This journal is©The Royal Society of Chemistry 2014 Chem. Commun., 2014, 50, 13735--13738 | 13735

Cite this:Chem. Commun., 2014,50, 13735

Halo-tag mediated self-labeling of fluorescentproteins to molecular beacons for nucleic aciddetection†

Daniel Blackstock and Wilfred Chen*

We report here the generation of a fluorescent protein (FP)-based

dual molecular beacon (MB) system for nucleic acid detection.

Halo-tag mediated conjugation was used for the site-specific

decoration of MBs with two different FP fusions, thereby enabling

easy detection of target sequences by fluorescence resonance

energy transfer or FRET. Enhanced intracellular delivery was

demonstrated by simply tethering a well-known TAT peptide

sequence to the N-terminus of the fusion proteins.

Green fluorescent protein (GFP) and its variants are widely used asin vitro and in vivo biosensors, and a variety of biological targetssuch as proteases, kinases, and Ca2+ have been successfullydetected.1–3 Although direct coupling of GFP variants to DNAhas been demonstrated,4,5 their use for biosensing remainsrelatively unexplored. Purification of DNA–GFP conjugates is oftentedious and expensive as a combination of ion-exchange and gel-filtration chromatography is required.4,5 To realize the use of GFPvariants for broader applications in DNA-based biosensing, amethod that provides site-specific decoration of DNA scaffoldswhile allowing simple purification is highly desirable.

One of the most intriguing DNA scaffolds for biosensing in thelast 20 years is based on molecular beacons (MBs). These arefluorescently labeled, single-stranded stem-loop DNA probes widelyused for sensitive and specific detection of nucleic acids.6 MBs havebeen used extensively for monitoring in vivo gene expression,7–9 aswell as specific interactions between proteins and nucleic acids.10

Functionalization of MBs with FPs is particularly desirable becausethis allows coupling of a variety of FP variants for multiplex sensing,higher photo-stability, and the ability to add on other proteinfunctionalities through simple genetic modifications. Our grouphas recently reported the use of FP–MBs for DNA sensing based onzinc finger protein (ZFP) guided assembly.11 However, this firstgeneration design lacks the required sensitivity due to the relative

low binding affinity of ZFPs. One promising candidate to addressthis problem is the self-labeling Halo tag based on a mutanthaloalkane dehalogenase, which forms a covalent bond to a suicidechlorohexane (CH) ligand.12 By genetically tethering the Halo tag toFPs, CH-modified DNA oligonucleotides can be site-specificallyconjugated onto different FP variants for biosensing applications.To simplify purification, an elastin like polypeptide (ELP) tag13 canbe incorporated to the N-terminus of the Halo–FP fusions to enableeasy recovery of fusion proteins and their conjugated forms by twocycles of precipitation and solubilization.

Our sensing scheme is based on two different MBs that aresite-specifically decorated with either CFP or YFP using the Halotag self-labeling method. CFP and YFP were chosen due to theirextensive success in detecting spatial variations by FRET, resis-tance to photobleaching, and their soluble expression in E. coli.14

By co-localizing the FP-modified MBs onto adjacent regions onthe same target, specific nucleic acid detection can be achieved viafluorescence resonance energy transfer (FRET) (Fig. 1). Two MBs

Fig. 1 A schematic representation of the dual FP–MB design and themechanism of action. The FP–MBs yield no FRET signal until the targetsequence is presented. The FP–MBs bind to the target and force the twoFPs into close proximity yielding a FRET response.

Department of Chemical and Biomolecular Engineering, University of Delaware,

Newark, Delaware 19716, USA. E-mail: wilfred@udel.edu; Tel: +1 302 831 6327† Electronic supplementary information (ESI) available: Materials and methods.See DOI: 10.1039/c4cc07118b

Received 10th September 2014,Accepted 17th September 2014

DOI: 10.1039/c4cc07118b

www.rsc.org/chemcomm

ChemComm

COMMUNICATION

Publ

ishe

d on

18

Sept

embe

r 20

14. D

ownl

oade

d by

Uni

vers

ity o

f D

elaw

are

on 1

4/09

/201

7 16

:17:

59.

