Table 1.

Rules for siRNA design

Design rule	References
General targeting rules
Select the target region preferably 50–100-nt downstream of the start codon	[21]
Avoid to target the middle of the coding sequence of the target gene	[64]
Pooling of four or five siRNA duplexes per gene	[64]
Nucleotide composition rules
Antisense strand with higher information content	[65]
Absence of any GC stretch >9 nt long	[66]
At least five A/U residues in the 5′ terminal one-third of the antisense strand	[49, 66, 67]
A higher “A/U” content in the 3′ end than that in the 5′ end (sense strand)	[68]
G/C content ranges: 32–58 %, 30–52 %, 32–79 %, 36–53 %, 35–73 %, 25–55 %	[21, 41, 68–71]
Absence of internal repeats	[41]
Presence of motifs “AAC,” “UC,” “UG,” “AAG,” “AGC,” “UCU,” “UCCG,” “CUU,” “CU,” “GUU,” “UCC,” “CG,” “AUC,” “GCG,” “UUU,” “ACA,” “UUC,” “CAA” in antisense strand	[49, 70–72]
Avoid motifs “CUU,” “CUA,” “GUU,” “GU,” “GAU,” “ACGA,” “GCC,” “GUGG,” “CCC,” “GGC,” “CCG,” “GGG,” “CAG,” “GAG,” “GCA,” “AUA,” “CUG,” “AG,” “GG,” “GGA” in antisense strand	[49, 70–72]
High content of “U” in antisense strand	[72]
Low content of “G” in antisense strand	[72]
Thermodynamics rules
Total hairpin energy <1	[69]
Antisense 5′ end binding energy <9	[69]
Sense 5′ end binding energy in range 5–9	[69]
Middle binding energy <13	[69]
Energy difference <0	[69]
Energy difference within −1 and 0	[69]
Significant ΔG difference between positions 1 and 18	[67]
High ΔG in positions 1–4, 5–8, and 13–14 in the antisense strand	[70]
Low ΔG in positions 18–19 in the antisense strand	[70]
Avoid folding of siRNA	[70]

The rules are classified in four different categories, three of which are summarized in this table (see also Fig. 1b). For each rule, or group of rules, references are given