Sequence specific fluorescence detection of double strand dna

Article abstract of DOI:10.1021/ja021011q

Methods for the fluorescent detection of specific sequences of double strand DNA in homogeneous solution may be useful in the field of human genetics. A series of hairpin polyamides with tetramethyl rhodamine (TMR) attached to an internal pyrrole ring were synthesized, and the fluorescence properties of the polyamide - fluorophore conjugates in the presence and absence of duplex DNA were examined. We observe weak TMR fluorescence in the absence of DNA. Addition of ≥1:1 match DNA affords a significant fluorescence increase over equimolar mismatch DNA for each polyamide - TMR conjugate. Polyamidefluorophore conjugates offer a new class of sensors for the detection of specific DNA sequences without the need for denaturation. The polyamide-dye fluorescence-based method can be used to screen in parallel the interactions between aromatic ring pairs and the minor groove of DNA even when the binding site contains a non-Watson - Crick DNA base pair. A ranking of the specificity of three polyamide ring pairs -Py/Py, Im/Py, and Im/Im - was established for all 16 possible base pairs of A, T, G, and C in the minor groove. We find that Im/Im is an energetically favorable ring pair for minor groove recognition of the T·G base pair.

Full text of DOI:10.1021/ja021011q

Published on Web 01/08/2003
Sequence Specific Fluorescence Detection of Double Strand
DNA
Victor C. Rucker, Shane Foister, Christian Melander, and Peter B. Dervan*
Contribution from the The DiVision of Chemistry and Chemical Engineering, California Institute
of Technology, Pasadena, California 91125
Received July 23, 2002 ; E-mail: dervan@caltech.edu
Abstract: Methods for the fluorescent detection of specific sequences of double strand DNA in homogeneous
solution may be useful in the field of human genetics. A series of hairpin polyamides with tetramethyl
rhodamine (TMR) attached to an internal pyrrole ring were synthesized, and the fluorescence properties of
the polyamide-fluorophore conjugates in the presence and absence of duplex DNA were examined. We
observe weak TMR fluorescence in the absence of DNA. Addition of g1:1 match DNA affords a significant
fluorescence increase over equimolar mismatch DNA for each polyamide-TMR conjugate. Polyamide-
fluorophore conjugates offer a new class of sensors for the detection of specific DNA sequences without
the need for denaturation. The polyamide-dye fluorescence-based method can be used to screen in parallel
the interactions between aromatic ring pairs and the minor groove of DNA even when the binding site
contains a non-Watson-Crick DNA base pair. A ranking of the specificity of three polyamide ring pairss
Py/Py, Im/Py, and Im/Imswas established for all 16 possible base pairs of A, T, G, and C in the minor
groove. We find that Im/Im is an energetically favorable ring pair for minor groove recognition of the T‚G
base pair.
Introduction
One example is the “molecular beacon” which consists of a
hairpin DNA labeled in the stem with a fluorophore and a
Interest in the detection of specific nucleic acid sequences in
homogeneous solution has increased due to major developments
in human genetics. Single nucleotide polymorphisms (SNPs)
are the most common form of variation in the human genome
and can be diagnostic of particular genetic predispositions
toward disease.1 Most methods of DNA detection involve
hybridization by an oligonucleotide probe to its complementary
single-strand nucleic acid target leading to signal generation.2-7
quencher.4 Binding to a complementary strand results in opening
of the hairpin and separation of the quencher and the fluoro-
phore. However, detection by hybridization requires DNA
denaturation conditions, and it remains a challenge to develop
sequence specific fluorescent probes for DNA in the double
strand form.8-10
Hairpin polyamides are a class of synthetic ligands that can
be programmed to recognize specific DNA sequences with
affinities and specificities comparable to DNA binding pro-
teins.11 The large number of DNA sequences which can be
targeted with hairpin polyamides suggests that polyamide-
fluorophore conjugates might be useful in fluorescence-based
detection of specific nucleic acid sequences. Within the context
of targeting sites in gigabase size DNA, Laemmli demonstrated
that polyamides with Texas Red or fluorescein at the C-terminus
specifically stain 5′-GAGAA-3′ repeats in Drosophila satellites,
as well as teleomeric repeats in insect and human chromosomes
* To whom correspondence should be addressed. Tel: (626) 395-6002.
