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Quantitative assessment of ratiometric bimolecular beacons as a tool for imaging single engineered RNA transcripts and measuring gene expression in living cells.

Zhang X, Song Y, Shah AY, Lekova V, Raj A, Huang L, Behlke MA, Tsourkas A - Nucleic Acids Res. (2013)

Bottom Line: The ability to acquire accurate measurements of RNA copy number in both HT-1080 cells and CHO cells also suggests that RBMBs can be used to image and quantify single RNA transcripts in a wide range of cell lines.Overall, these findings highlight the robustness and versatility of RBMBs as a tool for imaging RNA in live cells.We envision that the unique capabilities of RBMBs will open up new avenues for RNA research.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, University of Pennsylvania, 210 S. 33rd Street, 240 Skirkanich Hall, Philadelphia, PA 19104, USA, Department of Biology, University of Pennsylvania, 433 S. University Ave, 102 Leidy Laboratories, Philadelphia, PA 19104, USA and Integrated DNA Technologies, Inc., 1710 Commercial Park, Coralville, IA 52241, USA.

ABSTRACT
Recently, we developed an oligonucleotide-based probe, ratiometric bimolecular beacon (RBMB), which generates a detectable fluorescent signal in living cells that express the target RNA. Here, we show that RBMBs can also be used to image single RNA transcripts in living cells, when the target RNA is engineered to contain as few as four hybridization sites. Moreover, comparison with single-molecule fluorescence in situ hybridization confirmed that RBMBs could be used to accurately quantify the number of RNA transcripts within individual cells. Measurements of gene expression could be acquired within 30 min and using a wide range of RBMB concentrations. The ability to acquire accurate measurements of RNA copy number in both HT-1080 cells and CHO cells also suggests that RBMBs can be used to image and quantify single RNA transcripts in a wide range of cell lines. Overall, these findings highlight the robustness and versatility of RBMBs as a tool for imaging RNA in live cells. We envision that the unique capabilities of RBMBs will open up new avenues for RNA research.

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Related in: MedlinePlus

Schematic of RBMBs and the methodology used to assess RBMB performance in cells. (A) RBMBs are hairpin-forming oligonucleotide probes that are labeled with a reporter dye, quencher and reference dye. The reporter dye is held in close proximity to the reference dye in the absence of target, leading to a low fluorescent state. Hybridization of the loop-domain to complementary RNA causes the fluorescent dye and quencher to separate, resulting in the restoration of fluorescence. The reference dye remains unquenched regardless of the conformation of the RBMB. The double-stranded domain with a 3′-UU overhang drives nuclear export. (B) To evaluate the ability of RBMBs to quantify the number of RNA transcripts in single cells, cells are fixed following the delivery of RBMBs and single-molecule FISH is performed. FISH probes are represented in the schematic as short linear oligonucleotides with a green fluorescent dye, while RBMBs are drawn with a red reporter dye and magenta reference dye. The large number of RBMBs and FISH probes that bind to each RNA transcript results in bright punctate spots within cells, under fluorescence microscopy. Colocalization of RBMB-signal with the smFISH-signal indicates that the RNA transcript was successfully detected by RBMBs.
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gkt561-F1: Schematic of RBMBs and the methodology used to assess RBMB performance in cells. (A) RBMBs are hairpin-forming oligonucleotide probes that are labeled with a reporter dye, quencher and reference dye. The reporter dye is held in close proximity to the reference dye in the absence of target, leading to a low fluorescent state. Hybridization of the loop-domain to complementary RNA causes the fluorescent dye and quencher to separate, resulting in the restoration of fluorescence. The reference dye remains unquenched regardless of the conformation of the RBMB. The double-stranded domain with a 3′-UU overhang drives nuclear export. (B) To evaluate the ability of RBMBs to quantify the number of RNA transcripts in single cells, cells are fixed following the delivery of RBMBs and single-molecule FISH is performed. FISH probes are represented in the schematic as short linear oligonucleotides with a green fluorescent dye, while RBMBs are drawn with a red reporter dye and magenta reference dye. The large number of RBMBs and FISH probes that bind to each RNA transcript results in bright punctate spots within cells, under fluorescence microscopy. Colocalization of RBMB-signal with the smFISH-signal indicates that the RNA transcript was successfully detected by RBMBs.

