Introduction

The siRNA Target Designer analyzes an input sequence and find regions within that sequence which fit the siRNA Target Design Parameters. Each siRNA target region, its type (16), length, percent GC, and location relative to the input sequence, is displayed along with the oligonucleotide sequences needed to generate that siRNA in one of Promega's of DNA-directed RNA Interference systems:

• GeneClip™ U1 Hairpin Cloning System(Cat.#'s C8750, C8760, C8770, C8780, C8790)
• siLentGene™ U6 Cassette RNA Interference System (Cat.# C7800)
• T7 RiboMAX™ Express RNAi System (Cat.# P1700)
• siSTRIKE™ U6 Hairpin Cloning Systems (Cat.#'s C7890, C7900, C7910, C7920).
• siLentGene™-2 U6 Hairpin Cloning Systems (Cat.#'s C7860, C8060, C8070, C8080)

The context of each of the potential siRNAs found within the input sequence is displayed near the bottom of the output. Clicking on a displayed siRNA sequence will launch a new browser window with a BLAST search of that sequence against the entire Genebank database allowing users to evaluate the expected specificity of that sequence (15).

The siRNA Target Designer is copyrighted by Promega Corporation. 2003. All Rights Reserved

Using the siRNA Designer

To use the siRNA Target Designer, first click on the Find Targets link above. Follow these steps to generate target regions for your sequence.

1. Select the Promega RNA Interference System that you're using.
2. For hairpin applications, indicate the desired hairpin loop sequence.
3. Paste the sequence for which you wish to find siRNAs into the text box.
4. Indicate the target length of the siRNA desired.
5. Provide a name of the sequence (optional).
6. Indicate if you wish to have Type Ib siRNAs (16) displayed.
7. Use the Design Oligonucleotides Only checkbox to have the program design oligonucleotides with the appropriate promoter, terminator, loop and other sequences to generate the input sequence with the selected siRNA System.
8. Indicate the desired first and last base from which to find siRNAs if you do not wish to have the whole sequence analyzed. Note that there is a 199 siRNA maximum.

Every Type Ia (and type Ib if select) siRNA (16) from your sequence which also starts with a G (to allow initiation with the U6 promoter or T7 RNA Polymerase) will be displayed along with the oligonucleotides needed to produce that siRNA with the system selected.

We recommend that the effects of each siRNA be compared to those of a mock siRNA that has the same sequence composition as the active siRNA but having a sequence which is scrambled relative to the siRNA sequence (4). After generating targets, click on the Scramble button to generate three choices of scrambled sequences for each siRNA. It is recommended that you analyze each with a BLAST search and choose the one that has least sequence identity with other genes in your organism of interest. Note that mismatches near the center of the siRNA are much more effective at disrupting the RNAi effect than mismatches near the ends (5). If none of the three sequences offered is acceptable, you may generate as many more scrambled sequences as desired.

Find Targets

siRNA Target Design Parameters

When possible, the siRNA Designer will return Type Ia, and Type Ib if selected, siRNAs (16). Ui-Tei et al (2004) have shown that 38 out of 39 Type Ia siRNAs reduced target gene expression on the protein level by 70% or more. In addition the siRNA selection rules developed by Kumiko et al fit very well with other recent siRNA selection parameters developed in other labs (9, 11, 14).

Type Ia siRNAs have the following characteristics:

• The 5' terminal base of the sense strand is a G or C.

  All of Promega's systems use promoters which initiate transcription most efficiently on a G, so the siRNA designer will only choose siRNAs which have a 5' G.

• The 5' terminal base of the antisense strand (which is the 3' terminal base of the sense strand) is an A or U (T in the DNA sequence).
• Five of the 7 terminal base of the antisense strand are A or U (Four of 7 for Tybe Ib).

siRNAs which are the opposite of three rules listed above (Start with A or U, end either G or C, 5 of last 7 bases are G or C) are Type III. Type III siRNAs do not show significant RNAi effects.

• There are no runs of more than 9 G's or C'.

All siRNAs which do not fit the definitions of Type I or Type III are Type II. Type II siRNAs may or may not have significant RNAi effects.

Only those systems which express hairpin siRNAs can be used to generate Type I siRNAs. Systems in which each RNA strand must be transcribed individually must have a G at each 5' terminus and thus can not be Type I. These siRNAs are ranked by free energy of the 5' antisense region.

Additional parameters used:

• All siRNAs are sorted by the free energy of the 5' antisense region.

  Several recent papers(9, 11, 14) have shown that siRNA efficacy is correlated with the free energy of the 5' antisense region. The average free energy (delta G) of the 5 5' bases of the antisense strand is calculated (18) and those siRNAs which the highest free energy, those that are least stable at the 5' antisense end, are shown first. In addition, a chart of the delta G for each position is displayed.

