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Tools & Techniques: TALENs for microRNA Ablation, Ultrasound-guided siRNA Delivery, and More


Seeing a growing need for effective methods to study microRNA biogenesis and expression in cells, a research team from Korea's Cha University has developed a linear color-tunable molecular beacon probes for sensing the small, non-coding RNAs.

In a paper appearing in Scientific Reports, the investigators report on the probes' design and their use for detecting miR-9 during neurogenesis.

Molecular beacons typically comprise short stem loop-structured DNA oligonucleotides that have a quenching molecule and a fluorescent dye at the end of each molecule. Their loop sequences are complementary to specific DNA or RNA, and in the absence of target molecules their fluorescence is absorbed due to fluorescence resonance energy transfer between the fluorescent dye and the quencher. If a target oligo binds to the loop structure, the quencher separates from the dye, triggering a bright fluorescence signal.

Molecular beacon imaging systems have been developed for miRNAs, allowing visualization of their expression and function by providing "a highly specific on/off-tunable sensing system," the investigators wrote in their paper. "However, it is frequently difficult to distinguish a dynamic change in target molecules with an on/off-tunable [molecular beacon] sensor when fluorescence is weak in cells due to low transfection of the [beacons] or weak target molecule expression."

While color-tunable molecular beacons can overcome this by providing expression-dependent color change, these can produce nonspecific background signal or false positive results due to their stem-loop structures, which cause the quenching molecule to be attached even after hybridization with target molecules.

In an effort to address this issue, the research group developed a linear-structured color-tunable molecular beacon, focusing on miR-9 to demonstrate its applicability for miRNA visualization.

Dubbed ColoR9 MB, the beacon was synthesized by a partially double-stranded DNA oligonucleotide containing a miR-9 binding site and a reporter probe with Cy3/black hole quencher 1 at one end and a reference probe with Cy5.5 at the other end.

The molecular beacon visualized CHO and P19 cells with red color in the absence of miR-9 and yellow color in the presence of miR-9, according to the Scientific Reports study. In vivo imaging demonstrated that the green fluorescence recovery of the reporter probe from ColoR9 MB increased gradually during neuronal differentiation of P19 cells, whereas red fluorescence activity of the reference probe remained constant.

"These results showed the great specificity of sensing miR-9 expression- and neurogenesis-dependent color change," the researchers stated, and offer a solution to the problem of non-specific background signals and false positives encountered with stem loop-structured molecular beacons.

Taking a different tack, a group from South China Normal University constructed a rapid approach for miRNA detection based on base-stacking hybridization and magnetic microparticle enrichment technology.

The method also integrates electrochemiluminescent (ECL) technology — which has proven sensitive and specific for nucleic acid and protein detection — with microfluidics, which offers the "facile integration of multiple assay steps, the need for small sample volume, and … inexpensive reagents," the researchers wrote in Biosensors and Bioelectronics.

The team designed a sandwich-type hybridization model for the direct detection of
miRNAs using a capture probe and an ECL signal probe. "Under defined temperature conditions, the two probes cannot hybridize with each other stably due to their low melting temperatures," they wrote. "When the target microRNA is in the solution, the two probes and the target will form a ternary complex through stacking interactions."

The mixing of nucleic acid chains can be analyzed through a flow-controlled microchip system with ECL detection. Meantime, the use of magnetic microparticles as enrichment carriers eliminated the need for a laborious series of product collection steps and electrode surface modification, they added in their paper.

In testing their assay, they showed that it required less than an hour to complete. Additionally, although its sensitivity was in the low-femtomole range, this may be further improved using the gold nanoparticle-based signal amplification method. At the same time, because the assay is not reliant upon enzymatic amplification, amplification-related errors are avoided.

"Finally, the proposed microRNAs assay can be associated with a commercial ECL detection platform without significantly altering the assay procedure," the investigators wrote.

