A REVIEW ON THE ELECTROCHEMICAL BIOSENSORS FOR DETERMINATION OF microRNAs

Presented by Atufa kawan 08-Arid-1773 Ph.D 1st Semester Zoology DepartmentMicroRNAs and its detection methods1INTRODUCTIONRNA

RNA (Ribonucleic acid)is one of the three major macromolecules (along with DNA and proteins) in the body that are essential to all known forms of life. Like DNA, RNA is made up of a long chain of components called nucleotides.

There are several kinds of RNA in the body:

Messenger RNA(mRNA)Transfer RNA (tRNA) Ribosomal RNA( rRNA)Small nuclear RNA(snRNA)Other non- coding RNAs(ncRNA) Conti.. snRNA

A snRNA is a class of small RNA molecules that are found within the nucleus of eukaryotic cells.

They are transcribed by RNA polymerase and are involved in a variety of important processes such as regulation of transcription factor and maintaining the telomerase.ncRNA

A ncRNA is a RNA molecule that is not translated into a protein.

The number of ncRNAs encoded within the human genome is unknown;however recent transcriptomic and bioinformatics studies suggest the existence of thousands of ncRNAs.

Many ncRNAs show abnormal expression patterns in cancerous tissues .

MicroRNAOne category of ncRNA is MicroRNAs (miRNAs). MicroRNAs (miRNAs) are 22 nucleotide-long non coding RNA molecules which bind to target mRNAs, resulting in translational repression or degradation of messenger RNAs and are found in all eukaryotic cells.

Approximately 2200 miRNA genes have been reported to exist in the mammalian genome, from which over 1000 belong to the human genome.

Proximity to other genes in the genome and their locations in introns of coding genes, non coding genes and exons have been reported to have a major influence on the level of gene expressions in eukaryotic cells.

miRNAs are well conserved in eukaryotic system and are believed to be an essential and evolutionary ancient component of gene regulatory networks.

miRNAs Biogenesis

miRANS transcribed in nucleus by RNA polymerase II (RNA pol II) as a primary miRNA (pri-miRNA) transcript.

After being cleaved in the nucleus by the RNAse III ribonuclease, Drosha and its cofactor Pasha, the precursor miRNA (pre-miRNA) is exported to the cytoplasm by Exportin 5, where it is cleaved by a second RNAse III ribonuclease, Dicer.

This 16- to 29-nucleotide-long miRNA duplex is then unwound to free the mature strand for incorporation into a RNA-induced silencing complex (RISC) and, based on sequence complementarity, directs translational repression or cleavage of its mRNA target by binding to either the 3- or 5-untranslated (UTR) regions.

MicroRNAs Biosynthesis

Mechanisms of microRNA-mediated gene regulationtranslational repressionmRNA degradationIn translational repression,a majority of miRNAs bind to their targeted mRNAs at the 3 UTRs; however, some miRNAs can also bind to the 5 UTR (Zeng et al., 2002; Doench and Sharp, 2004). Although the precise mechanism of miRNA-mediated translational repression has not been elucidated, studies suggest that miRNA may hamper ribosome movement along the mRNAs, and repress protein translation (Carrington and Ambros,2003). mRNA degradation

In animals, there are also miRNAs which directly degrade their targeted mRNAs. miR-196directly cleaves the mRNA of HOXB8 play important role in animal development ROLE OF miRNAs IN DISEASESCANCEROver expressed miRNAs may function as both oncogenes and/ or regulator of cellular process such as cell differentiation. Unique miRNA expression profilings have been demonstrated for many types of cancer.

Over expression of MiR -210 was reported to be associated with the enhanced formation of capillary structures.

In another study the miRNA miR-155 has linked with MYC overexpression suggesting it may be playing a role in the regulation of this oncogenes.

Cardiovascular Disease

The discovery of microRNAs in recent years has made it evident that these RNA molecules have an important function in regulation of heart function and mammalian cardiovascular system in general.

The miRNA expression levels have been linked to deregulation of developmental processes and disease states, such as cardiac hypertrophy .

