MicroRNAs (miRNAs) are small non-coding RNAs 18~24 nucleotides in length that have been proven to play important roles on post-transcriptional regulation of the gene expression¹. Up to now, over 2000 miRNAs have been identified or predicted from human tissues or cells². Mature miRNAs are mainly existing in cytoplasm and, together with Argonaute (Ago) family of protein, packed into a protein complex known as miRNA-induced silencing complex (RISC)³. Base-paring to 3’ untranslated region (3’ UTR) of target mRNA, miRNA would induce translational repression or mRNA degradation due to the endonucleolytic activity of Ago, thus regulating gene expression and participants in many pivotal biological processes (Figure 1, right panel), including cell proliferation, differentiation, migration and apoptosis^4-6. To understand the regulatory mechanism of miRNA, miRNA gain of function and loss of function research are necessary. Thus, methods for inhibiting functional microRNAs in vitro and in vivo is wildly needed in miRNA researches^7-9.

Figure 1. Schematic representation of our project. Left panel shows the assembly strategy of miRNA. Right panel shows the mechanism how miRNA Lockers inhibit target miRNA activity.

Presently, loss-of-function phenotypes are mainly induced by means of chemically modified antisense oligonucleotides(ASO). Chemically modified antisense oligonucleotides includes 2’O-methyl, 2’O-methoxyethyl, locked nucleic acid (LNA) and others, which aims to pair with and block mature microRNAs through strictly sequence complementarity¹⁰. Antagomirs are a group of chemically modified antisense oligonucleotides, which are readily available tools wildly used for endogenous miRNA inhibition³. To improve the stability and miRNA binding affinity, different modifications are introduced to antagomir and optimized to achieve high fidelity, low toxicity, and improved stability. Although there are many advantages of ASO, limited scalability and off-target effects still limit the extensive usage of ASO in miRNA loss of function study.

Apart from ASO, two other approaches have been reported to be utilized in miRNA loss of function researches. One is called “miRNA sponge”, which stands for a class of competitive inhibitor of miRNA carrying multiple binding sites of miRNA¹¹. Another is termed “miRNA mask” ¹², which use oligonucleotides to perfectly complementary to mRNA, like a mask covering miRNA binding sites. Therefore, miRNA cannot bind to mRNA and induce subsequently miRNA-mRNA interaction. However, miRNA inhibition using miRNA sponge or miRNA mask are not widely used for miRNA loss of function researches ¹⁰.

Notably, previous reports have demonstrated that miRNA can efficiently bind to single strand DNA in a base-matching manner. Deng et al. and our project last year also showed that non-modified DNA with dumb-bell-shaped secondary structure and miRNA binding sites on the loop region presents high miRNA binding affinity and improved stability¹³ (see https://2016.igem.org/Team:NUDT_CHINA/Results ). Hence, we anticipate dumb-bell-shaped DNA may serve as miRNA inhibitors as well.

In our project, we demonstrated a dumb-bell-shaped DNA based design of miRNA inhibitor to achieve low-cost and high scalability with high inhibitory effect simultaneously. Our miRNA inhibitor, named as miRNA locker, can be easily assembled using modularized DNA parts from a set of chemically synthetic oligo DNA library (Figure 1, left panel).

Using miR-214 as a demo of our scheme, we assembled miR-214 specific Locker based on our oligo DNA library and validate the function of Locker as miRNA inhibitor in vitro. To be specific, we demonstrated that miRNA lockers could be efficiently recognized by ago-miRNA complex and further functioned by lowering the target miRNA level. Downstream gene expression changes and phenotypic changes consistent to commercialized miRNA inhibitors were also observed. Our results also showed a better inhibitory effect of miRNA Lockers comparing to the commercialized miRNA inhibitors. These results indicate that miRNA Locker might be a promising substitute miRNA inhibitor with similar or even stronger inhibitory effects comparing to existing ones. Also, we demonstrated how miRNA Lockers can be used in miRNA researches as a promising substitute of current miRNA inhibitors by identifying a new RNF8-targeting miRNA and establish its regulatory relationship with epithelial–mesenchymal transition (EMT).

With the unique advantage and potential on multi-targeting and convenience of miRNA Locker, we believe that our design might provide an alternative approach for miRNA inhibiting for research, diagnostic and therapeutic uses.