View Article OnlineView Journal | View Issue

http://crossmark.crossref.org/dialog/?doi=10.1039/c4cc07118b&domain=pdf&date_stamp=2014-09-23http://dx.doi.org/10.1039/c4cc07118bhttp://pubs.rsc.org/en/journals/journal/CChttp://pubs.rsc.org/en/journals/journal/CC?issueid=CC050089

13736 | Chem. Commun., 2014, 50, 13735--13738 This journal is©The Royal Society of Chemistry 2014

(a donor MB [dMB] and an acceptor MB [aMB], Fig. 1) weredesigned based on a previously reported dual K-ras MB systemtargeting a commonly mutated region of the K-ras gene involvedin many cancers.15 To incorporate the CH ligand, an amine groupwas incorporated to either the 50 or 30 end of the donor orthe acceptor K-ras MB, respectively. Highly efficient amine-N-hydroxysuccinimide (NHS) ester conjugation chemistry wasutilized to attach the amino-reactive NHS–CH ligand (Promega:P6751) to the donor and acceptor MBs. Complete derivatization ofMBs was achieved by coupling with 30 molar excess CH–NHS(Fig. S1, ESI†), and the unreacted ligands were removed using a3000 Da ultrafiltration column.

To attach the FPs to MBs, the Halo tag protein was geneti-cally tethered to the N-terminus of CFP and YFP such that bothFPs were fully exposed at the C-terminus. Production of full-length proteins was detected in cell lysates (Fig. S2, ESI†), andboth fusion proteins were easily purified by two cycles ofprecipitation and solubilization by utilizing the reversiblephase transition property of the N-terminus ELP tag (Fig. 2).Both fusion proteins retained their fluorescent properties asindicated by the bright fluorescence observed under UV light(Fig. S2, ESI†). Although the Halo tag was sandwiched betweenELP and FPs, purified MBs were easily conjugated to both Halofusions using a 3� molar excess MB (Fig. 2). Close to 100%conjugation was demonstrated by detecting a single, largerband corresponding to the protein–MB complex on SDS-PAGE.Formation of the protein–MB complex was further confirmed byobserving a higher mobility band using an electromobility shiftassay (EMSA) (Fig. 2). This very high labeling efficiency is notsurprising as it has been reported that the active site for CHconjugation is in the middle of the Halo tag and the potentialsteric effects on labeling is minimal.12 After labeling, excess MBwas removed in order to ensure no interference with thebiosensing assays. The thermally responsive nature of ELP wasagain exploited to purify the labeled proteins from the surplusMBs using one cycle of precipitation and solubilization. Allunbound MBs were removed while most of the protein–MBcomplexes was retained after purification (Fig. 2).

After confirming the formation of FP–MB conjugates,we next investigated the binding functionality. When theHalo–CFP:dMB and Halo–YFP:aMB conjugates were mixedtogether, only background FRET signal was detected (Fig. 3,black line). Upon the addition of 30 nM target (Fig. 3, blue line),however, a sharp decrease in the CFP emission peak at 480 nmand an increase in the YFP emission around 530 nm wasdetected, indicating a binding induced FRET response. Moreimportantly, the addition of a 10-fold higher concentration of anon-specific target resulted in no FRET change (Fig. 3, red line),confirming the highly specific nature of our MB design. Thesensitivity of the FP-modified dual MB system was furtherassessed by varying the target concentration from 0 to 300 nM.A typical S-shaped dose response curve was obtained with adetection limit of 5 nM (Fig. 4). This sensitivity is comparable tothe reported detection limit of traditional MBs,16 suggesting theincorporation of FPs has little or no inhibiting effect on targetrecognition and binding.

It has been reported that the gap size between the donor andacceptor fluorophores can significantly influence the FRETefficiency.17 A smaller gap size can result in interactions betweenfluorophores and ground-state quenching, while a larger gapsize reduces the energy-transfer efficiency. Since the FPs used

Fig. 2 SDS-PAGE and native PAGE analysis of FP labeling of MBs. PurifiedFPs without (�MB) or with (+MB) a 3-fold excess of the respective CH-modified MBs were analyzed. Reaction mixtures were purified by one cycleof precipitation and solubilization to remove the excess MB (pure).