Studies have shown that three mirRNAs (miR-1, miR-133, and miR-208) are highly expressed in the heart and are important regulators of heart development and myocyte differentiation. Functions of microRNAs in plants and AnimalsmiRNAs regulate organ development in plants

Loss-of function of the dcl1 gene reduced the expression level of mature miRNAs and consequently caused many developmental abnormalitiesThese developmental abnormalities include arrested embryos at early stages, altered leaf shape and morphology delayed floral transitionDicer-like enzyme 1 (DCL1)Processes pri-miRNA to pre miRNAs then to miRNA(DCL1)miRNAs control leaf development by regulating the expression of class-III (HD-ZIP) transcription factor genes which control leaf asymmetry pattern along adaxial/abaxial (upper/lower) axis .

PHABULOSA (PHB), HAVOLUTA (PHV), REVOLUTA (REV) are three closely related Arabidopsis HD-ZIP transcription factors. Mutations in any of these three transcription factor genes (phb, phv, and rev) results in radialization and adaxialization of leaf and vascular bundles in the stem homeodomain leucine zipper (HD-ZIP) transcription factor genes

miRNAs regulate animal developmentmiRNAs regulate animal development at multiple tissues and at multiple developmental stages. Emerging evidences suggest that miRNAs are essential for the normal development of almost all animal tissues.Specific miRNAs control specific tissue development at a specific developmental stage (Lagos-Quintana et al., 2002).Dicer mutant in zebrafish embryos developed normal at the beginning, but embryo development arrested 8 days after fertilization. This suggests that embryo development need appropriate expression of certain miRNAs. HOX is an important gene in animal development, and it is negatively regulate by miR-196 and miR-181. Misexpression of these miRNAs caused abnormal expression of HOX, and results in animal developmental abnormality.

Difficulties in miRNA Detection Cissell et al. (2009) described the challenges about miRNA detection such as small size of miRNA sensitivity of the assay.The concentration of cellular miRNA is around 1000 molecules per cell.For insitu detection,when a sample is a mixture of pre-and mature miRNA,the oligonucleotide probe can hybridize non specifically to pre-miRNA.This can cause false positive signal for expression levels of mature miRNA. DETECTION METHODS FOR miRNASAnalysis of miRNAs by solid-phase methodNorthern blotting method

Northern blot analysis is the most standardized and widely used method of detecting miRNAs. This technique allows examination of expression properties of target miRNAs, determination of their sizes.

Fig. 2. Schematic representation of the steps involved in Northern blotting analysis of miRNA expression from total RNA samples.

Conti..Mechanism

RNA ExtractionGel Electrophoresis (RNA separated by size)Northern blotting(RNA fixed on membrane by UV or heat).Membrane hybridized with radio labelled or fluorescent Probe.4. X-Ray film

Conti..Application

Examination of expression properties of target miRNAsDetermination of target miRNAsDetermination of their sizesAdvantageWidely used methodQuantitativeGood specificityLimitation Time-consuming Not practical in clinical studies in which the detection of a large number of miRNAs is usually required Its use in diagnostic is also limited by the relatively large amount of RNA sample required Multiple handling steps Detection limitFemtomolar range 2.Electrochemical detection of miRNAs

2.1Surface-enhanced Raman spectroscopyorsurface- enhanced Raman scattering (SERS)

SERS is an one of the variation of Raman spectrometry. SERs is an surface-sensitive technique that enhancesmoleculesadsorption on rough metalsurfaces or by nanostructures such as plasmonic-magnetic gold or silver nanotubes.

Raman spectroscopy is a powerful technique used to investigate the chemical states of the bonds in carbon materials.

This method displays excellent reproducibility and single-nucleotide specificity and does not require a label for detection and more useful for analysis of molecules deposited onto metal surfaces.

SERS has become a powerful technique for analyzing biological samples as it can rapidly and non-destructively provide chemical and in some cases structural information about molecules in aqueous environments.

Laser lightLensmonochromatorDetector

Fig. 4. Schematic representation describing the surface-enhanced Raman spectroscopy (SERS)-based assay for detection of miRNAsConti..Application

Direct detection of miRNAs in total RNA extracted from cell lines or tissues

Advantage

Excellent reproducibility and single-nucleotide specificityNot require a label for detectionMultiplexed-miRNA detection

Detection limitFemtomolar range

Drawback

sequences with overlapping peaks cannot be differentiated.

requires a sophisticated read-out system

convoluted data, interpretation and verification, rendering it rather impractical in most molecular biology laboratories.