Mentions: Perhaps, the most widely adopted probe for live cell imaging is the molecular beacon (MB). MBs are oligonucleotide-based probes that are labeled at one end with a fluorescent reporter and at the other end with a quencher (19,20). In the absence of complementary nucleic acid targets, the MB forms a hairpin structure, which serves to hold the fluorescent reporter and quencher in close proximity. In this configuration, the fluorescence is quenched. In the presence of complementary nucleic acids, hybridization between the central loop of the MB and the target leads to unfolding of the stem and separation of the fluorescent reporter and quencher. In this configuration, fluorescence is restored. Although a number of studies have shown that MBs can be used to detect mRNA in single living cells (21–24), there is growing evidence that the sensitivity of RNA detection is significantly hampered by the sequestration of MBs into the nucleus, where they emit false-positive signals (25–29), and by the large variations in cellular fluorescence that result from heterogeneous intracellular delivery (30). Recently, we developed a new probe for imaging RNA in living cells, ratiometric bimolecular beacons (RBMBs) (Figure 1A), to overcome the limitations of conventional MBs (31). Similar to MBs, RBMBs elicit an increase in reporter fluorescence on hybridization to complementary RNA. However, RBMBs were also designed to possess an 18-base pair double-stranded domain with a 3′-UU overhang and an unquenched reference dye. The unique structure of the RBMB facilitates nuclear export, which dramatically reduces the level of false-positive signals that are detected for at least 24 h, compared with conventional MBs. The reference dye allows for measurements of reporter fluorescence to be adjusted for cell-to-cell variability in RBMB delivery, which allows for more precise measurements of RNA hybridization.Figure 1.


Quantitative assessment of ratiometric bimolecular beacons as a tool for imaging single engineered RNA transcripts and measuring gene expression in living cells.

Zhang X, Song Y, Shah AY, Lekova V, Raj A, Huang L, Behlke MA, Tsourkas A - Nucleic Acids Res. (2013)

Schematic of RBMBs and the methodology used to assess RBMB performance in cells. (A) RBMBs are hairpin-forming oligonucleotide probes that are labeled with a reporter dye, quencher and reference dye. The reporter dye is held in close proximity to the reference dye in the absence of target, leading to a low fluorescent state. Hybridization of the loop-domain to complementary RNA causes the fluorescent dye and quencher to separate, resulting in the restoration of fluorescence. The reference dye remains unquenched regardless of the conformation of the RBMB. The double-stranded domain with a 3′-UU overhang drives nuclear export. (B) To evaluate the ability of RBMBs to quantify the number of RNA transcripts in single cells, cells are fixed following the delivery of RBMBs and single-molecule FISH is performed. FISH probes are represented in the schematic as short linear oligonucleotides with a green fluorescent dye, while RBMBs are drawn with a red reporter dye and magenta reference dye. The large number of RBMBs and FISH probes that bind to each RNA transcript results in bright punctate spots within cells, under fluorescence microscopy. Colocalization of RBMB-signal with the smFISH-signal indicates that the RNA transcript was successfully detected by RBMBs.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3753654&req=5

