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.
Predicted, not measured
- How good is it?
- A shortlist from published design rules, not a predicted knockdown level — no accuracy is claimed for the ranking. It does not fold the target, so it cannot see whether the site is accessible, and it does not search the transcriptome for off-targets. Order several and measure them.
- Only valid for:
- mRNA targets. The seed Tm is computed on siDirect's own RNA/RNA scale (Freier 1986 parameters, helix initiation A = -10.8, CT = 100 µM, 100 mM Na+) and is not comparable with the DNA/DNA Tm reported elsewhere in the toolkit.
How this was measured, and what it was fitted on
These are rule counts, not a regression, and no skill statistic is claimed for the ranking. Target-site accessibility is not modeled (no folding) and no transcriptome-wide off-target search is performed, so a top-ranked candidate is a starting point for a knockdown panel, not a predicted knockdown level.
Rule sets distilled from measured knockdown of small siRNA panels: Reynolds et al. 2004 (8 criteria) and Ui-Tei et al. 2004. The off-target flag uses the siDirect seed-duplex Tm threshold of Ui-Tei et al. 2008.
Paste a target transcript and design either siRNAs/shRNAs or antisense-oligonucleotide (ASO) gapmers. siRNA candidates are scored with the two most-cited design rule sets — Reynolds (an 8-criteria efficacy score) and Ui-Tei (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-based and thermodynamic; mRNA secondary-structure accessibility and transcriptome-wide off-target search are 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?
The Reynolds and Ui-Tei rules — the two most-cited siRNA design rule sets. Candidates are ranked by Reynolds score with the Ui-Tei pass and the seed-Tm flag factored in; a Reynolds score ≥6 is the common "good candidate" threshold, and each candidate's row shows which individual criteria it passed.
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 Reynolds/Ui-Tei/gapmer rules do not require folding, so this tool applies those rather than faking a folding model. Fold the region separately if accessibility matters to you.