Boronic acid-modified DNA that changes fluorescent properties upon carbohydrate binding

Article abstract of DOI:10.1039/b921163b

A long wavelength boronic acid-modified TTP (NB-TTP) has been synthesized and enzymatically incorporated into DNA. Such DNA shows intrinsic fluorescent changes upon carbohydrate addition.

Full text of DOI:10.1039/b921163b

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Boronic acid-modified DNA that changes fluorescent properties upon
carbohydrate bindingw
Xiaochuan Yang, Chaofeng Dai, Angie Dayan Calderon Molina and Binghe Wang*
Received (in Austin, TX, USA) 12th October 2009, Accepted 12th December 2009
First published as an Advance Article on the web 15th January 2010
DOI: 10.1039/b921163b
A long wavelength boronic acid-modified TTP (NB-TTP) has
been synthesized and enzymatically incorporated into DNA.
Such DNA shows intrinsic fluorescent changes upon
carbohydrate addition.
nuclear imaging, and cell division genomic DNA tracing.
Herein, we report the very first side chain functionalized
naphthalimide-based boronic acid-modified TTP (NB-TTP,
Fig. 1) that by itself shows significant intrinsic fluorescent
property changes upon carbohydrate binding and retains this
ability even after incorporation into DNA.
In addition to their critical roles in storing genomic information,
DNA molecules are also very important in the development of
aptamers,1 nanoelectronic assemblies, nanosensors, nano-
clusters, nanocomputing,2 and DNA rotary machines, to
name a few.3 DNA functionalization brings in additional
properties for broadened applications. For example, gold-
nanoparticle functionalized DNA assemblies have been used
in sensor development with extraordinary sensitivity;3 and
side-chain functionalized DNA has been used in aptamer
selection with improved affinity and properties.4,5
4-Amino-1,4-naphthalimide was chosen as the fluorophore
after careful consideration and evaluation.11–14 Many boronic
acids that change fluorescent properties have been reported.
However, not all fluorescent boronic acids retain their
fluorescent properties after incorporation into DNA. Such is
the case with quinolineboronic acid, which loses its ability to
show significant fluorescent intensity changes (over 40-fold)
in response to sugar binding after DNA incorporation.8
Furthermore, when we applied the criteria set out above, the
naphthalimide-based boronic acid moiety in Fig. 1 seemed
to represent the best choice because of its relatively long
excitation and emission wavelengths, stability, and ease of
synthesis. For example, under constant irradiation at 490 nm
for one hour, the fluorescence of boronic acid 7 (Scheme 1) did
not show noticeable changes (Fig. S1, ESIw). Thus, the
problem of photo bleaching faced by many fluorophores15,16
should not be a major issue in this case. In this design,
CuAAC (copper(I)-catalyzed azide-alkyne cycloaddition),17
which has been studied in previous boronic acid
synthesis,8,18,19 was used to conjugate the thymidine
triphosphate moiety with the boronic acid moiety. This was
done because of the difficulty in performing the triphosphoryl-
ation reaction in the presence of a boronic acid moiety
(hygroscopic) and the difficulty in installing a boronic acid
group in the presence of a triphosphate. The synthesis
of the boronic acid moiety is described in Scheme 1 and the
synthesis of M-TTP (Fig. 1) has been described previously.8
Our lab has had a long-standing interest in using the
boronic acid moiety, which is known to interact with diols/
carbohydrates, fluoride, cyanide, and other nucleophiles/
Lewis bases, as a recognition group for sensing and other
applications.6,7 Recently, we have reported the synthesis of a
quinoline boronic acid-modified thymidine triphosphate
(QB-TTP, Fig. 1),8 its enzymatic incorporation into DNA,
and discussed the feasibility of using it in DNA aptamer
selection. Along this line, we desire to incorporate a fluorescent
boronic acid into DNA that has the following properties:
(1) the excitation and emission wavelengths are longer than
the lmax of nucleobases; (2) shows fluorescent property
changes upon carbohydrate binding; (3) is stable under PCR
conditions; (4) is stable under copper-mediated ‘‘click’’
conditions;9,10 and (5) is relatively easy to synthesize. Such
boronic acid-modified DNA may have applications in
carbohydrate and glycoprotein sensing as well as the construction
of other DNA assemblies with unique properties.7 For example,
nucleic acid and peptide-templated multi-boronic acid
compounds are artificial lectin mimics; boronic acid-containing
compounds have been used in the preparation of polymers
with reversible properties; boronic acid-modified proteins can
be used as lectin mimics with improved affinity and for protein
purification; and boronic acid-containing polymers have been
used for the development of glucose-sensitive insulin release
systems. Beyond simple sensing, incorporation of fluorescent
boronic acid-modified thymidine into genomic DNA may
allow for the probe of changes in the nuclear environment,
Department of Chemistry and Center for Biotechnology and Drug
Design, Georgia State University, Atlanta, Georgia 30302-4098, USA.
