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April 30, 2014

MicroRNA: An Insight to miRNA-Based Microarrays, Diagnostics, and Therapeutics

Learn more about this rapidly evolving field.

MicroRNA: An Insight to miRNA-Based Microarrays, Diagnostics, and Therapeutics

The microRNA industry has been evolving quickly to cater to researchers, physicians, and patients worldwide.

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    microRNA (miRNA) is an RNA component that affects roughly a third of genetic expression. miRNA’s epigenetic properties have demonstrated potential therapeutic benefits for a variety of disorders including cancers, neurodegenerative diseases, and nephropathy. In response to their robust and versatile applications, miRNAs have been studied from all ends of R&D. Several companies are developing microarrays for better detection and analysis of miRNA in vivo, while others are utilizing miRNAs as diagnostics for a variety of diseases, and still others are exploring the world of therapeutics with miRNA repressors and reactivators. 

    With regard to diagnostics, one of the novel ways miRNAs are being investigated is for use as diagnostic biomarkers. According to a study done in 2013 (Verena, et al.), the purpose was to understand the differential stability of cell-free circulating miRNAs and investigate their potential uses as biomarkers. More specifically, researchers looked at miRNAs circulating in the blood, which were stabilized by forming a complex with proteins and/or additionally by encapsulation in lipid vesicles. Based on their findings, extracellularly circulating miRNAs have the potential to supplement current diagnostic and prognostic markers, not only establishing themselves as reliable biomarkers but also improving therapeutics for specific diseases.1

    Researchers have identified several properties that enhance an miRNA’s therapeutic potential. miRNA functions involve repressing or reactivating specific pathways while maintaining incredible stability. Researchers demonstrated this by studying miRNA stability in several environments and found that miRNA exhibits properties in both genetic suppression and activation as well as higher resistance to enzymatic degradation. These results gave researchers further incentive to investigate miRNA roles as a viable therapeutic. There are two ways in which miRNAs exist as therapeutics: antagonists and mimics.1

    miRNA antagonists are modified antisense oligonucleotides that contain the full or partial complementary reverse sequence of a mature miRNA, and are generated to inhibit endogenous miRNAs that show a gain-of-function in diseased tissues.2,3 These highly chemically modified miRNA passenger strands are often investigated for use in cancer therapies. The anti-miR, or antagamiR, binds with high affinity to the appropriate active miRNA strand.2 By chemically modifying miRNAs, researchers can engineer miRNA antagonists to meet certain property requirements including cell permeability, stability in vivo, possessing high specificity and affinity for binding, and resisting rapid excretion.3

    Another type of miRNA therapeutic in the works is miRNA mimicry. This method caters to creating a replacement, optimally functioning miRNA that the body cannot distinguish from the naturally, and often mutated, produced miRNA. Instead of inhibiting miRNAs that show a gain-of-function, mimicry is meant to address the miRNAs that exhibit a loss-of-function.2 It is considered to be a replacement therapy, aiming to re-introduce miRNAs into dysfunctioning cells that would otherwise have normally expressed the miRNA in question.2 By reintroducing these synthetic miRNAs back into the patient’s systems, they reactivate normal pathways indigenous to the life of the cell. Researchers have demonstrated that miRNA replacement therapy (by mimicking tumor suppressor miRNAs) has been linked to oncogenic pathway stimulation, apoptosis, and ultimate eradication of tumor cells.2

    Furthermore, companies are also investigating miRNA oligonucleotides, enhancing their properties for increased specificity and sensitivity, particularly for microarray analysis. Common ways, often used for miRNA antagonists, to engineer miRNAs are via chemical synthesis. There are three reactions that can be done and they include 2’-O-methyl-group (OMe)-modified oligonucleotides, locked nucleic acid (LNA)-modified oligonucleotides, and 2’-O-methoxyethyl (MOE) modified oligonucleotides. The difference between these three types of chemical modifications is the way in which the RNA is engineered. OMe contains the addition of a methyl group at the 2’ Oxygen while MOE contains a methoxyethyl group at the 2’-Oxygen. LNA features a distinctive bond between the 2’-O-oxygen and the 4’-carbon atom via a methylene linker that bridges this gap and literally locks the ribose structure in place.

    Another method that has been used for oligonucleotide creation is photolithographic synthesis. This technology is a light-directed oligonucleotide synthesis; more specifically, the methodology employs MeNPOC photo-activatable nucleoside monomers.4 With an ability to print individual 10 µm2 probe features at a density of 106 probes/cm2, this method facilitates the “widespread use in the hybridization-based detection and analysis of mutations manufacturing and polymorphisms.”4 Ultraviolet (UV) masking, in combination with light-directed chemical synthesis, selectively synthesizes probes located directly on the surface of the array.5 These probes contain a protecting group on their free end that is activated once in contact with the UV light.5 A photolithographic mask directs the UV light in such a way that exposure to specific nucleotides can be controlled.5 It works by deprotecting, and therefore activating, a series of hydroxyl groups.5 This effect initiates coupling with incoming protected nucleotides that attach to the activated sites.5 Although each probe on the chip requires four masks per round of synthesis, this method increases sensitivity and specificity compared to other technologies.

    Despite their well observed properties and oligonucleotide engineering capabilities, miRNAs are still not very well understood. Properties of high interest include the impact of differential association with lipids/vesicles on their stability and their use as biomarkers. With regard to stability, researchers have yet to understand the breadth of which miRNAs can be applied and affected. Chipping away and uncovering one detail after another, researchers have determined that miRNAs associated with vesicles appear to have greater stability and resistance to degradation by RNase A than serum miRNAs that are unaffiliated with vesicles.1 Furthermore, with regard to nonvesicle-affiliated miRNA, data indicated that hemolysis may be a useful method to achieve stabilization and preservation of extracellularly circulating miRNAs.1 From this study, researchers were able to conclude the following:1

    • miRNAs have different kinetics of decline in serum
    • RNase inhibitor (RI) reduces the loss of miRNAs from serum
    • Hemolysis improves the stability of circulating miRNAs
    • Incubation of serum leads to an increase of the portion of vesicle-associated miRNA (compared to nonvesicle-associated miRNA)
    • Vesicle-associated miRNAs are more resistant to RNase A than nonvesicle-associated miRNAs

    It is clear that microRNAs have many useful advantages and although there is still much to learn, researchers are eager to understand their properties for even more applications. With efforts being made in microarrays, diagnostic biomarkers, and alternative therapeutics, the miRNA industry has been rapidly evolving to cater to researchers, physicians and patients all around the world. Based on the amount of work that has already been done, miRNAs have established a solid reputation, and continue to show a bright future.

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