Design of a new fluorescent cofactor for DNA methyltransferases and sequence-specific labeling of DNA

Article abstract of DOI:10.1021/ja021106s

Sequence-specific labeling of DNA is of immense interest for analytical and functional studies of DNA. We present a novel approach for sequence-specific labeling of DNA using a newly designed fluorescent cofactor for the DNA methyltransferase from Thermus aquaticus (M·TaqI). Naturally, M·TaqI catalyzes the nucleophilic attack of the exocyclic amino group of adenine within the double-stranded 5′-TCGA-3′ DNA sequence onto the methyl group of the cofactor S-adenosyl-L-methionine (AdoMet) leading to methyl group transfer. The design of a new fluorescent cofactor for covalent labeling of DNA was based on three criteria: (1) Replacement of the methionine side chain of the natural cofactor AdoMet by an aziridinyl residue leads to M′TaqI-catalyzed nucleophilic ring opening and coupling of the whole nucleoside to DNA. (2) The adenosyl moiety is the molecular anchor for cofactor binding. (3) Attachment of a fluorophore via a flexible linker to the 8-position of the adenosyl moiety does not block cofactor binding. According to these criteria the new fluorescent cofactor 8-amino[1″-(N″-dansyl)-4″'-aminobutyl]-5′- (1-aziridinyl)-5′-deoxyadenosine (3) was synthesized. 3 binds about 4-fold better than the natural cofactor AdoMet to M′TaqI and is coupled with a short duplex oligodeoxynucleotide by M′TaqI. The identity of the expected modified nucleoside was verified by electrospray ionization mass spectrometry after enzymatic fragmentation of the product duplex. In addition, the new cofactor 3 was used to sequence-specifically label plasmid DNA in a M′TaqI-catalyzed reaction.

Full text of DOI:10.1021/ja021106s

Published on Web 02/26/2003
Design of a New Fluorescent Cofactor for DNA
Methyltransferases and Sequence-Specific Labeling of DNA
Goran Pljevaljcic, Marc Pignot,† and Elmar Weinhold*,‡
Contribution from the Max-Planck-Institut fu¨r molekulare Physiologie,
Abteilung Physikalische Biochemie, Otto-Hahn-Strasse 11, D-44227 Dortmund, Germany
Received August 21, 2002 ; E-mail: elmar.weinhold@oc.rwth-aachen.de
Abstract: Sequence-specific labeling of DNA is of immense interest for analytical and functional studies
of DNA. We present a novel approach for sequence-specific labeling of DNA using a newly designed
fluorescent cofactor for the DNA methyltransferase from Thermus aquaticus (M‚TaqI). Naturally, M‚TaqI
catalyzes the nucleophilic attack of the exocyclic amino group of adenine within the double-stranded 5′-
TCGA-3′ DNA sequence onto the methyl group of the cofactor S-adenosyl-L-methionine (AdoMet) leading
to methyl group transfer. The design of a new fluorescent cofactor for covalent labeling of DNA was based
on three criteria: (1) Replacement of the methionine side chain of the natural cofactor AdoMet by an aziridinyl
residue leads to M‚TaqI-catalyzed nucleophilic ring opening and coupling of the whole nucleoside to DNA.
(2) The adenosyl moiety is the molecular anchor for cofactor binding. (3) Attachment of a fluorophore via
a flexible linker to the 8-position of the adenosyl moiety does not block cofactor binding. According to
these criteria the new fluorescent cofactor 8-amino[1′′-(N′′-dansyl)-4′′-aminobutyl]-5′-(1-aziridinyl)-5′-
deoxyadenosine (3) was synthesized. 3 binds about 4-fold better than the natural cofactor AdoMet to M‚TaqI
and is coupled with a short duplex oligodeoxynucleotide by M‚TaqI. The identity of the expected modified
nucleoside was verified by electrospray ionization mass spectrometry after enzymatic fragmentation of the
product duplex. In addition, the new cofactor 3 was used to sequence-specifically label plasmid DNA in a
M‚TaqI-catalyzed reaction.