E-mail: wang@gsu.edu; Fax: +1 4044135543; Tel: +1 4044135545
w Electronic supplementary information (ESI) available: Experimental
details and spectroscopic data. See DOI: 10.1039/b921163b
Fig. 1 M-TTP, QB-TTP and NB-TTP.
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Table 1 Designed sequences for NB-TTP-DNA construction
Primer
30-GGTCGTTGGGCGAT-50
Template 0
Template 1
Template 2
Template 3
Template 4
Template 5
Template 6
Template 7
50-GGTTCCACCAGCAACCCGCTA-30
50-GGTTCCAACCAGCAACCCGCTA-30
50-GGTTCACACCAGCAACCCGCTA-30
50-GGTTACCACCAGCAACCCGCTA-30
50-GGTATCCACCAGCAACCCGCTA-30
50-GGATTCCACCAGCAACCCGCTA-30
50-GAGTTCCACCAGCAACCCGCTA-30
50-AGGTTCCACCAGCAACCCGCTA-30
full extension of the primer using natural dNTPs yielded
a DNA with m/z of 6520.4 (calculated [M+H]+: 6519.3)
as the major product peak in MALDI (Fig. S2.1, ESIw).
The minor peak of m/z 6850.6 could have resulted from a
non-specific extension with an extra nucleotide. In the
NB-TTP (replacing TTP) reaction, primer extension yielded
a DNA with m/z of 7142.8 as the major product peak
(Fig. S2.2, ESIw) and a new peak of m/z: 7160.6 after treatment
with H2O2 (Fig. S2.3, ESIw). Each was assigned as the deboryl-
ated (calc. [M+H-HBO2]+: 7142.5) and oxidative deborylated
NB-TTP-DNA sequence (calc. [M+H ꢂ HBO]+: 7158.5),
respectively. Thus, it was concluded that the boronic acid of
NB-TTP was intact after incorporation into the DNA sequence
and deborylation happened during the ionization process,
which is common with arylboronic acids.21
Scheme 1 Synthesis route of NB-TTP.
During the experiment, it was observed in steps (e) and (f)
(Scheme 1) that the solubility of boronic acids 6 and 7 in
methanol was very low if the solution was acidic and treatment
with K2CO3 brought the boronic acids into solution. The
synthesized NB-TTP has a solubility of about 170 mM in
water as determined using UV. This solubility is high enough
for most enzymatic reaction requirements.
The DNA from primer extension using NB-TTP was studied
using gel electrophoresis. The 14-nt primer was labeled with
32P at the 50-end using g-32P-ATP and T4 kinase (Fig. 2b,
Lane 1). Negative control reactions without the Klenow
fragment (Fig. 2b Lane 2) and without dNTPs (Fig. 2b, Lane 3)
showed no full length DNA sequence. On the other hand,
positive control reactions in the presence of both the enzyme
and natural dNTPs (Fig. 2b, Lane 4) showed full length DNA.
The shorter length of DNA in Lane 3 without dNTPs could
result from the 30 - 50 exonuclease activity of Klenow
fragment.20 Primer extension using NB-TTP and three
other natural dNTPs gave a full length of DNA sequence
(Fig. 2b, Lane 5). Its smear band could be due to the inter-
action of the boronic acid moiety with the gel matrix or the
DNA conformation disrupted by the large naphthalimide
moiety. Thus, both mass spectrometry and gel electrophoresis
results indicated that NB-TTP was recognized by DNA
polymerase as a substrate and incorporated into DNA.
