RNA visualization in live bacterial cells usingfluorescent protein complementationMaria Valencia-Burton1, Ron M McCullough1,2, Charles R Cantor1,3 & Natalia E Broude1

We describe a technique for the detection and localization

of RNA transcripts in living cells. The method is based on

fluorescent-protein complementation regulated by the

interaction of a split RNA-binding protein with its corresponding

RNA aptamer. In our design, the RNA-binding protein is the

eukaryotic initiation factor 4A (eIF4A). eIF4A is dissected into

two fragments, and each fragment is fused to split fragments of

the enhanced green fluorescent protein (EGFP). Coexpression of

the two protein fusions in the presence of a transcript

containing eIF4A-interacting RNA aptamer resulted in the

restoration of EGFP fluorescence in Escherichia coli cells. We also

applied this technique to the visualization of an aptamer-tagged

mRNA and 5S ribosomal RNA (rRNA). We observed distinct

spatial and temporal changes in fluorescence within single cells,

reflecting the nature of the transcript.

The study of the dynamics of transcription in single cells is achallenging and exciting enterprise, promising better understand-ing of this central biochemical process. Recently, several studieshave addressed the kinetics of gene expression at the single-celllevel1–5, by measuring the translation of reporter genes. However,only a few studies have attempted to answer this question bymeasuring RNA kinetics6–8. In these studies, the RNA of interestwas modified with multiple copies of the MS2-coat proteinrecognition sequence in tandem and was detected by expressingMS2 coat protein fused to a fluorescent protein in the same cell. Inthese experiments, a large multimeric complex of RNA-bindingprotein and fluorescent protein gets assembled onto the RNA6–8

and high background fluorescence coming from unbound fluores-cent protein limits the sensitivity of the method. An alternativetechnique using RNA-specific prelabeled probes called molecularbeacons has also been used for RNA localization in live cells9–12.This approach, however, requires invasive methods of probedelivery into the cell.

Protein complementation assay (PCA) is a comparatively newmethod developed for protein-interaction studies13. In PCA, amarker protein is split into two inactive fragments in such a waythat they cannot reassemble by themselves. But if these fragmentsare fused to two proteins that interact, this binding forces the two

fragments of the marker protein to come in close contact, reassem-bling a functional protein. PCA has several important advantagesover competing techniques. First, for the assay to work, theassembly of the functional marker-protein should depend onadditional interactions, which in PCA are provided by the twointeracting proteins. As a result, this assay is very specific anddisplays low background. Second, the marker protein reassemblyproceeds in an all-or-none manner, which results in signal develop-ment within a narrow set of conditions and with a great dynamicrange13. Recently, protein complementation was applied to thestudy of RNA-protein interactions in vivo14. In this approach, theRNA binding protein of interest is fused to a portion of a splitfluorescent protein, and the second protein fusion consists ofanother fragment of a split fluorescent protein and the MS2 coatprotein. The target RNA contains the MS2 binding motif, whichtethers one fragment of the fluorescent protein to the RNA via theMS2 protein-motif interaction. Protein complementation occurswhen an RNA-binding protein fused with the complementaryfluorescent protein fragment interact with a specific sequenceadjacent to the MS2 binding motif14. Fluorescent signal in thiscase is indicative of the interaction between the protein of interestand a specific sequence on the target RNA.

In the present study, we elaborated on this concept to make itapplicable for the localization and visualization of any RNA in livebacterial cells. In our design, a split fluorescent protein approach iscombined with high-affinity binding of a protein to an RNAaptamer. The RNA-binding protein we used was eIF4A, whichconsists of two globular domains. We split this protein into twofragments (the two domains) and separately fused each of these twofragments with two inactive fragments of a marker protein, EGFP.Simultaneous binding of the eIF4A fragments to an RNA aptameron a target RNA brings the two inactive fragments of EGFP in closeproximity, and results in reconstitution of fluorescence and detect-ion of the RNA of interest (Fig. 1).

Using quantitative time-lapse fluorescence microscopy, weobserved focal points of RNA concentration as well as changes influorescence over time and space in single bacteria. The fluctuationsin fluorescence observed with this labeling technique are reminis-cent of similar phenomena reported by others in Dictyostelium sp.

