essential for effective recognition and binding of RISC\* with mRNA target [39]. The mechanism of RNA-target cleavage (occurs between 10th and 11th nt relative to the 5'-end of the "guide" strand [40]) and the following dissociation are similar to that of the "passenger" strand (described above). Cleaved target RNA and the "passenger" strand are degraded by ribonucleases after dissociation, whereas RISC\* becomes available for acting in catalytic mode [7, 9, 17, 41]. Unlike antisense oligodeoxyribonucleotides (ODN), whose efficacy of action is determined by the efficacy of ODN hybridization with mRNA-target (fig. 3) [42], the formation of the duplex between the "guide" strand of siRNA and mRNA- target occurs due to the helicase activity of RISC\* and virtually independent from the hybridization properties of the oligoribonucleotide [43]. Silencing of gene expression by siRNAs is observed at the much lower ON concentrations than in the case of antisense ODN (IC50 for siRNAs is 100 – 1000 times lower, than IC50 for antisense ODNs) [43, 44].

Another mechanism of RISC activation - "bypass" route - employs the dissociation of the siRNA strands without preliminary cleavage of the sense strand by Ago2 [35]. This type of RISC activation is inherent in miRNAs, containing unpaired bases in the central part, critical for Ago2 endoribonuclease activity (fig. 4). Moreover, the "bypass" rout was observed for siRNAs with chemically modified nucleotides surrounding the cleavage site (between 9th and 10th nt of the "passenger" strand [45]) impeding Ago2 action [34, 35, 46]. Silencing of gene expression proceeds via the arrest of mRNA translation, since RISC\* binds to mRNA-

target together with translation suppressor proteins (for example, helicase RCK/p54 from DEAD box family) [47]. Complex of mRNA and proteins is deposited in "P-bodies" (processing bodies - distinct foci in the cytoplasm involved in mRNA turnover) [48]. In "Pbodies" mRNA decapping followed by ribonuclease cleavage occurs. In the other cases, mRNA complexed with proteins can be deposited in "P-bodies" for a long time without degradation (fig. 4). Later, mRNAs can escape from "P-bodies" and be involved in translation machinery or can be degraded in a described above manner [10].

190 Practical Applications in Biomedical Engineering

antisense ODNs) [43, 44].

PIWI domain that contains Mg2+ ion and basic amino acids.

of PIWI [26, 27]. Ago2 cleaves the one of the siRNA strand ("passenger" strand), another strand ("guide" strand) remains into the RISC\* and guides the target RNA recognition and cleavage (fig. 2 C). The structure of the complex containing siRNA and Ago2 effects the selection of the strand (sense or antisense) that incorporates into RISC\*, hence, determines the efficacy of mRNA-target cleavage. Evidently, the antisense strand that is homologous to the sequence of mRNA-target has to be "guide" strand in order to cleave mRNA. Ago2 replaces the Dicer-R2D2 dimer (fig. 2 B), interacting with Dicer via PAZ domain [28] whereas the phosphate group at the 5'-end of the strand from the Dicer side ("guide" strand) interacts with

The nucleotides located at 2 - 6 positions of the "guide" strand stabilizes the interaction between siRNA and Ago2 [29, 30, 31]. The presence of the phosphate at the 5'-end of the "guide" strand is essential for RISC\* assembly [32, 33]. This orientation of siRNA with respect to catalytic PIWI-domain provides the cleavage of the complementary strand ("passenger" strand) between 9th and 10th nucleotides that facilitates the strand dissociation (fig. 2 C) [34, 35]. Ago2 and the "guide" strand are the main components of RISC\* [36], however, other proteins could also interact with RISC\* [10]. Thus, the difference between thermodynamic stability of the duplex termini (thermodynamic asymmetry) determines the orientation of Dicer-R2D2 dimer and as a result, the structure of Ago2 siRNA complex. Therefore, thermodynamic properties of siRNA play a key role in the strand selection [20, 23, 24, 25] providing the preferential selection of the strand with low

thermodynamic stability at the 5'-end of the duplex as the "guide" strand [37, 38].

