DsDNA-triggered energy transfer and lanthanide sensitization processes. Luminescent probing of specific A/T sequences

Article abstract of DOI:10.1039/b927305k

Orthogonal attachment of a DOTA[Ln³⁺] complex or a coumarin fluorophore to appropriately functionalized bis-4-aminobenzamidines yields compounds that experience A/T-selective, dsDNA-dependent energy transfer processes, and elicit long wavelength emission of light. The Royal Society of Chemistry 2010.

Full text of DOI:10.1039/b927305k

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dsDNA-triggered energy transfer and lanthanide sensitization processes.
Luminescent probing of specific A/T sequenceswz
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Olalla Vazquez, Mateo I. Sanchez, Jose L. Mascarenas* and M. Eugenio Vazquez*
Received 5th January 2010, Accepted 9th April 2010
First published as an Advance Article on the web 11th May 2010
DOI: 10.1039/b927305k
Orthogonal attachment of a DOTA[Ln3+] complex or a
coumarin fluorophore to appropriately functionalized bis-4-
aminobenzamidines yields compounds that experience A/T-selective,
dsDNA-dependent energy transfer processes, and elicit long
wavelength emission of light.
The study of DNA has unfolded over the past two decades
from the initial structural and functional characterization in
living organisms, to what is now known as DNA technology,
encompassing the development of DNA as a tool in biological
sciences, and more recently as a structural and nanotechno-
logical scaffold.1,2 However, the development of DNA-based
functional and dynamic processes, particularly those employing
double stranded DNA (dsDNA), is still in its infancy.3 In this
context, finding new ways of codifying physicochemical
processes with dsDNA,4 and in particular the development
of efficient methods for selective sensing of dsDNA
sequences,5 might provide new opportunities for research
and discovery at the interface of biomedical and chemical
sciences. Herein, we report the first examples of small
molecules displaying intramolecular energy transfer processes
induced by specific dsDNA sites, in our case A/T-rich
sequences. The designed probes emit long wavelength light,
and might therefore be relevant for future applications in
dsDNA sensing and imaging.
Scheme 2 Synthesis of amino derivative 3, and lanthanide conjugates
5a/5b (Ln = Eu+3, Tb3+).
This project arose from our recent observation that bis-4-
aminobenzamidines such as 1 or 2 (Scheme 1) exhibit a
remarkable fluorescence emission enhancement upon binding
to A/T-rich sites in dsDNA (lexc = 329 nm, lem = 387 nm).6,7
We envisaged that appropriate attachment of an acceptor
fluorophore to the parent benzamidine might allow the
implementation of dsDNA-dependent energy transfer
processes. We first focused on lanthanide ions as it is well
known that they can exhibit long-lived excited states and
narrow, long wavelength emission bands,8,9 and are therefore
very attractive for luminescent biosensing applications.10
Towards this end we designed the DOTA[Eu3+] and [Tb3+
]
conjugates 5a and 5b, which were assembled as detailed in
Scheme 2. Coupling of bis-benzamidine 3, which features an
alkoxyaminopropane handle, with the tri-tBu protected
DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid)
monoacid 4, followed by deprotection and complexation with
the appropriate lanthanide trichloride in buffer solution,
afforded the desired macrocyclic conjugates 5a/5b.
Scheme 1 Structure of fluorescent bis-aminobenzamidines 1 and 2.
As expected, irradiation at 329 nm of a 0.5 mM solution of
compounds 5a or 5b in buffer did not result in significant
emission from the metal ions. Likewise, addition of a hairpin
oligonucleotide featuring a non-target GGCCC site, or a
sequence featuring a short A/T tract (AAT) did not affect
the luminescence intensity of the lanthanide complexes
(see Fig. S1 in ESIz). However, addition of the dsDNA hairpin
containing the consensus AATTT sequence induced an
increase in the emission bands of lanthanide ions in both 5a
(Fig. 1, top), and 5b (Fig. 1, bottom). Despite the similar
´
´
´
Dept. de Quımica Organica, Facultade de Quımica, Universidade de
Santiago de Compostela, 15782 Santiago de Compostela, Spain.
E-mail: joseluis.mascarenas@usc.es, eugenio.vazquez@usc.es;
Fax: (+ 34) 981-595-012; Tel: (+34) 981-595-012, ext. 14346
w This work is dedicated to Prof. Luis Castedo on the occasion of his
70th birthday.
z Electronic supplementary information (ESI) available: Synthesis and
characterization, DNA binding, and detailed spectroscopic experi-
ments. See DOI: 10.1039/b927305k
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Scheme 3 Synthesis of the coumarin conjugate 6.