enzymatic or chemical methods.6 Labeled deoxynucleoside
triphosphates can be incorporated into DNA by DNA poly-
Introduction
Sequence-specific labeling of native DNA still represents a
more or less unsolved problem. Difficulties mainly arise from
the necessity to combine two different functions: sequence-
specific recognition of DNA and covalent bond formation
between the label and DNA. Several systems, like triple helix-
forming oligodeoxynucleotides,2 polyamide nucleic acids,3
pyrrole-imidazole-hydroxypyrrole polyamides4 or zinc finger
proteins,5 are well suited to target many DNA sequences.
However, these systems generally lack a catalytic function for
covalent labeling. Labeling of DNA is often carried out using
merases (nick translation, random-primed labeling, PCR1) or
terminal deoxynucleotidyl transferases. Alternatively, chemical
techniques can be used, e.g. photochemical coupling of aromatic
azides with nucleobases (e.g., photobiotin) or bisulfite-catalyzed
exchange of the cytosine amino group against primary amines.
In addition, labeled oligodeoxynucleotides can be produced by
solid-phase DNA synthesis and incorporated at the ends of DNA
fragments by PCR. However, these enzymatic and chemical
methods are usually not suitable for sequence-specific internal
labeling of large DNA.
† Present address: Nanogen Recognomics GmbH, Industriepark Ho¨chst,
Geba¨ude G 830, D-65926 Frankfurt am Main, Germany.
In contrast to these systems and methods mentioned above,
DNA methyltransferases (MTases) are capable of recognizing
specific sequences within DNA and catalyzing covalent bond
formations. They naturally transfer the activated methyl group
from the cofactor S-adenosyl-L-methionine (1, AdoMet) to the
exocyclic amino group of adenine and cytosine or the 5-position
of cytosine within their individual recognition sequences
(Scheme 1, left).7 Unfortunately, the methyl group itself is a
very limited reporter group and only suitable for radioactive
labeling with tritium or carbon-14. We recently designed the
new cofactor N-adenosylaziridine (2) which is quantitatively,
base- and sequence-specifically coupled with the double-
‡ Present address: Institut fu¨r Organische Chemie der RWTH Aachen,
Professor-Pirlet-Str. 1, D-52056 Aachen, Germany.
(1) The following abbreviations are used: PCR, polymerase chain reaction;
MTase, methyltransferase; AdoMet, S-adenosyl-L-methionine; M.TaqI,
DNA methyltransferase from Thermus aquaticus; EDIA, N-ethyldiiso-
propylamine; M‚HhaI, DNA methyltransferase from Haemophilus
haemolyticus; M‚PVuII, DNA methyltransferase from Proteus Vulgaris;
DpnM, DNA methyltransferase from Streptococcus pneumoniae; M‚RsrI,
DNA methyltransferase from Rhodopseudomonas sphaeroides; M‚MboII,
DNA methyltransferase from Moraxella boVis; ESI-MS, electrospray
ionization mass spectrum/spectrometry; TLC, thin-layer chromatography.
(2) Thuong, N. T.; He´le`ne, C. Angew. Chem. 1993, 105, 697-723; Angew.
Chem., Int. Ed. Engl. 1993, 32, 666-690.
(3) Wittung, P.; Nielsen, P. E.; Buchhardt, O.; Egholm, M.; Norde´n, B. Nature
1994, 368, 561-563.
(4) White, S.; Szewczyk, J. W.; Turner, J. M.; Baird, E. E.; Dervan, P. B.
Nature 1998, 391, 468-471.
(5) Jamieson, A. C.; Kim, S.-H.; Wells, J. A. Biochemistry 1994, 33, 5689-
5695. Rebar, E. J.; Pabo, C. O. Science 1994, 263, 671-673. Wu, H.; Yang,
W.-P.; Barbas, C. F., III. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 344-
348. Greisman, H. A.; Pabo, C. O. Science 1997, 275, 657-661.
(6) Kessler, C. In Nonisotopic Probing, Blotting, and Sequencing; Kricka, L.
