3.2Me/PS10-LOOP.
50 - mGmUmCmAmCmC*mUmC*mAmG*mCmG*mUmA*-
mAmG*mUmG*mA*mUmG*mUmC*mGmUmGmAmC-3^0.
4.2Me/PSLOOP
50 - mGmUmCmAmCmC*mU*mC*mA*mG*mC*mG*mU*-
mA*mA*mG*mU*mG*mA*mU*mG*mU*mC*mGmUmG-
mAmC-3^0.
5.2Me/PSALT
50 - mG*mUmC*mAmC*mC*mUmC*mAmG*mCmG*mU-
mA*mAmG*mUmG*mA*mUmG*mUmC*mGmU*mGmA
*mC-3^0.
6.2Me/PSFULL
50 - mG*mU*mC*mA*mC*mC*mU*mC*mA*mG*mC*mG*
mU*mA*mA*mG*mU*mG*mA*mU*mG*mU*mC*mG*m
U*mG*mA*mC-3^0.Underlined letters indicate the MB stem. m represents 2^0 -O-
methyl RNA modification. * represents PS linkage modification.
The MBs are labeled with a Cy5 fluorophore at the 5^0 -end and an
Iowa Black®RQ-Sp quencher at the 3^0 -end. These MBs are com-
plementary to luciferase RNA but not to endogenous RNAs in
mammalian cells, and under ideal circumstances will remain closed,
and thus quenched, in the cellular environment.- Luciferase target RNA oligonucleotides
50 -GUCAGGACAUCACUUACGCUGAGUUU-3^0.
2.3 Microporation 1. Microporation system (e.g., Thermo Fisher Neon transfection
system).
- 1phosphate buffered saline (PBS), Mg2+- and Ca2+-free.
- Resuspension buffer R (Thermo Fisher).
- Electroporation buffer (Thermo Fisher).
- Electroporation Gold Tips (10μL size) (Thermo Fisher).
- Electroporation tube (Thermo Fisher).
- 8-well chambered cover glass (e.g., Nalgene Nunc Lab-Tek).
- 10μg/mL fibronectin.
- Refrigerated microcentrifuge.
2.4 Microinjection 1. Microinjection system (e.g., Eppendorf Femtojet and Inject-
man NI 2).
- Microinjection capillary (e.g., Eppendorf Femtotip I).
- Microloader.
Optimizing Molecular Beacons 245