Amid a lack of effective tools for fully blocking the expression of specific miRNAs, investigators from California State University and the University of California, San Francisco have created a strategy for bi-allelic miRNA ablation in human cells using TALENs.

To date, there are a number of technologies for miRNA inhibition including siRNAs, small molecules, antisense oligos, and miRNA sponges. But these all suffer key limitations including the transient nature of their effects and a high risk of off-target effects and resulting toxicity, the researchers wrote in RNA.

Transcription activator-like effector nucleases (TALENs), which create double-strand breaks in DNA sequences, are becoming a widely used tool for gene ablation, but the short and noncoding nature of miRNAs has made them a challenging target for the technology.

The disruption of miRNAs in human cells by TALENs has been reported, and it has been shown that TALENs alone can disrupt genes in the human genome at a frequency of about 2 to 40 percent, with an average of around 16 percent for mono-allelic disruptions, according to the RNA paper. But isolating cells carrying bi-allelic disruptions requires time-consuming single cell-derivation and subsequent screening.

Unlike other TALEN strategies, the research team's novel approach combines TALENs targeting to the miRNA seed region with a homologous recombination donor vector carrying a selectable marker. This, they wrote, enables "convenient positive selection," while the combination of non-homologous end joining with stem-loop deletions results in "efficient bi-allelic miRNA gene ablation, which is especially valuable for loci that may be difficult to target."

Further, by using homologous recombination donors, endogenous loci can be potentially modified with custom sequences to permit the functional assessment of endogenous gene expression and regulation, they added.

As a proof of concept, the researchers used their approach to target the human miR-21 seed region in cultured human HEK293 cells, successfully selecting bi-allelic miRNA knockouts with an efficiency of up to 87 percent.

Quantitative RT-PCR analysis of three independent clones confirmed complete loss of mature miR-21 expression, while phenotypical analysis confirmed increased protein levels of the miR-21 target gene PDCD4, reduced cell proliferation, and changes in global miRNA expression profiles.

"With the ease and efficiency of bi-allelic mutant generation, combined with the advantage of a stable phenotype, we envision that this approach will broaden our knowledge in deciphering the role of miRNAs in human physiology and disease," they wrote in the paper.

With efficient delivery still one of the key hurdles facing RNAi therapeutics, University of Utah researchers have developed an ultrasound-guided approach for carrying siRNAs into tumor tissue.

The team, which presented its findings in the Journal of Controlled Release, demonstrated their approach both in vitro and in vivo, showing that it could be used to deliver vascular endothelial growth factor (VEGF)-targeting siRNA into ovarian cancer cells.

Despite the efficacy and specificity of siRNAs, their use as anticancer agents is limited by their rapid degradation in serum. To address this, the University of Utah team combined two technologies that have been developed for RNAi delivery: cationic polymers — specifically arginine-grafted bioreducible polymers (ABPs) — and ultrasound-activating microbubbles (MBs).

The result is so-called siRNA-ABP-MB complexes, or SAMs, which can be formed by electrostatic interaction of negatively charged albumin MB and positively charged polyplexes. SAMs have a size range of 1 to 5 nanometers and a flexible zeta-potential that can be tuned from positive to negative depending on the concentration of polyplexes loaded on the outer shell of the MBs.

According to the report, SAM complexes are stable in serum-containing media for at least 60 minutes when compared to SAM stability in serum-free media. In combination with ultrasound, SAMs containing VEGF-targeting siRNAs showed significantly higher siRNA uptake and VEGF protein knockdown in vitro with serum-containing media when compared to naked siRNAs. In vivo, the SAMs triggered reductions in tumor size and slowed tumor growth in tumor-bearing mice as compared with controls.

"Our results indicate that the difficulties and challenges of siRNA delivery in the presence of serum can be successfully overcome using our novel, combined delivery system," the researchers wrote.

Additional studies are planned to optimize the delivery system for local administration at tumor sites, they noted.