Fig. 3 Fluorescence spectra of the dual FP–MB system with or without30 nM target or in the presence of 400 nM non-specific target.

Fig. 4 DNA target dose–response curve of the dual FP–MB system.

Communication ChemComm

Publ

ishe

d on

18

Sept

embe

r 20

14. D

ownl

oade

d by

Uni

vers

ity o

f D

elaw

are

on 1

4/09

/201

7 16

:17:

59.

View Article Online

http://dx.doi.org/10.1039/c4cc07118b

This journal is©The Royal Society of Chemistry 2014 Chem. Commun., 2014, 50, 13735--13738 | 13737

were significantly larger than the conventional organic fluoro-phores, a smaller gap size may also result in steric interferencebetween CFP and YFP fusions and less efficient binding. Tofurther characterize this new dual FP–MB system, we createdtarget sequences with a gap size ranging from 0 to 15 nucleotidesand investigated their effect on the resulting FRET efficiency. Asa reference, all prior experiments were carried out using a targetwith a 7-nucleotide gap size. For our system, a 4-nucleotidespacer proved to be the most effective in maximizing the FRETsignal (Fig. 5). As suspected, the 2-nucleotide gap size is probablytoo small to allow efficient dual binding of both FP–MBs,resulting in a lower FRET response. Considering the previousbinding experiments were carried out with the 7 nucleotidespacer target, it is likely that the sensitivity may actually beenhanced for targets with a smaller gap size.

Using the optimum spacing, the dual FP–MB system wasused to detect a RNA target containing a 4-nucleotide spacer.Even the presence of intermolecular hairpins had no effect onthe sensitivity as a similar detection limit was obtained as withDNA targets (Fig. 6). To ensure that the assay is equallyapplicable under harsh operating environments that reflect

in vivo conditions, a dose response curve was further obtainedby adding the probes and targets to HeLa cell lysates to mimicthe detection in a cell-like environment. Although the sensitivitywas slightly reduced (Fig. 6 inset), 10 nM RNA target was stilleasily detected. This result demonstrates that the presence ofcellular proteins and nucleic acids has little impact on the dualFP–MB system. Since the typical volume of mammalian cells isbetween 1000–25 000 mm3, the intracellular mRNA concentra-tions can be as low as 80 pM (1 copy in a 25 000 mm3 cell) or canexceed 2.2 mM (1000 copies in a 1000 mm3 cell).18 Based on thisinformation, our FP-based assay should be able to detect 4100copies per cell and further improvement to allow detection of10 copies per cell would necessitate the use of an improvedCFP–YFP pair with up to 10-fold enhancement in sensitivity.19

A key benefit of our protein-based MB assembly is the easeof attaching additional functionalities to the protein backbonewithout the use of conjugation chemistries. To demonstratethis feasibility, a well-known cell-penetrating TAT peptide20 wasadded to the N-terminus of ELP–Halo–YFP to enable efficientintracellular delivery of MBs as demonstrated previously.21

A monolayer of BGMK cells was incubated with TAT–FP–MBsfor 2 h and efficient delivery was clearly visible under afluorescence microscope (Fig. 7). We envision other targetingfunctionalities such as binding peptides or antibodies can besimilarly tethered to the FP–MBs, resulting in a highly modularand low-cost technology for targeted intracellular delivery.

To our knowledge, this is the first time that FPs have beencovalently linked to MBs and used to detect nucleic acids with asensitivity similar to traditional MBs. Beyond the detectioncapability, the results strongly indicate that our design is highlystable and effective in co-localizing two distinct proteins uponsensing a specific nucleic acid sequence. By simply replacingthe FP with any protein of interest, this protein–MB formatcould ultimately be applied for various applications in whichdistinct protein organization is necessary. Considering thetremendous success of FPs22 for in vivo applications, we believethis technology can be used for detecting mRNA expression. Wehave already provided this possibility by demonstrating thefunctionality in cellular lysate as well as the effective intra-cellular uptake of FP–MBs using the flanking TAT peptide.

We would like to acknowledge the funding support fromNSF (CBET1263774).

Fig. 5 Effects of nucleotide spacing between CFP and YFP on the FRETefficiency. The gap size between the two binding sites was varied asindicated. Experiments were carried out using 400 nM of each FP–MBand 100 nM of target.

