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Isolation and Identification of Post-Transcriptional Gene Silencing-Related Micro-RNAs by Functionalized Silicon Nanowire Field-effect Transistor.

Chen KI, Pan CY, Li KH, Huang YC, Lu CW, Tang CY, Su YW, Tseng LW, Tseng KC, Lin CY, Chen CD, Lin SS, Chen YT - Sci Rep (2015)

Bottom Line: 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.Next, we anchored viral p19 proteins, which bind the double-strand small RNAs (ds-sRNAs), on the SiNW-FET.After perfusing the total RNA mixture extracted from Nicotiana benthamiana across the device, this device could enrich the ds-sRNAs for sequence analysis.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, National Taiwan University, Taipei 106, Taiwan.

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.

No MeSH data available.


Related in: MedlinePlus

Discriminating the secondary structures of ds-sRNA by p19/SiNW-FET.(A) A flow diagram of a reusable p19/SiNW-FET using the GSH/GST association-dissociation. The process includes the immobilization of GST-p19 on a GSH/SiNW-FET to form p19/SiNW-FET, the application of ds-sRNA (miRNA/miRNA*) to bind p19, and the elution of the GST-p19-ds-sRNA complexes with ≥1 mM GSH. (B) The responses of p19/SiNW-FET to various forms of nucleic acids. The normalized ∆Gs were measured by introducing 1 μM of a 21-nucleotide solution in (i). perfectly matched ds-sRNA form (ds-sRNA-0, the structure depicted in (C)); (ii). ss-sRNA form (ss-sRNA-0); (iii). ds-sDNA form (ds-sDNA-0) to a p19/SiNW-FET; (iv). p19 replaced by a binding-deficient mutant (p19mut); and (v). p19 absent in the tests. The vertical red-dotted line indicates the addition of samples. (C) The dissociation constant (Kd) of p19 to various 21-nucleotide nucleic acids with different mismatch pairings. The ΔGs of p19/SiNW-FET to different concentrations of nucleic acids were used to determine the Kd values (Supplementary Fig. S5), which were then normalized to that of the ds-sRNA-0 (Krel). Nucleotides marked in red indicate the mispaired bases.
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f2: Discriminating the secondary structures of ds-sRNA by p19/SiNW-FET.(A) A flow diagram of a reusable p19/SiNW-FET using the GSH/GST association-dissociation. The process includes the immobilization of GST-p19 on a GSH/SiNW-FET to form p19/SiNW-FET, the application of ds-sRNA (miRNA/miRNA*) to bind p19, and the elution of the GST-p19-ds-sRNA complexes with ≥1 mM GSH. (B) The responses of p19/SiNW-FET to various forms of nucleic acids. The normalized ∆Gs were measured by introducing 1 μM of a 21-nucleotide solution in (i). perfectly matched ds-sRNA form (ds-sRNA-0, the structure depicted in (C)); (ii). ss-sRNA form (ss-sRNA-0); (iii). ds-sDNA form (ds-sDNA-0) to a p19/SiNW-FET; (iv). p19 replaced by a binding-deficient mutant (p19mut); and (v). p19 absent in the tests. The vertical red-dotted line indicates the addition of samples. (C) The dissociation constant (Kd) of p19 to various 21-nucleotide nucleic acids with different mismatch pairings. The ΔGs of p19/SiNW-FET to different concentrations of nucleic acids were used to determine the Kd values (Supplementary Fig. S5), which were then normalized to that of the ds-sRNA-0 (Krel). Nucleotides marked in red indicate the mispaired bases.

Mentions: To demonstrate the sequestration of the ds-sRNAs by p19, we modified the SiNW-FET with glutathione (referred to as GSH/SiNW-FET) to support the binding of glutathione S-transferase-conjugated p19 (GST-p19) (referred to as p19/SiNW-FET) (Fig. 2A)171824. We performed electric measurements in phosphate solution (containing 240 μM NaH2PO4 and 760 μM Na2HPO4 at pH 7.4 with NaOH) with a Debye-Hückel screening length of λD = 6.1 nm to sufficiently cover the p19-ds-sRNA complex, which is ~6 nm to the SiNW-FET surface2526. The binding of the negatively charged GST (pI ~ 6.72) or GST-p19 (pI ~ 5.9) to the n-type GSH/SiNW-FET caused prominent decreases in the normalized ΔG (0.19% and 0.55%, respectively) (Supplementary Fig. S2). It is noteworthy that in the biosensing measurements with SiNW-FETs, the charge redistribution helps the SiNW-FETs to detect target-receptor interactions taking place at the top end of surface linking molecules, which could be much longer than the Debye-Hückel screening length. With charge redistribution, SiNW-FETs are able to detect remote molecular interactions, which would otherwise not be possible27. In addition, by using an electrical field perpendicular to the SiNW surface, the structural ordering of surface molecules can be accomplished to improve the sensitivity, reliability, and reproducibility of SiNW-FET biosensors28.


