The BLOCK-iT™ RNAi Designer can design synthetic 19-mer short interfering RNA (siRNA) molecules that are specific to your nucleotide sequence of interest.
To begin, select the siRNA target design option at the top of the Designer page.
On the first page, you enter a sequence for the gene that you want to target or a database accession number for that sequence, select a database to BLAST search to identify unique regions of the sequence, specify a GC percentage range for the oligos, and select an algorithm to generate the designs.
You can copy and paste a nucleotide target sequence directly into the Nucleotide Sequence field, or you can enter a database accession number for the sequence in the Accession Number field. If you enter an accession number, the Designer will link to the Entrez search engine and search the crosslinked databases at the National Center for Biotechnology Information (NCBI), including GenBank and OMIM. Then the sequence associated with the accession number will be automatically downloaded into the Designer.
Note: If you enter an accession number in RefSeq format (e.g., NT_123456), the Designer will check the sequence for known single-nucleotide polymorphisms (SNPs). Designs that include SNPs will be rejected by the Designer. SNP identification will not be performed if you paste in nucleotide sequences or enter GenBank-format accession numbers.
Nucleotide Sequence: Type or paste your single-stranded target sequence into this box. The designer accepts sequences in text, FASTA, or GenBank format.
The sequence should be oriented from 5' to 3'. Because siRNA molecules must be highly specific, the input sequence should contain only the following 1-letter base code abbreviations: A, C, G, T, U (case insensitive). Any regions of the sequence containing ambiguous bases (e.g., N) or other letters will be skipped when generating the designs. If your sequence includes too many ambiguous bases or other letters, you will be prompted to re-enter the sequence. White spaces and numerals in the sequence will be ignored.
Note: If you are having difficulty generating siRNA designs, check your input sequence to make sure that it contains only the letters A, C, G, T, or U.
Accession Number: Enter the database accession number for your sequence in this field. The Designer will link to the Entrez search engine and search the crosslinked databases at NCBI, including GenBank and OMIM. The sequence associated with the accession number will be automatically downloaded into the Designer.
RefSeq-format accession numbers begin with two letters followed by an underscore and six digits (e.g., NT_123456). GenBank accession numbers are usually a combination of a letters and numbers, such as a single letter followed by five digits (e.g., U12345) or two letters followed by six digits (e.g., AF123456). If you enter an accession number in RefSeq format (e.g., NT_123456), the Designer will check the sequence for known single-nucleotide polymorphisms (SNPs). Designs that include SNPs will be rejected by the Designer. SNP identification will not be performed if you paste in nucleotide sequences or enter GenBank-format accession numbers.
If you entered an accession number in Step 1, in this step you specify the regions of the sequence for which you want to design siRNA molecules. The default selection of Open Reading Frame (ORF) is appropriate in most cases. If you have trouble generating designs for the ORF alone, select the 5' UTR and/or 3' UTR checkboxes and redesign the molecules.
Step 3. Choose Database for BLAST
In Step 3, select a species-specific database from the pulldown list that you want to perform a BLAST (Basic Local Alignment Search Tool) search on to identify unique regions in your target sequence. Areas of homology identified by the BLAST search will be "masked" in the sequence to ensure that the siRNA target designs are highly specific.
Each BLAST search is performed against a a nonredundant version of the UniGene database. The database contains representative gene sequences for the selected species.
Note: The databases used in this search contain one representation of each gene and do not include splice variants.
The Minimum GC Percentage and Maximum GC Percentage fields contain default values for the minimum and maximum percentage GC content of your siRNA molecules. For most sequences, this range is wide enough to generate several siRNA designs.
You can change this range if you are having difficulty generating siRNA designs, or if you know that the GC content of your siRNA molecules will fall outside this range based on the sequence. Designs outside the selected range will be rejected by the Designer.
In this step, you select the algorithm used to generate the siRNA designs. Select either the Proprietary algorithm or one or more of the Tuschl's motif patterns, and then click on the RNAi Design button. Up to 10 designs will be generated for your target sequence.
Note: The Default Motif Pattern design algorithm will give the best results in most cases, and is highly recommended. The results using this algorithm may include some Tuschl's patterns; these will be noted on the results page.
Default Motif Pattern: Select the option button next to Default Motif Pattern to use Invitrogen's proprietary design algorithm. This algorithm performs a statistical analysis of the target sequence—considering such factors as sequence composition, nucleotide content at 5' and 3' ends, and thermodynamic properties—and correlates it with data collected from multiple validated siRNA oligo sets tested on different gene targets.
Tuschl's Motif Pattern: To design RNA molecules using Tuschl's rules, select the option button next to Tuschl's Motif Pattern and then select one or more pattern checkboxes. The Designer will generate 19-nucleotide RNA sequences that match the selected pattern(s). For example, if you select the AA(N19)TT pattern, the Designer will search for an area of the sequence that begins with "AA" and ends with "TT," with 19 bases in between. Each pattern is listed using the following base code letters: A = Adenine; T = Thymine; R = Adenine or Guanine (Purines); Y = Thymine or Cytosine (Pyrimidines); N = Any
References for Tuschl's patterns.
For information about siRNA design results, see siRNA Design Results.