Achieving Efficient Delivery of Morpholino Oligos with Nucleofection®

› April, 2008 › Application Note page 01 of 04

Shannon T. Knuth, Jon D. Moulton and Paul A. Morcos, Biology and Customer Support, Gene Tools, LLC, Philomath, Oregon, USA

Morpholino oligos provide a stable, specific and effective antisense activity. Developmental biologists have long since used these properties by microinjecting or electroporating Morpholinos into embryos to allow for splice-blocking or gene knockdown. However, efficient delivery of Morpholinos in mammalian cell cultures can be a challenge. While complex formation with lipid-based reagents is ineffective due to the non-ionic Morpholino backbone, we show here that Nucleofection® can be used as an efficient delivery method. As such, Nucleofection® of Morpholinos offers a straightforward alternative to siRNA-mediated gene knockdown and miRNA maturation inhibition.

Introduction sequence, sterically inhibiting the movement of the translation initiation complex toward the Morpholino oligos (MOs), also known as start codon. This prevents the large subunit of phosphodiamidate Morpholino oligos, are desi- the ribosome from assembling and, as a conse- gned to bind to complementary RNA sequence quence, causing reduced gene expression. and sterically inhibit proteins from binding the Morpholinos are also used to modify pre-mRNA RNA. Morpholinos are commonly used to knock splicing by tar geting the oligo to a splice junc- down gene expression and alter mRNA splicing tion or regulatory site, sterically blocking in a variety of organisms, tissues or cells (for a binding of snRNPs or other splice factors1. searchable data base of references see Splice-blocking Morpholinos have great http://www.gene- tools. com/Publications/). For promise for therapeutic use correcting and/or gene knockdowns the MOs are designed to com- reducing the impact of the mutations that lead plement mRNA sequence in the 5’ untranslated to b-Thalessemia2, Duchenne muscular region and/or the first 25 bases of mRNA coding dystrophy3 and other diseases caused by

DNA RNA Morpholino

Figure 1: Comparison of the chemical structure of two-mers of Morpholino, RNA and DNA. Two significant differences in the Mor- pholino backbone compared to RNA and DNA are the non-charged phospho- diamiate linkage between the subunits and replacement of the five-membered sugar ring with the six-membered Morpholine ring.

mis-splicing4,5. Morpholinos have also been used the uncharged MO backbone (see Figure 1). to block microRNA maturation6 and microRNA While microinjection is suitable for delivery into targets7, block ribozyme activity8 and induce organs (embryos or brains), most commonly frameshifts9. used transfection methods for cultured cells are However, even more so than RNA and DNA scrape loading16, special delivery17, conjugated based oligos, Morpholinos are challenging to cell-penetrating peptides19, Endo-Porter delive- deliver into mammalian cells in culture. ry18 or electroporation14. Here, we present, the Standard lipid-based nucleic acid transfection results of our study using the Nucleofector® reagents rely on an ionic interaction with a Technology for delivering Morpholinos. charged back bone and are not effective with

Advantages of Morpholinos over DNA and RNA-based gene knockdown reagents:

› Very robust and stable molecules10, 11 due to the non-biological, chemically stable backbone structure (Figure 1) › Exquisite specificity: at low concentrations a Morpholino with just five mispairs along its length will have no antisense activity12 › Largely free of non-antisense effects as evidenced by rescue of the Morpholino phenotype via injection of the corresponding mRNA13 › Backbone is non-toxic › Predictable targeting › Steric block mechanism allows for additional applications such as altering mRNA splicing and blocking microRNA maturation

Material and Methods cuvette and transfected using Nucleofector® Program I-013. After Nucleofection®, 500 µL of The ON 705 HeLa cell line was grown to serum free RPMI medium was added to each 75-80% confluency in DMEM/F-12 media cuvette followed by transfer of contents to a (Invitrogen) with 10% FBS (Invitrogen). On the 6-well plate. In the ON 705 HeLa cell line, the day of the experiment the cells were treated delivery of the Morpholino corrects a splicing with 0.5% trypsin (Sigma), removed from the mutation, splicing out a stop codon and putting plate and spun down. The supernatant was luciferase in frame15. After 24 hours, the cells removed and the cells resuspended in 100 µl were lysed and luciferase (Promega) and Nucleofector® Solution R per manufacturer’s, protein (Bio-Rad) assays were carried out in direction. The indicated amount of MO was duplicate for each sample. Delivery success is added to each sample and mixed by vortexing. proportional to the light output and the data is The sample was transferred to a Nucleofection® normalized per protein.

