siDesign center user guide

The siDESIGN Center is an advanced, user-friendly tool for designing potent custom siRNAs. It employs an algorithm that considers various design criteria, such as avoiding motifs linked to toxicity, known microRNA seed sequences, and polymorphisms. Additionally, it offers advanced design options to discriminate transcript variants or add transcript targets from another gene or a different species.

Please note that the custom siRNAs designed with the siDESIGN Center are not functionally guaranteed. However, siRNAs designed using this tool are more likely to provide potent and specific knockdown than those designed with conventional methods.

Step 1: Choose an Identifier Type

The siDESIGN Center offers three options for identifying the target sequence of interest.

• Accession Number
  Accession numbers can be found in the NCBI Reference Sequence (RefSeq) database (link). This is a non-redundant, curated, and annotated collection of sequence records for major model organisms. RefSeq accession numbers use the systematic naming convention below.
  
  

  Accession Prefix	Molecule Type	Description and Notes
  NM_	mRNA	Protein-coding transcripts. Usually curated.
  XR_	mRNA	Computationally predicted model protein-coding transcript
  NR_	RNA	Non-protein-coding transcripts
  XR_	RNA	Computationally predicted model non-protein-coding transcript

   

• Nucleotide Sequence
  The Nucleotide box accepts FASTA-formatted sequence data up to 10,000 nucleotides. Only mature RNA or cDNA sequence should be entered. Sequences should be formatted using standard nucleic acid codes: A, C, G, T, and U. Any other alphanumeric entries will be ignored.
  FASTA format requires a ">" symbol and a line break prior to entering or pasting the nucleotide sequence. A name may also be designated following the “>” symbol and before the page break, as shown below:
  IMAGE HERE
• Gene ID
  This is the unique ID number assigned to each gene in the NCBI database, as you can find, for instance, on our website:
  
  • 672 (BRCA1)
  • 25 (ABL1)
  • 983 (CDC2)
  • 6419 (SETMAR)
• GI Number (GenInfo Identifier)
  A series of digits that are assigned consecutively to each sequence record processed by NCBI. The GI number bears no resemblance to the Accession number of the sequence record. If a sequence changes in any way, a new GI number will be assigned. Be sure the GI Number provided is for the nucleotide, not the protein sequence of interest.
  Example: 27436944 (Homo sapiens lamin A/C (LMNA), transcript variant 2, mRNA)

Advanced Options

It is important to note that the BLAST option (Step 4) must be checked and the proper species database selected, otherwise our tool will not take into consideration any advanced options.

• Selection of accessions
  
  The advanced options support more complex design requirements, such as isoform-specific siRNA designs. However, using these options can greatly reduce the number of siRNA designs generated due to the increased stringency of the design parameters.
  
  When the Accession number or Gene ID are entered in Step 1, selecting the Advanced Settings will display the additional accession numbers for all RefSeq variants associated with the target gene. As a default, all variants will be “required” except non-coding accessions, which will be denoted with the “NR” accession prefix.
  
  Select "Required" for any transcript variant that must be targeted by the siRNA designs. Note that at least one variant must remain "Required" for the design step to proceed.
  
  Select "Allowed" for any transcript variant that will neither be required nor excluded by resulting siRNA designs.

   

  Select "Excluded" for any transcript variant whose sequence should not be targeted. Use of Advanced Options to exclude a variant will require selection of a BLAST database in Step 4.
  
  
• Additional required targets
  
  This option supports the design of siRNAs that target multiple genes with homologous regions. For example, homologous genes across multiple species, or conserved gene family members within a species. Using this option may greatly reduce the number of siRNA designs generated due to the increased stringency of the design parameters.
  
  Up to 10 Accession numbers may be entered in the field provided. These must be separated by spaces or line breaks. Gene targets entered here will be "Required" by the design tool along with the original targeted accessions selected.

Step 2: Select desired regions for siRNA design

This step allows the user to specify which of the following regions to target:

• 5' UTR - check this box to include the 5' untranslated region (UTR) for design consideration
• ORF - check this box to include designs that target the Open Reading Frame or ORF. This is the default selection. Non-coding RNAs will be treated as an ORF region and no designs will be returned if ORF is not selected. Recommended.
• 3' UTR - Check this box to include the 3' untranslated region (UTR). Recommended.
• If nucleotide sequence was used for the input type in step 1, the sequence will be treated as the ORF and selecting 5’ UTR and 3’ UTR will not have any impact.
  