RECEIVED 25 AUGUST 2006; ACCEPTED 1 FEBRUARY 2007; PUBLISHED ONLINE 1 APRIL 2007; DOI:10.1038/NMETH1023

1Center for Advanced Biotechnology and Department of Biomedical Engineering, Boston University, 36 Cummington St., Boston, Massachusetts 02215, USA. 2Programof Molecular and Cellular Biology and Biochemistry, Boston University, 5 Cummington St., Massachusetts 02215, USA. 3Sequenom, Inc., 3595 John Hopkins Court,San Diego, California 92121, USA. Correspondence should be addressed to N.E.B. (nebroude@bu.edu).

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cells6. Thus, our results in E. coli also suggest complex organizationand regulation of transcription in a cell or cell populations thatwould merit further investigation. Finally, by testing our system ondifferent RNA species, an untranslatable RNA, 5S rRNA and anmRNA, we observed different cellular distributions of fluorescencedepending on the RNA target. This suggests that our detectiontechnique has the potential to detect and accurately localize anytranscript in a live cell.

RESULTSRNA detection schemeOur strategy for fluorescent labeling of RNA in vivo is shown inFigure 1. The probe interacting with the target RNA is eIF4A, asmall 29 kDa protein that acts as an ATP-dependent RNA helicaseworking in a complex with other initiation factors (4B, 4H, 4G and4F)15. We chose this RNA-binding protein because it consists of twoglobular domains, and its crystal structure resembles a dumbbell:compact N-terminal and C-terminal domains are connected via aflexible linker consisting of 11 amino acids16. These structuralfeatures make eIF4A a useful tool for domain dissection. Further-more, recent studies reported the isolation of several high-affinityaptamers that bind eIF4A with a binding constant in the nanomolarrange17. Others also showed that an aptamer sequence as short as58 nt could still preserve high-affinity binding17. Further, theseauthors showed using a nitrocellulose filter assay that neither of thedissected eIF4A domains can bind the aptamer alone but rather,

both domains of eIF4A need to be present for tight RNA bindingto occur17. Finally, the affinity of the eIF4A-aptamer interaction isin the same range as that of the MS2 coat protein–MS2 RNAinteraction used to monitor RNA within the cell6–8. Thus, itwas reasonable to think that eIF4A-aptamer interactions wouldsupport protein complementation in vivo. We selected EGFPas the marker protein because of the successful application ofsplit EGFP in studies aimed at finding putative protein-proteininteractions in vivo18–20.

RNA with aptamer can be detected in bacteriaWe made constructs for the coexpression of protein fusions andaptamer-containing RNA transcripts in E. coli BL21(DE3) cells(Fig. 2). We created a gene encoding a fusion of the C terminus ofthe EGFP fragment (residues 1–158) to the N terminus of the eIF4Afragment (residues 1–215) via a flexible polypeptide linker consist-ing of serine and glycine residues. We cloned this gene into the firstmultiple cloning site of vector pACYCDuet-1 (Novagen) designedfor the expression of two open reading frames from two T7promoters. Similarly, we created a gene encoding a fusion of theC terminus of the EGFP fragment (residues 159–238) to theN terminus of the eIF4A fragment (residues 216–406) via a flexiblepolypeptide linker, and cloned it in the second multiple cloning siteof the vector pACYCDuet-1 to create a construct yielding expres-sion of the two fusion proteins in approximately equimolaramounts. We used vector pETDuet-1 (Novagen) for the expressionof a 360-nt T7 transcript containing two copies of the eIF4A-interacting aptamer sequence in tandem. This small messageconsisted of a 33-nt leader sequence followed by two copies ofaptamer sequence and about 200 nt of the nuclease-resistant T7termination sequence.

We grew E. coli cells expressing the entire complementationcomplex and appropriate controls at room temperature (20–25 1C)in the presence of the inducer, isopropyl-b-D-thiogalactopyrano-side (IPTG) for coexpression of proteins and RNA. These growthconditions resulted in the best signal-to-background ratio. Whencultures reached an optical density of approximately 0.5 (OD600 ¼0.5), we analyzed them by flow cytometry (Fig. 2). Coexpression ofthe complementary fusion proteins along with the aptamer-con-taining RNA transcript resulted in a 10–20-fold increase in averagefluorescence (Fig. 2c). In the absence of aptamer-containingtranscript, however, E. coli cells bearing the complementary fusionproteins did not display fluorescence above background (Fig. 2a).Also, we did not observe a notable increase in fluorescence in cellsexpressing the protein components of the complementing complexalong with an untagged transcript (Supplementary Fig. 1 online).There was no difference in fluorescence yield when the T7 tran-script had or lacked a ribosome binding site, suggesting thattranslation of a message does not interfere with its detection.