Fig. 2 Fluorescence emission of 1 (0.5 mM in Tris-HCl buffer) with
9 equiv. of target dsDNA (AATTT, ’); fluorescence emission of
hybrid 6 0.5 mM in Tris-HCl buffer (J; the intensity of this spectrum
is presented multiplied by 10 for clarity), and in the presence of
9 equiv. of target dsDNA (AATTT, K). Insert shows fluorescence
cuvettes with 500 nM solutions of 6: (a) no dsDNA added; (b) 2 equiv.
of dsDNA AATTT. Excitation at 329 nm.
Fig. 1 Top. Emission spectra of DOTA[Eu3+] 5a showing the corre-
sponding electronic transitions. Bottom. Emission spectra of DOTA[Tb3+
]
5b. In both graphs: spectra in the absence of DNA (’), with 9 equiv. of
non-target dsDNA (GGCCC, J), with 9 equiv. of suboptimal dsDNA
(AATTC, &), and with 9 equiv. of target dsDNA (AATTT, K).
Hairpin oligonucleotide sequences (binding sites in italics): AATTT:
50-GGCG-AATTT-CGCTTTTTGCG-AAATT-CGCC-30; AATTC: 50-
GGCG-AATTC-AGCTTTTTGCT-GAATT-CGCC-30; GGCCC: 30-
GGCA-GGCCC-AGCTTTTTGCT-GGGCC-TGCC-30.
might make for a more efficient process.13 We therefore
prepared conjugate 6 by direct coupling of parent amino-
benzamidine
3 with PyAOP-activated 7-(diethylamino)-
coumarin-3-carboxylic acid (Scheme 3).
relative variation in the emission intensity of both conjugates,
the shorter emission wavelengths of terbium at 488, and
545 nm are significantly overlapped with the tail of the
bis-benzamidine band, yielding less clear spectra. Likewise,
incubation of 5a with a hairpin dsDNA containing an AATT
binding site also induced a response, albeit weaker than for the
consensus AATTT site (Fig. 1, top).11
As shown in Fig. 2, the fluorescence emission of 6 after
excitation at 329 nm is very weak, and is dominated by the
coumarin emission band at 472 nm. More importantly,
addition of the target dsDNA oligonucleotide containing the
AATTT target sequence induced a large increase (B60-fold) in
the fluorescence emission intensity of coumarin. The bright
emission of the coumarin fluorophore even allows the visual
detection of small quantities of DNA (less than 3 ng), as
shown in the inset.
For 5a we observed at least a four-fold increase in the
emission intensity of the typical Eu3+ bands. The lumines-
cence emission profile of Eu3+ in 5a was as expected for a
Therefore, appropriate conjugation of bis-benzamidine with
coumarin results in a bright fluorescent probe that displays a
remarkable fluorescence change upon binding to specific
dsDNA sites (AATTT), and an increased Stokes shift
(145 nm) relative to the parent compound 1. Moreover,
addition of the dsDNA containing the truncated target
sequence AAT induced significantly smaller increases in the
emission of the coumarin conjugate at 472 nm (Fig. 3 and
ESIz).
square-antiprismatic structure, typical of DOTA[Eu3+
]
complexes, which is mainly characterized by a DJ2 (5D0 - 7F2)
transition, known as the hypersensitive band, weaker than the
DJ1 band (Fig. 1).12 To our knowledge, this is the first
demonstration of
a sequence-selective dsDNA-promoted
environment-sensitive fluorescence enhancement coupled
to lanthanide sensitization, and sets the basis for further
developments of lanthanide-based dsDNA probes.
Having demonstrated the application of the bis-benzamidine
as lanthanide antenna for the DNA-triggered energy transfer,
we reasoned that other organic acceptors such as a coumarin,
with a large spectral overlap with the emitting bis-benzamidine,
In summary, we have described a sequence specific, dsDNA-
induced lanthanide sensitization process, with concomitant
long-wavelength luminescence, as well as a very efficient
Forster intramolecular energy transfer promoted by specific
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Fig. 3 Emission spectra of a 0.5 mM solution of 6 with increasing
amounts of dsDNAs. Left. with hairpin with the target sequence
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´
A/T rich dsDNA sequences. Studies to develop optimized
versions of these compounds that can lead to efficient and
practical dsDNA sequence-specific optical probes are
underway.
´
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We are grateful for the support given by the Spanish grants
SAF2007-61015, SAF2006-06868, CTQ2006-01339, Consolider
Ingenio 2010 CSD2007-00006, and the Xunta de Galicia
PGIDIT06PXIB209018PR, GRC2006/132, PGIDIT08CSA-
047209PR, INCITE09291084PR, and INCITE09209084PR.
O.V. and M.I.S. thank the Spanish MICINN for their PhD
fellowships. M.E.V. also thanks the Human Frontier Science
Program for their support with a Career Development Award
CDA0032/2005-C, and the Spanish MEC/MICINN for his
Ramon y Cajal contract.
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