siRNA Design Guidelines 【Ambion】

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For preparing SECs containing an H1 or U6 promoter by PCR using the Silencer Express Kits, (1) one or two DNA oligonucleotides encoding the siRNA sequence are designed and ordered, (2) the oligonucleotides are used as primers in one or more PCRs with the promoter-containing template included in the kit, and (3) the resulting PCR product is column purified. Ambion scientists typically use a loop sequence of 5'-UUUGUGUAG-3' for their SECs, although other loop sequences can be designed. As with vector insert design, a 5-6 T termination sequence is added to act as an RNA pol III terminator. For subsequent cloning convenience, EcoRI and HindIII restriction sites are also encoded by the primers. The detailed design parameters for the oligonucleotide primers used with the Silencer Express Kits can be found in the kits' Instruction Manual.

For cloning of functional Silencer Express Kit-derived SECs into vectors, the SEC and destination vector should be restricted with EcoRI and HindIII. Linearized destination vectors with neomycin, hygromycin and puromycin resistance genes, called pSEC Vectors, are available.

Selection of siRNA Targets

In addition to the algorithm developed by Cenix and our suggested procedure for selecting siRNA targets by scanning a mRNA sequence for AA dinucleotides and recording the 19 nucleotides immediately downstream of the AA, two other methods have been employed by other researchers. In the first method, the selection of the siRNA target sequence is purely empirically determined (4), as long as the target sequence starts with GG and does not share significant sequence homology with other genes as analyzed by BLAST search.

In the second report, a more elaborate method is employed to select the siRNA target sequences. This procedure exploits an observation that any accessible site in endogenous mRNA can be targeted for degradation by the synthetic oligodeoxyribonucleotide/RNase H method (5). Any accessible site identified in this fashion is then used as insert sequence in the U6 promoter-driven siRNA constructs.

Order of the Sense and Antisense Strands within the Hairpin siRNAs

A hairpin siRNA expression cassette is usually constructed to contain the sense strand of the target, followed by a short spacer, then the antisense strand of the target, in that order. One group of researchers has found that reversal of the order of sense and antisense strands within the siRNA expression constructs did not affect the gene silencing activities of the hairpin siRNA (6). In contrast, another group of researchers has found that similar reversal of order in another siRNA expression cassette caused partial reduction in the gene silencing activities of the hairpin siRNA (7). It is not clear what is responsible for this difference in observation. At the present time, it is still advisable to construct the siRNA expression cassette in the order of sense strand, short spacer, and antisense strand.

Length of the siRNA Stem

There appears to be some degree of variation in the length of nucleotide sequence being used as the stem of siRNA expression cassette. Several research groups including Ambion have used 19 nucleotides-long sequences as the stem of siRNA expression cassette (6-10). In contrast, other research groups have used siRNA stems ranging from 21 nucleotides-long (4-5) to 25-29 nucleotides-long (11). It is found that hairpin siRNAs with these various stem lengths all function well in gene silencing studies.

Length and Sequence of the Loop Linking Sense and Antisense Strands of Hairpin siRNA

Various research groups have reported successful gene silencing results using hairpin siRNAs with loop size ranging between 3 to 23 nucleotides (4, 6-9, 11). The following is a summary of loop size and specific loop sequences used by various research groups:

Presence of 5' Overhangs in the Hairpin siRNAs

Most research groups did not use a 5' overhang in their hairpin siRNA construct (4-8, 10-11). However, one research group included a 6 nucleotide 5' overhang in the hairpin siRNA constructs (9). These hairpin siRNAs with 5' overhangs were shown to be functional in gene silencing.

Chemical Synthesis of siRNA

Ambion synthesizes both customer designed siRNAs and siRNAs pre-designed using the Cenix algorithm.

To order a chemically synthesized siRNA for which you already have the design, you can either provide:

· the ~21 base mRNA sequence (starting with the AA dinucleotide) to which the siRNA will be directed

OR

· the sequence of each siRNA strand (This option is recommended if you wish your siRNA to have 3' termini other than dTdT or UU.)

Ambion will synthesize a complementary pair of siRNA oligonucleotides according to your sequence. By default, siRNAs for which you provide only the mRNA target sequence will be synthesized with dTdT 3' overhangs. If you wish, you can choose UU or other overhangs. Our scientists observe no functional difference in the potency of siRNA made with dTdT or UU overhangs. (Note: the 3' dTdT of the sense strand does not have to be complementary to the target gene.)

Currently, Cenix-designed siRNAs are available for >98% of all human, mouse, and rat genes in the RefSeq database maintained by NCBI. To order a pre-designed siRNA, search our siRNA database for your gene of interest, choose the design(s) you'd like to purchase, add them to your cart, and transfer the relevant information about each to our online oligo order form. See Designing a Better siRNA for information on the Cenix design algorithm.

Other Methods of siRNA Preparation

To prepare siRNA by in vitro transcription, siRNA expression vector, or PCR-generated siRNA expression cassette, appropriate templates must be prepared. Web-based tools for designing these templates are available for the following Ambion kits/products:

• Silencer siRNA Construction Kit [ Product Info ] [ Design Tool ]
• pSilencer siRNA Expression Vectors [ Product Info ] [ Design Tool ]
• Silencer Express siRNA Expression Cassette Kits [ Product Info ] [ Design Tool ]
• Silencer siRNA Cocktail Kit (Rnase III) [ Product Info ]

These tools are also accessible from the siRNA Target Finder described above.