Quantitation of RNA molecules in a bacterial cellFluorescence of cells expressing intact EGFP was about 30–50-foldhigher than that of cells with the complementation system(Fig. 2b,c). This difference is not surprising as there shouldbe a greater amount of full-length EGFP compared to thereassembled EGFP, whose concentration is determined by RNAconcentration. It is known that each RNA molecule is recycled byribosomes several times, which results in a molar ratio of protein toRNA larger than one.

A

F1

RNA aptamer

F2

B

Figure 1 | Design of RNA aptamer-based fluorescent protein complemen-

tation. Eukaryotic initiation factor eIF4A is split into two fragments, F1 and

F2, and fused with two fragments of a split EGFP (A and B). In the presence

of an RNA aptamer, the two fragments of eIF4A reassemble and bring together

two fragments of split EGFP. Reassembly of EGFP results in appearance of

intense fluorescence. The eIF4A ribbon structure was taken form reference 17.

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We next made use of EGFP calibration beads (Clontech) toevaluate the absolute average number of the reassembled EGFPmolecules per cell and thus the efficiency of our detection system.We found that cells expressing RNA from a pET vector with a copynumber of about 40, produced 500–600 molecules of reassembledEGFP, which corresponds to 1–2 mM RNA, if the cell volume is1.41 � 10�15 l (ref. 21). This RNA concentration correlates wellwith results obtained in E. coli from a vector with 50–70 copies/cell21. The concordance between expected and experimental resultssuggests that most molecules of aptamer-containing RNA aredetected by the reassembled protein complex. We tested thisassumption by increasing the copy number of the plasmid bearingthe RNA aptamer (from 40 to 100 copies/cell) and observed thatthese bacterial cells displayed higher fluorescence when comparedto the original construct (Supplementary Fig. 2 online). This resultsupports our conclusion that RNA molecules are detectedefficiently and quantitatively in our complementation system.

We then compared the fluorescence spectra of cells bearing thereassembled EGFP with those of cells expressing the full-lengthprotein. We found a maximum excitation at 470 nm for thereassembled complex (versus 490 nm for native EGFP), and amaximum emission about 520 nm (versus 508 nm for the nativeEGFP) (Supplementary Fig. 3 online). This difference can beexplained by possible changes in protein conformation betweenthe native EGFP and the reassembled EGFP-RNA complex. This

result also supports the notionthat fluorescent signal within the cell isdue to the formation of a nucleoproteincomplex. This is in agreement with ourin vitro experiments, in which we alsofound a similar red-shifted emissionspectrum (lmax ¼ 524 nm) for splitEGFP reassembled by appended comple-mentary oligonucleotides22.

Kinetics of fluorescence in single cellsWe used an epifluorescence microscope anda digital camera to observe the cells expres-sing the RNA labeling system (Figs. 2cand 3). In parallel, we recorded differentialinterference contrast (DIC) images to ana-lyze cell numbers and shapes. To allowenough time for complex formation andfluorescence development, we grew cellsovernight at room temperature. Coexpres-sion of the protein fusions and RNA tran-script containing aptamer resulted influorescent cells with bright fluorescentspots located at one or both poles of thecell (Figs. 2c and 3a). In contrast, expres-sion of the full-size EGFP producedstrongly fluorescent cells with a uniformdistribution of fluorescence throughoutthe cell (Fig. 2b). At the same time, expres-sion of protein fusions in the absenceof the aptamer expression did not lead tonotable fluorescence development (Fig. 2a),which confirms our previous flow cytome-try results.

Time-lapse microscopy revealed several remarkable features ofthe fluorescent particles as well as changes in total fluorescence ofthe cells. During a time-course experiment (Fig. 3a), cells in thesame field showed changes in fluorescence distribution over time(Fig. 3b). Total fluorescence in each cell gradually dropped duringthe first 2 h but later increased again. A decrease in fluorescenceresulted in the appearance of high-fluorescence particles at the cellpoles (Fig. 3c). This was followed by an increase in fluorescence anda more even distribution of fluorescence along the cell. Notably,several cells became fluorescent during the course of the experiment(Fig. 3a). The kinetics of these changes varied from experiment toexperiment, but the overall pattern of increase and decrease offluorescence over time and the synchronization of changes inindividual cells was very similar in six independent experiments(Supplementary Fig. 4 online).

