Quantitative and Multiplexed MicroRNA Sensing in Living Cells Based on Peptide Nucleic Acid and Nano Graphene Oxide (PANGO)

Abstract

MicroRNA (miRNA) is an important small RNA which regulates diverse gene expression at the post-transcriptional level. miRNAs are considered as important biomarkers since abnormal expression of specific miRNAs is associated with many diseases including cancer and diabetes. Therefore, it is important to develop biosensors to quantitatively detect miRNA expression levels. Here, we develop a nanosized graphene oxide (NGO) based miRNA sensor, which allows quantitative monitoring of target miRNA expression levels in living cells. The strategy is based on tight binding of NGO with peptide nucleic acid (PNA) probes, resulting in fluorescence quenching of the dye that is conjugated to the PNA, and subsequent recovery of the fluorescence upon addition of target miRNA. PNA as a probe for miRNA sensing offers many advantages including high sequence specificity, high loading capacity on the NGO surface compared to DNA and resistance against nuclease-mediated degradation. The present miRNA sensor allowed the detection of specific target miRNAs with the detection limit as low as ~1 pM and the simultaneous monitoring of three different miRNAs in a living cell.

Effect of chirality in gamma-PNA: PNA interaction, another piece in the picture

“…28,29 Switching elements can be introduced to produce sensing upon hybridization of the target DNA/ RNA, and some PNA-containing nanomaterials can act as sensing elements in cells. 30 In addition, as shown in the paper by Ly and co-workers, generation of a fluorescent signal from two pyrene modified PNA, and aggregation of microbeads can be regulated by the stereochemistry of the PNA duplex; in particular, for microbeads, positive aggregation of fluorescently labeled particles with g-PNA of different stereochemistry linked on the surface could be obtained; in this case, the PNA was modified in order to contain a mini-PEG residue on the g-side chain and at N-terminus, in order to prevent collapse on microparticles; association of small (2 microns) particles to larger ones (10 microns) through complementary PNA: PNA sequences only occurred when these were loaded with PNA of compatible stereochemistry. Thus, a new application of chirality or g-PNA, namely stereoorthogonality has been introduced, and this tool seems to be apt to simplify the design of micro-and nanoscale architectures.…”

Graphene-Based Nanomaterials in Bioimaging

“…For bioimaging applications, the multiplex roles of graphene and its derivatives have been gradually uncovered: i) as imaging contrast agents due to their intrinsic FL emission, Raman scattering, and NIR absorbance (12); ii) as carriers because they have two accessible sides with large specific surface area (theoretical value of 2,630 m 2 g −1 ) that offer high loading capacity of drugs, dyes, PSs and other inorganic nanomaterials by physical absorption, Van der Waals forces, electrostatic binding, or charge transfer interactions (105)(106)(107)(108); iii) as fluorescence quenchers because their sp 2 carbon structure is able to quench small molecule dyes, QDs and conjugated polymers via FL resonance energy transfer or charge transfer (109)(110)(111); iv) as wrapping materials because their flexible and amphiphilic structures make them suitable for wrapping or encapsulating insoluble nanoparticles, thus improving their water solubility and stability, biocompatibility, and preventing aggregation, degradation or toxicity in biological systems (112)(113)(114); v) as building blocks. Their ultra-high surface area and versatile surface functionalization promise the synthesis of graphene-based composites, opening a new avenue for new materials construction (115)(116)(117)(118).…”

Intracellular and Organic miRNA In Situ Detection

“…The application of nanosized GO (NGO) in detection of multiple miRNA targets inside the living cells has been reported. Dal-Hee Min and co-workers [16] develop a biosensor for quantitative monitoring of target miRNA expression levels in living cells. This strategy is based on tight binding of NGO with peptide nucleic acid (PNA) probes, resulting in fluorescence quenching of the dye that is conjugated to the PNA, and subsequent recovery of the fluorescence upon addition of target miRNA by forming a DNA/miRNA heteroduplex ( Fig.…”