. 2019 Apr 26;10:444. doi: 10.3389/fphar.2019.00444

Table 1.

The effect of chemical modifications on siRNA properties.

Modification	_ΔT_m duplex per modi-fication	Impact on the efficiency of RNAi	Other properties of modification (effect on ribose conformation, nuclease resistance, toxicity, etc.)
SUGAR MODIFICATIONS
2′-O-methyl (2′O-Me)	+0.5–1.5°C	Two or more consecutive 2′O-Me inhibits RNAi (Czauderna et al., 2003; Prakash et al., 2005; Akinc et al., 2008; Manoharan et al., 2011). However, siRNAs possessing biological activity, containing 75–82% 2′O-Me, are described (Ray et al., 2017; Foster et al., 2018).	Stabilizes 3′endo ribose conformation. ≥5–30% of 2′O-Me increase nuclease resistance in vitro (Jackson et al., 2006; Volkov et al., 2009; Petrova Kruglova et al., 2010; Takahashi et al., 2012) and in vivo (Liu et al., 2014; Chernikov et al., 2017). 2′O-Me analogs of A, G and U reduce the immune response (Judge et al., 2006).
2′-fluoro (2′F)	+1.5–4°C	2′F analogs in all siRNA positions only slightly reduces the activity of RNAi (Deleavey et al., 2010).	Stabilizes 3′endo ribose conformation. ≥50% 2′F increase nuclease resistance in vitro (Cuellar et al., 2014) and in vivo (Viel et al., 2008; Manoharan et al., 2011). 2′F analogs of adenine (≥7%) reduce the immune response in vitro (Fucini et al., 2012). >50% of the 2′F in siRNA may cause toxicity (Ohrt and Schwille, 2008; Shen et al., 2015; Garber, 2016).
2′F-arabinonucleic acid (2′FANA)	+1.2°C	100% 2′FANA in the sense chain reduce the efficiency of RNAi. ≥30% 2′FANA in the antisense chain inhibits RNAi (Dowler et al., 2006; Deleavey et al., 2010).	Stabilizes 2′endo ribose conformation. ≥50% 2′FANA increases nuclease resistance in vitro (Deleavey et al., 2010); more effectively than 2′F protect siRNA from the action of exoribonucleases (Damha et al., 2001).
2′-O-methoxyethyl (2′O-MOE)	+0.9–1.7°C	2′-MOE at the flanks of the sense strand and the central part (6–11) of the antisense strand are tolerable for RNAi (Prakash et al., 2005; Manoharan et al., 2011). Replacement of 9th or 10th nucleotides from the 5′ end to 2′O-MOE analogs of nucleotide increases the probability of entry in RISC (Song et al., 2017).	Stabilizes 3′endo ribose conformation. ≥15% 2′O-MOE at the ends of the siRNA sense chain increases nuclease resistance in vitro (Lima et al., 2012).
Locked nucleic acid (LNA)	+2–8°C	≥40% LNA in the sense chain inhibit RNAi by 5–20% (Elmen et al., 2005). >20% LNA in the antisense chain, or the first LNA nucleotide at the 5′ end completely inhibit RNA (Braasch et al., 2003; Elmen et al., 2005; Mook et al., 2007; Schyth et al., 2012). LNA can change thermal asymmetry of the duplex, increasing the efficiency of siRNA (Elmen et al., 2005).	Reduces the conformational flexibility of nucleotides, fixing the C3′endo conformation of the ribose (Julien et al., 2008). ≥10–20% LNA in siRNAs increase nuclease resistance in vitro (Elmen et al., 2005) and in vivo (Mook et al., 2010).
Unlocked nucleic acid (UNA)	−5–8°C	>15% UNA inhibit RNAi (Laursen et al., 2010). UNA can change thermal asymmetry of the duplex, increasing the efficiency of siRNA (Mook et al., 2010; Vaish et al., 2011; Snead et al., 2013).	Increases conformational flexibility of nucleotides and reduces the melting point of the duplex. UNA at the 3′ends of the duplex protect siRNA from 3′ exoribonucleases in vitro and in vivo (Laursen et al., 2010; Mook et al., 2010; Pasternak and Wengel, 2011). 4′-thioribonucleosides (4′S)
4′-thioribonucleosides (4′S)	+1°C	>7–15% 4′S in the antisense strand inhibit RNAi (Hoshika et al., 2005, 2007; Dande et al., 2006).	>10–15% 4′S at the ends of the strands increase the nuclease resistance in vitro (Dande et al., 2006; Takahashi et al., 2012).
4′-C-aminomethyl-2′-O-methyl	−1°C	>2 analogs in the sense or >1 analog in the antisense strand inhibit RNAi (Gore et al., 2012).	≥2 modifications at the 3′ ends increase nuclease resistance in vitro (Gore et al., 2012).