gkt561-F1: Schematic of RBMBs and the methodology used to assess RBMB performance in cells. (A) RBMBs are hairpin-forming oligonucleotide probes that are labeled with a reporter dye, quencher and reference dye. The reporter dye is held in close proximity to the reference dye in the absence of target, leading to a low fluorescent state. Hybridization of the loop-domain to complementary RNA causes the fluorescent dye and quencher to separate, resulting in the restoration of fluorescence. The reference dye remains unquenched regardless of the conformation of the RBMB. The double-stranded domain with a 3′-UU overhang drives nuclear export. (B) To evaluate the ability of RBMBs to quantify the number of RNA transcripts in single cells, cells are fixed following the delivery of RBMBs and single-molecule FISH is performed. FISH probes are represented in the schematic as short linear oligonucleotides with a green fluorescent dye, while RBMBs are drawn with a red reporter dye and magenta reference dye. The large number of RBMBs and FISH probes that bind to each RNA transcript results in bright punctate spots within cells, under fluorescence microscopy. Colocalization of RBMB-signal with the smFISH-signal indicates that the RNA transcript was successfully detected by RBMBs.
Mentions: Perhaps, the most widely adopted probe for live cell imaging is the molecular beacon (MB). MBs are oligonucleotide-based probes that are labeled at one end with a fluorescent reporter and at the other end with a quencher (19,20). In the absence of complementary nucleic acid targets, the MB forms a hairpin structure, which serves to hold the fluorescent reporter and quencher in close proximity. In this configuration, the fluorescence is quenched. In the presence of complementary nucleic acids, hybridization between the central loop of the MB and the target leads to unfolding of the stem and separation of the fluorescent reporter and quencher. In this configuration, fluorescence is restored. Although a number of studies have shown that MBs can be used to detect mRNA in single living cells (21–24), there is growing evidence that the sensitivity of RNA detection is significantly hampered by the sequestration of MBs into the nucleus, where they emit false-positive signals (25–29), and by the large variations in cellular fluorescence that result from heterogeneous intracellular delivery (30). Recently, we developed a new probe for imaging RNA in living cells, ratiometric bimolecular beacons (RBMBs) (Figure 1A), to overcome the limitations of conventional MBs (31). Similar to MBs, RBMBs elicit an increase in reporter fluorescence on hybridization to complementary RNA. However, RBMBs were also designed to possess an 18-base pair double-stranded domain with a 3′-UU overhang and an unquenched reference dye. The unique structure of the RBMB facilitates nuclear export, which dramatically reduces the level of false-positive signals that are detected for at least 24 h, compared with conventional MBs. The reference dye allows for measurements of reporter fluorescence to be adjusted for cell-to-cell variability in RBMB delivery, which allows for more precise measurements of RNA hybridization.Figure 1.

Bottom Line: The ability to acquire accurate measurements of RNA copy number in both HT-1080 cells and CHO cells also suggests that RBMBs can be used to image and quantify single RNA transcripts in a wide range of cell lines.Overall, these findings highlight the robustness and versatility of RBMBs as a tool for imaging RNA in live cells.We envision that the unique capabilities of RBMBs will open up new avenues for RNA research.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, University of Pennsylvania, 210 S. 33rd Street, 240 Skirkanich Hall, Philadelphia, PA 19104, USA, Department of Biology, University of Pennsylvania, 433 S. University Ave, 102 Leidy Laboratories, Philadelphia, PA 19104, USA and Integrated DNA Technologies, Inc., 1710 Commercial Park, Coralville, IA 52241, USA.

ABSTRACT
Recently, we developed an oligonucleotide-based probe, ratiometric bimolecular beacon (RBMB), which generates a detectable fluorescent signal in living cells that express the target RNA. Here, we show that RBMBs can also be used to image single RNA transcripts in living cells, when the target RNA is engineered to contain as few as four hybridization sites. Moreover, comparison with single-molecule fluorescence in situ hybridization confirmed that RBMBs could be used to accurately quantify the number of RNA transcripts within individual cells. Measurements of gene expression could be acquired within 30 min and using a wide range of RBMB concentrations. The ability to acquire accurate measurements of RNA copy number in both HT-1080 cells and CHO cells also suggests that RBMBs can be used to image and quantify single RNA transcripts in a wide range of cell lines. Overall, these findings highlight the robustness and versatility of RBMBs as a tool for imaging RNA in live cells. We envision that the unique capabilities of RBMBs will open up new avenues for RNA research.

Show MeSH
Related in: MedlinePlus