siRNA / shRNA / ASO Designer — Reynolds/Ui-Tei siRNA & 5-10-5 Gapmers
Design knockdown reagents against an mRNA — Reynolds/Ui-Tei-scored siRNAs with ready shRNA cassettes, or 5-10-5 ASO gapmers screened for known liabilities.
🌐 Runs on the SeqBench API — also callable via REST & MCP, and in bulk from the batch tools
Paste a target transcript and design either siRNAs/shRNAs or antisense-oligonucleotide (ASO) gapmers. siRNA candidates are scored with the two most-cited rational-design rule sets — Reynolds et al. 2004 (an 8-criteria efficacy score) and Ui-Tei et al. 2004 (four asymmetry/seed rules) — and flagged for seed-region off-target risk using a siDirect-style seed-duplex melting temperature (≥21.5 °C). Each siRNA comes with its sense and guide strands (with UU 3' overhangs) ready to order and a fully-assembled shRNA cassette (sense–loop–antisense–Pol III terminator). ASO mode builds each antisense oligo in the standard 5-10-5 gapmer layout (chemically-modified wings, central DNA gap for RNase H1) and screens for the classic liabilities: G-quadruplex motifs, CpG immunostimulation, self-complementarity, and GC extremes. All scoring is sequence/thermodynamic and runs in your browser; mRNA secondary-structure accessibility and transcriptome-wide off-target search are honestly out of scope for this version.
How to use the siRNA / shRNA / ASO Designer tool
- 1Paste the target mRNA/cDNA and choose siRNA/shRNA or ASO gapmer mode.
- 2For siRNA: review candidates ranked by Reynolds score and Ui-Tei pass, check the seed-Tm off-target flag, and copy the sense/guide oligos or the ready shRNA cassette.
- 3For ASO: review the 5-10-5 gapmers, avoid ones flagged for G-quadruplex/CpG/self-structure, and copy the antisense sequence.
- 4Validate genomic/transcriptomic specificity separately — this tool does not run a genome-wide off-target search.
Frequently asked questions
Which siRNA design rules are used?
Reynolds et al. 2004 (an 8-criterion score: GC 30–52%, A/U-rich 3' end of the sense strand, specific position preferences, no long GC runs) and Ui-Tei et al. 2004 (A/U at the guide 5' end, G/C at the guide 3' end, an A/U-rich seed, no long GC stretch). Candidates are ranked by Reynolds score with the Ui-Tei pass and seed-Tm flag factored in. A Reynolds score ≥6 is the common "good candidate" threshold.
How is off-target risk assessed?
With a siDirect-style seed-duplex melting temperature — the Tm of guide positions 2–8, the region that drives miRNA-like off-target silencing. A seed Tm ≥21.5 °C is flagged as higher off-target risk. This is a sequence-thermodynamic proxy, not a genome-wide search: it does not check how many transcripts share the seed. Run a dedicated off-target/BLAST search before committing.
What does the shRNA cassette contain?
Sense strand – loop (default TTCAAGAGA) – antisense strand – a Pol III terminator (TTTTT), assembled as DNA ready to clone into a U6/H1 expression vector. The loop is configurable via the API.
What about the ASO gapmer chemistry?
The tool designs the base antisense sequence in the standard 5-10-5 architecture — a 10-nt central DNA gap (for RNase H1 cleavage) flanked by 5-nt modified wings (2'-MOE or LNA), on a phosphorothioate backbone. The specific wing chemistry is a synthesis choice shown as an annotation; the tool's job is the sequence and screening it for G-quadruplex motifs, CpG dinucleotides, self-complementarity and GC extremes.
Does it model mRNA secondary structure / target accessibility?
Not in this version — the established Reynolds/Ui-Tei/gapmer rules don't require folding, and this tool implements those honestly rather than faking a folding model. A structure-aware accessibility upgrade (using a real RNA-folding engine) is planned as a follow-on.
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