Silencing of microRNA families by seed-targeting tiny LNAs

Abstract

The challenge of understanding the widespread biological roles of animal microRNAs (miRNAs) has prompted the development of genetic and functional genomics technologies for miRNA loss-of-function studies. However, tools for exploring the functions of entire miRNA families are still limited. We developed a method that enables antagonism of miRNA function using seed-targeting 8-mer locked nucleic acid (LNA) oligonucleotides, termed tiny LNAs. Transfection of tiny LNAs into cells resulted in simultaneous inhibition of miRNAs within families sharing the same seed with concomitant upregulation of direct targets. In addition, systemically delivered, unconjugated tiny LNAs showed uptake in many normal tissues and in breast tumors in mice, coinciding with long-term miRNA silencing. Transcriptional and proteomic profiling suggested that tiny LNAs have negligible off-target effects, not significantly altering the output from mRNAs with perfect tiny LNA complementary sites. Considered together, these data support the utility of tiny LNAs in elucidating the functions of miRNA families in vivo.

Chemical modification and design of anti-miRNA oligonucleotides

“…40 Shorter AMOs may also invade miRISC easily, which may contribute to the efficacy of the tiny LNA class of compounds. 39 Along these lines, AMOs with 3¢ nucleotide truncations were seen to improve efficacy in one study, potentially by avoiding hypothetical structural constraints associated with the Piwi-binding domain, thereby allowing improved access of the AMO to the targeted miRNA. 26 Specificity is an important consideration when designing AMOs and the degree of stringency desired can vary with the experimental goals.…”

“…It is also important to remember that the seed region drives the miRNAtarget mRNA interaction so it is essential that the AMO:miRNA duplex structure includes these critical bases; this is best evidenced by the surprising efficacy of the tiny LNAs that bind only to the seed region. 39 Additional factors may contribute to the functional potency of an AMO. In particular, it may be advantageous for the AMO to invade miRISC and inactivate pre-existing miRNA:miRISC complexes.…”

“…miRNA family members tend to regulate the same genes through their shared seed region sequences. To address this, Obad et al 39 recently described the use of short 8mer fully-LNA-PSmodified AMOs (Table 1), which bind solely to the seed region of the targeted miRNA. These 'tiny LNAs' were validated to effectively inhibit the function of both individual miRNAs (miR-21) as well as entire families of miRNAs (miR-221/222 and let-7) in cell culture using a luciferase-based detection method.…”

“…For example, a high-affinity AMO with reduced specificity (such as the LNA/2¢OMe mixmer), which is the reverse complement to a fulllength 'consensus sequence' , could target an entire miRNA family. Alternatively, the clever approach used by Obad et al 39 effectively utilized the high-affinity LNA modifications in short AMOs complementary to the seed region to knockout the let-7 and miR-221/222 families of miRNAs. This strategy obviates the challenges associated with designing a full-length AMO nonspecific enough to include all of the intended miRNA family members whereas specific enough to eliminate other off-target events.…”

Epigenetics and ncRNAs in Brain Function and Disease: Mechanisms and Prospects for Therapy

“…Deficiency in expression of a miRNA may be compensated by delivery of a miRNA mimic [24]. Conversely, miRNA inhibition strategies may be effective in cases where the expression of a miRNA is a causative factor in a disease [25]. Together with epigenetic mechanisms, noncoding RNAs like miRNAs form a regulatory network that modulate multiple phenotypic traits, or the pathology of complex diseases such as neurological disorders and cancer.…”