This work was supported by the National Science Foundation CAREER Award (0953583) and the National Institute of Health NCI/R21-CA116102, NCI/R21-CA125088, NIBIB/R01-EB012065, NCI/R01-CA157766.

In this protocol, one oligonucleotide (RBMB1) was labeled at its 5&#8217;-end with a CF640R reporter dye and has the sequence: 5&#8217;-mCmUmUmC mGmUmC mCmAmC mAmAmA mCmAmC mAmAmC mUmCmC mU mGmAmAmG mGmAmC mGmGmC mAmGmC mGmUmG mCmAmG mCmUmC mUmU-3&#8217;. Self-complementary domains, which drive the formation of the hairpin structure, are in bold. The second oligonucleotide (RBMB2) was labeled at the 3&#8217;-end with an Iowa Black RQ-Sp quencher and has the sequence: 5&#8217;-mGmAmG mCmU...

<ol>
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F., Lilie, H., Koumoutsakos, P., Helenius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-particle+tracking+of+murine+polyoma+virus-like+particles+on+live+cells+and+artificial+membranes.">Single-particle tracking of murine polyoma virus-like particles on live cells and artificial membranes.</a> Proc Natl Acad Sci USA. 102 (42), 15110-15115 (2005).</li><li>Forrest, K. M., Gavis, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+endogenous+RNA+reveals+a+diffusion+and+entrapment+mechanism+for+nanos+mRNA+localization+in+Drosophila.">Live imaging of endogenous RNA reveals a diffusion and entrapment mechanism for nanos mRNA localization in Drosophila.</a> Curr Biol. 13 (14), 1159-1168 (2003).</li><li>Weil, T. T., Forrest, K. M., Gavis, E. 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The ability to image single engineered RNA transcripts in living cells using a conventional wide-field microscope requires a bright and photostable fluorescent signal to be associated with each RNA transcript and a low fluorescent background emanating from unbound fluorescent probes. In this method, a bright fluorescent signal is achieved by hybridizing multiple (up to 96) oligonucleotide-based fluorescent probes, i.e. RBMBs, onto each RNA transcript. However, as few as four bindings sites are sufficient16<...

The expression and regulation of RNA transcripts is a complex and dynamic process that is largely responsible for controlling cell behavior and fate.&#160; Although the importance of RNA in dictating cell function has been known for some time, it is often difficult to draw clear connections between the two since most RNA analysis tools lack the spatial and temporal resolution required to capture important regulatory events such as transcription bursting, RNA trafficking, and localized RNA processing. This has led to the advent of several techniques that allow individual RNA transcripts to be visualized in living cells in real-time1. Perhaps, the most prominent of these techniques utilizes a GFP-MS2 fusion protein to target RNA that has been engineered to contain tandem repeats of the MS2 binding site in the 3&#8217;-UTR2,3. By bringing multiple GFP molecules into close proximity, individual RNA transcripts appear as bright fluorescent spots via fluorescence microscopy. The GFP-MS2 system has provided unprecedented insight into RNA behavior, including direct visualization and measurements of transcriptional bursting4,5, detection of unique sub-cellular localization and processing6-9, and real-time imaging of RNA transport10. However, despite the tremendous potential of the GFP-MS2 system, unbound GFP-MS2 fusion proteins can create a high background fluorescent signal that limits the versatility and dynamic range of this technique.&#160; Several approaches have been developed to limit this background signal, including subcellular compartmentalization of unbound GFP-MS210, protein fragment complementation11, and alternative RNA binding proteins and targets12.&#160; However, all of these approaches remain sensitive to the relative and total expression of the RNA target and GFP-MS2 fusion protein.