Nevertheless, this methodology is of interest for application in multiplexed miRNA detection, and may be well suited for routine miRNA expression profiling in clinical settings and diagnostics.2.2 Electrocatalytic oxidation of guanine

Lusi et al. (2010) detect miRNA by using electrochemical biosensor, based on guanine oxidation consequent to the hybrid formation between the miRNA and its inosine substitute capture probe .This label-free detection method, already described for nucleic acids detection had never been used for microRNAs.

microRNA Inosine modified guanine free probe

Hybrid formation (RNA/DNA)Oxidation of guanineOn electrodeElectrical signal producedEvaluated by voltameter24

Electrochemical detection of the hybridization between the inosine- modified Probe capture and the target (miR122)Conti..Application

Direct detection of miRNA in total RNA from tissue and cells

Advantage

label-free methodsimpleless time consuming feasible for a routine microRNAs detection in serum and other biological samples

Detection limit

0.1 pmol2.3 Silicon Nanowires Biosensor

Zhang et al. (2008) reported a label-free direct detection of miRNAs with silicon nanowire biosensors .

Peptide nucleic acids

Electrical measurements for the sensing experiments were carried out by detecting the resistance change of SiNWs before and after PNA miRNA hybridization.Recognize miRNA miRNA is immobilized on SiNW device

Fig. 1. Schematic illustration of the label-free direct hybridization assay developed for ultrasensitive detection of miRNA.

Fig. 4. Response of the PNA-functionalized SiNW biosensors to the complementary miRNA of varying concentrations.Application

Detecting miRNA in total RNA

Advantage label-free hybridization method suitable for the demand for early detection of miRNA in cancer diagnostics.

Detection limit1 fMConclusion

The mature miRNAs control gene expression interacting with as specific mRNA either inducing its degradation or blocking the translation process.

It is now predicted that as much as 4050% of mammalian mRNA could be regulated at great impact in biological processes. Therefore there is an urgent need to reliable and ultrasensitive test for miRNAs.

At the present time miRNAs are detected with techniques such as Northern blot using many commercial kits.Because the inherent superiorities of electrochemical transduction methods such as excellent compatibility with advanced semiconductor technology, miniaturization and low cost,nucleic acid biosensors based on electrochemical detection are able to provide high performance at low cost with simple miniaturised redout and thus are exempt from the problems encountered in the other detection systems .

REFERENCES

Shanmukh, S., Jones, L., Zhao, Y.-P., Driskell, J.D., Tripp, R.A., Dluhy, R.A., 2013. Anal. Bioanal. Chem. 390, 15511555. P. Negri, G. Chen, A. Kage, A. Nitsche, D. Naumann,B. Xu, and R. A. Dluhy, Anal. Chem. 84, 55015508 (2012).P. Negri, A. Kage, A. Nitsche, D. Naumann, and R. A. Dluhy, Chem. Commun. 47, 86358637 (2011).J. D. Driskell and R. A. Tripp, Chem. Commun. 46, 32983300 (2010).S. Shanmukh, L. Jones, Y. P. Zhao, J. D. Driskell, R. A. Tripp, and R. A. Dluhy, Anal Bioanal Chem 390, 15511555 (2008).J. D. Driskell, S. Shanmukh, Y.-J. Liu, S. Hennigan, L. Jones, Y.-P. Zhao, R. A. Dluhy, D. C. Krause, R. A. Tripp, IEES Sens. J. 8, 863870 (2008).Zhang, B.,Wang, Q., Pan, X., 2007. J. Cell Physiol. 210 (2), 279289. S. Shanmukh, L. Jones, J. Driskell, Y. P. Zhao, R. Dluhy, and R. A. Tripp, Nano Lett. 6, 26302636 (2006). J. Barenfanger, C. Drake, N. Leon, T. Mueller, and T. Troutt, J. Clin. Microbiol. 38, 28242828 (2000). B. Bartel, Nat. Struct. Mol. Biol. 12, 569571 (2005).D. P. Bartel, Cell 116, 281297 (2004).33