Next we wanted to know whether the changes observed(Fig. 3a,b and Supplementary Fig. 4), corresponded with realfluctuations in RNA concentration instead of being an artifact ofthe labeling technique. To examine this, we extracted total RNAfrom cell culture at different time points and used real competitivePCR (rcPCR) to determine the absolute concentration of the targetRNA relative to the expression of a housekeeping gene, mreB, usedfor normalization.

rcPCR uses a serially diluted synthetic DNA competitor added tothe sample at known concentrations to act as a standard23. We

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Figure 2 | Protein complementation of split EGFP detects RNA in live bacterial cells. (a) Expression of two

protein fusions each containing fragment of a split eIF4A and a split EGFP does not result in a fluorescent

signal. (b) Expression of full-length EGFP results in uniformly fluorescent E. coli. (c) Coexpression of two

protein fusions and the RNA transcript with aptamer results in a fluorescent signal often localized to the

cell poles. Molecular constructs expressed in E. coli from the indicated plasmids expressing components of

the complementation complex: pMB33 expresses two protein fusions, pMB38 expresses full-length EGFP,

pMB23 expresses eIF4A-specific aptamer within an untranslatable message. The graphs show fluorescence

distributions of cells expressing EGFP complementing complexes obtained by flow cytometry. The images

at the bottom are fluorescence micrographs (left) and DIC images (right) of E. coli expressing

corresponding components of the complementing complexes. Scale bars, 2 mm.

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Figure 3 | Fluorescence changes within a single bacterium. (a) Time-lapse

fluorescence micrographs of E. coli expressing RNA aptamer–eIF4A

complementing complex at 30-min intervals. Eight cells out of 21 cells in the

field are shown. The arrows mark cells that became fluorescent in the course

of the experiment. Phase-contrast micrographs did not change during the

experiment. Scale bar, 2 mm. (b) Fluorescence changes in different randomly

chosen cells. (c) Changes in fluorescence distributions in real-time in a single

bacterial cell measured along the long axis of the cell. Scale bar, 2 mm.

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designed a competitor that contains only a single-base differencefrom the gene of interest for both mreB and the aptamer-containingmessage. Each PCR tube contained four targets that were amplifiedin parallel: mreB gene (internal control), aptamer-containing geneand two competitors of known concentrations (external controls).This assay design has been shown to result in robust performancemitigating PCR fluctuations and allowing detection of relativelysmall changes in RNA concentrations23. We detected PCR productsby exploiting a molecular-weight difference between extensionproducts using matrix-assisted laser desorption/ionization–timeof flight mass spectrometry (MALDI-TOF MS). We quantifiedthe genes by generating a least-squares regression line from the plotof the known log10 competitor concentrations against the log10

ratio of the gene to the competitor signal. Interpolation of theregression line at the equivalence point, when the competitor signalequals the gene signal, gives the concentration of the gene of interestin the sample assayed.

Analysis of the aptamer mRNA levels using software TitrationAnalyzer for interpolating RNA/DNA concentration, TITAN24,

shows statistically significant changes in aptamer-containingRNA concentration (Fig. 4 and Table 1), in contrast withthat for the control gene, mreB, for which the concentra-tion remained constant over time. Thus, analysis of RNAconcentration in a cell population confirms the changes in fluor-escence seen at a single-cell level detected by our RNAimaging technique.

Localization of a reporter mRNA and 5S rRNA in E. coliTo test our RNA detection system on other target RNAs, we taggeda reporter gene (LacZ) and the 5S rRNA at the 3¢ end with theeIF4A aptamer sequence. We cloned the coding sequencefor b-galactosidase and the partial rrnB operon from plasmidpKK5-1 (ref. 25) in pET-Duet vector and then coexpressed themalong with the protein fusions in BL21(DE3) cells. The fluorescencesignal in both cases was analyzed by flow cytometry and byfluorescence microscopy (Fig. 5).

Figure 4 | Changes in RNA concentration as determined by MALDI TOF MS.

Interpolated values for the aptamer and mreB RNA concentrations at each

time point were determined by a linear regression model. Each time point

represents three different competitor concentrations replicated four times

resulting in a total of twelve separate PCRs. This analysis was repeated twice

from a single RNA isolation from one cell culture for a total of n ¼ 24 PCRs.

Time points marked with an open circle are statistically different (P ¼ 0.05)

from the aptamer concentration at zero time point. None of the mreB time

points were statistically different from the zero time point. The zero time

point corresponds to the start of the experiment. The cells were induced by

IPTG overnight, and the samples were then collected for RNA isolation at

indicated times.

