PNA-Based MicroRNA Detection Methodologies

Abstract

MicroRNAs (miRNAs or miRs) are small noncoding RNAs involved in the fine regulation of post-transcriptional processes in the cell. The physiological levels of these short (20-22-mer) oligonucleotides are important for the homeostasis of the organism, and therefore dysregulation can lead to the onset of cancer and other pathologies. Their importance as biomarkers is constantly growing and, in this context, detection methods based on the hybridization to peptide nucleic acids (PNAs) are gaining their place in the spotlight. After a brief overview of their biogenesis, this review will discuss the significance of targeting miR, providing a wide range of PNA-based approaches to detect them at biologically significant concentrations, based on electrochemical, fluorescence and colorimetric assays.

Keywords: colorimetric detection; electrochemical biosensors; fluorescence; light-triggered; microRNA; nanoparticles; peptide nucleic acid (PNA); templated reactions.

Figure 1

Schematic representation of the principal biogenetic pathways of microRNA (miR) formation and activity.

Figure 2

Illustration of some of the principal electrochemical methodologies based on hybridization for miR detection, relying on peptide nucleic acids (PNAs). (A) Field-effect transistor (FET)-based biosensor introduced in [59]. (B) Silver nanofoam (AgNF)-based biosensor decorated with acpcPNAs shown in [60]. (C) A 384-channel array on the Au/Cr surface based on the oxidation current of ferricyanide, described in [62].

Figure 3

Illustration of the nanopore-based methodologies for miR detection relying on PNAs. (A) Nanopore sensing of let-7b, adapted with permission from [65]. Copyright 2013 American Chemical Society (B) Nanopore-based sensing of miR-21, adapted with permission from [66]. Copyright 2019 American Chemical Society (C) AuNP-displacement based biosensor described in [68].

Figure 4

Illustration of the signal amplification and PNA-based electrochemical methodologies for miR detection. (A) Dual-mode detection of target miR-145, as presented in [71]. (B) Illustration of the miR-guided PAn biosensor proposed in [72] (C) Scheme of the biosensor described in [73].

Figure 5

Illustration of some of the principal fluorescence-based methodologies for miR detection relying on PNAs hybridization. (A) Functioning of PNA–nano-graphene-oxide (PANGO) multiplexed analysis, adapted with permission from [82]. Copyright 2013 American Chemical Society. (B) Hydrogel-coated microneedle patch for sampling and detection of miR. Adapted with permission from [85]. Copyright 2019 American Chemical Society.

Figure 6

Illustration of some of the principal oligonucleotide templated reactions for miR detection relying on PNAs. (A) In situ fluorescence labelling of dicysteine PNAs for miR detection, through unmasking of FlAsh, described in [88] (B) Michael addition of a thiol-containing PNA to an α,β-unsaturated ketone of a nonfluorescent coumarin precursor. Adapted with permission from [90]. Copyright 2016 American Chemical Society. (C) Ruthenium (II)-based light-triggered reaction, freeing a quenched fluorophore upon light-mediated reduction of a pyridinium linker, as shown in [93]. (i) The reaction was conducted in cellulo (ii, [94]) and in vivo (iii, adapted with permission from [95]. Copyright 2016 American Chemical Society) for miR imaging.

Figure 7

Illustration of some of the principal fluorescence-based methodologies for miR detection relying on PNAs, featuring a signal amplification strategy. (A) RCA-based detection of miR, adapted with permission from [98]. Copyright 2016 American Chemical Society. (B) Quadratic amplification applied for miR detection and release of coumarin, adapted with permission from [99]. Copyright 2019 American Chemical Society.

Figure 8

Illustration of some of the principal colorimetric methodologies for miR detection relying on PNAs. (A) Multiplexed, lateral flow strip detection of miR bladder cancer markers as illustrated in [103]. (B) Detection based on the peroxidase-like activity of graphene-AuNP nanohybrids shown in [107]. (C) Colorimetric/fluorescence-based detection of miR, relying on cPT:PNA:miR triplex formation, as described in [110].

Figure 9

Illustration of the diselenide–selenoester ligation to a selenocysteine, templated by a target miR, used in a lateral flow strip assay as proposed in [112]. Published by the Royal Society of Chemistry.

Figure 10

Illustration of other miR-detection assays based on PNAs. (A) DQAMmir analysis as adapted from [114]. Copyright 2016 American Chemical Society. (B) Bio-labeling of a PNA:miR-21 duplex with a biotinylated reactive base described in [115]. (C) Detection of miR-208a using the SPR-based methodology, adapted from [44]. Published by the Royal Society of Chemistry. (D) Illustration of the QCM-based methodology adapted from [116]. Published by the Royal Society of Chemistry.