Abstract
Many transcribed RNAs are non-coding RNAs, including microRNAs (miRNAs), which bind to complementary sequences on messenger RNAs to regulate the translation efficacy. Therefore, identifying the miRNAs expressed in cells/organisms aids in understanding genetic control in cells/organisms. In this report, we determined the binding of oligonucleotides to a receptor-modified silicon nanowire field-effect transistor (SiNW-FET) by monitoring the changes in conductance of the SiNW-FET. We first modified a SiNW-FET with a DNA probe to directly and selectively detect the complementary miRNA in cell lysates. This SiNW-FET device has 7-fold higher sensitivity than reverse transcription-quantitative polymerase chain reaction in detecting the corresponding miRNA. Next, we anchored viral p19 proteins, which bind the double-strand small RNAs (ds-sRNAs), on the SiNW-FET. By perfusing the device with synthesized ds-sRNAs of different pairing statuses, the dissociation constants revealed that the nucleotides at the 3′-overhangs and pairings at the terminus are important for the interactions. After perfusing the total RNA mixture extracted from Nicotiana benthamiana across the device, this device could enrich the ds-sRNAs for sequence analysis. Finally, this bionanoelectronic SiNW-FET, which is able to isolate and identify the interacting protein-RNA, adds an additional tool in genomic technology for the future study of direct biomolecular interactions.
Highlights
Many transcribed RNAs are non-coding RNAs, including microRNAs, which bind to complementary sequences on messenger RNAs to regulate the translation efficacy
The expressions of RNAs are under strict regulation and usually in a small quantity except those house-keeping genes
reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the most sensitive technique to identify the expression of a specific RNA from a mixture owing to its precise amplification procedure
Summary
The expressions of RNAs are under strict regulation and usually in a small quantity except those house-keeping genes. MiR168 presents in the total extracted RNA, but not in the eluted complexes These results demonstrate the specificity of the DNAprobe/SiNW-FET in recognizing the target miRNA and we could elute the bound oligonucleotides for qPCR amplification or sequencing analysis. To verify p19/SiNW-FET is useful in enriching the miRNA/miRNA* duplexes in the total RNA extracted from N. benthamiana, we dissociated the GSH-GST association to release the p19-ds-sRNA complexes from the SiNW-FET surface and used a generation sequencing (NGS) technique to analyze the sequence of each ss-sRNA in the total (Input) and captured (Eluted) samples (Fig. 3C). After p19 sequestration, the miRNA/miRNA* ratio tends to approaching unity, i.e. 79/36, 49/39, and 172/117, respectively, for the three predicted structures These results further support that the p19/SiNW-FET binds ds-sRNAs with high affinity and specificity. This bionanoelectronic device, which is able to isolate and identify the interacting protein-RNA, adds an additional tool in genomic technology for the future study of direct biomolecular interactions
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