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Target accessibility and signal specificity in live-cell detection of BMP-4 mRNA using molecular beacons.

Rhee WJ, Santangelo PJ, Jo H, Bao G - Nucleic Acids Res. (2008)

Bottom Line: The ability to visualize mRNA in single living cells and monitor in real-time the changes of mRNA level and localization can provide unprecedented opportunities for biological and disease studies.The beacon specificity was further confirmed using blocking RNA and in situ hybridization.This has significant implications to MB design for live cell mRNA detection.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.

ABSTRACT
The ability to visualize mRNA in single living cells and monitor in real-time the changes of mRNA level and localization can provide unprecedented opportunities for biological and disease studies. However, the mRNA detection specificity and sensitivity are critically dependent on the selection of target sequences and their accessibility. We carried out an extensive study of the target accessibility of BMP-4 mRNA using 10 different designs of molecular beacons (MBs), and identified the optimal beacon design. Specifically, for MB design 1 and 8 (MB1 and MB8), the fluorescent intensities from BMP-4 mRNA correlated well with the GFP signal after upregulating BMP-4 and co-expressing GFP using adenovirus, and the knockdown of BMP-4 mRNA using siRNA significantly reduced the beacon signals, demonstrating detection specificity. The beacon specificity was further confirmed using blocking RNA and in situ hybridization. We found that fluorescence signal from MBs depends critically on target sequences; the target sequences corresponding to siRNA sites may not be good sites for beacon-based mRNA detection, and vice versa. Possible beacon design rules are identified and approaches for enhancing target accessibility are discussed. This has significant implications to MB design for live cell mRNA detection.

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Effect of target-sequence shifting of MB8 on BMP-4 mRNA detection. (A) Hybridization sites of MB8, MB8a and MB8b. Two molecular beacons, MB8a and MB8b were designed so that their hybridization domains are shifted to the left and right, respectively from that of MB8. (B–D) BMP-4 mRNA detection in HDF cells using respectively 0.2 μM of MB8 (B), 1 μM of MB8a (C) and 1 μM MB8b (D). Note that higher (5×) concentrations of MB8a and MB8b were used in this experiment as it was difficult to detect any signal with the same exposure time when a low concentration (0.2 μM) of MB8a or MB8b was used.
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Figure 8: Effect of target-sequence shifting of MB8 on BMP-4 mRNA detection. (A) Hybridization sites of MB8, MB8a and MB8b. Two molecular beacons, MB8a and MB8b were designed so that their hybridization domains are shifted to the left and right, respectively from that of MB8. (B–D) BMP-4 mRNA detection in HDF cells using respectively 0.2 μM of MB8 (B), 1 μM of MB8a (C) and 1 μM MB8b (D). Note that higher (5×) concentrations of MB8a and MB8b were used in this experiment as it was difficult to detect any signal with the same exposure time when a low concentration (0.2 μM) of MB8a or MB8b was used.

Mentions: To address this issue, we evaluated the accessibility of MBs that target sequences adjacent to MB8-targeting sequence on BMP-4 mRNA. As shown in Figure 8A (also Table 1), two MBs, MB8a and MB8b, were designed so that their target sequences share respectively 8 and 10 overlapping bases with the target sequence of MB8. Specifically, MB8a has 8 of its 17-base sequence overlapping with the 5′ end of the MB8 sequence, and MB8b has 10 of its 14-base sequence overlapping with the 3′ end of the MB8-targting sequence (Figure 8A). As shown in Figure 8C and D, MB8a and MB8b produced much lower fluorescence signal compared with that of MB8 (Figure 8B), although the GFP signal remained the same. This result further demonstrates that the fluorescence signal from MB8 was critically dependent on the specific target sequences; any change to the target sequence, even a few bases, could significantly reduce the target accessibility. Therefore, MBs need to be designed properly with very specific sequences in order to have good target accessibility in live-cell mRNA detection.Figure 8.