• Target sequence contains no runs of four or more of the same nucleotide.

  Runs of 4 or more T's (or A's on the other strand) serve as terminator sequences for RNA polymerase III, which is the polymerase which transcribes from the U6 promoter. In addition, it has been reported that regions with a run of any single base should be avoided (5).

In addition to the parameters used by the siRNA Designer, there are a number of additional parameters which can be evaluated for siRNA target design. These include:

• Evaluation of homology to other known sequences in the organism of interest

  siRNAs can potentially affect non-target sequences to which they have sufficient homology. This effect can be reduced by eliminating any sequences which have too much sequence identity with other sequences in the database. For that reason, the siRNA sequence displayed is directly linked to a BLAST search of that sequence. This search is done using an expect value of 1000 and a word size of 7. These are the NCBI-recommended parameters for short sequences. Note that mismatches near the center of the siRNA are much more effective at disrupting the RNAi effect that mismatches near the ends (5).

• Distance from the start codon

  Elbashir et al (7) recommend choosing siRNAs which are at least 50 to 100 bases downstream of the start codon. This parameter seems to be completely theoretical and is meant to avoid regions in which regulatory proteins might bind. More recent work (17, 19) has not shown any preference for a specific region of the message.

• Evaluation of accessibility of targeted region using predictions of RNA folding

  Accessibility of the targeted region of the mRNA to base pairing seems to be important for siRNA efficacy (1, 12, 17) but current mRNA secondary structure prediction algorithms do not appear to be able to predict this accessibility (1, 8, 12). At this time the siRNA Designer does not predict or use any RNA secondary structure information in siRNA design.

• The exact loop sequence used for hairpin siRNAs

  The exact sequence of the loop may make a difference to siRNA efficacy. The characteristics which make one sequence better than another are not known, but several different loops have been shown to work (2, 3, 10).

References

1. Bohula, E. et al. (2003) The Efficacy of Small Interfering RNAs Targeted to the Type 1 Insulin-like Growth Factor Receptor (IGF1R) Is Influenced by Secondary Structure in the IGF1R Transcript J. Biol. Chem. 278, 15991-15997.
2. Brummelkamp, T.R.et al. (2002) A system for stable expression of short interfering RNAs in mammalian cells. Science 19, 550-553.
3. Castanotto, D.et al. (2002) Functional siRNA expression from transfected PCR products. RNA 8, 1454-60.
4. Chi, J.et al. (2003) Genomewide view of gene silencing by small interfering RNAs. Proc. Natl. Acad. Sci. U.S.A. 100, 6343-6.
5. Czauderna, F. et al. (2003) Structural variations and stabilising modifications of synthetic siRNAs in mammalian cells. Nucl. Acids Res. 31, 2705-16.
6. Dykxhoorn, D. et al. (2003) Killing the messenger: short RNAs that silence gene expression. Nature Reviews 4, 457-467.
7. Elbashir, S. et al. (2002) Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods 26, 99-213.
8. Holen, T.et al. (2002) Positional effects of short interfering RNAs targeting the human coagulation trigger Tissue Factor. Nucl. Acids Res. 30, 1757-66.
9. Hohjoh H. (2004) Enhancement of RNAi activity by improved siRNA duplexes. FEBS Lett. 557, 193-8
10. Kawasaki, H. and Taira, K. (2003) Short hairpin type of dsRNAs that are controlled by tRNA(Val) promoter significantly induce RNAi-mediated gene silencing in the cytoplasm of human cells. Nucleic Acids Res. 15, 700-707.
11. Khvorova A. et al. (2003)Functional siRNAs and miRNAs exhibit strand bias. Cell 115, 209-16.
12. Lee, N. et al. (2002) Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nat. Biotechnol. 20, 500-5.
13. Scherr, M. et al. (2003) Gene silencing mediated by small interfering RNAs in mammalian cells.
    Curr. Med. Chem. 10, 245-56.
14. Schwarz D. S. et al. (2003)Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199-208.
15. Semizarov, D. et al. (2003) Specificity of short interfering RNA determined through gene expression signatures. Proc. Natl. Acad. Sci. U.S.A. 100, 6347-52.
16. Ui-Tei, K. et al. (2004) Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference Nucleic Acids Res. 32, 936-948
17. Vickers, T. et al. (2003) Efficient reduction of target RNAs by small interfering RNA and RNase H-dependent antisense agents. A comparative analysis.J. Biol. Chem. 278, 7108-18.
18. Xia T. et al. (1998)Thermodynamic parameters for an expanded nearest-neighbor model for formation of RNA duplexes with Watson-Crick base pairs. Biochemistry 37, 14719-35.
19. Yu, J. et al. (2002) RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 99, 6047-52.