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Cells expressing the tagged LacZ mRNA showed a fourfoldincrease in cell fluorescence as compared with cells expressinguntagged RNA (Fig. 5a). Unlike our previously tagged unstrans-lated message, the LacZ mRNA signal was more evenly distributedalong the bacterial cell as expected for a nonlocalized mRNA(Fig. 5b). Time-lapse fluorescence microscopy also revealed oscil-lations in fluorescent signal that were very similar to the changesobserved before for a short untranslated message (Fig. 5c).

We also wanted to use our RNA detection technique to assay thelocalization of 5S rRNA in E. coli. For this, we used a partial rrnBoperon that contains the sequence for 5S rRNA. Expression of thetagged 5S rRNA showed a twofold increase in cell fluorescence ascompared with cells expressing untagged 5S rRNA. In this case, weobserved a remarkable array of fluorescent particles distributedalong the cell or at the cell poles (Fig. 5d) in stark contrast with

control cells expressing the untagged message. These results sup-port a specific localization of ribosomes in E. coli that agrees withstudies of localization of ribosomal proteins in Bacillus subtilis26,27.

DISCUSSIONThere are several lines of evidence that the fluorescence changes in asingle bacterial cell reported here accurately reflect changes in targetRNA concentration. First, the generation of fluorescence is com-pletely dependent on target RNA expression (Fig. 2). Second, anincrease in RNA concentration leads to an increase in total cellfluorescence (Supplementary Fig. 2). Finally, a direct estimation ofRNA concentration over time agrees with the observed pattern offluorescence variations (Fig. 4 and Table 1). As with previousprotein complementation assays19,20, however, we cannot rule outthe possibility that fluorescent protein complexes may persist in thecell even after the disappearance of the interacting RNA substrate.This will create some background fluorescence that needs to beaccounted for if this technique is to be used in RNA dynamicstudies. The use of an inducible promoter for RNA expressionwould help to make this method more sensitive for monitoringgene activity in vivo.

Time-lapse microscopy revealed changes of fluorescence in cellsexpressing both an untranslated RNA and an mRNA. Thesevariations in RNA levels were not related to cell division, as mostof the cells were not dividing during the course of the experiment.These changes do not appear to be an artifact of cell exposure to UVlight as we did not observe such changes in cells expressing full-sizeEGFP (data not shown).

The nature of fluorescent particles, which are visible at the cellpoles when the untranslated transcript is expressed and concentra-tion of RNA is decreasing, is unclear. The group who studieduntranslated RNA in E. coli using MS2-based technique alsoreported fluorescent particles at the cell poles7. These authors alsosaw particles in the middle of the cells, which are expected if thespots are sites of replication of plasmid origins7. Our results arenot consistent with the dynamics of plasmid replication and the

Table 1 | Aptamer and mreB mRNA transcript levels

Time (min)

mreB Aptamer

Equivalence point

(M x 1014) R2

Equivalence point

(M x 1011) R2

0 2.67 0.995 4.52 0.965

60 2.45 0.990 5.49 0.985

120 2.57 0.992 4.76 0.987

150 2.42 0.960 4.47 0.986

180 2.54 0.984 5.33 0.992

210 2.69 0.983 3.73 0.993

240 2.06 0.983 3.51 0.993

270 2.48 0.978 4.14 0.994

Concentration values for each time point are interpolated values from a linear regression line atthe equivalence point between the competitor and the gene of interest. TITAN24, a TitrationAnalyzer for interpolating RNA/DNA concentrations from mass spectrometry data, providedstatistics for the data analysis using a least-squares regression with linear terms and backwardsselection by Wald Test (P ¼ 0.05). All analyzed data were o0.05 for residuals and lack-of-fitvalues; these values indicate variability between replicate reactions and variability betweendata groups from the regression line, respectively. R2 values are for each gene and time pointand indicate variability that can be explained by the regression line.

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Figure 5 | Localization of LacZ mRNA and 5S rRNA in live bacterial cell. (a) Coexpression of LacZ tagged with aptamer and two protein fusions results in a

fluorescent signal (red) exceeding control (LacZ without aptamer) about tenfold (green). Black, uninduced sample. (b) Fluorescence (left) and DIC (right) images

of E. coli cells expressing LacZ mRNA tagged with aptamer. (c) Total fluorescence changes in cells expressing LacZ tagged with aptamer. Total fluorescence of all

cells in the field (n ¼ 16) for each time point was calculated three times with slightly different background threshold, and the result was averaged. Error bars,

s.d. (d) Fluorescence (left) and DIC (right) images of representative E. coli expressing 5S rRNA tagged with aptamer. Scale bars, 2 mm.