J., Ed.; Academic Press: San Diego, 1995; pp 41-109.
(7) Cheng, X. Annu. ReV. Biophys. Biomol. Struct. 1995, 24, 293-318.
9
3486
J. AM. CHEM. SOC. 2003, 125, 3486-3492
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Sequence-Specific Labeling of DNA
A R T I C L E S
Scheme 1. Reactions Catalyzed by DNA MTasesa
Figure 1. Model of N-adenosylaziridine (2) bound in the cofactor binding
site of M‚TaqI. The enzyme is shown as an electrostatic surface potential
structure (red denotes negative potential, blue denotes positive potential,
and white is neutral). The extrahelical target adenine residue and the cofactor
are presented as stick models. The overall model was prepared by replacing
the 2-aminoethylthio side chain of the bound cofactor analogue 5′-[2-
(amino)ethylthio]-5′-deoxyadenosine from the ternary complex structure12
with aziridine. The solvent-exposed 8-position of the cofactor adenine ring
is indicated by a white star.
a DNA MTases activate nitrogen 6 of adenine, nitrogen 4 or carbon 5 of
cytosine within their recognition sequences for nucleophilic attack onto the
methyl group of AdoMet (1) resulting in methyl-group transfer (left) or for
nucleophilic ring opening of the protonated aziridine group of N-adenosyl-
aziridine (2) leading to sequence-specific coupling of the whole cofactor
with DNA (right).
stranded 5′-TCGA-3′ DNA sequence by the adenine-specific
DNA MTase from Thermus aquaticus (M‚TaqI) (Scheme 1,
right).8 We have extended this work and now report on the
design and synthesis of a fluorescent derivative of N-adenosyl-
aziridine (2) which is used in combination with M‚TaqI to
sequence-specifically deliver a fluorophore to DNA.
This prediction was tested by synthesizing the N-adenosyl-
aziridine derivative 3 which contains the dansyl fluorophore
connected to the 8-position via a diaminobutyl linker (Scheme
2). 8-Bromo-2′,3′-O-isopropylideneadenosine (4) was prepared
according to a literature procedure13 and treated with 1,4-
diaminobutane to yield the primary amine 5. Coupling of the
dansyl fluorophore to the amine 5 was achieved by transient
protection of the 5′-hydroxyl group using trimethylsilyl chloride
followed by reaction with dansyl chloride. Activation of the
5′-hydroxyl group of the fluorescent nucleoside 6 using mesyl
chloride led to mesylate 7 which was deprotected in aqueous
formic acid. Finally, nucleophilic substitution of the deprotected
mesylate 8 with aziridine yielded the fluorescent N-adenosyl-
aziridine derivative 3.
Binding of the Fluorescent N-Adenosylaziridine Derivative
3 to M‚TaqI. Binding of 3 to M‚TaqI was investigated by direct
fluorescence titration. To a buffered solution of 3 increasing
amounts of M‚TaqI were added, and the dansyl fluorescence
was monitored. With rising protein concentrations the fluores-
cence intensity of the dansyl group increased about 2.5-fold until
saturation was observed (data not shown). Fitting the real
solution of the quadratic equation for one binding site14 to the
obtained titration data yielded a dissociation constant KD of 0.47
( 0.05 µM. In comparison with the natural cofactor AdoMet
(1), which binds to M‚TaqI with a KD-value of about 2.0 µM,11
the fluorescent N-adenosylaziridine derivative 3 binds about
4-fold better to M‚TaqI. This result is in line with our design
and shows that modification of the 8-position of the cofactor 2
does not hinder but, in contrast, enhances binding to the enzyme.
Since 3 is more hydrophobic than AdoMet (1), this enhanced
binding affinity might not result from an additional specific
interaction of the dansyl group with the enzyme but could well
be the consequence of a hydrophobic effect.
