Development of novel cell-permeable DNA sensitive dyes using combinatorial synthesis and cell-based screening

Article abstract of DOI:10.1039/b303960a

A novel cell-permeable DNA fluorescence sensor was developed based on combinatorially-created styryl dyes and cell-based localization screening.

Full text of DOI:10.1039/b303960a

Development of novel cell-permeable DNA sensitive dyes using
combinatorial synthesis and cell-based screening†
Jae Wook Lee,a Michelle Jung,a Gustavo R. Rosaniab and Young-Tae Chang*a
a Department of Chemistry, New York University, New York, New York 10003, USA.
E-mail: yt.chang@nyu.edu; Fax: 1-212-260-7905; Tel: 1-212-998-8491
b Department of Pharmaceutics, College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109,
USA. E-mail: grosania@umich.edu; Fax: 1-734-615-6162; Tel: 1-737-763-1032
Received (in Cambridge, UK) 10th April 2003, Accepted 5th June 2003
First published as an Advance Article on the web 26th June 2003
A novel cell-permeable DNA fluorescence sensor was
developed based on combinatorially-created styryl dyes and
cell-based localization screening.
Compound B crystallized out in ethyl acetate. The condensation
with aldehydes (A) and compound B was performed by
refluxing with piperidine for 2 h in EtOH. After cooling to room
temperature, the crystallized compounds were filtered and
washed with ethyl acetate. With these purified compounds, we
tested the fluorescence intensity change upon addition of DNA.
Out of 8 nuclear localizing compounds, only compound 1
showed a strong fluorescence increase.
Compound 1 is an orange solid that exhibits an excitation
wavelength of l = 413 nm and an emission wavelength of l =
583 nm (Table 1). A linear fluorescence response was observed
in the 0.05–100 µM range (in PBS: phosphate-buffered saline)
without self-quenching or shifts in emission or excitation
wavelengths. With a series of concentrations of dsDNA (double
stranded DNA) added to compound 1, a linear increase in the
fluorescence intensities was observed (Fig. 2). At the highest
DNA-sensitive fluorescent probes have been widely used for
cell imaging1 and DNA sequencing on gels.2 As most of the
commonly used dyes, such as ethidium bromide3 and Sytox
Green,4 are not cell permeable, these cell imaging processes
require damage to the membrane or separation of the DNA from
the cell in order to stain the nucleic acids. Only a couple of cell
permeable dyes, such as Hoechst 332585 and DAPI,6 are
available, and they permeate the membrane and localize in the
nuclei of living cells.7 The development of novel DNA sensitive
dyes with higher selectivity and sensitivity has been of great
interest.
In our previous research, we developed a unique combinato-
rial approach to the synthesis of styryl dyes (574 compounds)
and their subcellular localization including nuclear, mitochon-
drial, cytosolic, vesicular, granular and reticular structures.8
The nuclear staining of the dyes may have two different
mechanisms: (1) binding to DNA or other nucleic targets with
high affinity, and/or (2) increasing their fluorescence intensity
upon binding to DNA. We envisioned that the latter case would
provide novel DNA sensors. Herein, we present the discovery
of novel DNA sensitive styryl dyes by extended combinatorial
synthesis, cell-based screening and the fluorescence property
measurement upon binding to DNA.
An extended styryl dye library, composed of 855 compounds,
was synthesized (for details see ESI†) and screened for the
subcellular localization in live UACC-62 human melanoma
cells on glass bottom 96-well plates by the previously reported
procedure (Scheme 1).8,9 Only 8 out of 855 compounds showed
strong nuclear localization (Fig. 1). The compounds were
resynthesized on large scale for further study.
Fig. 1 Eight nuclear localizing compounds and three derivatives of 1.
Table 1 Spectrophotometric properties of the styryl dyes
The synthesis of compounds B was achieved by refluxing
with the pyridine derivatives and iodomethane for 2 h.
free
DNA
DNA
free
lmax
nm
/
lem
nm
/
lem
nm
/
fF
fF
/
free
DNA
Dye
fF
fF
1
2
3
413
366
370
583
553
491
566
520
502
0.00024
0.0051
0.0024
0.0032
0.022
0.0037
13.3
4.3
1.5
Scheme 1 General procedure of the synthesis and screening of the styryl
dyes in 96-well format.
† Electronic supplementary information (ESI) available: experimental
section. See http://www.rsc.org/suppdata/cc/b3/b303960a/
Fig. 2 Fluorometric titration of compound 1 with dsDNA in a buffer
solution (lex = 394 nm, compound 1 (5 µM)).
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concentration of DNA tested (50 µg mL21), the fluorescence
emission reached up to 13.3 times higher than that of the free
compound (Fig. 3). A blue shift of 17 nm in the emission
wavelength upon DNA addition was observed, without a
significant excitation wavelength shift. The structure of com-
pound 1 includes a 2,4,5-trimethoxy group from the benzalde-
hyde moiety and a unique adamantyl pyridinium function-
ality.
Different trimethoxy isomers, 2 (3,4,5-trimethoxy) and 3
(2,3,4-trimethoxy), were synthesized to compare the positional
effects of the methoxy groups in compound 1 (Fig. 1). While the
responses of compounds 2 and 3 to DNA treatment were similar
to that of compound 1, the fluorescence emission increase was
much smaller in 2 (4.3 fold) and 3 (1.5 fold). It is noteworthy
that the intrinsic fluorescence intensities of compounds 2 and 3
are higher than that of compound 1, but DNA treated samples
showed comparable quantum yields (Table 1). Compound 4
was also resynthesized and tested to study the structural
importance of the adamantyl group in compound 1.
Fig. 4 Nuclear staining of compounds 1, 2, and 3 (500 µM).
Interestingly, the simple exchange of the adamantyl with a
methyl group significantly reduced the DNA response in
compound 4. Therefore, both 2,4,5-trimethoxy groups and the
adamantyl group are important in the specific interaction of
compound 1 and DNA.
The three related compounds 1, 2, and 3 were incubated in
live UACC-62 human melanoma cells to compare their nuclear
localization properties (Fig. 4). In comparison to compound 1 in
the same concentration, compounds 2 and 3 showed stronger
fluorescence backgrounds and spread throughout the cyto-
plasm. However, compound 1 clearly stains the nucleus of live
cells more selectively.
In summary, a combined combinatorial synthesis of styryl
dyes and cell-based screening provided a novel structural motif
for a DNA sensor, compound 1. Compound 1 is one of the rare
cell permeable nuclear staining dyes and would be useful for
live cell imaging purposes. The general strategy developed in
this study will be applied to novel organelle specific sensor
development.
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Fig. 3 Absorption and fluorescence spectra of compounds 1–3. Dye 1, 2, 3
(50 µM), dsDNA (50 µg mL21).
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