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Tools & Techniques: TALENs for miRNA Manipulation; CLASH for miRNA-Target Analysis; and More

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With microRNAs emerging as key regulators of biological processes and disease states, there is a need for tools to manipulate their expression and function in human cells. Antisense oligos are widely used for this purpose, but these are limited to certain cell types that can take them up with high efficiency and require constant delivery of fresh miRNA antagonists.

To address this, researchers from the University of Utah have developed a novel approach to target and disrupt human miRNAs based on engineered transcription activator-like effectors, or TALEs, which are a class of DNA-binding proteins derived from Xanthomonas plant pathogens.

According to a paper appearing in PLoS One, TALEs have recently been shown to bind to DNA in a highly sequence-specific manner, and to mediate gene modifications based upon their fusion to transactivation, repression, or nuclease domains.

“Importantly, because TALE proteins are made up of modules, with each interchangeable module recognizing specific DNA bases, TALEs can theoretically be engineered to bind virtually any DNA sequence,” the scientists wrote. “Just recently TALE proteins have been shown to function in human cells indicating that this technology can be used to modify specific human genes.”

The team developed custom TALEs called TALENs — short for TALEs that function as nucleases — to target four miRNAs with established functional importance: miR-155, miR-155*, miR-146a, and miR-125b.

In all experiments, the TALENs were able to trigger sequence deletions within the target genes that include disruptions to the miRNA seed sequence, according to the paper. Additionally, the researchers were able to achieve complete miR-155 hairpin removal by using two TALEN pairs together.

“Furthermore, we observe bi-allelic modifications indicating that TALENs can disrupt both miRNA gene alleles within a human cell,” they added.

The findings suggest that TALENs make “excellent candidates to achieve miRNA gene targeting and manipulation in a variety of relevant human cell types, including those with important therapeutic applications, such as stem cells, neurons, and primary tumors,” the authors concluded.


As miRNA research continues, a set of canonical rules of miRNA/target interactions has emerged, yet there is growing evidence that there are exceptions to the rules, creating a need for new tools to fully examine nucleic acid interactomes.

Research over the past decade suggests that, among other things, miRNA/target interactions are mediated by the small RNA’s seed region, nucleotides paired outside of this region stabilize interactions but don’t impact miRNA efficacy, and functional miRNA targets are localized close to the extremes of the 4’ UTRs of protein-coding genes in mostly unstructured regions.

But this is not always the case. For example, in Caenorhabditis elegans, the well-studied lin-

4/lin-14 interaction involves bulged nucleotides, whereas the let-7/lin-41 interaction involves wobble G-U pairing, according to a paper published last month in Cell by a team from the University of Edinburgh.

Previously, the researchers developed a method called CLASH — short for crosslinking, ligation, and sequencing of hybrids — to enable direct, high-throughput mapping of RNA-RNA interactions. Now, they have adapted it to allow direct observation of miRNA-target pairs as chimeric reads in deep-sequencing data.

In their paper, the investigators reported data sets of more than 18,000 high-confidence miRNA-mRNA interactions, finding that while the binding of most miRNAs includes the 5’ seed region, around 60 percent of seed interactions are non-canonical, containing bulged or mismatched nucleotides.

“Moreover, seed interactions are generally accompanied by specific, non-seed base pairing,” they wrote in Cell. Eighteen percent of miRNA-mRNA interactions were found to involve the miRNA 3’ end with “little evidence” for 5’ contacts, and some were functionally validated.

Analyses of miRNA/mRNA base pairing showed that miRNA species systematically differ in their target RNA interaction, and “strongly over-represented motifs were found in the interaction sites of several miRNAs.”

Overall, there is great potential for the use of CLASH in future miRNA research, the investigators concluded.

“As an example, analyses of miRNA association reveal comparable distributions of miRNAs associated with the four mammalian AGO homologs, but it is less clear whether all miRNAs target the same mRNAs when bound to different AGOs,” they stated. “Similarly, closely related paralogs exist for many human miRNAs, but it has been difficult to determine their relative efficiencies in mRNA targeting.”

Meanwhile, the distribution of non-templated terminal U residues among miRNAs has been determined, but it is not clear how this related to targeting in vivo. “More generally, the spectrum of miRNA-mRNA interactions is expected to rapidly change during differentiation and viral infection and following metabolic shifts or environmental insults.

“All of these can potentially be addressed using CLASH,” the team wrote.