As an alternative to the GFP-MS2 systems, molecular beacons have also been used to detect engineered RNA transcripts with tandem repeats of the complementary binding site in the 3&#8217;-UTR13,14. Molecular beacons are hairpin-forming oligonucleotide probes that are labeled at one end with a quencher and at the other end with a fluorescent reporter. When not bound to target RNA the fluorescent reporter and quencher remain in close proximity, resulting in a low-fluorescent state. Upon hybridization, the fluorescent reporter and quencher are forced apart and fluorescence is restored. Similar to the GFP-MS2 system, the binding of multiple molecular beacons onto a single RNA transcript results in a bright fluorescent spot that can be identified by fluorescence microscopy; however, the background fluorescence is expected to be much lower, due to the quenched configuration of unbound molecular beacons. Unfortunately, despite the clever activation mechanism that is incorporated into the molecular beacon design, there is now growing evidence that unhybridized molecular beacons do not remain in the hairpin conformation when introduced into living cells.&#160; Consequently, they generate a false-positive signal that significantly reduces the signal-to-background. To overcome this shortcoming, we recently developed a new synthetic probe for imaging RNA in living cells, ratiometric bimolecular beacons (RBMBs; Figure 1A)15,16. RBMBs are composed of two 2&#8217;-O-methyl oligonucleotide strands that form a hybrid structure with features from both short hairpin RNA (shRNA) and molecular beacons.&#160; The loop and fluorescence activation mechanism is similar to the molecular beacon, while the long double stranded domain with a 3&#8217;-UU overhang is more characteristic of shRNA.&#160; The shRNA features are designed to drive nuclear export, which we have found increases intracellular lifetime to &#62;24 hr, with minimal observable degradation, and prevents non-specific opening of the loop. As a result RBMBs exhibit a significantly higher signal-to-background than molecular beacons.

It should be noted that RBMBs cannot be prepared using DNA oligonucleotides, since DNA-based probes do not possess the same nuclear export capabilities as RNA-based probes. Structurally, the RBMB loop is typically designed to be between 15 and 21 bases long, to create a balance between specificity and selectivity upon RNA hybridization.&#160; The short stem that forms from the two self-complementary domains is usually designed to be 4 bases.&#160; If a longer stem is selected, the rate of hybridization between the RBMB loop and target RNA is significantly slowed17,18. Conversely, when a shorter stem sequence is selected the melting temperature is often too low to sustain the stem-loop structure at 37 &#176;C, leading to a high background signal. The specificity of the RBMB is also reduced as the stem length is shortened. Since the sequence of the RBMB stem-loop can also influence RBMB performance, it must be carefully selected19. In particular, ideal loop sequences should have minimal secondary structure, hybridize to RNA sequences with minimal secondary structure, avoid protein binding sites, and avoid off-target binding. Predictions of both RBMB and RNA secondary structure can be obtained using software such as mfold20. Complementary off-target sites can identified using a nucleotide Basic Local Assignment Search Tool (BLAST).&#160; However, because of inconsistencies in model predictions and the difficulty in identifying protein-biding sites, the specificity of all RBMBs must ultimately be validated experimentally.

If desirable, an unquenched reference dye that is insensitive to the hybridization state can be added to the RBMB15.&#160; The addition of a reference dye can provide a marker for probe delivery and be used for ratiometric imaging, if more accurate measurements of total cellular fluorescence are required.&#160; The reference dyes allows measurements to be adjusted for differences in background owing to cell-to-cell variations in delivery.&#160; However, when imaging individual RNA transcripts the reference dye is not necessary. Notably, some reference dyes can interfere with the export of RBMBs from the nucleus, leading to a slightly higher background signal in the nucleus.

When designed properly, RBMBs can be used to image individual RNA transcripts in single living cells, if the target RNA is engineered to contain at least four RBMB binding sites (Figure 1B)16. RBMBs can be efficiently delivered into a wide range of cells types via microporation, with little to no effect on cell viability21, and quantitative measurements of gene expression can be acquired within 30 min.&#160; Moreover, the methodology is fairly insensitive to RBMB concentration and target RNA levels, since fluorescence from unbound RBMBs is efficiently quenched. Here we provide a detailed description of the methodology used to prepare and purify RBMBs, as well as a general procedure for the delivery of RBMBs into live-cells via microporation and the imaging of single RNA transcripts in real-time.

