microRNA biosensors: Opportunities and challenges among conventional and commercially available techniques
Introduction
Ever since its discovery dates back to 1990s, researchers have paved a long way in the field of microRNAs (miRNAs), a large class of small (ranging in length 18ā25Ā bp) non-coding RNA that were unappreciated before the investigation of their roles in lin-4 and lin-14 genes by Ambros and his colleagues who showed the posttranscriptional regulation of lin-14 protein synthesis by a 22-nt transcript (Lee et al., 1993). After its discovery several follow-up work and discovery also summarized by Fig. 1 have enlightened the action of mechanism of these gene regulators and lead miRNAs to burst onto medical diagnosis field. Among all other functions they possess, the main function is believed to be post-transcriptional regulation of proteins. The abundance of miRNAs in both invertebrates and vertebrates has been proven in 2001. According to the current database (http://www.mirbase.org), there are total 1881 annotated human miRNA precursor genes that processes 2588 mature miRNAs. Although miRNAs abundant in tissues, it has also been shown that, there are also circulating miRNAs in body fluids such as plasma, urine, saliva, peritoneal fluid, pleural fluid, seminal fluid, tears, amniotic fluid, breast milk, bronchial lavage, cerebrospinal fluid, and colostrum. The amount of total RNA found in plasma is in 6ā300Ā ng/mL and miRNA fraction is known to be only a few percent of total RNA (Weber et al., 2010). Although the exogenous miRNAs are comparatively small amount in plasma, they are also stable as endogenous miRNAs which exist either inside micro vesicles in an encapsulated form or bound to RNA binding proteins such as Ago2 to be protected from degradation (Vickers et al., 2011).
Although miRNAs have various critical roles in wide range of biological processes including embryonic development (Virant-Klun et al., 2016), proliferation, apoptosis, hematopoiesis and link between their expression to some human diseases, genetic disorders and cancer onset, the underlying principle of their function is RNA-mediated gene silencing through RNA interference (RNAi)-like pathways (Bhaskaran and Mohan, 2014, Farazi et al., 2013, Montagner et al., 2014, Moreno-Moya et al., 2014, Piubelli et al., 2014). Initially, a large piece of mRNA includes the specific miRNA stem-loop structure that is also known as pri-miRNA is transcribed by RNA polymerase II in the nucleus. After transcription, Drosha, an RNAse III endonuclease cleaves pri-miRNA into stem loop structure named as pre-miRNA. After its transportation from nucleus to cytoplasm via Exportin-5 protein, pre-miRNA is cleaved by another RNAse III enzyme, Dicer and maturates to miRNA (Bhaskaran and Mohan, 2014, Dong et al., 2013, Moreno-Moya et al., 2014, Zhuo et al., 2013). miRNAs regulate genes by interfering with intracellular messenger RNA (mRNA) either directly through cleavage mRNA or indirectly through repression of translation. In both cases, miRNA base pairs with its target mRNA from its 3ā² untranslated region (3ā² UTR). Depending on the complementarity ā complete or incomplete, between two, target mRNA is either degraded or its translation is blocked.
To date, huge variety of miRNAs have been identified in animals, plants, microorganisms and over 4000 miRNAs have been identified in humans (http://www.mirbase.org/), which is estimated to target more than 30% of the human genome that play important roles in cellular processes. Among their association to diseases like myocardial infarction, single point mutations, neurological and autoimmune diseases, their specific expression signatures in various cancer types have provided hope that miRNAs can be great candidates for early cancer diagnosis (Ardekani and Naeini, 2010). Regarding cancer, miRNAs possess two different characteristic feature as being either tumor suppressors or oncogenic miRNAs (Di Leva et al., 2014). First category comprises of miRNAs like miR-34, miR-let7, miR 143, miR 145, miR-133b, and miR- 126 whose expression is down regulated in cancer cells while oncogenic miRNAs are up regulated due to down regulation of tumor suppressor genes. miRNA-155, miRNA-21, miRNA-372, miRNA-373, miRNA-15a, miRNA-16-1, miRNA-34s, are some miRNAs that have been recently associated with various cancers (Negrini et al., 2007). Recent finding also shows the association between altered levels of certain circulating miRNAs with various diseases (Bostjancic et al., 2009a, Di Leva et al., 2014, Farazi et al., 2013, Islam et al., 2017, Montagner et al., 2014, Moreno-Moya et al., 2014, Piubelli et al., 2014, Rupaimoole and Slack, 2017, Virant-Klun et al., 2016, Weber et al., 2010, Yi et al., 2013, Zhuo et al., 2013).
