Design of Effective Primary MicroRNA Mimics With Different Basal Stem Conformations

Fiona T van den Berg; John J Rossi; Patrick Arbuthnot; Marc S Weinberg

doi:10.1038/mtna.2015.53

Design of Effective Primary MicroRNA Mimics With Different Basal Stem Conformations

Mol Ther Nucleic Acids. 2016 Jan 12;5(1):e278. doi: 10.1038/mtna.2015.53.

Authors

Fiona T van den Berg¹, John J Rossi², Patrick Arbuthnot¹, Marc S Weinberg^{1

3

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Affiliations

¹ Wits/SAMRC Antiviral Gene Therapy Research Unit, Department of Molecular Medicine and Haematology, School of Pathology, University of the Witwatersrand, Johannesburg, South Africa.
² Department of Molecular and Cellular Biology, Beckman Research Institute of City of Hope, Duarte, California, USA.
³ HIV Pathogenesis Research Unit, Department of Molecular Medicine and Haematology, School of Pathology, University of the Witwatersrand, Johannesburg, South Africa.
⁴ Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA.

Abstract

Primary microRNA (pri-miRNA) mimics are important mediators of effective gene silencing and are well suited for sustained therapeutic applications. Pri-miRNA mimics are processed in the endogenous miRNA biogenesis pathway, where elements of the secondary RNA structure are crucial for efficient miRNA production. Cleavage of the pri-miRNA to a precursor miRNA (pre-miRNA) by Drosha-DGCR8 typically occurs adjacent to a basal stem of ~11 bp. However, a number of pri-miRNA structures are expected to contain slightly shorter or longer basal stems, which may be further disrupted in predicted folding of the expressed pri-miRNA sequence. We investigated the function and processing of natural and exogenous RNA guides from pri-miRNAs with various basal stems (9-13 bp), where a canonical hairpin was predicted to be well or poorly maintained in predicted structures of the expressed sequence. We have shown that RNA guides can be effectively derived from pri-miRNAs with various basal stem conformations, while predicted guide region stability can explain the function of pri-miRNA mimics, in agreement with previously proposed design principles. This study provides insight for the design of effective mimics based on naturally occurring pri-miRNAs and has identified several novel scaffolds suitable for use in gene silencing applications.