1600 Luciferase activity Figure 2: Dose-dependent delivery of 1400 Morpholinos into ON 705 HeLa cells. Cells were transfected with indicated 1200 concentrations of splice-correcting MO. 1000 In the ON 705 HeLa cell line, the 800 Mor pholino corrects a splicing mutation 600 splicing out a stop codon and putting

400 luciferase in frame. After 24 hours, the cells were lysed and luciferase activity 200 and protein amount were determined. 0 Delivery success is proportional to the light output (normalized to total protein 01 248 amount). MO concentration ( M)

Results/Discussion be slightly higher with Nucleofection® compared to activities commonly achieved with Morpholinos were delivered uniformly in nearly Endo-Porter. Effective MO amounts needed for 100% of the cells when imaged by fluorescence Nucleofection® seem to be lower than those microscopy (data not shown). To further analy- published for classical electroporation14. This ze delivery efficiency we used our established suggests that Nucleofection® could be advanta- splice correction assay with a luciferase repor- geous in terms of effective amounts due to its ter stably expressed in ON 705 HeLa cells. The direct nuclear delivery or to smaller reaction luciferase activity produced at the 1-8 µM range volumes (higher concentrated cell suspension). of MO reveals a consistent increase in MO In an additional experiment (Figure 3), the MO activity (Figure 2). Generally, as shown by the concentration was fixed at 2 µM and we compa- efficacy of cytosolic microinjection of splice- red delivery of Morpholinos with different 3’ modifying Morpholinos into embryos, delivery end modifications. The carboxyfluorescein (Fl) to the cytosol is sufficient to give nuclear spli- has two negative charges at physiological pH ce-correcting activity of Morpholinos. However, while the lissamine (Li) (sulforhodamine B) is a average luciferase activities (Figure 2) seem to zwitterion with no net charge.

Figure 3: Modifications of 3' end do 700 Luciferase activity not alter delivery efficiency. 600 Cells were transfected with 0.2 nmol 500 (2 µM) of a standard MO, a carboxy- 400 fluorescein (Fl) or a lissamine (Li) 300 labeled MO. 24 hours post 200 Nucleofection®, delivery efficiency 100 was analyzed by activity of splice- 0 corrected luciferase (normalized to Std. control Fl. std. control Li. std. control MO concentration ( M) total protein amount).

amaxa AG amaxa Inc. Europe/World USA Scientific Support Scientific Support +49 (0)221-99199-400 (888) 632-9110 (toll free) [email protected] › www.amaxa.com [email protected] WTB-1013_2008-04-05 › In conclusion, Nucleofection quantitative shiftinthe effectively inC2C12 Other product andcompany names mentionedherein are thetrademarks of theirrespective owners. Li-COR Inc., OligoEngine isatrademark of OligoEngineInc. BD Pharmingenisatrademark of Becton, Dickinson andCompany, Li-COR and Odyssey are trademarks of amaxa AG intheU.S. and/or Germany and/or othercountries. amaxa, Nucleofector, Nucleofection, Nucleocuvette, maxGFPand96-well Shuttle are registered trademark of and/or patent pendingrights owned by amaxa AG. In ourlab, Nucleofection groups compared to MOswithunmodifiedends. diff We found thatthere were nosignificant Solutions, The Nucleofector 19. 18 17. 16. 15. 14. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. References April, 2008 . Wu Wu Summerton (2005) AnnNYAcad Sci 1058: 62-75. Morcos (2001) Genesis 30(3): 94-102. Partridge Schmajuk Heasman Summerton (1999) Youngblood Hudziak Howard Yen Choi Kloosterman Ugarte Scaffidi andMisteli (2006) Science 312(5776): 1059-63. Yokota Kole Morcos (2007) Matter andKonig (2005) NucleicAcids Res 33(4): e41. erences indelivery of theMOswith3’end et al. et et al. et et al. et et al. et Nucleofector et al. et et al. et et al. et et al. et (2007) NucleicAcids Res 35(15): 5182-91. mx Gamaxa Inc. USA Scientific Support (888)632-9110 (toll free) scientific-support +49 (0)221-99199-400 Scientific Support Europe/World amaxa AG (2004) Nature 431(7007):(2004) 471-6. et al. et et al. et et al. et (2004) (2007) Science 318: 271-4. ® et al. et (2007) OpinBiol Ther Expert 7(6):831-42. (2007) AmJHumGenet 81(6). et al. et Technology, comprising Nucleofection (2004) RNA10(10):1653-61. (1996) Antisense NucleicAcid DrugDev 6(4):267-72. Biochem Biophys Res Commun 358(2): 521-7. (2000) Dev Biol 222(1): 124-34. (1996) Antisense NucleicAcid DrugDev 6(3):169-75. (1999) JBiol Chem274(31): 21783-9. ® muscle cells to produce a (2007) Bioconjug Chem18(1):50-60. Oligonucleotides 14(1):65-74. (2007) PLoS Biol 5(8):e203. 96-well Shuttle Biochim Biophys Acta 1489(1): 141-58. splicing of dystrophin. ® @ has also beenused amaxa.com ® has beenshown › plcto oepg 4of 04 page 04 Application Note ® System andNucleocuvette › w.mx.o [email protected] www.amaxa.com ® substrates. for otherDNA- orRNA-based geneknockdown types andprimarycells aspotent alternative now beexplored indifficult-to-transfect cell Nucleofection in cultured mammaliancells. Thus, by using to allow efficient delivery of Process, Nucleofector ® plates andmodules iscovered by patent ® for for delivery, Morpholinos can ® Device, Nucleofector Morpholino oligos ®