It was shown, that the perfect match between 2 - 12 nt of the "guide" strand (corresponding to one turn of dsRNA helix) and the target mRNA is essential for effective recognition and binding of RISC\* with mRNA target [39]. The mechanism of RNA-target cleavage (occurs between 10th and 11th nt relative to the 5'-end of the "guide" strand [40]) and the following dissociation are similar to that of the "passenger" strand (described above). Cleaved target RNA and the "passenger" strand are degraded by ribonucleases after dissociation, whereas RISC\* becomes available for acting in catalytic mode [7, 9, 17, 41]. Unlike antisense oligodeoxyribonucleotides (ODN), whose efficacy of action is determined by the efficacy of ODN hybridization with mRNA-target (fig. 3) [42], the formation of the duplex between the "guide" strand of siRNA and mRNA- target occurs due to the helicase activity of RISC\* and virtually independent from the hybridization properties of the oligoribonucleotide [43]. Silencing of gene expression by siRNAs is observed at the much lower ON concentrations than in the case of antisense ODN (IC50 for siRNAs is 100 – 1000 times lower, than IC50 for

Another mechanism of RISC activation - "bypass" route - employs the dissociation of the siRNA strands without preliminary cleavage of the sense strand by Ago2 [35]. This type of RISC activation is inherent in miRNAs, containing unpaired bases in the central part, critical for Ago2 endoribonuclease activity (fig. 4). Moreover, the "bypass" rout was observed for siRNAs with chemically modified nucleotides surrounding the cleavage site (between 9th and 10th nt of the "passenger" strand [45]) impeding Ago2 action [34, 35, 46]. Silencing of gene expression proceeds via the arrest of mRNA translation, since RISC\* binds to mRNA-

**Figure 3.** Silencing of gene expression by antisense ODNs: **A.** The degradation of the duplexes: ODN / mRNA or ODN / pre-mRNA by RNase H. **B.** Inhibition of mRNA translation or pre-mRNA splicing by ODNs those are not RNase H substrates [43].

The "bypass" mechanism of RISC activation is less efficient than the "classical" one, since in this case RISC\* does not act in a catalytic mode. The efficiency of RISC\* assembly also is limited by the low rate of ATP-dependent dissociation of intact siRNA strands [35, 33, 39].

Thermodynamic stability siRNA duplex is an additional parameter determining the efficiency of RISC\* assembly, hence, the efficiency of RNAi. Earlier developed algorithms for the selection of active siRNA sequences suggest selection of mRNA targets with approximately 50% GC-content. In the later studies, the percentage of G+C nucleotides in the structure of effective siRNAs varied from 30 to 50% [49, 50]. It was shown that low thermodynamic stability of the central part of the duplex (from 9th to 14th nt counting from 5'-end of the antisense strand) arising from AU-rich sequences or the presence of mismatches is a hallmark of active siRNAs [37]. The average difference between Gibbs energy of central parts of active and inactive siRNAs was found to be about 1.6 kcal/mol. Unlike thermodynamic asymmetry, unstable center of duplex is not a sufficient criteria for selection of active siRNA [37].

Structural features of mRNA-target also effects the RNAi efficacy [51, 52, 53, 54]. The availability of the mRNA sequence for binding with siRNA (the absence of the hairpins in the secondary structure or overlapping with the binding regions of regulatory factors) as well as in the case of the ribozymes and the antisense ODN [55] can influence the silencing efficacy. In order to prove this, siRNAs targeted to mRNA sequences with different binding availability were synthesized [53]. It was shown that siRNAs targeted to the region of the initiation of translation or the 3'-end of mRNA-target were inactive. Whereas siRNAs targeted to the regions forming the hairpins displayed low and average silencing activity. As expected highperformance siRNAs were those targeted to unstructured regions of mRNA [51, 52, 54].

Thus, the analysis of the secondary structure of mRNA-target and thermodynamic properties of siRNA duplex play important role in the selection of active inhibitors of gene expression.

Structure - Functions Relations in Small Interfering RNAs 193

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

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

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

melting temperature (ΔTm) increases approximately 1 0С per modification [65].

*3.1.1.1. Substituents in furanose* 

silencing were detected [59].

stability insignificantly [76].

**Figure 4.** Scheme of RNAi with the "bypass" rout of RISC\* assembly (miRNA-like action). RISC\* binds with mRNA-target (n is equal 1, if the antisense strand is fully complementary to mRNA) [10].