(1) (a) Wang, D. G.; Fan, J.-B.; Siao, C.-J.; Berno, A.; Young, P.; Sapolsky,
R.; Ghandour, G.; Perkins, N.; Winchester, E.; Spencer, J.; Kruglyak, L.;
Stein, L.; Hsie, L.; Topaloglou, T.; Hubbell, E.; Robinson, E.; Mittmann,
M.; Morris, M. S.; Shen, N.; Kilburn, D.; Rioux, J.; Nusbaum, C.; Rozen,
S.; Hudson, T. J.; Lipshutz, R.; Chee, M.; Lander, E. S. Science 1998,
280, 1077-1082. (b) Hacia, J. G. Nature Genetics Suppl. 1999, 21, 42-
47. (c) Ihara, T.; Chikaura, Y.; Tanaka, S.; Jyo, A. Chem. Commun. 2002,
2152-2153. (d) Frutos, A. G.; Pal, S.; Quesada, M.; Lahiri, J. J. Am. Chem.
Soc. 2002, 124, 2396-2397. (e) Kukita, Y.; Higasa, K.; Baba, S.;
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2002, 74, 3421-3428. (g) Yamashita, K.; Takagi, M.; Kondo, H., Takenaka,
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A.; Booth, J. A.; Brown, T. Nucleic Acids Res. 2000, 28, 3752-3761.
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Natl. Acad. Sci. U.S.A. 1991, 88, 7276-7280. (b) Woo, T. H. S.; Patel, B.
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and oxozole yellow dimers, see: (a) Rye, H. S.; Yue, S.; Wemmer, D. E.;
Quesada, M. A.; Haughland, R. P.; Mathies, R. A.; Glazer, A. N. Nucleic
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J. R. N.; McNamee, J. P.; Scaiano, J. C. Photochem. Photobiol. 2001, 73,
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S.; Bratu, D. P.; Kramer, F. R. Nat. Biotechnol. 1998, 16, 49-53. (c)
Kostrikis, L. G.; Tyagi, S.; Mhlanga, M. M.; Ho, D. D.; Kramer, F. R.
Science 1998, 279, 1228-1229.
(9) (a) Seifert, J. L.; Connor, R. E.; Kushon, S. A.; Wang, M.; Armitage, B.
A. J. Am. Chem. Soc. 1999, 121, 2987-2995. (b) Wang, M.; Silva, G. L.;
Armitage, B. A. J. Am. Chem. Soc. 2000, 122, 9977-9986.
(10) For distamycin-Hoechst hybrids, see: (a) Satz, A. L.; Bruice, T. C. Bioorg.
Med. Chem. 2000, 8 (8), 1871-1880. (b) Satz, A. L.; Bruice, T. C. J. Am.
Chem. Soc. 2001, 123 (11), 2469-2477. (c) Satz, A. L.; Bruice, T. C.
Bioorg. Med. Chem. 2002, 10, 241-252.
(5) (a) Jenkins, Y.; Barton, J. K. J. Am. Chem. Soc. 1992, 114, 8736-8738.
(b) Rajir, S. B.; Robles, J.; Wiederholt, K.; Kiumelis, R. G.; McLaughlin,
L. W. J. Org. Chem. 1997, 62, 523-529.
(6) Ranasinghe, R. T.; Brown, L. J.; Brown, T. Chem. Commun. 2001, 1480-
1481.
(11) Dervan, P. B. Bioorg. Med. Chem. 2001, 9, 2215-2235.
9
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J. AM. CHEM. SOC. 2003, 125, 1195-1202
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Rucker et al.