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tethering of RNA to the template DNA. Rather, we see a stablelocalization of the particles at the cell poles. Thus, we think theseparticles can be better explained as sites for RNA storage or RNAprocessing in bacteria28. Further studies will be needed to testthis hypothesis.

Finally, our results show for the first time different localizationof fluorescent signal in a bacterial cell depending on the nature ofthe transcript. When we compared the localization patternobserved for a short untranslated transcript, an mRNA and 5SrRNA, we observed distinct patterns of fluorescence. For instance,visualization of the 5S rRNA in E. coli cells resembles patterns oflocalization for the ribosomal proteins L1 and S2 in B. subtilis26,27.Also, the diffuse cell fluorescence observed for the LacZ mRNA isconsistent with the distribution of a nonlocalized, nonintegralmessage in the cell. Finally, the accumulation of fluorescenceat the cell poles was the distinct feature in the case of theunstranslatable message7. We think this phenomenon couldbe a reflection of a mechanism of storage of abundant, unusedRNAs in bacteria28.

In conclusion, the technology based on fluorescent proteincomplementation, presents a robust tool for RNA tracking invivo. Its major advantage is lower background signal compared tothe one generated by the use of full-length EGFP6–8 (comparefluorescence of the cells expressing full-size EGFP and our completefluorescent complex; Fig. 2). Additionally, a relatively small proteincomplex is assembled on the target RNA, which apparentlydoes not interfere with RNA function and localization. Single-cellRNA profiling based on protein complementation should findvaluable applications in different types of cells and in a widerange of studies related to transcription, translation, RNA proces-sing and network modeling.

METHODSGrowth conditions and induction. We cotransformed BL21(DE3)cells with pMB33 (expressing the protein chimeras) and pMB23,pMB84 or pMB99 (expressing different target RNAs, see Supple-mentary Methods online). We incubated the cells with shakingfirst at 37 1C in LB medium supplemented with antibiotics for3–4 h. Then, we diluted the cultures into fresh medium containing1 mM IPTG and allowed growth overnight at room temperature(20–25 1C). The OD600 of the cultures was between 0.4 and 0.6 atthe time of examination.

Flow cytometry. To measure fluorescence of a cell population,we used a Becton-Dickinson FACSCalibur flow cytometer with a488-nm argon excitation laser and a 515–545 nm emission filter(FL1). We washed cells once with 1� PBS before assaying. Wemeasured fluorescence of 100,000 cells in each sample.

Microscopy analysis. We immobilized bacterial cells in culturebetween a cover slip and a thin slab of 0.8% agarose in 1� PBS.We next analyzed cells at room temperature using a Nikon Eclipse80i inverted microscope equipped with an epifluorescence systemX-Cite 120 and a Nikon Cool Snap, HQ black and white digitalcamera (12 bit, 20 mHz).

Additional methods. A description of DNA constructs andbacterial strains used in this study, details of imaging acquisitionand analysis, and full details of real competitive PCR and MALDITOF MS are available in Supplementary Methods.

Note: Supplementary information is available on the Nature Methods website.

ACKNOWLEDGMENTSWe thank A. Randhawa for technical assistance, C. Witte-Hoffmann for suggestingthe use of eIF4A, C. Proud (University of Dundee, UK) for the eIF4A clone, P. Moore(Yale University) for pKK5-1 clone, S. Mazzei (Cleveland Clinic Foundation) forBD EGFP beads, V. Demidov for measuring cell fluorescence spectra and constantsupport, A. Gershteyn for help with preparation of figures, I. Keren and G. Balaszifor discussions and support. We thank J.J. Collins for reading the manuscript andfor valuable suggestions, and all members of the Center for AdvancedBiotechnology for help, insightful discussions and suggestions. This work wassponsored by Hamilton Thorne Biosciences and supported in part by a Charles E.Culpeper Biomedical Pilot Grant from Goldman Philanthropic Partnership to N.E.B.

AUTHOR CONTRIBUTIONSM.V.-B. performed cloning, flow cytometry and microscopy analysis, R.M. performedrcPCR and MALDI TOF analysis, C.R.C. and N.E.B. were responsible for projectplanning. N.E.B. drafted the paper. M.V.-B., R. M., C.R.C. and N.E.B. discussed theresults and extensively revised the manuscript.

COMPETING INTERESTS STATEMENTThe authors declare no competing financial interests.

Published online at http://www.nature.com/naturemethods/Reprints and permissions information is available online athttp://npg.nature.com/reprintsandpermissions

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