Deoxyribonucleotide (dNMP)	−0.5°C	>50% dNMP inhibits RNAi (Parrish et al., 2000; Elbashir et al., 2001; Ui-Tei et al., 2008). dNMP can change thermal asymmetry of the duplex, increasing the efficiency of siRNA in vitro (Ui-Tei et al., 2008).	Protects against exoribonucleases (Parrish et al., 2000).
Cyclohexenyl nucleic acids (CeNA)	+1.5°C	5% CeNA in siRNA are tolerated by RNAi (Herdewijn and Juliano, 2007; Nauwelaerts et al., 2007). CeNA can change thermal asymmetry of the duplex, increasing the efficiency of siRNA in vitro (Herdewijn and Juliano, 2007; Fisher et al., 2009).	Stabilizes 3′endo ribose conformation (Ovaere et al., 2011). ≥25% CeNA analogs increase serum nuclease resistance (Wang et al., 2001).
Hexitol nucleic acids (HNA)	+0.85°C	15% HNA in siRNA are tolerated by RNAi (Fisher et al., 2009). HNA can change thermal asymmetry of the duplex, increasing the efficiency of siRNA in vitro (Herdewijn and Juliano, 2007).	Slightly increases siRNA resistance to nucleases in serum (Fisher et al., 2009).
PHOSPHATE BACKBONE MODIFICATIONS
Phosphorothioate (PS)	−0.7°C	PS inhibits RNAi when introduced in the central part of the antisense strand (Amarzguioui et al., 2003; Schwarz et al., 2004; Prakash et al., 2005; Eckstein, 2014).	PS protects siRNAs from the action of exoribonucleases in vitro and in vivo (Soutschek et al., 2004). >50% PS cause toxicity in vitro (Harborth et al., 2003) and in vivo (Henry et al., 2002; Iannitti et al., 2014). Dimethylethylenediamine (DMEDA)
Dimethylethylenediamine (DMEDA)	−0.7–3.4°C (shown only for thymidine)	10% DMEDA in the sense strand are tolerated by RNAi (Vlaho et al., 2017).	The effect on nuclease resistance of siRNA was not shown.
Tert-butyl-S-acyl-2-thioethyl (tBu-SATE)	No data.	25% tBu-SATE are tolerated by RNAi (Meade et al., 2014).	≥20–40% tBu-SATE in siRNA increase nuclease resistance in vitro and in vivo (Meade et al., 2014). Increases hydrophobicity of siRNA. Cleaved by thioesterase in the cytoplasm of the cell giving a phosphodiester bond (Meade et al., 2014).
Boranophosphate (BP)	+0.4–1°C (<50% of siRNA) −0.8–2.5°C (>50% of siRNA)	>50% PB inhibit RNAi, the central part of the antisense strand is the most sensitive to modifications (Hall et al., 2004).	Approximately two times more effectively protect against ribonucleases than PS, but do not cause toxicity in vitro (Hall et al., 2004, 2006).
Amide linker	−0.3 to +0.9°C	In some siRNA positions, a single substitution for an amide linker is tolerated by RNAi (Mutisya et al., 2017).	The introduction of two amide linkers from the 3′ ends of the duplex increases the nuclease resistance of siRNA in serum (Iwase et al., 2007; Selvam et al., 2011).
5′-PHOSPHATE MODIFICATIONS
5′C-methyl (S-isomer)	−3.2°C	One (S) 5′C-methyl at the 5′ end of the antisense strand is tolerated by RNAi (Prakash et al., 2015).	(S) 5′C-methyl protect siRNA from exonucleases two times more efficiently than PS (Kel'in et al., 2016).
5′ (E)-vinylphosphonate	No data.	5′(E)-vinylphosphonate 2-folds improves siRNA interaction efficiency with Ago2 (Elkayam et al., 2017). Does not change the biological activity of siRNA in vitro (Haraszti et al., 2017).	Stabilizes 5′ phosphate, protect from the action of phosphatases and exonucleases. Improves the pharmacokinetics (Elkayam et al., 2017; Haraszti et al., 2017). 5′ methylenephosphonate
5′ methylenephosphonate	No data.	5′ methylenephosphonate at the 5′ end of the antisense strand reduces the biological activity of siRNA by ~10-folds (Lima et al., 2012; Prakash et al., 2015).	No data.
BASE MODIFICATIONS
2′ thiouridine (s²U)	0–2°C	7% s²U are tolerated by RNAi (Sipa et al., 2007). s²U can change thermal asymmetry of the duplex, increasing the efficiency of siRNA in vitro (Sipa et al., 2007; Peacock et al., 2011).	s²U slightly increases nuclease resistance in vitro.
Pseudouridine (Ψ)	−1 to +1°C	One Ψ is tolerated by RNA(Sipa et al., 2007).	Stabilizes 3′endo ribose conformation. Reduces the PKR-induced interferon response (Anderson et al., 2010).