Short-hairpin RNA (shRNA) is an RNA molecule that contains sense and antisense sequences connected by a short spacer of nucleotides that enables the molecule to form a loop structure. shRNA is expressed in mammalian cells from a vector with a Pol III-type promoter, and is processed by the Dicer enzyme in vivo into 21-23 nucleotide siRNA duplexes.
If you have identified a synthetic siRNA that is active in triggering knockdown of your target gene, you can generate a corresponding shRNA molecule using the RNAi Designer. Select the siRNA to shRNA design option at the top of the Designer page. Then enter the siRNA sequence that will form the "sense" portion of the shRNA oligo, select or enter the loop sequence, and select the Gateway®-adapted entry vector for expressing your shRNA. The Designer will generate the complete shRNA design with appropriate overhangs for directional cloning.
In the Sense siRNA Sequence field, enter the siRNA sequence that you want to convert into the sense part of the shRNA molecule (the resulting shRNA structure will be sense-loop-antisense).
Select one of the predesigned 4-nucleotide loop sequences from the Default Loop Sequence pulldown list, or enter a custom loop sequence of 4–11 nucleotides in the Custom Loop Sequence field. Note that short loop sequences (4–7 nucleotides) are usually more efficient. Avoid using a loop sequence containing thymidines (T’s) as they may cause early termination. This is particularly true if the target sequence ends in a T residue.
Next, select the Gateway®-adapted entry vector from the Vector pulldown list. Select either the pENTR™/U6 entry vector for constitutive shRNA expression or the pENTR™/H1/TO entry vector for inducible expression. Note that if you select pENTR™ /U6, the shRNA oligo designs will be compatible with both vectors.
Click on Design shRNA Oligo to begin the conversion process. The BLOCK-iT™ RNAi Designer will generate the shRNA oligo from your parameters. See shRNA Final Designs for more information.