nihms122794.pdf

Inorganic-Organic Hybrid Luminescent Binary Probe for DNA

Detection Based on Spin-Forbidden Resonance Energy Transfer

Angel A. Marti

†

Cindy A. Puckett

‡

Joanne Dyer

†

Nathan Stevens

†

Steffen Jockusch

†

Jingyue Ju

||,

⊥

Jacqueline K. Barton

‡

, and

Nicholas J. Turro

†,

⊥

†

Department of Chemistry, Columbia University, New York, NY 10027

⊥

Department of Chemical Engineering, Columbia University, New York, NY 10027

‡

Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena,

California 91125

Columbia Genome Center, Columbia University College of Physicians and Surgeons, New York,

NY 10032

Abstract

We describe the design of new fluorescent binary probe sensors for DNA detection based on spin-

forbidden resonance energy transfer (SF-RET). Binary probes consist of a donor and acceptor

fluorophores that are attached to two different oligonucleotides and serve as resonance energy transfer

(RET) donor-acceptor pair when hybridized to adjacent sites of a target sequence. In the absence of

target, excitation of the donor results in fluorescence only from the donor, but when the probes

hybridize to target, the fluorophores are brought into close proximity favoring RET, yielding

fluorescence mainly from the acceptor fluorophore. These new binary probes use the metal complex

Ru(bpy

′

)(DIP)

as the energy donor and an organic fluorophore (Cy5) as the energy acceptor.

Energy transfer from the MLCT state of the Ru complex to singlet Cy5 is spin forbidden and produces

a delayed fluorescence of Cy5. This paper demonstrate that fluorescence delay of Cy5 can be used

to time resolve the emission of the probe from the intense fluorescence background of a model system

for cellular background; this provides the reported system to overcome intense autofluorescence, an

important and general advantage over “classical” spin-allowed steady-state probes.

The design of new fluorescent sensors for DNA has applications in following transcription,

developing sensitive diagnostics,

and monitoring biological processes in the cell.

Among the

most effective sensors for DNA detection are binary probes,

which have donor and acceptor

fluorophores that are attached to two different oligonucleotides and serve as a resonance energy

transfer (RET) donor-acceptor pair when hybridized to adjacent sites of a target sequence.

the absence of target, excitation of the donor results in fluorescence only from the donor, but

when the probes hybridize to target, the fluorophores are brought into close proximity favoring

RET, yielding fluorescence mainly from the acceptor fluorophore. Thus the selective

hybridization of the probes to a target produces a unique fluorescence signal that can be used

for detecting target DNA

in vitro

and

in vivo

Binary probes show target specificity, high

sensitivity and reliability and avoid false positive signals.

Nonetheless, the sensitive detection

of DNA in living cells is often obscured by a high autofluorescence background.

In this report,

we describe the design of new binary probes based upon

spin-forbidden resonance energy

njt3@columbia.edu.

Supporting Information Available: Experimental procedures, steady-state and lifetime data and determination of optimum time window

for TRE spectra. This material is available free of charge via the Internet at http://pubs.acs.org.

NIH Public Access

Author Manuscript

J Am Chem Soc

. Author manuscript; available in PMC 2009 September 21.

Published in final edited form as:

J Am Chem Soc

. 2007 July 18; 129(28): 8680–8681. doi:10.1021/ja0717257.

NIH-PA Author Manuscript

transfer

(SF-RET). These probes allow sensitive fluorescence detection of DNA in highly

fluorescent cell media.

We earlier reported the utilization of long-lived pyrene binary probes that show monomer

emission (390 nm) when free in solution and excimer emission (480 nm) when bound to target.

The long fluorescence lifetime of these probes allowed the pyrene signal to be time-resolved

from short-lived emission that is typical of cellular autofluorescence. Notwithstanding the

inherent advantages of the pyrene probe, its UV excitation (350 nm) and its relatively short

lifetime in aqueous solution (~40 ns) challenged us to design probes with properties suited to

detect DNA targets in living cells. To meet this challenge, we have now utilized the metal

complex Ru(bpy

′

)(DIP)

as the energy donor and an organic fluorophore (Cy5) as the energy

acceptor (Figure 1).

A characteristic of ruthenium(II) polypyridyl complexes is their long lived luminescent metal

to ligand charge transfer (MLCT) excited states.

Energy transfer from the MLCT state to

singlet Cy5 is spin forbidden (SF-RET). Although SF-RET occurs at a slower rate, the very

long emission lifetime of the Ru-complex (1.8

s) easily compensates, leading to high RET

efficiency.

Oligonucleotide probes were synthesized to examine SF-RET. Ru(bpy

′

)(DIP)

tethered to

the 5

′

-end was used as the energy donor and oligonucleotides containing 3

′

-tethered Cy5 were

used as acceptors; three of these acceptor oligonucleotides were synthesized with variable

sequences to produce different distances between the Ru and Cy5 probes when hybridized to

target.

Figure 2 shows the luminescence spectra of one probe set with and without target by

steady-state luminescence and time resolved emission (TRE). When the probes are free in

solution, only emission from the ruthenium complex is observed (

Ex.

= 440 nm). In the

presence of target, Ru(bpy

′

)(DIP)

and Cy5 are brought into close proximity, a condition

favorable for RET, and mainly Cy5 emission is observed.