6-base pair DNA match sequence (Figure 3). Tetramethyl
rhodamine-5-maleimide was coupled as a thioether to an internal
pyrrole ring by a cysteine linker. This point of attachment was
chosen to direct the fluorophore into aqueous solution (away
from the DNA helix) to minimize possible unfavorable steric
interactions when the hairpin is bound in the minor groove
(Figure 1). The synthetic details for conjugates 1-6 (as well as
an unusual conjugate 7 containing an Im/Im pair for a
subsequent mismatch study) are described in the Supplemental
Section. The purity and identity of conjugates 1-7 were verified
by reversed phase analytical HPLC and MALDI/TOF mass
spectrometry (Figure 2).
The parent hairpins typically bind with equilibrium associa-
tion constants Ka g 109 M-1 11
To determine the energetic
.
consequences for covalent attachment of fluorophores to the
N1 position of at internal pyrrole ring in the 8-ring hairpin, the
equilibrum association constants for conjugates 1-6 at six sites
were determined by quantitative footprinting titration. The
affinities of conjugates 1-6 at DNA match sites are reduced
by a factor of 10-50 from the parents (Table 1A).11 Despite
the energetic penalty, the expected sequence specificities are
maintained Ka (match) > Ka (single mismatch) > Ka (multiple
mismatch) (Table 1B).
The fluorescence properties of the polyamide-TMR conju-
gates 1-6 in the presence and absence of short segments of
duplex DNA (17-mer) were examined (Figure 4). The absorption
maxima of conjugates 1-6 (∼560 nm) are red-shifted and are
attenuated relative to the absorption maximum (550 nm) of the
TMR fluorophore 8 alone (Figure 4a), presumably due to
electronic interactions between the fluorophore and the poly-
amide in the ground state.16 In the absence of DNA, the
fluorescence intensities of the polyamide-TMR conjugates are
significantly diminished relative to the TMR control 8 (pH 7.0,
TKMC buffer). The addition of increasing amounts of match
DNA to the conjugate affords an increase of fluorescence until
a 1:1 DNA:conjugate stoichiometry is reached (Figure 4bc).
Using polyamide 3 as an example, fluorescence data from the
addition of an increasing amount of a 17 base pair (17-mer)
DNA duplex (0.05-1.0 µM concentration) containing the match
sequence 5′-tAGTACTt-3′ to an aqueous solution of 1 µM
concentration of match conjugate 3 results in an increase in
fluorescence intensity characteristic of all conjugates 1-6.
Fluorescent enhancement is greatest when the DNA duplex
contains the polyamide’s match sequence (Table 2). Addition
of 17-bp DNA duplex containing a match sequence beyond the
1:1 DNA:polyamide stoichiometry does not lead to further
significant increase of emission intensity which indicates the
occurrence of a single binding event between the conjugate and
each DNA duplex (Figure 4c).
Figure 1. (Top) Hairpin polyamide-fluorophore conjugate is weakly
fluorescent in the absence of DNA, but affords fluorescence enhancement
in the presence of match duplex DNA. (Bottom) Model of polyamide-
TMR conjugate 1 bound to match site.
(5′-TTAGG-3′ and 5′-TTAGGG-3′, respectively).12 Similarly,
Trask has demonstrated the use of polyamide-dye conjugates
to fluorescently label specific repetitive regions on human
chromosomes 9, Y and 1 (TTCCA repeats) for discrimination
in cytogenetic preparations and flow cytometry.13
We report here that hairpin polyamide-fluorophore conju-
gates are capable of detecting specific sequences within short
segments of double helical DNA in homogeneous solution
(Figure 1). The fluorescence of our polyamide-tetramethyl
rhodamine (TMR) conjugates is largely quenched in the absence
of DNA. Addition of duplex DNA containing a match site to a
polyamide-TMR conjugate affords a g10-fold increase in
fluorescence. Attenuated signal results when mismatch DNA
is added at the same concentration. This fluorescence detection
method allows us to analyze the specificity of hairpin poly-
amides that target non-Watson-Crick base pairs.
Results and Discussion
For the most effective discrimination, the stabilities of the
different polyamide-DNA complexes should be match > single
base mismatch > multiple base mismatch as determined from
our footprinting study (Table 1). The ability of each conjugate
1-6 to distinguish one match from an ensemble of six different
DNA sequences by fluorescence was examined by direct laser
excitation scanning through the bottom of polystyrene plates.