After incorporating NB-TTP into the DNA sequence, we
were interested in studying whether sugar addition to the
DNA solution would still cause fluorescence property
changes. Since dNTPs and oligonucleotides are known to
quench fluorescence,22–24 the retention of NB-TTP’s fluorescent
property in DNA was uncertain. Fructose in the concentration
range of 1 to 100 mM was used as a model sugar. Fig. 3
shows that the fluorescence intensity of NB-TTP-DNA
increased by about 1.5-fold upon fructose addition at its
highest concentration used (100 mM) (lex: 490 nm), which
demonstrated that the fluorescent properties of NB-TTP were
retained. Again, this is the very first example of side-chain
functionalized DNA showing intrinsic fluorescent property
changes upon sugar binding and such changes do not rely
on FRET.
Since the NB-TTP compound was designed for its ability to
change fluorescent properties, we studied its binding with a
model sugar, fructose. As shown in Fig. 2a, the fluorescence
intensity of NB-TTP increased upon D-fructose addition in a
concentration dependent fashion (lex: 490 nm). The Ka was
determined to be 73 ꢁ 5 Mꢂ1 (see ESI for detailsw). Such
results demonstrate that even after conjugation with a
nucleobase (thymidine), the naphthalimide-based boronic acid
maintains its ability to change fluorescent properties upon
sugar binding.
Next we studied whether this NB-TTP could be incorporated
into DNA in an enzyme-catalyzed reaction. In this study,
NB-TTP was incorporated into a DNA sequence of a 14-nt
primer (Table 1) using a 21-nt template (Template 0, Table 1)
through Klenow fragment catalyzed primer extension.20 The
primer and template sequences were designed in such a way
that the first base to be incorporated is a T, which led to a
‘‘none-or-all’’ case in the extension. In the control reaction,
Fig. 2 (a) Fluorescent binding test of NB-TTP with D-fructose.
(b) Primer extension analyzed on 15% PAGE: (1) primer only;
(2) reaction without enzyme; (3) reaction without dNTPs; (4) reaction
with natural dNTPs; (5) reaction with NB-TTP and other dNTPs.
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We appreciate the financial support by the Georgia Cancer
Coalition, Georgia Research Alliance, the National Institutes
of Health (Grants CA123329, CA113917, GM086925 and
GM084933), and the GSU MBD program. We also thank
Dr. Siming Wang, Director of GSU MS Facilities, for her
extensive help with the mass spectrometry work.
Notes and references
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Fig. 3 Fluorescent binding tests of NB-TTP-DNA with D-fructose.
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of the fluorophore. Therefore, Templates 1–7 were designed in
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The same model sugar, fructose, was chosen for the
fluorescent test. Double stranded NB-TTP-DNA sequences
were generated through primer extension reactions with a
primer/template ratio of 1 : 1 and no heat denaturing before
the binding test. The 21-nt template denoted as Template 0
was used as the control (Table 1). From the testing results
(Fig. S3.1, ESIw), it can be seen that all boronic acid-modified
DNA showed fluorescent intensity changes upon sugar
addition. Such results indicate that having more than one
fluorophore in the DNA sequence did not affect its ability to
fluoresce. The same studies were conducted using the ssDNA.
Again, the boronic acid fluorophore retained its ability to
change fluorescence (Fig. S3.2, ESIw). It should be noted that
the fructose studies were meant to test the intrinsic ability for
the fluorophore in question to change fluorescence. This is not
a ‘‘sensor’’ for fructose or any other sugar.
A novel fluorescent boronic acid conjugated TTP analogue
(NB-TTP) was successfully synthesized. NB-TTP was readily
recognized by the Klenow fragment and incorporated into DNA.
NB-TTP modified DNA showed fluorescence intensity increases
upon binding with fructose. Spacing two boronic acids in various
positions does not fundamentally affect its ability to change
fluorescence upon sugar binding. The boronic acid-modified
thymidine will be very useful for the preparation of DNA-based
sensing and molecular recognition applications and for genomic
DNA incorporation to probe specific properties in the nucleus.
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This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 1073–1075 | 1075