Table 1. Significant siRNA chemical modifications to address siRNA drawbacks. Table 1. Significant siRNA chemical modifications to address siRNA drawbacks. Table 1. Significant siRNA chemical modifications to address siRNA drawbacks. Table 1. Significant siRNA chemical modifications to address siRNA drawbacks. Table 1. Significant siRNA chemical modifications to address siRNA drawbacks.
siRNA modified moiety siRNA chemical modification Example Functions/comments Ref.
Sugar 2ʹ-O-Me ALN-VSP02 (ASC-06), ALN-HBV, ONPATTRO (Patisiran), ARO-HBV, ALN-HBV02, Atu027 Enhancing binding affinity, melting temperature (Tm), and nuclease stability; reducing immune activation (CHIU & RANA, 2003; Weng, Xiao, Zhang, Liang, & Huang, 2019)
2ʹ-O-MOE This alteration commonly has been employed in the 3ʹ-overhangs of siRNA (employed just in the sense strand); enhancing melting temperature (Tm) and nuclease stability; reducing immune activation (Behlke, 2008; Jackson et al., 2006)
2ʹ-F ALN-HBV, ONPATTRO (Patisiran), ARO-HBV, ALN-HBV02 In every part of both sense and antisense strands can be partially modified, and there are studies of active siRNAs, which are completely changed with 2ʹF-RNA; enhancing binding affinity, melting temperature (Tm), and nuclease stability; reducing immune activation (Weng et al., 2019)
2ʹ-O-cyanoethyl Improving interaction affinity and nuclease resistance (Sekine, 2018)
2ʹ-O-acetalester Can be employed to develop protected siRNA molecules (Biscans et al., 2015)
2ʹ-esterified units (levulinates) Can be employed to develop protected siRNA molecules (Khvorova & Watts, 2017)
2ʹ-O-DNP Improving interaction affinity and resistance to nucleases while in some cases, somewhat reducing activity (Wu et al., 2016)
4ʹ-S Enhancing binding affinity and nuclease stability; is compatible with siRNA activity when placed close to the terminal of siRNA duplexes (Hoshika, Minakawa, Kamiya, Harashima, & Matsuda, 2005; Hoshika et al., 2007)
Simultaneous application of 2ʹ-O-MOE with 4ʹS-RNA and 2ʹ-O-Me Improving potency and serum stability (Dong, Siegwart, & Anderson, 2019)
2ʹF-ANA Can be tolerated in completely modified sense strands and partially modified antisense strands siRNA; improving binding affinity and nuclease resistance (Watts, Katolik, Viladoms, & Damha, 2009)
4ʹS-2ʹF-ANA Does not hinder siRNA activity at different positions in both strands. 2ʹF-ANA modifications in the sense strand are synergistic with 4ʹS-2ʹF-ANA in the antisense strand; limited modifications can be applied following the reduction of interaction affinity. (Watts et al., 2007)
LNA Improving binding affinity to RNA, which results from conformational rigidity (Vester & Wengel, 2004)
UNA Reducing binding affinity to RNA (Langkjær, Pasternak, & Wengel, 2009)
tc-DNA Can improve silencing activity when placed in the overhangs (Ittig, Schümperli, & Leumann, 2008)
CeNA Can improve the potency of siRNA (Chernikov, Vlassov, & Chernolovskaya, 2019)
ANAs Can increase the potency and duration of silencing activity when placed at the proper position (Chernikov et al., 2019)
HNAs Improving the potency of siRNA (Fisher et al., 2009)
Morpholino Can be employed in the sense strand and on the overhangs; can suppress silencing activity in the antisense strand; can eliminate backbone charges (P. Kumar et al., 2019)
Backbone Linkage Modifications PS ALN-VSP02 (ASC-06), ALN-HBV, ARO-HBV, ALN-HBV02 Enhancing nonspecific protein binding (Weng et al., 2019)
Amide-linked Enhancing thermodynamic stability and nuclease resistance of siRNA duplex (Dong et al., 2019)
Phosphonoacetate can eliminate backbone charges via esterification leading to cellular uptake without transfection reagent (Sheehan et al., 2003)
Phosphorothioate Can increase potency of siRNA (Jahns et al., 2015)
