Shan Li 092111 with help from Dr. Guihua Sun (Rossi Group)
Glackin Group
How to design an shRNA
Generate duplex:
http://www1.infosci.coh.org/hpcdispatcher/
RNA folding
http://mfold.bioinfo.rpi.edu/
http://mfold.rna.albany.edu/
Notes for selecting deplexes:

11th position as U/A

Relative accessibility score bigger better (look at the 2nd structure of mRNA)

3’ of 5 sequence entry point as U/A better

Duplex _end differential >1

dG5” antisense not too stable (as with each other) not too unstable (as
targeting the target sequence)
order primers to be annealed with sticky ends and cloned into pcDNA3-U6m2 (J.
Rossi’s group) vector.

ONLY 4 nucleotide in between the promoter sequence and the transcription
initiation site (BglII)
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Shan Li 092111 with help from Dr. Guihua Sun (Rossi Group)
Glackin Group
For example:
shTwist duplex:
418
0.75
GGACAAGCUGAGCAAGAUUca
AAUCUUGCUCAGCUUGUCCga
5.6
-6.1
-11.7
Use
5’ end: BglII over hang: gatc
3’ end: SpeI over hang: ctag
loop: TTTGTGTAG or GATGTGTTT
U6 termination seq: ttttt
BglII
A’GATCT
TCTAG’A
SpeI
A’CTAGT
TGATC’A
5B-Tw418,GATCGGACAAGCTGAGCAAGATTCATTTGTGTAGTGAATCTTGCTCAGCTTGTCCTTTTT
3S-Tw418,CTAGAAAAAGGACAAGCTGAGCAAGATTCACTACACAAATGAATCTTGCTCAGCTTGTCC
5B-Tw418,GATCGGACAAGCTGAGCAAGATTCATTTGTGTAGTGAATCTTGCTCAGCTTGTCCTTTTT
CCTGTTCGACTCGTTCTAAGTAAACACATCACTTAGAACGAGTCGAACAGGAAAAAGATC,3S-Tw418
2
Shan Li 092111 with help from Dr. Guihua Sun (Rossi Group)
Glackin Group
Target sequence
siRNA
5B-Tw418,GATCGGACAAGCTGAGCAAGATTCATTTGTGTAGTGAATCTTGCTCAGCTTGTCCTTTTT
Seed seq
Clone U6-shRNA fragment into pENTR shuttle vector to be delivered to pBLOCKiT6
(Invitrogen) for lentiviral packaging.
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