Modified DNA analogues that sense light exposure with color changes

Article abstract of DOI:10.1021/ja046910o

We report the discovery of a new class of light-sensing molecules. These light sensors are composed of fluorophore oligomers assembled on a DNA backbone. A combinatorial library of tetrafluorophores consisting of over 14000 compounds was synthesized and s

Full text of DOI:10.1021/ja046910o

Published on Web 09/18/2004
Modified DNA Analogues That Sense Light Exposure with Color Changes
Jianmin Gao, Soichiro Watanabe, and Eric T. Kool*
Department of Chemistry, Stanford UniVersity, Stanford, California 94305-5080
Received May 25, 2004; E-mail: kool@stanford.edu
Molecular sensors are becoming increasingly useful for detection
and identification of biomolecules and biophysical conditions in
aqueous solution.1 The large majority of sensors are designed
compounds that include a recognition function and a reporting
functionality.2 While such design-based approaches are attractive
and have enjoyed considerable successes, the trial-and-error, one-
at-a-time approach can be heavily time consuming. For this reason,
combinatorial approaches to sensor discovery may offer significant
advantages.2c,3 Here we report on the rapid combinatorial screening
and discovery of color-changing sensors of light exposure from a
library of over 14 000 DNA-analogous compounds. Their oligo-
nucleotide-based structure and synthesis allows for ready conjuga-
tion to DNAs and RNAs for biochemical and biophysical appli-
cations.
Our combinatorial approach uses the DNA phosphodiester
backbone as a scaffold for arranging fluorescent aromatic com-
Figure 1. (a) Structures of the deoxyriboside monomers in this study. (b)
Structure of one tetramer (5′-EEYS) assembled on a DNA backbone.
pounds in a potentially stacked oligonucleotide-like arrangement.
This close proximity of fluorophores results in useful changes in
fluorescence properties such as large Stokes shifts and tuned
absorption and emission properties.4 In addition, the DNA backbone
maintains water solubility for compounds that otherwise would be
poorly soluble, and the automated synthesizer makes preparation
of such oligomeric compounds quite simple. We previously reported
static fluorescence properties of a small test library of 256
compounds.4 We now describe a new application of this approach,
with the synthesis of a much larger library, and demonstrate a
screening strategy that can be applied to changes in fluorescence
rather than to static properties alone. This enables sensing of
biophysical conditions in solution (in the present case, exposure to
light).
We prepared 11 monomeric deoxyribosides as components of a
Figure 2. (a) Sample image of a tetrafluor library composed of 14 641
library (Figure 1 and Supporting Information). The monomers were
chosen to have varied redox and fluorescence properties. Both R-
and â-glycosidic anomers of fluorophores on deoxyribose were
included, for synthetic convenience and structural diversity. These
were then assembled into tetrameric strands on PEG-polystyrene
beads using a DNA synthesizer; standard split-and-mix methods
were used to yield all possible combinations (114, 14 641 total) of
the 11 monomers. The chemical encoding methodology of Still6
was used to aid in later identification of sequences. Based on the
weight and loading of the library, we estimate >10x overcoverage
of the combinations. Images of the library under the microscope
revealed a large variation in fluorescence color and intensity (Figure
2).
To evaluate the ability of these various molecules on beads to
respond to light, we used a mercury light source combined with
filters to illuminate the beads at 340-380 nm over a period of 1 h
and gathered digital images of the visible emission over time. Digital
subtraction of later images from the time-zero image revealed beads
that were strongly sensitive to light exposure (Figure 2b), whereas
others were more resistant. Selected beads of the most sensitive
ones were decoded to yield sequences of the tetrameric fluoro-
members (excitation 340-380 nm). (b) Digital subtraction images of the
tetrafluor library after 10 min (top) and 60 min (bottom) of light exposure.
Bright beads indicate a drop in emission intensity.
phores. We then resynthesized a number of these candidate
sequences on a preparative scale and characterized them for their
chemical integrity, their static fluorescence, and their responsiveness
to light exposure. Interestingly, at least three of these were found
to respond to light exposure by apparent color changes, rather than
by simple photobleaching.
Three examples of the DNA-like color-changing photosensors
were studied in further detail (Table 1). The data showed that the
selected compounds were fluorescent and exhibited large Stokes
shifts of ca. 130 nm. We evaluated color responses in aqueous
solution, using the Xe lamp in a fluorescence spectrometer as the
light source being sensed. Figure 3 shows spectra of the tetrafluors
before and after light exposure; the data showed that all three
changed their colors markedly. For example, compound 5′-SBBB
originally showed a broad (green) emission band at 510 nm. This
band decreased in intensity over a few minutes, and a shorter
wavelength band (412 nm) increased simultaneously. This blue-
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10.1021/ja046910o CCC: $27.50 © 2004 American Chemical Society

C O M M U N I C A T I O N S
memory storage is also conceivable. Simpler applications are also
possible, including such uses as molecular timers or calendars (by
measuring hours or days of light exposure), or even as color-
changing indicators in applications such as sunscreens.
Our experiment confirms the utility of combinatorial fluorophore/
sensor libraries built on the DNA backbone. The phosphodiester
scaffold allows for ready water solubility in fluorophores that would
otherwise not be useful in aqueous applications, and the DNA
backbone allows for ease of construction and conjugation to
molecules and surfaces. The DNA scaffold facilitates face-to-face
interactions in the fluorescent monomers, yielding useful new
wavelengths of emission and possibly leading to the origin of the
sensing color change in the present application.
Finally, the results illustrate the ease of combinatorial screening
not only for static fluorescence properties but also for sensing of a
physical condition in solution. Future applications of such oligo-
meric molecules might be developed for responses to other physical
properties such as temperature, pH, or ionic strength. In addition,
combinatorial searches for responsiveness to molecular species
might also be envisioned. A number of these experiments are
underway.
Figure 3. Three tetrafluors that respond to light exposure. (a) Fluorescence
emission spectra of the polyfluors before (black lines) and after (red lines)
light exposure. (b) Images of the same compounds in aqueous solution before
(left) and after (right) light exposure.
Table 1. Static Photophysical Data for Color-Changing Oligomers
in Aqueous Solutiona
λem (nm)
λabs (nm)
λ
em (nm)
(after light exposure)
Φ(before)
Acknowledgment. This work was supported by the U.S. Army
Research Office and the NIH (GM067201). J.G. acknowledges a
Stanford Graduate Fellowship. S.W. acknowledges Yamada Science
Foundation for financial support.
SBBBSS
EEYSS
378, 397
329, 346,
418, 445
418, 444
412, 435, 510
375, 576
412, 435
375, 488
0.09
0.07
EESSSS
447, 476, 567
447, 476
0.03
a Extra spacers (S) were added to insure solubility. All samples were
prepared in phosphate buffered saline, pH ) 7.2.
Supporting Information Available: Details of monomer and
library synthesis and fluorescence methods (PDF). This material is
available free of charge via the Internet at http://pubs.acs.org.
violet band closely resembles the emission of the monomeric benzo-
[a]pyrene deoxyriboside (data not shown).
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Subsequent experiments showed that addition of an oxygen
scavenger, Trolox,7 greatly diminished the rate of the color change,
suggesting that an irreversible reaction with oxygen caused the drop
in intensity of the longer wavelength band. We hypothesize that
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Other mechanisms may also be possible; more work is underway
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Similar results were also seen for sequences 5′-EESS and 5′-
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