<table border="0" cellpadding="0" cellspacing="0" width="977">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Nuclease-free Water</td>
			<td style="width:207px;">Life Technologies</td>
			<td style="width:220px;">AM9932</td>
			<td style="width:244px;">no DEPC-treated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">DPBS</td>
			<td style="width:207px;">Life Technologies</td>
			<td>14190-144</td>
			<td>No Ca2+ and Mg2+</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Cary 100 UV-Vis Spectrophotometer</td>
			<td style="width:207px;">Agilent Technologies</td>
			<td style="width:220px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Potassium Phosphate Dibasic</td>
			<td style="width:207px;">Fisher Scientific</td>
			<td style="width:220px;">P290-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Potassium Phosphate Monobasic</td>
			<td style="width:207px;">Fisher Scientific</td>
			<td style="width:220px;">P284-500&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Sodium Phosphate Monobasic</td>
			<td style="width:207px;">Fisher Scientific</td>
			<td style="width:220px;">S381-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Microcentrifuge tubes</td>
			<td style="width:207px;">Eppendorf</td>
			<td style="width:220px;">22364111</td>
			<td>1.5mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Chromatography Column</td>
			<td style="width:207px;">Kimble Chase Life Science</td>
			<td style="width:220px;">420400-0720</td>
			<td>0.7x20cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Superdex 75 Prep Grade</td>
			<td style="width:207px;">GE healthcare</td>
			<td style="width:220px;">17-1044-01&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Syringe Pump</td>
			<td style="width:207px;">Braintree Scientific</td>
			<td style="width:220px;">BS-300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Syringe</td>
			<td style="width:207px;">BD</td>
			<td style="width:220px;">301035</td>
			<td>60mL, Luer-lok tip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Centrifugal Filter Units</td>
			<td style="width:207px;">Millipore</td>
			<td style="width:220px;">UFC501096</td>
			<td>Mw 10,000 cutoff</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Centrifuge 5418</td>
			<td style="width:207px;">Eppendorf</td>
			<td style="width:220px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Poly-D-lysine</td>
			<td>Sigma-Aldrich</td>
			<td style="width:220px;">P7280-5MG</td>
			<td>lyophilized powder, g-irradiated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">8-well chambered coverglass</td>
			<td style="width:207px;">Fisher Scientific</td>
			<td style="width:220px;">155409</td>
			<td>Working volume 0.2-0.5mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">HT1080</td>
			<td style="width:207px;">ATCC</td>
			<td style="width:220px;">CCL-121</td>
			<td>Human Fibrosarcoma cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Cell Culture Flask</td>
			<td style="width:207px;">Corning</td>
			<td style="width:220px;">430639</td>
			<td>25cm2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">DMEM</td>
			<td style="width:207px;">Life Technologies</td>
			<td style="width:220px;">11965084</td>
			<td>High glucose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">DMEM without Phenol Red</td>
			<td style="width:207px;">Life Technologies</td>
			<td style="width:220px;">21063029</td>
			<td>High glucose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Fetal Bovine Serum</td>
			<td style="width:207px;">Sigma-Aldrich</td>
			<td style="width:220px;">F2442-500ML&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Penecillin/Streptomycin</td>
			<td style="width:207px;">Life Technologies</td>
			<td style="width:220px;">15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Trypsin</td>
			<td style="width:207px;">Life Technologies</td>
			<td style="width:220px;">25300054</td>
			<td>0.05% Trypsin-EDTA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Neon transfection system</td>
			<td style="width:207px;">Life Technologies</td>
			<td style="width:220px;">MPK5000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neon transfection system kit&#160;</td>
			<td style="width:207px;">Life Technologies</td>
			<td style="width:220px;">MPK1096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Hemacytometer</td>
			<td style="width:207px;">Fisher Scientific</td>
			<td style="width:220px;">02-671-10&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoescht 33342</td>
			<td style="width:207px;">Life Technologies</td>
			<td style="width:220px;">H1399</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">IX-81 Inverted fluorescence microscope&#160;</td>
			<td style="width:207px;">Olympus</td>
			<td style="width:220px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">SOLA light engine</td>
			<td style="width:207px;">Lumencor</td>
			<td style="width:220px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Metamorph</td>
			<td style="width:207px;">Molecular Devices</td>
			<td style="width:220px;"></td>
			<td>Software controlling microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immersol oil 518F</td>
			<td style="width:207px;">Fisher Scientific</td>
			<td style="width:220px;">12-624-66B&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

real-time imaging of single engineered rna transcripts in living cells using ratiometric bimolecular beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Shortly following the microporation of HT1080 cells in the presence of RBMBs, individual RNA transcripts that were engineered to contain multiple RBMB binding sites in their 3&#8217;-UTR appear as bright fluorescent spots when imaged by wide-field fluorescence microscopy (Figure 2). While individual RNA transcripts with as few as four RBMB binding sites can be imaged in live-cells, the more RBMB binding sites in the 3&#8217;-UTR, the stronger the fluorescent signal.&#160; Moreover, the more RBMBs that ar...

Watch this Scientific Journal Video about Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons at JoVE.com

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

real-time-imaging-single-engineered-rna-transcripts-living-cells

Research

JoVE Journal

Biology

11.7K visualizações.  University of Pennsylvania.  Beacons bimoleculares proporcional (RBMBs) pode ser usado para imagens simples transcrições de RNA projetados em células vivas. Aqui, nós descrevemos a preparação e purificação de RBMBs, entrega de RBMBs em células de formação de microporos e de imagem fluorescente de transcritos de ARN em tempo real. 