About the tools

siSPOTR - siRNA Design Tool

This simple interface allows a user to paste or upload a candidate mRNA sequence (plain or FASTA format) intended for knockdown, and produces an output of candidate si/shRNAs sorted with the lowest POTS (off-targeting potential) at the top. The output provides the actual sense and antisense sequences to order, making sequence modifications to improve loading of the appropriate strand.


The output provides the following information regarding the candidate sequence:

Rank

Rank position of the siRNA in regards to POTS. Lower=less off-target potential.

Start Position

Shows the location of the siRNA binding site in the input sequence.

% G/C Content

Shows what percent of the siRNA sequence is composed of G's and C's. Default criteria set a generous range of 20-70%. Potent siRNAs have been found with G/C content in this range.

POTS (Probability of Off-Targeting Score)

This is a representation of the off-targeting potential of a given sequence that combines seed complement frequencies as well as estimates of their probability of being off-targeted if an experiment overexpressing that siRNA. Probabilities are assigned by first categorizing seed matches based on rules used by the Targetscan algorithm used for predicting targets for endogenous miRNAs:

In increasing order of potency:
  • 6mer: Seed match only to nt 2-7
  • 7mer-1A: In addition to a seed match to nt 2-7, an "A" nt is found in the target across from nt1.
  • 7mer-M8: Seed match extends to nt 2-8.
  • 8mer: A combination of a 1A and M8 site

Microarray data were used to estimate the probability that a gene containing a seed match of a particular type would be down-regulated at least 0.3 log2 fold relative to the control conditions. These probabilities are applied to each predicted target, and used to calculate an overall score for a particular seed sequence.

See the Targetscan documentation and literature for more information.

Percentile (% worse)

The percent of all 7mer sequences with a worse (higher) POTS value

Seed Sequence

This is the seed sequence for the siRNA used for calculating POTS.

miRNA seed

If the seed of the siRNA matches a known endogenous miRNA, the matching miRNA family is presented here. We suggest avoiding sequences with miRNA seeds, as many predicted miRNA binding sites have evolved to facilitate miRNA accessibility. Consequently, work developing miRNA seed targeting rules demonstrated a much higher probability of being targeted than we have predicted from artificial siRNA expression data.

miRNA Conservation

This represents the conservation of the miRNA with a matching seed sequence. H(uman), M(ouse) and D(og) are given as representative species. Sequences with only one of the three letters shows that the miRNA is specific to that species or evolutionary lineage. H,M says that orthologus miRNAs are found in both human and mouse genomes. H,M,D means that the miRNA is at the very least conserved in those 3 species, and are often much more conserved than that. The more conserved a miRNA is, the more likely conserved, functional target sites exist.


siRNA POTS Lookup

This tool allows researchers to determine the off-targeting potential for siRNAs they have already designed. Input can be a list of sequences, separated by a line break, corresponding to the antisense sequence. Output not only provides the POTS for the siRNA, but also a percentile score that shows the percent of seed sequences with worse POTS values.

Run siRNA POTS Lookup

siRNA antisense sequences (limit 20)
NOTE: For multiple sequences, enter one per line.

The output provides the following information:

Input Order

The order the sequences were entered.

POTS (Probability of Off-Targeting Score)

This is a representation of the off-targeting potential of a given sequence that combines seed complement frequencies as well as estimates of their probability of being off-targeted if an experiment overexpressing that siRNA. Probabilities are assigned by first categorizing seed matches based on rules used by the Targetscan algorithm used for predicting targets for endogenous miRNAs:

In increasing order of potency:
  • 6mer: Seed match only to nt 2-7
  • 7mer-1A: In addition to a seed match to nt 2-7, an "A" nt is found in the target across from nt1.
  • 7mer-M8: Seed match extends to nt 2-8.
  • 8mer: A combination of a 1A and M8 site

Microarray data were used to estimate the probability that a gene containing a seed match of a particular type would be down-regulated at least 1.5 fold relative to the control conditions. These probabilities are applied to each predicted target, and used to calculate an overall score for a particular seed sequence.

See the Targetscan documentation and literature for more information.

Percentile (% worse)

The percent of all 7mer sequences with a worse (higher) POTS value

Seed Sequence

This is the seed sequence for the siRNA used for calculating POTS.

miRNA seed

If the seed of the siRNA matches a known endogenous miRNA, the matching miRNA family is presented here. We suggest avoiding sequences with miRNA seeds, as many predicted miRNA binding sites have evolved to facilitate miRNA accessibility. Consequently, work developing miRNA seed targeting rules demonstrated a much higher probability of being targeted than we have predicted from artificial siRNA expression data.

miRNA Conservation

This represents the conservation of the miRNA with a matching seed sequence. H(uman), M(ouse) and D(og) are given as representative species. Sequences with only one of the three letters shows that the miRNA is specific to that species or evolutionary lineage. H,M says that orthologus miRNAs are found in both human and mouse genomes. H,M,D means that the miRNA is at the very least conserved in those 3 species, and are often much more conserved than that. The more conserved a miRNA is, the more likely conserved, functional target sites exist.




Additional Features

These options are available in both tools by clicking the "Off-Target Details" button on the result of interest.

Details of POTS score:
This provides summary information on the total number of predicted off-targets and the distribution of the various "site-types" found in those predicted targets. The POTS value is calculated by analyzing the potential off-target genes for seed matches conforming to known endogenous microRNA targeting rules. "8mer" sites tend to be the most potent followed by (in decreasing order) 7mer-M8, 7mer-1A and 6mer.

Potential off-target list:
A list of probable off-target genes rank-ordered by individual transcript Probability of Off-Target Score (tPOTS). tPOTS is calculated based on the number and type of seed matches found in that transcript. The number of 8mer, 7mer-M8, 7mer-1A and 6mer sites are also shown. The entire list of off-targets can be exported to a CSV file to analyze as a simple spreadsheet.

Run BLAST search:
This is a link to the NCBI nucleotide blast page. In addition to seed-mediated off-targets, siRNAs can have more extensive binding to mRNAs outside of the 3'UTR. The option to search the full 21-mer, or the core 18-mer are provided (p1 and p20/21 mismatches often show negligible impact on silencing efficacy). Previous studies have suggested avoiding hits with 0 or 1 mismatches, although 2 and 3 mismatches can still mediate silencing. In addition, this feature can be used to look for potentially desirable "on-targets". Search for hits to known gene paralogs or to see if your sequence would likely target the orthologous transcript in another species.