Detection of Specific DNA Sequences in Homogeneous
Solution. As a model study, six hairpin pyrrole-imidazole (Py-
Im) polyamides with TMR14 attached at an internal pyrrole ring
were synthesized (Figure 2). Each eight-ring polyamide 1-6
was programmed according to the pairing rules11 for a different
(12) (a) Janssen, S.; Durussel, T.; Laemmli, U. K. Mol. Cell 2000, 6 (5), 999-
1011. (b) Maeshima, K.; Janssen, S., Laemmli, U. K. EMBO J. 2001, 20,
3218-3228.
(15) Trauger, J. W.; Dervan, P. B. Methods Enzymol. 2001, 340, 450-466.
(16) (a) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Plenum
Press: New York, 1983. (b) Johansson, M. K.; Fidder, H.; Dick, D.; Cook,
R. M. J. Am. Chem. Soc. 2002, 124, 6950-6956. (c) Rabinowitch, E.;
Epstein, L. F. J. Am. Chem. Soc. 1941, 63, 69-78.
(13) Gygi, M. P.; Ferguson, M. D.; Mefford, H. C.; Kevin, P. L.; O’Day, C.;
Zhou, P.; Friedman, C.; van den Engh, G.; Stobwitz, M. L.; Trask, B. J.
Nucleic Acids Res. 2002, 30, 2790-2799.
(14) Kubin, R. F.; Fletcher, A. N. J. Luminescence 1982, 27, 455-462.
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A R T I C L E S
Figure 2. Structures of conjugates 1-7 and control TMR fluorophore 8.
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A R T I C L E S
Rucker et al.
Table 1. (A) Equilibrium Association Constants (M-1) for Conjugates 1-6 at Sequences Shown on Left (B) Normalized Values of Relative
Affinities of Match vs Mismatch Sites from Table 1Aa
A
1
2
3
4
5
6
taGGCCtt
taGTATtt
taGTACtt
taGTGTtt
taGGTAtt
taGCGCtt
2.1 × 109
<107
<107
2.0 × 107
e5.2 × 108
e4.2 × 108
1.4 × 109
1.10 × 108
3.0 × 107
e2.8 × 107
<107
<107
2.5 × 108
5.0 × 107
1.0 × 108
1.0 × 108
<107
5.2 × 107
1.7 × 109
<107
<107
e5.4 × 107
e1.4 × 108
e2.0 × 108
<107
<107
3.2 × 108
e4.5 × 107
e9.7 × 108
e8.7 × 107
<107
<107
<107
<107
<107
e4.9 × 108
B
1
2
3
4
5
6
taGGCCtt
taGTATtt
taGTACtt
taGTGTtt
taGGTAtt
taGCGCtt
1
<0.05
<0.01
0.03
1
<0.01
<0.01
<0.01
0.01
e0.03
<0.01
0.33
e0.05
1
e0.02
e0.11
e0.29
e0.41
<0.02
1
<0.01
1
e0.37
e0.30
1
0.08
0.02
<0.01
<0.01
<0.01
<0.01
0.20
0.40
0.40
<0.05
e0.09
a The assays were carried out at 22 °C at pH 7.0 in the presence of 10 mM Tris-HCl, 10 mM KCl, 10 mM MgCl2, and 5 mM CaCl2.
conjugate 1-6. We note the agreement between the normalized
specificities determined by DNase I footprinting titration (Table
1B) and those we observe from this new optical assay (Table
2).