The steady-state luminescence spectra for all three oligonucleotide sets are similar (Supporting

Information). However, time-resolved lifetime measurements show important differences. The

lifetime of Cy5 is ca. 2 ns when free in solution, but seems to increase in the presence of target

with excitation of Ru. For the three sets, with increasing probe separation, the lifetimes are 45,

46, and 69 ns. The delayed fluorescence of Cy5 results from the slow SF-RET rate constant,

which decreases with longer distance. Figure 2b shows the TRE spectrum for one

oligonucleotide set.

The TRE spectrum is similar to its steady-state counterpart with a

slight improvement in the signal to background ratio (S/B).

This improvement is attributed

to the near depopulation of directly excited Cy5 on the time scale of the TRE measurement.

To mimic cellular autofluorescence, we tested the probes in Dulbecco’s cell medium, adding

the fluorophore Red-X to further intensify the medium’s fluorescence.

The steady-state

spectrum is dominated by the brightly fluorescent background and changes only modestly upon

the addition of target (Figure 2c). The S/B is reduced compared to the probes in buffer (Figure

2a) from 14 to 2.5. In comparison, the TRE spectrum (Figure 2d),

even in the presence of

strongly fluorescent cell medium

, shows a spectral profile that closely resembles that in buffer.

Integrating the emission signal from 59–77 ns allows measurement of a TRE spectrum after

most of the background fluorescence has decayed. Although some background remains, it is

dramatically reduced by TRE, with the S/B increasing from 2.5 to 10.

To further demonstrate the advantages of SF-RET probes over standard spin-allowed RET

(SA-RET) probes, we replaced the Ru complex with Alexa 488, a singlet energy donor (Figure

1).

Figure 3a shows that, as expected, the Alexa 488 fluorescence intensity decreases and the

Cy5 fluorescence intensity increases when the target is added as a result of RET. The TRE

spectrum successfully reproduces the fluorescence emission profiles in buffer, but fails in

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Dulbecco’s cell medium supplemented with RedX (Figure 3b, d). The background fluorescence

of the cell medium has a fluorescence lifetime similar to that of the probes (2 ns), making

impossible the discrimination of the RET signal from background using TRE.

Figure 4 shows the direct comparison for the two systems (SF versus SA-RET). As is evident,

the longer time scale for SF-RET permits sufficient time for background autofluorescence to

decay.

In summary, we have designed, synthesized and successfully demonstrated the utility of SF-

RET to target DNA using a hybrid inorganic-organic probe set with a ruthenium complex as

donor and Cy5 as acceptor. With TRE, the delayed fluorescence of the Cy5 acceptor due to

SF-RET can be employed to distinguish target signal, from intense, but short-lived background

emission such as cell autofluorescence. Additionally, Ru complexes possess tunable absorption

and emission profiles, by using different ligands, an advantage for use with fluorescence

microscopes. SF-RET may be useful for the detection of DNA and RNA in highly fluorescent

media

in vitro

and

in vivo

. Further studies are under progress to evaluate the effectiveness of

these probes inside living cells.

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

Acknowledgments

This work was supported by the Center of Excellence in Genomic Science Grant P50 HG002806 from the NIH and

by the NSF under grant NSF CHE-04-15516. J.K.B. thanks the NIH (GM33309) for financial support.

References

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SV, Ju J, Turro NJ. Nucleic Acids Res 2006;34:3161–3168. [PubMed: 16769776]

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New York: 1992. p. 106-164.

10. Turro, NJ. Modern molecular photochemistry. University science books; Sausalito, CA: 1991. p.

338-339.

11. Ru-probe/Alexa-probe used here: 5

′

-AAG TTG ATC AAG TTG GT-Ru (or Alexa488)-3

′

; Cy5-

Probe-3: 5

′

-Cy5-TTC ACT GGA TGA-3

′

. Other Cy5 probe sequences are in Supporting Information.

For synthesis see Holmlin RE, Dandliker PJ, Barton JK. Bioconjugate Chem 1999;10:1122–

1130.1130 Details and characterization are in Supporting Information.

12. The procedure to determine the optimum integration time window is described in Supporting

Information.

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13. Cy5-Probe-3 was selected for TRE since it showed the greatest degree of delayed Cy5 fluorescence.

14. S/B = (Em

667

with target/Em

667

without target)/(Em

610

with target/Em

610

without target)

15. Yang CJ, Jockusch S, Vincens M, Turro NJ, Tan W. Proc Natl Acad Sci U S A 2005;102:17278–

17283. [PubMed: 16301535]

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Figure 1.

(a) Spin-forbidden and (b) spin-allowed binary probes.

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Figure 2.

(a) Steady-state luminescence spectra and (b) TRE (59–77 ns after the excitation pulse) of Ru-

probe and Cy5-probe in buffer with (

) and without (

) target. (c) Steady-state luminescence

spectra and (d) TRE (59–77 ns after the excitation pulse) of Ru-probe and Cy5-probe in RedX-

containing cell medium with (

) and without (

) target.

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Figure 3.

(a) Steady-state luminescence spectra and (b) TRE (1.0–6.2 ns after the excitation pulse) of

Alexa 488-probe and Cy5-probe in buffer with (

) and without (

) target. (c) Steady-state

luminescence spectra and (d) TRES (1.0–6.2 ns after the excitation pulse) of Alexa 488-probe

and Cy5-probe in RedX-containing cell medium with (

) and without (

) target.

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Figure 4.

Fluorescence decay traces of the spin-allowed (

), spin-forbidden (

) binary probes, and

medium (

) showing the optima integration time windows.

Marti et al.

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