Abstract
Morpholino oligomers are effective antisense molecules to regulate gene expression and the US FDA has approved a Morpholino drug for the treatment of Duchenne muscular dystrophy. However, it has been observed that the antisense activities of aqueous solutions of some Morpholinos decrease over time. We hypothesize that the decreased activity is caused by the formation of soluble aggregates of the Morpholinos. Here, we analyzed three Morpholino sequences by size exclusion chromatography and found two of them have over time formed soluble aggregates in water. The degree of aggregation is sequence-, temperature-, and time-dependent. We describe a simple procedure for detecting and breaking down the aggregates to return the Morpholinos to their monomeric forms.
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References
Burrer R, Neuman BW, Ting JP, Stein DA, Moulton HM, Iversen PL, Kuhn P, Buchmeier MJ (2007) Antiviral effects of antisense morpholino oligomers in murine coronavirus infection models. J Virol 81:5637–5648
Rajsbaum R, Versteeg GA, Schmid S, Maestre AM, Belicha-Villanueva A, Martinez Romero C, Patel JR, Morrison J, Pisanelli G, Miorin L et al (2014) Unanchored K48-linked polyubiquitin synthesized by the E3-ubiquitin ligase TRIM6 stimulates the interferon-IKKepsilon kinase-mediated antiviral response. Immunity 40:880–895
Tripathi S, Pohl MO, Zhou Y, Rodriguez-Frandsen A, Wang G, Stein DA, Moulton HM, DeJesus P, Che J, Mulder LC et al (2015) Meta- and orthogonal integration of influenza "OMICs" data defines a role for UBR4 in virus budding. Cell Host Microbe 18:723–735
Geller BL, Marshall-Batty K, Schnell FJ, McKnight MM, Iversen PL, Greenberg DE (2013) Gene-silencing antisense oligomers inhibit acinetobacter growth in vitro and in vivo. J Infect Dis 208:1553–1560
Greenberg DE, Marshall-Batty KR, Brinster LR, Zarember KA, Shaw PA, Mellbye BL, Iversen PL, Holland SM, Geller BL (2010) Antisense phosphorodiamidate morpholino oligomers targeted to an essential gene inhibit Burkholderia cepacia complex. J Infect Dis 201:1822–1830
Shen N, Ko JH, Xiao G, Wesolowski D, Shan G, Geller B, Izadjoo M, Altman S (2009) Inactivation of expression of several genes in a variety of bacterial species by EGS technology. Proc Natl Acad Sci U S A 106:8163–8168
Xiao G, Wesolowski D, Izadjoo M, Altman S (2010) Morpholino oligonucleotides do not participate perfectly in standard Watson-Crick complexes with RNA. RNA 16:2218–2225
Summerton J, Weller D (1997) Morpholino antisense oligomers: design, preparation, and properties. Antisense Nucleic Acid Drug Dev 7:187–195
Lambert JD, Johnson AB, Hudson CN, Chan A (2016) Dpp/BMP2–4 mediates signaling from the d-quadrant organizer in a spiralian embryo. Curr Biol 26(15):2003–2010
Amantana A, Moulton HM, Cate ML, Reddy MT, Whitehead T, Hassinger JN, Youngblood DS, Iversen PL (2007) Pharmacokinetics, biodistribution, stability and toxicity of a cell-penetrating peptide-morpholino oligomer conjugate. Bioconjug Chem 18:1325–1331
Hudziak RM, Barofsky E, Barofsky DF, Weller DL, Huang SB, Weller DD (1996) Resistance of morpholino phosphorodiamidate oligomers to enzymatic degradation. Antisense Nucleic Acid Drug Dev 6:267–272
Youngblood DS, Hatlevig SA, Hassinger JN, Iversen PL, Moulton HM (2007) Stability of cell-penetrating peptide-morpholino oligomer conjugates in human serum and in cells. Bioconjug Chem 18:50–60
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Chow, G., Morcos, P.A., Moulton, H.M. (2017). Aggregation and Disaggregation of Morpholino Oligomers in Solution. In: Moulton, H., Moulton, J. (eds) Morpholino Oligomers. Methods in Molecular Biology, vol 1565. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6817-6_3
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DOI: https://doi.org/10.1007/978-1-4939-6817-6_3
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-6817-6
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