The observation that the fluorescence of the TMR fluorophore
is significantly diminished when attached by a short tether to
an internal pyrrole ring position in a series of hairpin poly-
amide-TMR conjugates was not expected. We have found that
other fluorophores, such as fluorescein, when conjugated to an
internal pyrrole of hairpin polyamides behave similarly. Presum-
ably, the short linker holds the fluorophore in very close
proximity or in direct contact with the hairpin polyamide. After
excitation, the polyamide-fluorophore decays from the excited
state via a nonradiative pathway. Incorporation of the aliphatic
â-alanine (â) residues leads to a decrease in fluorescence
quenching. The more flexible aliphatic â may allow increased
conformational freedom to the conjugates 4 and 6, possibly
disrupting the association of dye and polyamide. The perturbed
ground-state absorption spectrum of the fluorophore suggests a
molecular association between polyamide and fluorophore that
allows quenching to occur.17
The simplest model is that, upon binding DNA, the hairpin
is sequestered in the minor groove moving the TMR fluorophore
away from the polyamide and into the surrounding aqueous
environment. One can imagine two conformations for the
polyamide-dye conjugate: polyamide and TMR are in close
proximity when free in solution (fluorescence quenched), and
polyamide and TMR are separated in an extended conformation
with the TMR projecting away from the polyamide when bound
to DNA (fluorescence unquenched).
Figure 3. Ball and stick model of six different polyamide-TMR conjugates
1-6 which code for six different match sites within a 17-mer DNA duplex.
Im and Py residues are shown as black and white circles, respectively;
â-alanine is diamond. TMR is covalently attached at the pyrrole containing
the asterisk.
Both Laemmli and Trask have demonstrated the utility of
polyamide-dye conjugates in the context of gigabase size DNA
as sequence specific chromosome “paints”.12,13 This report
extends the properties of polyamide-dye molecules for specific
DNA sequence detection in solution within short segments of
DNA. The short segments of DNA are not immobilized on a
chip nor is there a need for end labeling. It may be that
applications might be related to “SNP typing” in which the
identity of the SNP containing sequence is known. In a formal
The data were acquired on a Molecular Dynamics Typhoon
optical scanner employing a 532 nm 10-20 mW excitation laser
and a 580 nm emission filter ((15 nm fwhm) suitable for TMR
fluorescence detection. The emission data for hairpin polyamide
conjugates 1-6 interacting with six different double stranded
17-mers is presented in Figure 5a. Polyamide-TMR conjugates
are mostly quenched, except in the presence of increasing
concentrations of a match DNA sequence. The data were
normalized and plotted against the concentration of DNA titrant
(0.02 µM to 1 µM) added to a 1 µM solution of conjugate
(Figure 5b). The 1:1 stoichiometry end point provides a measure
of the relative sequence specificity of each polyamide-TMR
(17) Addition of a four-ring polyamide to TMR (intermolecular association)
affords a 10 nm bathochromic absorption maximum shift of TMR, similar
to that observed for conjugates 1-6 (vs control TMR 7). Fluorescence
emission data in the presence of linearly increasing polyamide concentration
affords a linear Stern-Volmer plot.16
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A R T I C L E S
Table 2. Normalized Fluorescence Intensity for Polyamide-TMR
Conjugates 1-6 at 1 µM Concentration Interacting with Six
Different DNA Duplexes at 1 µM Concentration
1
2
3
4
5
6
taGGCCtt
taGTATtt
taGTACtt
taGTGTtt
taGGTAtt
taGCGCtt
1
0.04
1
0.22
0.30
0.13
0.02
0.03
0.09
1
0.01
0.03
0.05
0.04
0.30
0.12
1
0.09
0.07
0.05
0.20
0.01
0.08
1
0.08
0.10
0.27
0.18
0.07
1
0.04
0.02
0.04
0.05
0.07
0.04
a The assays were carried out at 22 °C at pH 7.0 in the presence of 10
mM Tris-HCl, 10 mM KCl, 10 mM MgCl2, and 5 mM CaCl2.
