Limits...
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

p19/SiNW-FET sequestration enriches the ds-sRNAs.We perfused the p19/SiNW-FET with sRNA (Total) from synthesized ds-sRNA-0 (A) or extracted RNA from Arabidopsis (B) or Nicotiana benthamiana (C); we then eluted the bound sRNA (Eluted) from p19/SiNW-FET and analyzed the relative amounts of the designated sRNA by (A,B) RT-qPCR or (C) a deep sequencer. (A) The relative amounts of sense and anti-sense strands of ds-sRNA-0 in the total and eluted fractions. (B) The relative amounts of miR168 (sense) and miR168* (anti-sense) in the extracted and bound fractions. (C) Proportions of the analyzed sRNAs with different read counts. The number of each sRNA sequenced was counted (read counts) and binned with a power of 2; the number of sRNAs in each binning group was normalized to the total number of sRNA counted. (D) Predicted structures of three pre-miRNAs yielding the corresponding paired ds-sRNAs captured on p19/SiNW-FET. According to the genome of N. benthamiana, nine genes (Table S1) might transcribe pre-miRNAs which have 2nd structures yielding paired ds-sRNAs (bases in bold) identified from the Elute fraction. The red lines indicate the matured miRNA forms. The digits beside each sRNA segment (bases in bold) are the counts of the corresponding sRNA segment appearing in the Input (I) or Eluted (E) sRNA sample. The data (mean ± standard deviations) were the averages of three independent experiments and ** indicates the p-value < 0.01. A magnified image of Fig. 3D is shown in Supplementary Fig. S7 for easier reading.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4663627&req=5

f3: p19/SiNW-FET sequestration enriches the ds-sRNAs.We perfused the p19/SiNW-FET with sRNA (Total) from synthesized ds-sRNA-0 (A) or extracted RNA from Arabidopsis (B) or Nicotiana benthamiana (C); we then eluted the bound sRNA (Eluted) from p19/SiNW-FET and analyzed the relative amounts of the designated sRNA by (A,B) RT-qPCR or (C) a deep sequencer. (A) The relative amounts of sense and anti-sense strands of ds-sRNA-0 in the total and eluted fractions. (B) The relative amounts of miR168 (sense) and miR168* (anti-sense) in the extracted and bound fractions. (C) Proportions of the analyzed sRNAs with different read counts. The number of each sRNA sequenced was counted (read counts) and binned with a power of 2; the number of sRNAs in each binning group was normalized to the total number of sRNA counted. (D) Predicted structures of three pre-miRNAs yielding the corresponding paired ds-sRNAs captured on p19/SiNW-FET. According to the genome of N. benthamiana, nine genes (Table S1) might transcribe pre-miRNAs which have 2nd structures yielding paired ds-sRNAs (bases in bold) identified from the Elute fraction. The red lines indicate the matured miRNA forms. The digits beside each sRNA segment (bases in bold) are the counts of the corresponding sRNA segment appearing in the Input (I) or Eluted (E) sRNA sample. The data (mean ± standard deviations) were the averages of three independent experiments and ** indicates the p-value < 0.01. A magnified image of Fig. 3D is shown in Supplementary Fig. S7 for easier reading.

Mentions: To verify that p19/SiNW-FET could selectively sequester ds-sRNAs from an RNA mixture, we perfused the p19/NW-FET with synthesized ds-sRNA-0 (Fig. 3A) or extracted RNA from Arabidopsis (Fig. 3B) and analyzed the relative amount of sRNA (the sense vs. anti-sense strands of ds-sRNA-0) by RT-qPCR. Perfusion of the synthesized ds-sRNA-0 or total extracted RNA across the p19/NW-FET induced a decrease in the conductivity (Supplementary Fig. S6). Figure 3A shows that the normalized amounts of the sense and anti-sense strands of ds-sRNA-0 in the input mixture were 100 and 67.8%, respectively; whereas the relative amounts in the eluted sample were 21.7 and 20.6%, respectively. These results revealed that the sense and anti-sense strands of ds-sRNA-0 are not all paired in the input mixture; however, after p19 sequestration, the amounts of the sense and anti-sense strands are almost of equal amounts suggesting that the sRNA are mostly paired. Unlike the synthesized ds-sRNA-0, endogenous ds-sRNAs degraded quickly; so the total RNA (input) contains very few miR168* (4.9%) relative to the miR168 (regarded as 100%) as shown in Fig. 3B; however, the eluted sRNAs contains 1.8 and 1.1% of miR168 and miR168*, respectively. The nearly equal amounts of sense and anti-sense miRNAs in the eluted samples demonstrate the binding specificity of p19 to ds-sRNA.


