An aptamer targeting the apical-loop domain modulates pri-miRNA processing

MicroRNAs (miRNAs) are short noncoding RNAs that recognize complementary bases on target mRNAs, thereby triggering either inhibition of translation initiation or mRNA degradation. They have unique expression patterns and are involved in almost every important biological process, including cell proliferation, differentiation, and apoptosis. In turn, deregulation of miRNA expression patterns is a key condition in the onset and progression of tumor development. Following the synthesis of the primary transcript (primiRNA), the maturation process of miRNAs comprises several steps. First, the pri-miRNA is hydrolyzed by the microprocessor complex, consisting of Drosha/DGCR8, to release hairpin-shaped precursor RNAs (pre-miRNAs). Subsequently, the pre-miRNA is exported into the cytoplasm and further processed by the type III ribonuclease Dicer to produce mature miRNAs. Owing to the prominent role of miRNAs in regulating gene expression, considerable efforts have been made to develop selective tools that will allow the direct targeting of miRNAs affecting either their biogenesis or function. One such class of tools is represented by the so-called antagomirs, short single-stranded 2’-methoxy-modified oligonucleotides. Antagomirs recognize mature miRNAs by complementary bases, thereby preventing miRNA–mRNA association. Here, we introduce another class of nucleic acid based molecular tools to interfere with miRNA activity, namely, RNA aptamers that specifically recognize the loop domains of a pri-miRNA and modulate its processing. Aptamers are short single-stranded nucleic acids that fold into well-defined three-dimensional structures that facilitate specific target recognition. Aptamers can be isolated by an in vitro selection process, and a wide variety of target molecules, such as proteins, cells, small molecules, and nucleic acids have been already applied for aptamer identification. Especially in the latter case, the interaction between the aptamer and the target RNA has been proven to rely on fitting three-dimensional shapes, going beyond mere recognition through complementary base pairing. We sought to elucidate whether RNA aptamers could be used as an alternative nucleic acid based molecular tool to specifically interfere with the biogenesis of individual miRNAs. Here we describe the isolation and characterization of an RNA aptamer that specifically targets the pri-miRNA polycistron 17~ 18a~ 19a~ 20a~ 19b-1~ 92. We show that the aptamer binds inter alia to the apical-loop domain of primiR18a and thereby inhibits the biogenesis of all miRNAs 1719b-1 within this cluster. Our results show that aptamers can be applied as agents that modulate pri-miRNA processing and as tools for elucidating mechanisms of this process. Furthermore, the ability to modulate the maturation of miRNA by targeting the apical-loop domain supports the importance of these domains during pri-miRNA processing. To obtain aptamers that specifically target pri-miR17~ 18a~ 19a~ 20a~ 19b-1~ 92, we applied an in vitro selection scheme in which the 791 nucleotide (nt) miRNA polycistron comprising pri-miR17 ~ 18a ~ 19a~ 20a ~ 19b-1 was biotinylated at its 5’-end and immobilized on streptavidin-coated magnetic beads (Figure 1). The beads were incubated with an RNA library comprising a 25nt random region. To avoid the participation of the constant regions of the RNA library in pri-miRNA binding, we sequestered these regions by hybridization to complementary oligodeoxynucleotides (ODNs), leaving the random nucleotides free for independent folding. After removal of all unbound RNA sequences, the retained RNAs were eluted by adding EDTA. This step essentially favors the release of those molecules that require Mg ions for RNA binding over those that exclusively rely on complementary base pairing. After seven selection cycles, enhanced pri-miRNA binding was detectable, which could be further improved by five succeeding cycles of selection and amplification (Figure S1A in the Supporting Information). The RNA library obtained from selection cycle 12 was cloned and sequenced. Amongst 17 analyzed sequences, nine revealed the consensus motif I, 5’-AACACCUC, comple[*] C. E. L nse, C. S. Hopp, Dr. A. Rentmeister, Prof. M. Famulok, Prof. G. Mayer Life and Medical Sciences (LIMES), University of Bonn Gerhard-Domagk-Strasse 1, Bonn (Germany) Fax: (+49)228-734-809 E-mail: m.famulok@uni-bonn.de gmayer@uni-bonn.de