Discriminating a Single Nucleotide Difference for Enhanced miRNA Detection Using Tunable Graphene and Oligonucleotide Nanodevices

Abstract

In this study we have reported our efforts to address some of the challenges in the detection of miRNAs using water-soluble graphene oxide and DNA nanoassemblies. Purposefully inserting mismatches at specific positions in our DNA (probe) strands shows increasing specificity against our target miRNA, miR-10b, over miR-10a which varies by only a single nucleotide. This increased specificity came at a loss of signal intensity within the system, but we demonstrated that this could be addressed with the use of DNase I, an endonuclease capable of cleaving the DNA strands of the RNA/DNA heteroduplex and recycling the RNA target to hybridize to another probe strand. As we previously demonstrated, this enzymatic signal also comes with an inherent activity of the enzyme on the surface-adsorbed probe strands. To remove this activity of DNase I and the steady nonspecific increase in the fluorescence signal without compromising the recovered signal, we attached a thermoresponsive PEGMA polymer (poly(ethylene glycol) methyl ether methacrylate) to nGO. This smart polymer is able to shield the probes adsorbed on the nGO surface from the DNase I activity and is capable of tuning the detection capacity of the nGO nanoassembly with a thermoswitch at 39 °C. By utilizing probes with multiple mismatches, DNase I cleavage of the DNA probe strands, and the attachment of PEGMA polymers to graphene oxide to block undesired DNase I activity, we were able to detect miR-10b from liquid biopsy mimics and breast cancer cell lines. Overall we have reported our efforts to improve the specificity, increase the sensitivity, and eliminate the undesired enzymatic activity of DNase I on surface-adsorbed probes for miR-10b detection using water-soluble graphene nanodevices. Even though we have demonstrated only the discrimination of miR-10b from miR-10a, our approach can be extended to other short RNA molecules which differ by a single nucleotide.

A dual discrimination mode for improved specificity towards let‐7a detection via a single‐base mutated padlock probe‐based exponential rolling circle amplification

“…Some reports have illustrated that, by intentionally introducing a base mutation into the DNA probe strand, the discrimination capability against the target oligonucleotide over other highly homologous family sequences can be improved . Inspired by this finding, we postulated that, by introducing a base mutation into the let‐7a recognition region of the fully complementary padlock probe C, the discrimination power of the assay towards base mismatch would be enhanced.…”

Graphene-Based Strategies in Liquid Biopsy and in Viral Diseases Diagnosis

“…A new GO-polymer-oligonucleotide (nGO-PEGMA/M2) and enzyme ( DNase I ) system able to detect miR-10b in an RNA pool taken from metastatic breast cancer cells were reported by Robertson et al [ 90 ]. The insertion of a specific mismatch fragment into the probe sequence induced an increase of specificity towards miR-10b, an oligonucleotide overexpressed in breast cancer.…”

Progress in miRNA Detection Using Graphene Material–Based Biosensors

“…Robertson et al found that the addition of DNase I to a nanodevice mixture could improve sensitivity, but because of enzyme activity on the surface of adsorption probes, unwanted background signals were generated . The attachment of (poly(ethylene glycol) methyl ether methacrylate) PEGMA polymers eliminated the undesired enzymatic activity and steady increase in background fluorescence by blocking the access of DNase I to the nanodevice surface.…”