Isolation and Identification of Post-Transcriptional Gene Silencing-Related Micro-RNAs by Functionalized Silicon Nanowire Field-effect Transistor.

Chen KI, Pan CY, Li KH, Huang YC, Lu CW, Tang CY, Su YW, Tseng LW, Tseng KC, Lin CY, Chen CD, Lin SS, Chen YT - Sci Rep (2015)

Discriminating the secondary structures of ds-sRNA by p19/SiNW-FET.(A) A flow diagram of a reusable p19/SiNW-FET using the GSH/GST association-dissociation. The process includes the immobilization of GST-p19 on a GSH/SiNW-FET to form p19/SiNW-FET, the application of ds-sRNA (miRNA/miRNA*) to bind p19, and the elution of the GST-p19-ds-sRNA complexes with ≥1 mM GSH. (B) The responses of p19/SiNW-FET to various forms of nucleic acids. The normalized ∆Gs were measured by introducing 1 μM of a 21-nucleotide solution in (i). perfectly matched ds-sRNA form (ds-sRNA-0, the structure depicted in (C)); (ii). ss-sRNA form (ss-sRNA-0); (iii). ds-sDNA form (ds-sDNA-0) to a p19/SiNW-FET; (iv). p19 replaced by a binding-deficient mutant (p19mut); and (v). p19 absent in the tests. The vertical red-dotted line indicates the addition of samples. (C) The dissociation constant (Kd) of p19 to various 21-nucleotide nucleic acids with different mismatch pairings. The ΔGs of p19/SiNW-FET to different concentrations of nucleic acids were used to determine the Kd values (Supplementary Fig. S5), which were then normalized to that of the ds-sRNA-0 (Krel). Nucleotides marked in red indicate the mispaired bases.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4663627&req=5

f2: Discriminating the secondary structures of ds-sRNA by p19/SiNW-FET.(A) A flow diagram of a reusable p19/SiNW-FET using the GSH/GST association-dissociation. The process includes the immobilization of GST-p19 on a GSH/SiNW-FET to form p19/SiNW-FET, the application of ds-sRNA (miRNA/miRNA*) to bind p19, and the elution of the GST-p19-ds-sRNA complexes with ≥1 mM GSH. (B) The responses of p19/SiNW-FET to various forms of nucleic acids. The normalized ∆Gs were measured by introducing 1 μM of a 21-nucleotide solution in (i). perfectly matched ds-sRNA form (ds-sRNA-0, the structure depicted in (C)); (ii). ss-sRNA form (ss-sRNA-0); (iii). ds-sDNA form (ds-sDNA-0) to a p19/SiNW-FET; (iv). p19 replaced by a binding-deficient mutant (p19mut); and (v). p19 absent in the tests. The vertical red-dotted line indicates the addition of samples. (C) The dissociation constant (Kd) of p19 to various 21-nucleotide nucleic acids with different mismatch pairings. The ΔGs of p19/SiNW-FET to different concentrations of nucleic acids were used to determine the Kd values (Supplementary Fig. S5), which were then normalized to that of the ds-sRNA-0 (Krel). Nucleotides marked in red indicate the mispaired bases.
Mentions: To demonstrate the sequestration of the ds-sRNAs by p19, we modified the SiNW-FET with glutathione (referred to as GSH/SiNW-FET) to support the binding of glutathione S-transferase-conjugated p19 (GST-p19) (referred to as p19/SiNW-FET) (Fig. 2A)171824. We performed electric measurements in phosphate solution (containing 240 μM NaH2PO4 and 760 μM Na2HPO4 at pH 7.4 with NaOH) with a Debye-Hückel screening length of λD = 6.1 nm to sufficiently cover the p19-ds-sRNA complex, which is ~6 nm to the SiNW-FET surface2526. The binding of the negatively charged GST (pI ~ 6.72) or GST-p19 (pI ~ 5.9) to the n-type GSH/SiNW-FET caused prominent decreases in the normalized ΔG (0.19% and 0.55%, respectively) (Supplementary Fig. S2). It is noteworthy that in the biosensing measurements with SiNW-FETs, the charge redistribution helps the SiNW-FETs to detect target-receptor interactions taking place at the top end of surface linking molecules, which could be much longer than the Debye-Hückel screening length. With charge redistribution, SiNW-FETs are able to detect remote molecular interactions, which would otherwise not be possible27. In addition, by using an electrical field perpendicular to the SiNW surface, the structural ordering of surface molecules can be accomplished to improve the sensitivity, reliability, and reproducibility of SiNW-FET biosensors28.

Bottom Line: 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.Next, we anchored viral p19 proteins, which bind the double-strand small RNAs (ds-sRNAs), on the SiNW-FET.After perfusing the total RNA mixture extracted from Nicotiana benthamiana across the device, this device could enrich the ds-sRNAs for sequence analysis.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, National Taiwan University, Taipei 106, Taiwan.

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.

No MeSH data available.


Related in: MedlinePlus