Bifunctional small molecule-oligonucleotide hybrid as microRNA inhibitor

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Abstract

miRNAs are key regulators of various biological processes. Dysregulation of miRNA is linked to many diseases. Development of miRNA inhibitor has implication in disease therapy and study of miRNA function. The biogenesis pathway of miRNA involves the processing of pre-miRNA into mature miRNA by Dicer enzyme. We previously reported a proximity enabled approach that employs bifunctional small molecules to regulate miRNA maturation through inhibiting the enzymatic activity of Dicer. By conjugating to an RNA targeting unit, an RNase inhibitor could be delivered to the cleavage site of specific pre-miRNA to deactivate the complexed Dicer enzyme. Herein, we expanded this bifunctional strategy by showing that antisense oligonucleotides (ASOs), including morpholinos and γPNAs, could be readily used as the RNA recognition unit to generate bifunctional small molecule-oligonucleotide hybrids as miRNA inhibitors. A systematic comparison revealed that the potency of these hybrids is mainly determined by the RNA binding of the targeting ASO molecules. Since the lengths of the ASO molecules used in this approach were much shorter than commonly used anti-miRNA ASOs, this may provide benefits to the specificity and cellular delivery of these hybrids. We expect that this approach could be complementary to traditional ASO and small molecule based miRNA inhibition and contribute to the study of miRNA.

Introduction

MicroRNAs (miRNAs) are short non-coding RNAs. They play a major regulatory role in orchestrating various biological processes.1 Dysregulation of miRNAs is associated with many diseases. They have been considered as a new type of therapeutic target.2 Therefore, a lot of efforts have been dedicated in developing approaches for regulating the function or biogenesis of miRNAs.3, 4, 5 These methods are expected to find application in disease therapy and the study of miRNA function.

One promising direction for miRNA regulation is focused on developing small molecule inhibitors to regulate the biogenesis of miRNA. The pathway starts with the transcription of miRNA gene followed by RNase Drosha-mediated processing of the primary transcript (pri-miRNA) to produce hairpin looped structure, precursor miRNA (pre-miRNA). pre-miRNA was then processed by RNase Dicer to give the mature miRNA. Small molecules targeting these processes could potentially serve as miRNA inhibitors. For this purpose, intense researches have been focusing on identification of small molecules targeting miRNA precursors in order to disrupt miRNA maturation.3, 4, 6, 7, 8, 9 Despite the significant progresses in this field, it remains challenging to design or identify small molecules that selectively recognize the target RNAs while at the same time provide satisfactory biological activities.10, 11

To address the issues that an RNA binder lacks desired activity, we recently reported a proximity enabled approach that employs bifunctional small molecules to regulate miRNA maturation through inhibiting the enzymatic activity of Dicer.12, 13, 14 These bifunctional molecules separate the functions of RNA recognition and RNA inhibition into two distinct structural units (i.e., a pre-miRNA binding unit and a Dicer inhibiting unit). The recognition of a target pre-miRNA by the RNA binding unit brings the conjugated weak Dicer inhibitor unit into the proximity to the pre-miRNA cleavage site of the bound Dicer molecule, thus blocking the enzymatic activity of Dicer and inhibiting the biogenesis of the target miRNA. We demonstrated that this bifunctional strategy significantly enhanced the inhibitory activity of the identified pre-miRNA binding small molecule (i.e., neomycin) against the maturation of miR-21, a well-known oncogenic miRNA overexpressed in many cancers.12, 14 However, aminoglycosides including neomycin are known to have poor selectivity in RNA recognition.10, 15 As a result, the neomycin-based pre-miRNA binding unit in the reported bifunctional molecules would not likely to provide desired selectivity.

Chemically modified antisense oligonucleotides (ASOs) have been developed to recognize and block the function of RNAs, including miRNAs, and have made significant contributions on RNA functional studies and therapeutic development for RNA-related diseases.16, 17, 18, 19, 20, 21, 22, 23, 24 Significant benefits of using ASOs for RNA targeting include enhanced recognition specificity and their sequence-based design that allows rapid generation of RNA binders/inhibitors when compared to the small molecule-based approaches. To provide a pre-miRNA binding unit with enhanced RNA recognition specificity in our bi-functional approach, in this work, we investigated the possibility of using the ASO to replace a small molecule as the RNA targeting unit (Fig. 1). We designed bifunctional molecules with different ASOs conjugated to weak Dicer inhibitors to achieve the inhibition of miR-21 maturation through Dicer inhibition.

Section snippets

Bifunctional small molecule-oligonucleotide hybrid inhibits miR-21 biogenesis

Morpholino ASOs bind to complementary RNAs in high affinity, are resistant to nucleases and have been widely used for knocking down gene expression, modifying RNA splicing and inhibiting miRNA function and maturation.25, 26, 27, 28 These favorable characteristics make morpholino ASO a promising candidate to be used as the pre-miRNA targeting unit in our bifunctional design. Pre-miRNAs typically fold to form stem-loop structures, which provide exposed single-stranded loop regions to be targeted

Conclusion

We previously demonstrated that using bifunctional small molecules to achieve enzymatic inactivation of Dicer can be a novel way for miRNA inhibition. By conjugating to an RNA targeting unit, an RNase inhibitor could be delivered to and locally enriched at the cleavage site of specific pre-miRNA to block Dicer-mediated pre-miRNA processing. To expand on this bifunctional strategy, we showed that ASO molecules, including morpholinos and γPNAs, could be readily used as the RNA targeting unit to

Chemicals and instrumentation

Morpholino ASOs were purchased from GeneTools, LLC. Other reagents were purchased from Sigma-Aldrich or Alfa Aesar. Column chromatography was carried out on silica gel (pore size 60 Å, 200–425 mesh particle size, Sigma Aldrich). HPLC was performed using a Thermo Scientific UltiMate 3000 semi-preparative system. 1H and 13C NMR spectra were recorded on a Bruker Avance III 300 spectrometer. Chemical shifts are reported in parts per million (ppm, δ) referenced to the residual 1H resonance of the

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This research was supported by NIH R21CA202831 (F.-S.L.).

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