It is now well known that aberrant miRNA expression is linked to cancer, and miRNAs have an important role in cancer occurrence, progression and metastasis. Therefore, tumor suppressors miRNAs or the antagomirs of oncomirs might be used as effective cancer therapeutics (Rupaimoole and Slack, 2017). The usage of miRNAs as therapeutics could be done in either of two strategies: for the purpose of either suppressing tumors via targeting oncomirs or replacement of tumor suppressor miRNAs 'miRNA replacement therapy'. Oncomir targeting could be done either direct strategies where oligonucleotides antimiRs, antagomirs or locked nucleic acids (LNAs) are used or indirect strategies based on virus-based constructs. For both the direct and indirect methods of oncomir targeting, the purpose is to block the expression of oncogenic miRNA by preventing its binding to RISC complex. These strategies could be shown to be effective also sensitizing tumors to therapy as in the example of tamoxifen where antagonizing miR-221/mir-222 may further sensitize cells to the drug (Miller et al., 2008). In the case of 'miRNA replacement therapy', tumor suppressor miRNAs could be replaced to restore loss function due to downregulation of certain miRNAs. For instance, due to decreased levels of miR 34a and let-7a in various cancers, several laboratories have been working on for the investigation of therapeutic benefit of "miRNA replacement therapy" (Miller et al., 2008, Wiggins et al., 2010).
Paradigm shifting genome editing tool, clustered regulatory interspaced short palindromic repeats (CRISPR)/cas9 system have been taken great attention in recent years. It has also been shown that, this technology can be applied to non-coding genes such as miRNA genes. It has been reported that, generation of knockouts for miRNAs such as miR-21, miR-29a could be achieved (Chang et al., 2016, Ho et al., 2015).
For further reading on therapeutic role of miRNAs reference paper should be followed (Kasinski and Slack, 2011).
Owing to their crucial roles in several diseases, especially in cancers, understanding the function and detection of expression levels of miRNAs is considered as very important non-invasive biomarkers and raised great attention not only by the academia but also by the industry where some miRNA based diagnostic tools/kits like mirVana, miRNAQrt-PCR kits,SmartFlare, etc. have already been launched by various companies. Despite current commercial products and intense work on miRNA research, there is still need for a standardized and medically reliable method for miRNA detection and quantification that can overcome the handicaps of current bias-prone technologies and challenges of miRNAs detection. Regarding to their intrinsic properties, miRNAs are difficult to be amplified due to their short length and sometimes excessively being lost in conventional RNA isolation procedures. Additionally, they are highly homologues in sequence similarity that makes detection challenging in terms of selectivity. Also, there is still a gap in miRNAs detection including performing multiplex analysis, detection of circulating miRNAs, performing in vivo analysis and enabling single-nucleotide specificity (Rosenwald et al., 2010).
Over the past decade various methodologies have been implemented for sensitive, selective and high-throughput detection of miRNAs. These methodologies can roughly be subgrouped as; conventional miRNA detection methods, electrochemical methods, optical methods and other emerging technologies as summarized in Fig. 2.
Among them biosensors are rapid, sensitive, selective, low-cost analytic devices comprise two elements; a biological recognition element to capture target miRNAs and a transducer able to convert the signal of recognition reaction into a measurable signal, have taken great attention and designed in a wide variety of geometry and transduction method. Compared to conventional techniques used for miRNAs detection such as Northern blotting, qRT-PCR, miRNA microarrays and cloning, biosensors have superior features in sample preparation, assay time, cost, portability and complexity. Although the classification of miRNA biosensors can be categorized according to the type of transducer they use; electrochemical (Bartosik et al., 2014, Jamali et al., 2014, Keshavarz et al., 2015a, Labib and Berezovski, 2015, Zhao et al., 2015) and optical (Almlie et al., 2015, Gao et al., 2014a, Li et al., 2016a, Li et al., 2014a, Å ĆpovĆ” et al., 2010, Wang et al., 2016a), there are also some novel platforms combine different conventional methods with various biosensor designs that can quantify miRNAs in a sensitive manner.