sense, recognition and detection in these bifunctional polyamide-
dye molecules can be tuned separately; the polyamide module
is programmed for a desired DNA sequence by the pairing rules
and the dye is chosen for optimal absorption/emission proper-
ties.18
Polyamides as Molecular Calipers for Non-Watson-Crick
Base Pairs in the DNA Minor Groove. Our fluorescence DNA
detection method allows us to screen, in parallel, how a hairpin
polyamide will behave when it is forced to bind over a non-
Watson-Crick base pair. To date, the energetics of polyamide
binding over non-Watson-Crick base pairs has not been
examined, mainly due to the labor intensive effort required for
the preparation of special end-radiolabeled DNA fragments
containing discrete non-Watson-Crick base pairs for use in
quantitative footprinting titration analysis. Three hairpin poly-
amide-fluorophore conjugates 3, 5, and 7 were used to screen
Py/Py, Im/Py, and Im/Im ring pairs, respectively, against all
possible 16 pairing permutations of the nucleic acid bases A,
T, G, and C (Figure 6). Polyamide 7 contains an unusual Im/
Im ring pairing, previously shown to be energetically unfavor-
able for recognition of any of the four Watson-Crick base
pairings.19 However, Wang and co-workers have reported an
NMR structure for the polyamide dimer (ImImImDp)2 that
indicates binding with a minor groove site containing G‚T and
T‚G base pairs under the Im/Im pair.20 Im/Im recognition of a
G‚T and T‚G wobble mismatch is the first example of ring pairs
for specific recognition of non-Watson-Crick base pairs.21
Hairpin-forming oligonucleotides (37-mer) were synthesized
and annealed (pH 7.0, tris-EDTA buffer). Each duplex was
designed to allow for base pair variance at a single unique X/Y
position within the context of a 6-base pair hairpin match site.
Assays were conducted in 30 µL solutions with the concentration
of polyamide conjugate fixed at 1 µM, and the concentration
of oligonucleotide was varied from 0.1 µM to 1 µM. After 4 h
incubation (22 °C), fluorescence intensities at the 16 variable
positions were acquired in parallel on the optical scanner (16
X/Y position × 5 concentrations).
Figure 6 shows the data collected by direct excitation
fluorescence and the normalized data represented in bar graph
format. Polyamide 3 and 5 reveal strong fluorescence intensity,
and hence affinity, for a sequence containing a match site as
dictated by the pairing rules, i.e., Py/Py prefers A‚T and T‚A
Figure 4. (A) Absorption spectra of 1-6, 8. All conjugates 1-6 exhibit
a ∼10 nm bathochromic shift and absorption attenuation relative to control
compound 8. (B) Fluorescence emission profiles for conjugate 3 at 1 µM
concentration in the presence of increasing concentrations of DNA (0.05-1
µM) containing the polyamide’s match site 5′-taGTACtt-3′ (10 mM Tris-
HCl (pH 7.0), 10 mM KCl, 10 mM MgCl2, and 5 mM CaCl2). Solutions
were allowed to equilibrate for 4 h at 22 °C. Excitation wavelength is 545
nm. (C) Direct excitation data of 3 with various equivalent of match DNA
was collected on a Typhoon optical scanner as described in the Experimental
Section. Addition of more than one equivalent of match dsDNA generates
no appreciable increase in fluorescence for conjugates as shown for 3. The
two lines intersect at 0.96:1 polyamide:DNA yielding the stoichiometry of
a single binding event.
(18) Although polyamide-Hoechst hybrids10 have the property of enhanced
fluorescence when bound to match DNA, the minor groove binding Hoechst
dye requires an A/T tract adjacent to the DNA target site and hence is not
as versatile.
(19) White, S.; Baird, E. E.; Dervan, P. B. Chem. Biol. 1997, 4, 569-578.
(20) Yang, X.-L.; Hubbard IV, R. B.; Lee, M.; Tao, Z.-F.; Sugiyama, H.; Wang,
A. H.-J. Nucleic Acids Res. 1999, 27, 4183-4190.
(21) Baird, E. E.; Dervan, P. B. J. Am. Chem. Soc. 1996, 118 (26), 6141-
6146.
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Rucker et al.
