siAbasic: a comprehensive database for potent siRNA-6Ø sequences without off-target effects

Abstract

Small interfering RNA (siRNA) is widely used to specifically silence target gene expression, but its microRNA (miRNA)-like function inevitably suppresses hundreds of off-targets. Recently, complete elimination of the off-target repression has been achieved by introducing an abasic nucleotide to the pivot (position 6; siRNA-6Ø), of which impaired base pairing destabilizes transitional nucleation (positions 2–6). However, siRNA-6Ø varied in its conservation of on-target activity (∼80–100%), demanding bioinformatics to discover the principles underlying its on-target efficiency. Analyses of miRNA–target interactions (Ago HITS-CLIP) showed that the stability of transitional nucleation correlated with the target affinity of RNA interference. Furthermore, interrogated analyses of siRNA screening efficiency, experimental data and broadly conserved miRNA sequences showed that the free energy of transitional nucleation (positions 2–5) in siRNA-6Ø required the range of stability for effective on-target activity (−6 ≤ ΔG[2:5] ≤ −3.5 kcal mol−1). Taking into consideration of these features together with locations, guanine-cytosine content (GC content), nucleotide stretches, single nucleotide polymorphisms and repetitive elements, we implemented a database named ‘siAbasic’ that provided the list of potent siRNA-6Ø sequences for most of human and mouse genes (≥ ∼95%), wherein we experimentally validated some of their therapeutic potency. siAbasic will aid to ensure potency of siRNA-6Ø sequences without concerning off-target effects for experimental and clinical purposes.

AGO-accessible anticancer siRNAs designed with synergistic miRNA-like activity

“… 22 However, not all perfectly complementary sequences are equally effective; thus, siRNA sequences were generally screened by the gene-walking method that could comprehensively identify the most potent sequence for therapeutic purposes. Bioinformatics that considers the reported features of potent siRNA can assist in this rigorous approach, 25 but its application is often limited. Since siRNAs are also AGO-dependent, AGO-bound regions analyzed by CLIP were the most efficiently targeted by RNAi.…”

“… 22 To this end, chemical modifications such as abasic pivot substitution 23 , 24 have been developed to avoid miRNA-like off-targeting and maintain on-target activity. 25 …”

The double edge of anti-CD40 siRNA therapy: It increases renal microcapillar density but favours the generation of an inflammatory milieu in the kidneys of ApoE−/− mice

“…To reduce these unwanted effects a number of strategies have been devised, as reducing the concentration of siRNA used [28], modifying the structure of the siRNA backbone [29], introducing chemical modifications to the seed sequence [30] or introducing sequential information and structural features of the sequences flanking the seed sequence [31]. Furthermore, a number of algorithms [32, 33] and databases [34, 35] have been established to facilitate the development of more specific siRNAs with less off-targets effects.…”

AGO CLIP-based imputation of potent siRNA sequences targeting SARS-CoV-2 with antifibrotic miRNA-like activity

“…siRNAs loaded on AGO can also function similarly to miRNAs, suppressing hundreds of off-target transcripts wherever the seed region can make a match 18 . Thus, miRNA-like off-targets must be evaluated and controlled 9 , where chemical modifications, including abasic pivot substitution 19 , 20 , have successfully prevented the off-targeting 21 . However, a miRNA-mimicking activity could be advantageously arranged in siRNAs by containing the same seed sequences of miRNAs with specific functions 22 , as shown in the development of synergistic anticancer siRNA drugs 23 .…”