Up to now, some review articles on miRNA detection have been published (Campuzano et al., 2017; Catuogno et al., 2011; Dong et al., 2013; Hamidi-Asl et al., 2013; Islam et al., 2017; Jamali et al., 2014; Labib and Berezovski, 2015; Tian et al., 2015). However, the fast evolving, wide 'miRNA sensing field' needs to be updated. The purpose of this review is to explain the technologies available for miRNA analysis, from research based applications to commercially available methods, to make readers understand the challenges and advantages associated with each method and make comparison between them to be chosen according to the need of the work conducted. The review, along with the miRNA biosensors, summarizes the current miRNA detection methods (2005ā2017 years) with an emphasis on electrochemical detection methods. Before jumping to miRNA biosensor section, the conventional methods like northern blotting, qRT-PCR and microarray technology were briefly explained. In miRNA biosensors section that is in accordance with Table 1, electrochemical, optical and other miRNA biosensors (Photoelectrochemical miRNA biosensors, Localized Surface Plasmon Resonance (LSPR), Paper-based, Electromechanical) were detailed by giving examples from recently published papers. In the last section of the review paper, commercially available miRNA analysis tools were exemplified. As a conclusion remark, future trends in miRNAs detection are discussed considering the needs of biomedical field.
Section snippets
Conventional miRNA detection methods
Although the emphasis of this review is 'miRNA biosensors', we would like also to include conventional miRNA detection methods to give insights about the field and ease the comparison between past and future trends of miRNA detection tools in general. In this regard, Northern blotting, qRT-PCR and microarray assays will be exemplified.
Electrochemical biosensors developed for miRNA detection
Electrochemical methods has superior advantages due to their low cost, selectivity and capability for miniaturization compared to most of the analytical techniques and thus they had a widespread use in food, agricultural, medical, and environmental fields (Erdem, 2008). Transduction often relies on an electrochemically-active reporter species, whose activity is used to infer changes in electrode properties. When it comes to miRNA detection, electrochemical genosensors show great promise for
Optical biosensors developed for mirna detection
Similar to electrochemical biosensors, optical biosensors designed for miRNA detection transduce the optical signal in the form of absorbance or fluorescence due to hybridization of target miRNA with a probe nucleic acid tagged with an optically-active reporter. A reporter could either be a dye-label or quantum dots (Jamali et al., 2014). In following section, some optical miRNA biosensors will be reviewed. For more detailed information references cited in the text and in Table 1 should be
Other miRNA biosensors
Although the majority of miRNA biosensors comprised of electrochemical and optical ones, various novel miRNA biosensors depending on different sensing mechanisms and/or platforms have been designed. Among them some hybrid techniques as combinations of conventional ones have taken attention. For instance, electrochemiluminescence (ECL) biosensors, measuring the chemiluminescence directly/indirectly produced as a result of conversion between electrochemical and radiative energy at the electrode
Commercially available miRNA analysis tools
Due to its value on early diagnosis, miRNAs have taken great attention from industries by time. There are three major product type for miRNA analysis; qRT-based MicroRNA Assays, miRNA microarrays and next-generation sequencing tools.
Concluding remarks with future prospectives
Applications of technology at nanomedicine have paved a long way over the last decade and considered as one of the epochal areas of research that is giving rise to well established and commercially available products to be used for self-testing and clinical analysis. Significant amount of work have been conducted on miRNA detection because of their specific signatures and clinical importance in early detection of cancer. At the present time, molecular biology tools, biosensors and hybrid
Acknowledgement
Dr. Kilic acknowledges the Horizon 2020, Marie SkÅodowska-Curie actions, EPFL fellows international postdoctoral fellowship program co-funded by Marie Curie.
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