RNA accessibility impacts potency of Tough Decoy microRNA inhibitors

Abstract

MicroRNAs (miRNAs) are small RNA molecules that post-transcriptionally regulate gene expression through silencing of complementary target mRNAs. miRNAs are involved in many biological processes, including cell proliferation, differentiation, cell signaling and cellular defense responses to infection. Strategies that allow for strong and stable suppression of specific microRNA activity are needed to study miRNA functions and to develop therapeutic intervention strategies aimed at interfering with miRNA activity in vivo. One of these classes of miRNA inhibitors are Tough Decoys (TuD) RNAs, which comprise of an imperfect RNA hairpin structure that harbors two opposing miRNA binding sites. Upon developing TuDs targeting Epstein-Barr virus miRNAs, we observed a strong variation in inhibitory potential between different TuD RNAs targeting the same miRNA. We show that the composition of the ‘bulge’ sequence in the miRNA binding sites has a strong impact on the inhibitory potency of the TuD. Our data implies that miRNA inhibition correlates with the thermodynamic properties of the TuD and that design aimed at lowering the TuD opening energy increases TuD potency. Our study provides specific guidelines for the design and construction of potent decoy-based miRNA inhibitors, which may be used for future therapeutic intervention strategies.

Enhancement of gene expression noise due to nonspecific transcription factor binding

“…While this contribution focuses on TFs binding to genomic decoy sites, these results are applicable to other classes of proteins, for example RNA-binding proteins binding to sites on RNA. [112][113][114][115] Our results show that degradation of decoy-bound TFs critically impacts both the mean and noise levels of the free TF pool. More specifically, while the average number of free (available) TFs monotonically decrease with increasing decoy abundance, the noise levels can sharply increase at low/intermediate decoy numbers before attenuating to Poisson levels as N → ∞ (Fig 2).…”

LidNA, a miRNA inhibitor constructed with unmodified DNA, requires an xxxA insertion sequence in miRNA binding site for its potent inhibitory activity

“…TuD RNA is a potent miRNA inhibitor that harbors two miRNA binding sites 18,20. TuDs can be expressed intracellularly via vector‐based delivery.…”

“…TuDs have similar insertion sequences in the miRNA binding sites. Some insertion sequences cause high miRNA inhibitory activity, while other insertion sequences cause low or no activity 20. Hooykaas et al .…”

“…These miRNA binding regions contain mismatched insertion sequences in the center of each miRNA binding region. For example, the mismatched insertion sequence of TuD RNA expressed from a DNA vector was essential for higher inhibitory activity 18,20. The insertion sequences affected inhibitory activity, some insertion sequences caused high activity, whereas other insertion sequences caused low or no activity 20.…”

“…For example, the mismatched insertion sequence of TuD RNA expressed from a DNA vector was essential for higher inhibitory activity 18,20. The insertion sequences affected inhibitory activity, some insertion sequences caused high activity, whereas other insertion sequences caused low or no activity 20. Interestingly, synthetic TuD consisting of 2′‐ O ‐methylated RNA with no insertion had a higher activity than the insertion variant 21.…”