Fig. 6 RNA target dose response. (Inset) RNA target binding response inHeLa cell lysates.

Fig. 7 Intracellular delivery of TAT–ELP–Halo–YFP–MB conjugates.BGMK cells were incubated with conjugates (750 nM) for 2 h before phaseand fluorescent images were captured.

ChemComm Communication

Publ

ishe

d on

18

Sept

embe

r 20

14. D

ownl

oade

d by

Uni

vers

ity o

f D

elaw

are

on 1

4/09

/201

7 16

:17:

59.

View Article Online

http://dx.doi.org/10.1039/c4cc07118b

13738 | Chem. Commun., 2014, 50, 13735--13738 This journal is©The Royal Society of Chemistry 2014

Notes and references1 B. A. Pollok and R. Heim, Trends Cell Biol., 1999, 9, 57–60.2 Q. Ni, D. V. Titov and J. Zhang, Methods, 2006, 40, 279–286.3 K. Truong, A. Sawano, H. Mizuno, H. Hama, K. I. Tong, T. K. Mal,

A. Miyawaki and M. Ikura, Nat. Struct. Biol., 2001, 8, 1069–1073.4 V. Lapienc, F. Kukolka, K. Kiko, A. Arndt and C. M. Niemeyer,

Bioconjugate Chem., 2010, 21, 921–927.5 C. B. Rosen, L. B. KodalAnne, J. S. Nielsen, D. H. Schaffert, C. Scavenius,

A. H. Okholm, N. V. Voigt, J. J. Enghild, J. Kjems, T. Tørring and K. V.Gothelf, Nat. Chem., 2014, 6, 804–809.

6 W. Tan, K. Wang and T. J. Drake, Curr. Opin. Chem. Biol., 2004, 8, 547–553.7 Y. Li, X. Zhou and D. Ye, Biochem. Biophys. Res. Commun., 2008, 373,

457–461.8 H.-Y. Yeh, M. V Yates, A. Mulchandani and W. Chen, Proc. Natl.

Acad. Sci. U. S. A., 2008, 105, 17522–17525.9 X.-H. Peng, Z.-H. Cao, J.-T. Xia, G. W. Carlson, M. M. Lewis,

W. C. Wood and L. Yang, Cancer Res., 2005, 65, 1909–1917.10 T. Heyduk and E. Heyduk, Nat. Biotechnol., 2002, 20, 171–176.

11 D. Blackstock, Q. Sun and W. Chen, Biotechnol. Bioeng., DOI:10.1002/bit.25441.

12 G. Los and L. Encell, ACS Chem. Biol., 2008, 3, 373–382.13 Q. Sun, B. Madan, S.-L. Tsai, M. P. DeLisa and W. Chen, Chem.

Commun., 2014, 50, 1423–1425.14 A. Miyawaki, Dev. Cell, 2003, 4, 295–305.15 P. J. Santangelo, B. Nix, A. Tsourkas and G. Bao, Nucleic Acids Res.,

2004, 32, e57.16 Y. Kim, D. Sohn and W. Tan, Int. J. Clin. Exp. Pathol., 2008, 1, 105–116.17 A. Hillisch, M. Lorenz and S. Diekmann, Curr. Opin. Struct. Biol.,

2001, 11, 201–207.18 C. Ragan, M. Zuker and M. A. Ragan, PLoS Comput. Biol., 2011, 7, 11.19 A. W. Nguyen and P. S. Daugherty, Nat. Biotechnol., 2005, 23, 355–360.20 H. Brooks, B. Lebleu and E. Vivès, Adv. Drug Delivery Rev., 2005, 57,

559–577.21 H. Y. Yeh, M. V. Yates, A. Mulchandani and W. Chen, Chem.

Commun., 2010, 46, 3914–3916.22 N. C. Shaner, P. A. Steinbach and R. Y. Tsien, Nat. Methods, 2005, 2,

905–909.

Communication ChemComm

Publ

ishe

d on

18

Sept

embe

r 20

14. D

ownl

oade

d by

Uni

vers

ity o

f D

elaw

are

on 1

4/09

/201

7 16

:17:

59.

View Article Online

http://dx.doi.org/10.1039/c4cc07118b