Figure 5. (A) Plate experiments conducted at 1 µM of polyamide-TMR in the presence of 0.02 µM to 1 µM concentration gradient of six different 17-mer
DNA duplexes. Each polyamide-TMR conjugate 1-6 reveals a preference for one of the six sequences of DNA. The “minus” DNA well is shown for
measurement of background fluorescence. Fluorescence intensity correlates with the dark color of a well. (5B) Normalized data for the 54-well experiment
using the background as the lowest observable fluorescence intensity.
over the 14 other base pairs; Im/Py prefers G‚C over the 15
other base pairs. Polyamide 8 with the Im/Im pair shows a slight
preference for G/C over A/T, but does not discriminate G‚C
from C‚G as expected for a symmetrical ring pair. Remarkably,
the Im/Im pair binds T‚G with the highest affinity validating
the Lee-Sugiyama-Wang model.20 However, in our study Im/
Im distinguishes T‚G from G‚T, a result not anticipated in the
NMR structure, which may be due to different sequence contexts
used in the two experiments. It is noteworthy that a minor
groove binding polyamide can target a single non-Watson-
Crick base pair out of 12 unique possibilities.
In summary, polyamide-fluorophore conjugates have been
used to target sequence repeats in chromosomes for interrogation
of sequence location and estimation of repeat length.12,13 In this
study, we extend the use of polyamide-fluorophore conjugates
to distinguish match and mismatch sequences within short
segments of DNA in solution. The correlation of sequence
identity with fluorescence enhancement may be useful in other
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A R T I C L E S
Figure 6. (A) Design of hairpin forming duplex oligonucleotides with polyamide match sites for conjugates 3, 5 and 7 which vary at a single internal X/Y
position all possible 16 base pairs of A, T, G, and C. (B-D) Fluorescence intensities acquired on an optical scanner for the interaction of 3, 5, and 7 (1 µM
concentration) with 0.1-1 µM of each of 16 different oligonucleotides listed above. Plotted below is normalized graphical representation of polyamides
pairing preference as reported by the optical assay.
applications such as screening for sequence variation in specific
segments of genomic DNA.
elution 1.25% acetonitrile/min. Preparatory reversed phase
HPLC was performed on a Beckman HPLC with a Waters
DeltaPak 25 × 100 mm, 100 µm C18 column equipped with a
guard, 0.1% (wt/v) TFA/0.25% acetonitrile/min. Water was from
either a Millipore MilliQ water purification system or RNase
free water from a USB. All buffers were 0.2 µm filtered prior
to storage. Oligonucleotides were synthesized and purified by
The Caltech Biopolymer Synthesis and Analysis Resource
Center and Genbase Inc., San Diego. Enzymes for molecular
biology were purchased from either New England Biolabs or
Boeringer-Mannheim. [R-32P] adenosine triphosphate and
[R-32P] thymidine triphosphate were purchased from New
Experimental Section
Materials. UV spectra were measured in water on a Beckman
model DU 7400 spectrophotometer. MALDI-TOF mass spec-
trometry data was collected by the Protein and Peptide Mi-
croanalytical Facility at The California Institute of Technology.
HPLC analysis was performed using a Beckman Gold Nouveau
system using a Rainin C18, Micosorb MV, 5 µm, 300 × 4.6
mm reversed phase column using 0.1% (wt/v) TFA with
acetonitrile as eluent and a flow rate of 1.0 mL/min, gradient
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A R T I C L E S
Rucker et al.
England Nuclear. L-Boc(Trt)-Cys-OH was purchased from
Bachem. Tetramethyl rhodamine-5-maleimide was from Mo-
lecular Probes. Polystyrene plates were from VWR Scientific.
Fluorescence spectrophotometer measurements were made at
room temperature on an ISS K2 spectrophotometer employing
5 nm emission and excitation slits in conjunction with a Hg
lamp. Polystyrene plate measurements were made on a Molec-
ular Dynamics Typhoon employing a 532 nm 10-20 mW
excitation laser. The emission filter employed is 580 ( 15 nm
filter for observation of tetramethyl rhodamine fluorescence.
