RNAi Designer

The tools here support the Clontech Knockout RNAi product. Two applications are provided:
 
RNAi Target Sequence Selector
Sequence selector uses the 19-mers selection rules found in the literature and supported by our own studies to provide suggestions for design of siRNA for the particular target sequence.
 
shRNA Sequence Designer
Oligo designer takes a sense sequence selected for a siRNA target site and provides the sequences of the two complementary oligonucleotides that need to be synthesized for use with Clontech pSIREN vectors.

Design of shRNAs

This application generates a list of potential 19mer sequences for use as siRNAs or shRNAs targeted against an mRNA of interest. The mRNA target sequence can be provided either by giving an accession number or by simply pasting the sequence of interest into the analysis window. The choice of 19bp for use as shRNA stems is based upon previous studies, which have shown efficient knockdown using shRNAs with 19bp stems (Brummelkamp, T.R., Bernards, R. & Agami, R. A System for Stable Expression of Short Interfering RNAs in Mammalian Cells. Science 296, 550-553 (2002)).

The current tool implements a multi-step procedure to identify potential siRNAs/shRNAs including: analysis of GC content, presence or absence of internal hairpins, differential thermal stability of ends, sequence complexity, and various position-specific criteria. Default settings are based on the results of our own analysis into the efficacy of several hundred different shRNAs. However, the user is also able to alter these settings to fit their own specific requirements.

The following criteria of siRNA sequence selection were implemented:

Sequence similarity to non-target genes

Note that the tool does not perform any sequence similarity searches to ensure specificity of the siRNA. A link is given to the NCBI Blast server enabling the comparison of the suggested siRNA with sequences from the organism of interest.

Design of DNA oligonucleotides for shRNA cloning

Once an siRNA sequence of interest has been identified, it is possible to obtain the sequence of the DNA oligo pair that must be ordered in order to clone the shRNA of interest into one of the Clontech pSIREN vectors by simply clicking on the sequence itself. Each oligonucleotide pair includes the following elements: a Bam HI overhang on the 5'end of the duplex; the 19 nucleotides of shRNA sense strand; a loop sequence (top strand: 5' TTCAAGAGA); the 19 nucleotides of the shRNA antisense strand; a Poll III termination site of 6 consecutive thymidine residues; an Mlu I site to verify cloned inserts; and an Eco RI overhang on the 3' end of the duplex. If the desired 19-nucleotide shRNA sequence does not start with a guanine or adenine (required for Pol III transcription initiation) an extra guanine residue is added to the 5' end of the shRNA sense strand, and this 20-nucleotide sense-strand is then used in place of the 19 base sequence as a basis for oligo design.