When the calculation is complete, an html (hypertext markup language) page is generated with links to tables containing predicted siRNA efficacy data and thermodynamic binding data. In the siRNA efficacy table (), the sequences of siRNA candidates are ranked in the output list by their probabilities of being efficient siRNA. The probabilities are predicted by a SVM embedded in the web server for selecting efficient siRNA. The classification model (16
) used in the SVM was trained with a publically available database (10
), using thermodynamic and sequence features of siRNA candidates. The position number of each siRNA candidate is also listed in the table as the index of the 5′ most base in the target-binding region.
A table of siRNA candidates generated by OligoWalk server. The sequences of siRNA are ranked from top to end by their probabilities of being efficient (antisense efficacy larger than 70%).
In addition, the predicted equilibrium thermodynamics table is generated as a reference for advanced users. In the table, the position number and sequence of each siRNA candidate appear with thermodynamic terms. ‘Overall’ (in kcal/mol) is the overall free energy change of oligonucleotide-target binding,
when all contributions are considered, including breaking target and oligonucleotide self-structures (18
). A more negative value indicates tighter binding. It is affected by the oligonucleotide concentration. ‘Duplex’ (in kcal/mol) is the free energy change of hybridized duplex between oligonucleotide and target (antisense–sense duplex),
. The value is independent of oligonucleotide concentration because it is a standard free energy change. ‘Tm-Dup’ (in °C) is the melting temperature in degrees for the duplex formation of oligonucleotide and target. ‘Break-targ’. (in kcal/mol) is the free energy cost to open the intramolecular target base pairs for oligonucleotide binding,
. A more negative number indicates higher free energy cost, which is unfavorable for oligonucleotide-target binding. ‘Intraoligo’ (in kcal/mol) is the free energy change of intramolecular oligonucleotide structure,
. It usually has a negative value or, if there is no favor-able intramolecular structure, it is zero. ‘Interoligo’ (in kcal/mol) is the free energy change of intermolecular oligonucleotide structure,
. A negative number indicates a stable antisense–antisense bimolecular structure, which decreases the oligonucleotide-target (antisense–sense) binding affinity. ‘End_diff’. (in kcal/mol) is the free energy difference between the 5′ and 3′ end of the antisense strand of siRNA, with windows of two base pairs. Functional siRNA prefer to have an unstable 5′ end (3
), which means a positive End_diff. ‘Prefilter_score’ is the score calculated with a method based on the empirical rules by Reynolds et al.
). All the scores are calculated in the same way as Reynolds et al.
), except for the melting temperature of intramolecular oligonucleotide self-structure because the free energy (21
) and enthalpy parameters (22
) used by OligoWalk are more recent. When calculating the prefilter score, 57°C is used as the cutoff of the intramolecular oligonucleotide melting temperature, as suggested in another study (23
As an example, the prediction of the webserver is compared with experimental results in . In the experiment (3
), siRNA were tested for efficacy against the target mRNA, Human Cyclophilin (Genbank ID: M60857), at 37°C. The inhibition efficacy of each siRNA is defined as 100% minus the percentage of mRNA level after siRNA application as compared to matched control. The prediction result is the probability of being efficient (having inhibition efficacy larger than 70%), which is calculated with the server. In , most of the siRNA with high inhibition efficacy are predicted to have high probability of being efficient.
Figure 2. A comparison between the prediction and experimental result. In blue is the experimental result (inhibition efficacy) for an mRNA target, Human Cyclophilin (Genbank ID: M60857), at 37°C (3). The inhibition efficacy is defined as 100% minus the (more ...)