Target accessibility and signal specificity in live-cell detection of BMP-4 mRNA using molecular beacons.

Rhee WJ, Santangelo PJ, Jo H, Bao G - Nucleic Acids Res. (2008)

Effect of target-sequence shifting of MB8 on BMP-4 mRNA detection. (A) Hybridization sites of MB8, MB8a and MB8b. Two molecular beacons, MB8a and MB8b were designed so that their hybridization domains are shifted to the left and right, respectively from that of MB8. (B–D) BMP-4 mRNA detection in HDF cells using respectively 0.2 μM of MB8 (B), 1 μM of MB8a (C) and 1 μM MB8b (D). Note that higher (5×) concentrations of MB8a and MB8b were used in this experiment as it was difficult to detect any signal with the same exposure time when a low concentration (0.2 μM) of MB8a or MB8b was used.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2275124&req=5

Figure 8: Effect of target-sequence shifting of MB8 on BMP-4 mRNA detection. (A) Hybridization sites of MB8, MB8a and MB8b. Two molecular beacons, MB8a and MB8b were designed so that their hybridization domains are shifted to the left and right, respectively from that of MB8. (B–D) BMP-4 mRNA detection in HDF cells using respectively 0.2 μM of MB8 (B), 1 μM of MB8a (C) and 1 μM MB8b (D). Note that higher (5×) concentrations of MB8a and MB8b were used in this experiment as it was difficult to detect any signal with the same exposure time when a low concentration (0.2 μM) of MB8a or MB8b was used.
Mentions: To address this issue, we evaluated the accessibility of MBs that target sequences adjacent to MB8-targeting sequence on BMP-4 mRNA. As shown in Figure 8A (also Table 1), two MBs, MB8a and MB8b, were designed so that their target sequences share respectively 8 and 10 overlapping bases with the target sequence of MB8. Specifically, MB8a has 8 of its 17-base sequence overlapping with the 5′ end of the MB8 sequence, and MB8b has 10 of its 14-base sequence overlapping with the 3′ end of the MB8-targting sequence (Figure 8A). As shown in Figure 8C and D, MB8a and MB8b produced much lower fluorescence signal compared with that of MB8 (Figure 8B), although the GFP signal remained the same. This result further demonstrates that the fluorescence signal from MB8 was critically dependent on the specific target sequences; any change to the target sequence, even a few bases, could significantly reduce the target accessibility. Therefore, MBs need to be designed properly with very specific sequences in order to have good target accessibility in live-cell mRNA detection.Figure 8.

Bottom Line: The ability to visualize mRNA in single living cells and monitor in real-time the changes of mRNA level and localization can provide unprecedented opportunities for biological and disease studies.The beacon specificity was further confirmed using blocking RNA and in situ hybridization.This has significant implications to MB design for live cell mRNA detection.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.

ABSTRACT
The ability to visualize mRNA in single living cells and monitor in real-time the changes of mRNA level and localization can provide unprecedented opportunities for biological and disease studies. However, the mRNA detection specificity and sensitivity are critically dependent on the selection of target sequences and their accessibility. We carried out an extensive study of the target accessibility of BMP-4 mRNA using 10 different designs of molecular beacons (MBs), and identified the optimal beacon design. Specifically, for MB design 1 and 8 (MB1 and MB8), the fluorescent intensities from BMP-4 mRNA correlated well with the GFP signal after upregulating BMP-4 and co-expressing GFP using adenovirus, and the knockdown of BMP-4 mRNA using siRNA significantly reduced the beacon signals, demonstrating detection specificity. The beacon specificity was further confirmed using blocking RNA and in situ hybridization. We found that fluorescence signal from MBs depends critically on target sequences; the target sequences corresponding to siRNA sites may not be good sites for beacon-based mRNA detection, and vice versa. Possible beacon design rules are identified and approaches for enhancing target accessibility are discussed. This has significant implications to MB design for live cell mRNA detection.

Show MeSH
Related in: MedlinePlus