Delivery remains the key hurdle for the development of RNAi-based therapeutics, and while much focus has been placed on systemic approaches, the barriers facing local delivery are considerably lower.

As such, a group of researchers from the Massachusetts Institute of Technology has published the details of a novel ultrathin, electrostatically assembled coating for the localized delivery of siRNAs.

Despite the broad range of diseases that can be addressed with systemic RNAi, local delivery can limit potential side effects and maintain the highest drug load possible in a targeted area before clearance, they wrote in ACS Nano.

Using layer-by-layer assembly — which involves the sequential adsorption of materials onto a surface using various complementary interactions and has successfully been used for local delivery of biomolecules — the scientists constructed film architectures containing siRNA-bearing calcium phosphate nanoparticles.

The nanoparticles are intrinsically negatively charged and remain intact after their incorporation into layer-by-layer assemblies, but dissociate upon maturation of the endosome when pH levels drop.

The film was applied to a commercially available woven nylon bandage commonly used for surgical applications and showed minimal impact on the viability of cells exposed to it. It was also shown to induce significant knockdown of target protein expression in multiple cell types for more than a week in vitro.

“The capability to load siRNA into an ultrathin polymer coating for safe and effective delivery of siRNA over an extended period of time provides a significant advance in the existing capabilities of RNA interference,” the MIT group concluded in their paper. “The film described in this work has great potential in many applications ranging from coatings for medical implants and tissue engineering constructs to uses in molecular biology and basic research.”


Given their small size and relatively low levels in clinical samples, miRNAs remain difficult to detect.

In an effort to overcome this challenge, a team of Turkish researchers has developed an electrochemical biosensor based on protein 19 for the small, non-coding RNAs, demonstrating its use with miR-21 in a paper appearing in Biosensors and Bioelectronics.

Protein 19, or p19, is a homodimeric, carnation Italian ringsport virus-encoded fusion protein that acts as an RNA silencing suppressor. “By pairing up the siRNA/miRNA recognition capability of p19 protein with electrochemistry … [the] biosensor offers sensitive detection in relatively short assay time without using any labels,” according to the paper.

Specifically, p19 acts as a molecular caliper of dsRNA, sequestering miRNAs in a size-dependent, sequence-independent manner, the scientists wrote. “Hybridization of a miRNA probe and its target creates dsRNA structure and this formation firmly binds p19 protein. Additionally, p19 protein does not interact with DNA due to lack of 2’-OH groups in the DNA structure.”

When used for miR-21, detection of the miRNA was achieved in picomolar sensitivity through the changes of intrinsic p19 oxidation signals, and the specificity of the sensor was proven in control studies.

The approach promises a cost-effective and sensitive alternative for miRNA detection that has a short assay time, and requires no pre-amplification prior to hybridization or labeling, the team wrote.


Despite its utility for exploring gene function, hairpin RNA-based RNAi requires constructs that can be time-consuming to create. Now, scientists from South China Agricultural University have developed an alternative approach they say is more streamlined and flexible.

Several modification methods have been proposed for making hairpin RNAs, including one involving the direct amplification of intron-containing hairpin RNA constructs in one tube directly from genomic DNA, the scientists wrote in Planta.

And while this method is simple and rapid, it still requires traditional cloning steps, and is unsuitable for making constructs for intron-less genes or those with only expressed sequence tags available.

“Recently, a single-reaction, isothermal in vitro recombination system has been shown [to be] highly efficient in seamless assembly of multiple overlapping DNA molecules up to several hundred kilobases via the collaborative actions of the 5’-T5 exonuclease, the Phusion polymerase, and the Taq DNA ligase,” they wrote.

In this technique, known as Gibson assembly, the overlapping ends of DNA fragments are chewed back at the 3’ ends by the DNA exonuclease, exposing complementary single-stranded DNA overhangs. The ssDNA gaps are filled in by the DNA polymerase, while the nick is sealed by the DNA ligase.

The Chinese team adapted Gibson assembly for the construction of hairpin RNA so that two PCR products of a target gene sequence are generated. Each is complementary to each other and to the ends of a linearized vector, and each is fused to the vector head-to-head or tail-to-tail.

Using the approach, they created a hairpin RNA construct for the Arabidopsis PDS3 gene, and confirmed that it knocked down its target and created a mutant phenotype.

The new method, the investigators concluded, eliminates the need for engineering restriction enzyme cutting sites in target DNA fragments and is ligation-independent, making it a promising tool for functional genomics research.

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