2-shRNA Oligo Designer

How to use 2-shRNA?

1. Use Dharmacon siDESIGN Center to find functional siRNA sequences;
2. Input the siRNA sequence here to get the stem-loop oligos;
3. Order desalted oligos from Integrated DNA Technologies or other companys;
4. Do "wet" experiments.

Useful Information

I. Criteria for identifying functional siRNA described by Reynolds and colleagues

• from Rational siRNA design for RNA interference Nature Biotechnology 2004 22: 326-330
• low G/C content
• a bias towards low internal stability at the sense strand 3'-terminus
• lack of inverted repeats
• sense strand base preferences (positions 3, 10, 13 and 19)

II. Algorithm of Dharmacon score

1. 30%-52% GC content - Add 1 point for satisfying this criterion
2. Three or more A/Us at positions 15-19 (sense) - Add 1 point for each A/U for a total up to 5 points. At least 3 points are required to be scored as positive ("+") in the final output
3. The absence of internal repeats or hairpins as measured by a T_m < 20 degrees C. - Add 1 point for satisfying this criterion
4. A at position 19 (sense) - Add 1 point for satisfying this criterion
5. A at position 3 (sense) - Add 1 point for satisfying this criterion
6. U at position 10 (sense) - Add 1 point for satisfying this criterion
7. No G/C at position 19 (sense) - Subtract 1 point for not satisfying this criterion
8. No G at position 13 (sense) - Subtract 1 point for not satisfying this criterion

III. Asymmetry for siRNA function

• from Asymmetry in the Assembly of the RNAi Enzyme Complex Cell 2003 115: 199-208
  

IV. Melting temperature predicted by Expanded Nearest-Neighbor Model

• from Thermodynamic Parameters for an Expanded Nearest-Neighbor Model for Formation of RNA Duplexes with Watson-Crick Base Pairs Biochemistry 1998 37: 14719-14735
• Set a total strand concentration of 0.2 mM
• RNA thermodynamic parameters in 1M NaCl, pH 7