- 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:
- Presence of Internal Hairpins: Looks for potential hairpin structures within the 19mer stems. The default setting is 45C. We have found that shRNA sequences containing internal hairpins have a very low probability of working effectively in RNAi.
- Differential Thermal Stability: It has been shown that sequences with higher thermal stability at the 5'-end of the sense strand compared to that at the 3' end are better able to direct entry of the minus (targeting) strand of an siRNA into the RISC complex, and so promote more effective RNAi (Schwarz, D.S. et al. Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199-208 (2003)) while presumably decreasing the likelihood of off-target silencing. The current application compares the Tms of the last 4 bases at each end and selects those which are a given number of degrees more stable at the 5'end than at the 3' end. The default setting is 1.
- GC Content: We and others have found GC content to have a strong impact on shRNA efficacy. The application chooses sequences with GC content ranging from 30 to 53%. Our own analysis suggests that moderate GC content (between 36 and 48%) is optimal. For this reason, the default setting is 45 (best score value) and the cutoff limits are 30 and 53.
- PolyN sequences: The application looks for and ignores sequences containing defined stretches of the same nucleotide. The default setting we have used blocks all sequences with stretches of the same base longer than 4.
Low complexity filter:
By default, the application searches for and removes sequences that are
of low complexity. This filter eliminates sequences of alternating
nucleotides (e.g., ACACACAC or AACCAACC). In addition, all 4 bases must
appear at least once in any oligonucleotide.
Based on our own analysis of several hundred shRNAs, position-specific
rules appear, in general, to have little significant impact on shRNA
efficacy. Nonetheless, for completeness and flexibility of design, we
have included the ability to select siRNA sequences that conform to
various position-specific rules that have been described. In particular,
we have included the original rule of Tuschl et al. (Elbashir, S.M.,
Lendeckel, W. & Tuschl, T. RNA interference is mediated by 21- and
22-nucleotide RNAs. Genes Dev 15, 188-200 (2001)), that limits shRNAs
chosen to those that follow 'AA' dinucleotide. This rule is included for
compatibility with our original shRNA design tool, which implemented
this rule. However, currently, it is not thought that this criterion has
any bearing on successful shRNA design.
We have also included several of the rules suggested by Reynolds et al. (Reynolds, A. et al. Rational siRNA design for RNA interference. Nat Biotechnol 22, 326-330 (2004)).
Default settings include presence of at least one A or T at residues 18 or 19, and an A at position 6, both of which had reasonable statistical support from our own studies.Sequences that pass the selection rules are ranked according to differential thermal stability, GC-content, Tm of sense-antisense RNA duplex and Tm of any internal RNA hairpins. Overall, the ranking system is based on the penalties for deviation from the optimal values. The scoring system does not strictly enforce selection of oligonucleotides within the optimal parameters, but rather provides a basis for compromise when no 'ideal' sequence can be found.
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.