MicroRNAs are non-coding RNA molecules that act as endogenous inhibitors of gene expression. MicroRNAs are commonly transcribed by type II RNA polymerases as a long-acting precursor called primary-microRNA (pre-microRNA). They are then processed in the nucleus by an enzymatic complex, (RNase III) and Pasha / DGCR8, and converted to a liberated hairpin referred to precursor-microRNA (pre-microRNA) with approximately 60–80 nucleotides in length. Pre-microRNA is then transported from the nucleus to the cytoplasm by exportin-5 protein, cleaved by the RNase III enzyme called Dicer, and converted to a double-stranded 18–24 nucleotide RNA molecule. The RNA molecule is located in the RNA-induced silencing complex (RISC) and, depending on the relative thermodynamic stability of the 5'-end, one of the strands remains as the guide strand or mature microRNA and another strand is degraded. MicroRNA guides the RISC to its target mRNA, cleaving the mRNA or preventing translation [1–2]. MicroRNAs are widely used in biological processes, including cell proliferation and apoptosis. Aberrant expression of microRNAs can lead to disorders and malignancies in humans, including cancer. In addition, altered microRNA expression levels are associated with the type and stage of cancers [1]. These molecules regulate gene expression through specific interaction with 3'-untranslational region (3'-UTR) of mRNA after transcription. The discovery of microRNAs as the main regulators of genes opens up a new avenue to develop more powerful and effective ways to control and treatment of diseases [3]. Due to the potential of microRNAs in targeting a large number of mRNAs, these molecules are involved in many biological phenomena, and therefore, many of the researches have been conducted to use microRNAs in the diagnosis and treatment of diseases [4–6]. For example, the normal expression of microRNAs that have been removed from the genome or their expression has been reduced can be compensated by microRNA restoration based-therapy through microRNA-mimic or microRNA-mimetic [7]. Therefore, the expression of microRNAs in both in vivo and in vitro can be regulated by the synthesis of pre-microRNA molecules or antisense oligonucleotides, which is a promising prospect for the treatment of a variety of diseases. Pre-microRNA gene cloning is one of the strategies of this technology. In this strategy, the pre-microRNA gene is cloned into an expression vector. This method has been widely used in many studies to detect microRNAs, detecting their target genes, investigating cell signaling pathways, and generating model cells expressing specific microRNAs [8]. Common cloning methods usually involve using restriction endonucleases to produce DNA fragments with complementary ends that ligate together by a DNA ligase. This process involves preparing the insert (microRNA precursor gene) and the expression vector. These DNAs are cleaved by two specific restriction enzymes that identify the restriction sites on either side of the insert sequence and the multiple cloning site of the vector. By using two different restriction enzymes, two different ends are produced in each of these DNAs, which leads to the directional binding of the insert sequence to the expression vector. Directional cloning is very important for maintaining an open reading frame as well as the activity of regulatory factors [9]. In addition, non-directional cloning can be performed using a restriction enzyme. In this case, a screening step is needed to determine the correct direction of the gene and to prevent the self-ligation, the vector should be dephosphorylated, which reduces the efficiency of the cloning reaction [10].
As mentioned above, common cloning methods depend on the presence of suitable restriction sites in vectors and the insert sequences. Restriction enzymes used in these methods are often expensive [11]. In addition, the low efficiency of enzymatic digestion for PCR products leads to the failure of the common cloning protocol. T-A cloning technology can solve this problem, but this technique is time-consuming and expensive. In addition, using the TA enzyme recognition sites adds extra nucleotides to the insert sequence and may disturb the open reading frame [12]. Therefore, it is better to place the recognition sites of the restriction enzymes on the primers. These limitations are the result of the need for enzymatic digestion to produce sticky ends in the insert sequence. In order to overcome these limitations, Li et al. proposed a cloning method independent of the restriction sites, which is based on homologous recombination and single-stranded annealing under in vitro conditions [13]. In addition, there are several reports of restriction enzyme-independent cloning strategies, including CPEC (circular polymerase extension cloning), SLiCE (seamless ligation cloning extract), RSFC (restriction site-free cloning), SLIC (sequence- and ligation-independent cloning), and Gibson assembly [13–15, 18]. Despite the effectiveness of these methods, they are complex and dependent on other special enzymes and laborious and time-consuming protocols. Therefore, designing a simple, fast and accurate method that eliminates the need to use restriction enzymes for producing PCR products with appropriate sticky ends paves the way for using pre-microRNA cloning strategy for the diagnostic and treatment.
In the present study, we decided to provide a new generation of precursor microRNA cloning method without need to restriction enzymes for producing PCR products with appropriate sticky ends (insert sequence). In the proposed strategy, by engineering an expression vector's sequence and designing two intelligent primer sets for two consecutive PCR reactions, the pre-microRNA sequence with appropriate sticky ends counterpart with the expression vector were produced, without restriction enzymes (Fig. 1). The present method can be a fast, convenient, inexpensive and reliable method for microRNA precursor cloning by providing a high-performance protocol.