PNA Enhancing thermodynamic stability, hydrophobicity, and nuclease resistance of siRNA duplex; can eliminate backbone charges (Nielsen, Egholm, & Buchardt, 1994; Potenza et al., 2008)
2ʹ,5ʹ-linked Reducing the potency of siRNA (Prakash, Kraynack, Baker, Swayze, & Bhat, 2006)
Base Modifications 5-Me-U Enhancing siRNA stability and effective gene silencing by siRNA (Terrazas & Kool, 2008)
5-Me-C Enhancing siRNA stability and effective gene silencing by siRNA (Terrazas & Kool, 2008)
GNA ALN-HBV02, ALN-AGT Improving thermal stability; enhancing siRNA stability against snake venom phosphodiesterase; increasing siRNA potencies (Schlegel et al., 2017; Weng et al., 2019)
Diaminopurine Can improve the strength of A-U base pairs (Chiu & Rana, 2002)
2-thiouracil Improving binding affinity, potency, and specificity of siRNA (Sipa et al., 2007)
Pseudouracil Can improve the strength of A-U base pairs (Sipa et al., 2007)
2,4-difluorobenzene Can be tolerated in specific positions of siRNA; in some cases, can increase the specificity of siRNA (Somoza, Silverman, Miller, Chelliserrykattil, & Kool, 2008)
2,4-dichlorobenzene Can be tolerated in specific positions of siRNA; in some cases, can increase the specificity of siRNA (Somoza et al., 2008)
Terminal Conjugates Inverted abasic end cap ARO-HBV, AMG 890, ARO-ANG3 Can improve exonuclease stability; can be used in biophysical/biochemical studies as a result of biotin or fluorescent dyes conjugation (Weng et al., 2019)
Cholesterol conjugated Can protect siRNA duplex from HSV-2 after intravaginal administration (Shmushkovich et al., 2018)
Abbreviation: 2′-O-Me, 2′-methoxy group substitution; 2′-F, 2′- fluoro substitution; 2ʹ-O-DNP, 2′-O-dinitrophenyl ethers; 2ʹF-ANA, HNAs, hexitol nucleic acids; 2′-deoxy-2′- fluoroarabinonucleic acids; LNA, locked nucleic acid; UNA, unlocked nucleic acid; CeNA, cyclohexenyl nucleic acids; ANAs, altritol nucleic acids; PS, Phosphorothioate; PNA, peptide nucleic acid; GNA, glycol nucleic acid; 2′-O-MOE, 2′-O-methoxyethyl; tc-DNA, tricyclo-DNA modification Abbreviation: 2′-O-Me, 2′-methoxy group substitution; 2′-F, 2′- fluoro substitution; 2ʹ-O-DNP, 2′-O-dinitrophenyl ethers; 2ʹF-ANA, HNAs, hexitol nucleic acids; 2′-deoxy-2′- fluoroarabinonucleic acids; LNA, locked nucleic acid; UNA, unlocked nucleic acid; CeNA, cyclohexenyl nucleic acids; ANAs, altritol nucleic acids; PS, Phosphorothioate; PNA, peptide nucleic acid; GNA, glycol nucleic acid; 2′-O-MOE, 2′-O-methoxyethyl; tc-DNA, tricyclo-DNA modification Abbreviation: 2′-O-Me, 2′-methoxy group substitution; 2′-F, 2′- fluoro substitution; 2ʹ-O-DNP, 2′-O-dinitrophenyl ethers; 2ʹF-ANA, HNAs, hexitol nucleic acids; 2′-deoxy-2′- fluoroarabinonucleic acids; LNA, locked nucleic acid; UNA, unlocked nucleic acid; CeNA, cyclohexenyl nucleic acids; ANAs, altritol nucleic acids; PS, Phosphorothioate; PNA, peptide nucleic acid; GNA, glycol nucleic acid; 2′-O-MOE, 2′-O-methoxyethyl; tc-DNA, tricyclo-DNA modification Abbreviation: 2′-O-Me, 2′-methoxy group substitution; 2′-F, 2′- fluoro substitution; 2ʹ-O-DNP, 2′-O-dinitrophenyl ethers; 2ʹF-ANA, HNAs, hexitol nucleic acids; 2′-deoxy-2′- fluoroarabinonucleic acids; LNA, locked nucleic acid; UNA, unlocked nucleic acid; CeNA, cyclohexenyl nucleic acids; ANAs, altritol nucleic acids; PS, Phosphorothioate; PNA, peptide nucleic acid; GNA, glycol nucleic acid; 2′-O-MOE, 2′-O-methoxyethyl; tc-DNA, tricyclo-DNA modification Abbreviation: 2′-O-Me, 2′-methoxy group substitution; 2′-F, 2′- fluoro substitution; 2ʹ-O-DNP, 2′-O-dinitrophenyl ethers; 2ʹF-ANA, HNAs, hexitol nucleic acids; 2′-deoxy-2′- fluoroarabinonucleic acids; LNA, locked nucleic acid; UNA, unlocked nucleic acid; CeNA, cyclohexenyl nucleic acids; ANAs, altritol nucleic acids; PS, Phosphorothioate; PNA, peptide nucleic acid; GNA, glycol nucleic acid; 2′-O-MOE, 2′-O-methoxyethyl; tc-DNA, tricyclo-DNA modification