Three of the numbers a designer wants most are not printed here: whether the oligonucleotide folds on itself, whether the site on the transcript is open enough to be bound, and how many other places in the transcriptome it could bind. Each of those has a published model, and each needs either a folding calculation over thousands of bases or an alignment against the whole of the pre-messenger RNA. Neither is arithmetic over the pasted sequence, so neither runs on this page. What follows is what the literature says about each, in its own words.
01
Why designs sit between sixteen and twenty bases
However, the specificity for the cognate sequence versus all other RNAs varies inversely with length to at least 18 to 20 nucleotides. Theoretical analyses and experimental results have shown that higher-affinity PS ASOs have optimal affinity and specificity between 16 and 18 nucleotides (64). The propensity of PS ASOs to form self-structures that inhibit binding to cognate sequences and other effects increases when ASO length exceeds 20 to 22 nucleotides, resulting in current approaches being generally limited to 16 to 20 nucleotides (1, 33).
[R10]
Shorter and an oligonucleotide struggles to bind one transcript and only that transcript; longer and it begins to fold on itself. The tool prints the length of whatever is pasted and no verdict on it.
02
What a strong candidate's energies look like
To achieve optimal statistical preference, the values for self-interaction should be (DeltaG(o)37) > or = -8 kcal/mol for inter-oligonucleotide pairing and (DeltaG(o)37) > or = -1.1 kcal/mol for intra-molecular pairing.
[R07]
The same analysis, over more than a thousand experiments, puts an effective candidate at -30 kcal per mol or stronger against its target [R07]. This tool computes the duplex free energy and computes neither self-interaction figure, so it prints the published targets and scores nothing against them, the duplex figure included: a free energy is a statement about a model and a salt concentration as much as about a sequence, and two of them are comparable only when both were computed the same way.
03
Why no off-target score is printed
We have observed examples where different accessibility most likely accounts for the differences in off-target activity, e.g. in the case of BBS9 and FGF5 which both have two mismatches in the same position but differ by almost 12-fold in EC50
[R09]
Two candidates with the same number of mismatches can differ more than tenfold in how much they knock down the wrong transcript, so a mismatch count is not a safety ranking. That group's own search was a full alignment against a genomic database with the pseudogenes removed [R09], not a quick scan, and it covered introns: a cleavage-dependent oligonucleotide acts on the transcript before it is spliced, so a search that reads only mature messenger RNA is reading the smaller half of the problem [R08] [R09].
04
A melting temperature is a statement about conditions
The millimolar concentration of salt [Na+] can be entered and will adjust the salt adjusted and nearest neighbor melting temperature calculations. The default value is 50 mM. The nanomolar concentration of primer in the hybridization solution can also be entered and will adjust the nearest neighbor melting temperature.
[R06]
Change the salt or the strand concentration and the number changes, so a melting temperature printed without both beside it is not a melting temperature. The same paper puts the nearest-neighbor method at its most accurate on oligonucleotides of the length this catalogue sells, and says that a chemically modified oligonucleotide falls outside it [R06].
Which duplex, and why it matters
An antisense oligonucleotide pairs with RNA, not with DNA, and the two duplexes have different parameter tables. The table this tool uses was measured for that pairing at a salt concentration close to a cell's rather than at the one molar of a laboratory standard, which is what its authors set out to correct [R01]. Its parameters were revised the year after publication, and this tool uses the revised ones [R02]. The three other cases are named in the tool with the paper that measured each, and none of them is computed here.
Every one of those measurements was made on unmodified strands. So no melting temperature is printed for a modified oligonucleotide under any framing, and that includes the chemistry AUMsilence™ sdASO™ is made in: a number from a table that does not cover the material would be a false statement in a unit, and this tool prints the published guidance instead.