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)

p19/SiNW-FET sequestration enriches the ds-sRNAs.We perfused the p19/SiNW-FET with sRNA (Total) from synthesized ds-sRNA-0 (A) or extracted RNA from Arabidopsis (B) or Nicotiana benthamiana (C); we then eluted the bound sRNA (Eluted) from p19/SiNW-FET and analyzed the relative amounts of the designated sRNA by (A,B) RT-qPCR or (C) a deep sequencer. (A) The relative amounts of sense and anti-sense strands of ds-sRNA-0 in the total and eluted fractions. (B) The relative amounts of miR168 (sense) and miR168* (anti-sense) in the extracted and bound fractions. (C) Proportions of the analyzed sRNAs with different read counts. The number of each sRNA sequenced was counted (read counts) and binned with a power of 2; the number of sRNAs in each binning group was normalized to the total number of sRNA counted. (D) Predicted structures of three pre-miRNAs yielding the corresponding paired ds-sRNAs captured on p19/SiNW-FET. According to the genome of N. benthamiana, nine genes (Table S1) might transcribe pre-miRNAs which have 2nd structures yielding paired ds-sRNAs (bases in bold) identified from the Elute fraction. The red lines indicate the matured miRNA forms. The digits beside each sRNA segment (bases in bold) are the counts of the corresponding sRNA segment appearing in the Input (I) or Eluted (E) sRNA sample. The data (mean ± standard deviations) were the averages of three independent experiments and ** indicates the p-value < 0.01. A magnified image of Fig. 3D is shown in Supplementary Fig. S7 for easier reading.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: p19/SiNW-FET sequestration enriches the ds-sRNAs.We perfused the p19/SiNW-FET with sRNA (Total) from synthesized ds-sRNA-0 (A) or extracted RNA from Arabidopsis (B) or Nicotiana benthamiana (C); we then eluted the bound sRNA (Eluted) from p19/SiNW-FET and analyzed the relative amounts of the designated sRNA by (A,B) RT-qPCR or (C) a deep sequencer. (A) The relative amounts of sense and anti-sense strands of ds-sRNA-0 in the total and eluted fractions. (B) The relative amounts of miR168 (sense) and miR168* (anti-sense) in the extracted and bound fractions. (C) Proportions of the analyzed sRNAs with different read counts. The number of each sRNA sequenced was counted (read counts) and binned with a power of 2; the number of sRNAs in each binning group was normalized to the total number of sRNA counted. (D) Predicted structures of three pre-miRNAs yielding the corresponding paired ds-sRNAs captured on p19/SiNW-FET. According to the genome of N. benthamiana, nine genes (Table S1) might transcribe pre-miRNAs which have 2nd structures yielding paired ds-sRNAs (bases in bold) identified from the Elute fraction. The red lines indicate the matured miRNA forms. The digits beside each sRNA segment (bases in bold) are the counts of the corresponding sRNA segment appearing in the Input (I) or Eluted (E) sRNA sample. The data (mean ± standard deviations) were the averages of three independent experiments and ** indicates the p-value < 0.01. A magnified image of Fig. 3D is shown in Supplementary Fig. S7 for easier reading.
Mentions: To verify that p19/SiNW-FET could selectively sequester ds-sRNAs from an RNA mixture, we perfused the p19/NW-FET with synthesized ds-sRNA-0 (Fig. 3A) or extracted RNA from Arabidopsis (Fig. 3B) and analyzed the relative amount of sRNA (the sense vs. anti-sense strands of ds-sRNA-0) by RT-qPCR. Perfusion of the synthesized ds-sRNA-0 or total extracted RNA across the p19/NW-FET induced a decrease in the conductivity (Supplementary Fig. S6). Figure 3A shows that the normalized amounts of the sense and anti-sense strands of ds-sRNA-0 in the input mixture were 100 and 67.8%, respectively; whereas the relative amounts in the eluted sample were 21.7 and 20.6%, respectively. These results revealed that the sense and anti-sense strands of ds-sRNA-0 are not all paired in the input mixture; however, after p19 sequestration, the amounts of the sense and anti-sense strands are almost of equal amounts suggesting that the sRNA are mostly paired. Unlike the synthesized ds-sRNA-0, endogenous ds-sRNAs degraded quickly; so the total RNA (input) contains very few miR168* (4.9%) relative to the miR168 (regarded as 100%) as shown in Fig. 3B; however, the eluted sRNAs contains 1.8 and 1.1% of miR168 and miR168*, respectively. The nearly equal amounts of sense and anti-sense miRNAs in the eluted samples demonstrate the binding specificity of p19 to ds-sRNA.

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