  

Step 3: GC content

This value is calculated using the core 19 nucleotide target sequence and does not include the dinucleotide overhangs. Early research suggested that siRNA should have a G/C content of approximately 50%. For genes that are G/C poor or rich, the range of G/C content be expanded to include other ranges. The default and recommended range is between 30% and 64% GC content.

Step 4: Choose BLAST Options

The siDESIGN Center offers the option to BLAST the target sequences against one or two species in order to remove the designs which have significant risk of presenting cross-targets.

• No BLAST: Choose if there is no BLAST database available through this tool for the desired species, if specificity is less of a concern compared to functionality, or if you have entered a nucleotide sequence (at Step1) which is present in the targeted organism. (In this case, selecting “no BLAST” but doing a BLAST analysis on the siRNA designs through NCBI is recommended.)
• BLAST: Select this option to perform a BLAST analysis to ensure specificity of the siRNA candidates and/or because you have entered advanced options. Select the host species from the drop-down menu.

The following species-specific protein-coding transcript databases (RefSeq) are available for BLAST analyses:

Note that 3 databases encompass non-coding transcripts in addition to protein-coding transcripts. These are available for Human, Mouse and Rat. You may select those if you wish to exclude some non-coding variants (Step1, advanced options) or if you are concerned about targeting any non-coding transcript.

Two combined databases are also available to support cross-species designs:

Please use these databases if you have added transcripts from a different species with the advanced options of Step 1.

If your species does not appear in this list, we recommend performing a BLAST analysis at NCBI with the candidate siRNAs target sequences. The siRNA sequences can be found by adding the siRNA designs to your cart and then selecting “More” from the line item in the cart.

Results

A list of up to 50 of the top-scoring candidates is presented along with details which will help you select the best siRNAs. The candidates are sorted by the score. Here are explanations of each column:

• Select - Click to choose siRNA designs that will be added to your shopping cart. When items are in your cart, be sure to click on “Edit” to select additional options, such as the type of siRNA, required processing, and desired quantity.
• Region - Region where the design aligns to one of the indicated targeted transcripts. This field will indicate "N/A" if the targeted gene is a non-protein-coding gene or if a nucleotide sequence was provided as the Identifier Type in Step 1.
• Start Position - Position of the siRNA design relative to transcription starting site of one of the indicated targeted transcripts.
• GC% - Percentage of the 19-base target sequence that is G or C.
• Score - Algorithmic indicator of the likelihood of successful silencing with this siRNA design. This score does not quantify expected percent knockdown but is a prediction based on characteristics of the sequence.
• Low Seed Frequency - Yes/No indicator of whether the target sequence has a low seed sequence frequency within the 3'UTR of the target or host genome. siRNA with low frequency seed regions will have reduced risk of off-target effects relative to higher frequencies¹. When possible, choosing target sequences with a “Yes” here is recommended.
• Min # mismatches (Antisense) - The lowest number of mismatches of the 19-base antisense strand to a sequence record other than the required targets, as determined by BLAST analysis. For example, a score of 3 in this field would indicate that the antisense strand has no more than a 16/19 base match to any non-required transcript accession in the BLAST database.
• Min # mismatches (Sense) - Similar to above but for the sense strand.
• Requires Sense Modification - This is a yes/no indicator of whether the siRNA design should be synthesized with a chemical modification to inhibit sense-strand uptake. This requirement is indicated when (a) The sense strand has fewer than 2 mismatches to non-targeted sequence record in the BLAST database or (b) analysis indicates that the design favors sense strand over antisense strand entry into RISC. If "No BLAST" was selected, only criteria (b) will be applied.
• Start over – this will clear the input so that new design parameters can be entered.
• Modify – this will go back to the design steps with the original design parameters pre-filled to allow adjustments.

References

1. Anderson, E, et al., "Low Genomic 3' UTR Seed Complement Frequency Predicts siRNA Target Specificity". Poster at Keystone Symposia: MicroRNAs and siRNAs: Biological Functions and Mechanism. (January 2007)

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