Manipulating Levels of Specific MicroRNAs in Mammalian Cells - MicroRNAs as Tools in Biopharmaceutical Production - page 114

8.2 Transient miRNA Manipulation with Synthetic Mimics

and Inhibitors

Synthetic, double stranded RNA (dsRNA) oligonucleotides are often transfected
into cells to imitate or mimic an endogenous miRNA. These dsRNA oligos resemble
siRNAs, except that they are usually comprised of the natural mature miRNA
paired with its partial complement instead of two fully complementary strands.
The sequences of all known mature miRNAs and their hairpin precursors are freely
available from miRBase (Kozomara and Griffiths-Jones 2011 ) and can be accessed
at http://www.mirbase.org/. Although only two Chinese hamster miRNAs had
been submitted to miRBase at the time this chapter was written (cgr-miR-21 and
cgr-miR-7) (Barron et al. 2011 ; Gammell et al. 2007 ), miRBase 18, contained
1,157 mouse and 1,921 human mature miRNA sequences, many of which are con-
served. Both the Borth (Hackl et al. 2011 ) and Lee (Hammond et al. 2012 ) groups
have published sequencing results for conserved miRNAs expressed in several
CHO cell lines, which can be cross-checked to verify sequence conservation for
miRNAs of interest. Furthermore, the Borth group computationally identified gene
loci and precursor miRNA sequences for several hundred conserved CHO miRNAs
and deposited the sequences in miRBase for addition to future updates (Hackl et al.
2011 ).
As with siRNAs, various modifications to the dsRNA structure have been shown to
improve efficacy and/or stability. Two nucleotide extensions on one or both 3 -ends,
either the natural 2 nucleotides, 2 uridines, or 2 thymidines, have been reported to
improve performance. An amino group at the 5 -end of the partially complementary
(i.e., non-miRNA) strand may inhibit loading into the RISC complex in favor of
the mature miRNA sequence, and 2 -O-methyl or other modified nucleotides may
improve stability (Czauderna et al. 2003 ). Alternatively, miRNA mimics are commer-
cially available from several sources. Each has their own proprietary miRNA mimic
design and synthesis processes, and will custom design mimics for any miRNA se-
quence. These companies also sell libraries or panels of individual mimics for all
known human or mouse miRNAs. As an alternative approach to profiling miRNAs
in cell lines by microarray or sequencing, libraries or panels of miRNAs can be
transfected into cells in a medium or high throughput format to screen for desired
phenotypes and identify miRNA targets for cell line engineering.
Whereas some miRNAs may enhance recombinant protein production when up-
regulated, others may interfere with functions essential for good production, and
therefore, need to be inhibited for biopharmaceutical applications. Synthetic miRNA
inhibitors, also called antimiRs, are usually single-stranded RNA (ssRNA) antisense
to the miRNA (i.e., the reverse complement sequence). Modified nucleotides, such
as 2 -O-methyl and locked nucleic acids (LNAs), are often substituted for standard
nucleotides to improve stability and targeting (Davis et al. 2006 ).
Synthetic miRNA inhibitors are commercially available from most of the same
sources as mimics. In addition, as with miRNA mimics, libraries of miRNA inhibitors
can be screened to identify miRNAs whose inhibition gives a desirable phenotype.