Synthesis of Polyamide-Fluorophore Conjugates. Poly-
amides 1-7 were synthesized by solid-phase methods. A new
monomer, 4-[(t-butoxycarbonyl)amino]-1-(phthalimidopropyl)-
pyrrole-2-carboxylic acid (15) was incorporated in the usual
manner by DCC/HOBt activation (DMF, 4 h., 37 °C). (See
Supplemental Figures S1 and S2.) Polyamide 3 was simulta-
neously deprotected and cleaved from the resin by aminolysis
with 3-(dimethylamino)-propylamine (60 °C, 10 h.). Purification
by reversed phase HPLC afforded a diamine which was allowed
to react with one equiv of L-Boc(Trt)-Cys-OH activated with
DCC/HOBt. The polyamide-cysteine conjugate 10 was char-
acterized by analytical HPLC and MALDI-TOF mass spec-
trometry. The L-cysteine modified polyamides in DMF were
allowed to react with one equivalent of TMR-5-maleimide in a
minimal volume of DMF. This reaction was monitored by
analytical HPLC and was usually complete in less than 15 min.
The products were purified by reversed phase preparatory
HPLC. All products were analyzed by MALDI/TOF MS. For
Optical Characterization. All measurements were performed
in TKMC buffer {10 mM Tris-HCl (pH 7.0), 10 mM KCl, 10
mM MgCl2, and 5 mM CaCl2}. The concentration of poly-
amide-TMR conjugate was 1 µM and the volume of solution
used for each measurement was 150 µL. In the fluorimeter,
polyamides 1-6 were excited at 545 nm and measured over
the interval of 570 nm to 680 nm. Emission maxima are 575
nm for 1-8. 1 µM solutions of each conjugate were irradiated
in the presence and absence of 1 µM of the appropriate match
DNA to generate the fluorescence enhancements reported in
Supplemental Figure S4. Absorption spectra for 1-6 and 8 were
recorded under the same conditions as emission with 1-6 at
higher concentration.
96-Well Plate Characterization. In the dark, 150 µL
solutions were made by titrating together a fixed concentration
of 1 µM conjugate against 20 nM to 1 µM of each DNA 17-
mer. The solutions were gently swirled for a few minutes and
then allowed to sit for 4 h before measurements were made.
No changes in fluorescence intensity were observed for longer
equilibration times. Plates containing polyamides 1-7 were
excited at 532 nm, and data were collected at 580 ((15 nm
fwhm) nm. ImageQuant software (Molecular Dynamics) was
used to analyze the fluorescence intensity of each titration
experiment. From these data, histograms were constructed to
assist in determining DNA sequence preference for each
conjugate. The normalized data used in each histogram is
polyamide specific assuming the background to be the lowest
observable fluorescence intensity, which is subtracted in the
denominator from the highest observed fluorescence intensity,
that of the match sequence at 1:1 conjugate:duplex as given by
the relation (Fobs - Fmin)/(Fmax - Fmin).
1 (monoisotopic) [M + H] 1853.6 (1853.8 calcd for C89H105
-
N28O16S); 2 (monoisotopic) [M + H] 1850.54 (1850.81 calcd
for C92H108N25O16S); 3 (monoisotopic) [M + H] 1851.74
(1851.81 calcd for C91H107N26O16S); 4 (monoisotopic) [M +
H] 1749.9 (1749.79 calcd for C85H105N24O16S); 5 (monoisotopic)
[M + H] 1851.8 (1851.81 calcd for C91H107N26O16S); 6
Acknowledgment. We are grateful to the National Institutes
of Health for grant support (GM 27681), training grant support
for V.C.R. (GM19789-02) and a postdoctoral fellowship to
C.M., and to the National Science Foundation for a graduate
research fellowship to S.F. We thank G.M. Hathaway for
MALDI-TOF mass spectrometry.
(monoisotopic) [M + H] 1751.6 (1751.78 calcd for C83H103
-
N26O16S); 7 (monoisotopic) [M + H] 1852.7 (1852.63 calcd
for C90H106N27O16S); 8 (monoisotopic) [M + H] 617.4 (617.2
calcd for C32H34N5O6S). For 1-3, 5, 7 UV (H2O) λmax(ꢀ) 312
(68 800), 4 and 6, UV (H2O) λmax(ꢀ) 300 (51 600), 1-6, Vis
(H2O) λmax(ꢀ) 563 (65 700), 8 vis (H2O) λmax(ꢀ) 550 (95 000).
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1202 J. AM. CHEM. SOC. VOL. 125, NO. 5, 2003