Results and Discussion
Design and Synthesis of a New Fluorescent Cofactor for
M‚TaqI. We planned to use the cofactor 2, which already
contains the reactive aziridine group for DNA MTase-catalyzed
coupling and the adenosyl moiety for cofactor binding,9 as a
delivery system for a fluorescent reporter group. The most
critical part for the design of a new fluorescent cofactor appeared
to be the choice of a suitable attachment position where the
fluorophore attached via a flexible linker would not sterically
interfere with binding to the enzyme. The three-dimensional
structures of M‚TaqI in complex with the natural cofactor
AdoMet (1),10,11 as well as with DNA and a cofactor analogue,12
were determined by X-ray analysis. Figure 1 shows the cofactor
binding site of M‚TaqI with the extrahelical target adenine
residue and N-adenosylaziridine (2) which was modeled in by
replacing the cofactor analogue from the ternary complex
structure. From this model it appears that the adenine 8-position
of the bound cofactor is not recognized by M‚TaqI and is
accessible from the solvent. Thus, it is expected that attaching
a reporter group at the 8-position of N-adenosylaziridine (2)
should not interfere with cofactor binding.
(8) Pignot, M.; Siethoff, C.; Linscheid, M.; Weinhold, E. Angew. Chem. 1998,
110, 3050-3053; Angew. Chem., Int. Ed. 1998, 37, 2888-2891.
(9) Pignot, M,; Pljevaljcic, G.; Weinhold, E. Eur. J. Org. Chem. 2000, 549-
555.
(10) Labahn, J.; Granzin, J.; Schluckebier, G.; Robinson, D. P.; Jack, W. E.;
Schildkraut, I.; Saenger, W. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 10957-
10961.
(11) Schluckebier, G.; Kozak, M.; Bleimling, N.; Weinhold, E.; Saenger, W. J.
Biol. Chem. 1997, 265, 56-67.
(12) Goedecke, K.; Pignot, M.; Goody, R. S.; Scheidig, A. J.; Weinhold, E.
Nat. Struct. Biol. 2001, 8, 121-125.
(13) Ikehara, M.; Tada, H.; Kaneko, M. Tetrahedron 1968, 24, 3489-3498.
(14) Reinstein, J.; Vetter, I. R.; Schlichting, I.; Ro¨sch, P.; Wittinghofer, A.;
Goody, R. S. Biochemistry 1990, 29, 7440-7450.
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A R T I C L E S
Pljevaljcic et al.
Scheme 2. Synthesis of the Fluorescent N-Adenosylaziridine
Derivative 3a
Scheme 3. M‚TaqI-Catalyzed Coupling of the N-Adenosylaziridine
Derivative 3 with a Short Duplex Oligodeoxynucleotidea
a The recognition sequence of M‚TaqI is printed in bold face (AMe
N6-methyl-2′-deoxyadenosine).
)
anion-exchange HPLC. Almost complete conversion to a new
fluorescent product with a retention time of 8.0 min was
observed (Figure 2B). In the absence of M‚TaqI no conversion
of the duplex 9‚10 could be detected (data not shown), indicating
that the observed reaction is enzyme-catalyzed. Interestingly,
the fluorescent product duplex 9‚11 eluted in complex with
M‚TaqI which was inferred from the small retention time and
a reduced UV-absorption ratio (260 to 280 nm, not shown).
Such a stable complex formation is not surprising because the
M‚TaqI-catalyzed reaction should lead to a covalently linked
product which should bind much more tightly to the enzyme
than the individual products of the natural methyl-group transfer
reaction. The fluorescent duplex 9‚11 was released from the
complex by fragmentation of M‚TaqI with proteinase K. As
can be seen from the anion-exchange chromatogram shown in
Figure 2C, the fluorescent peak at 8.0 min disappeared
completely upon proteinase K treatment, and the fluorescent
duplex 9‚11 eluted with a retention time of 27.5 min. For further
characterization the duplex 9‚11 was isolated and treated with
an enzyme mix consisting of DNaseI, phosphodiesterase from
Crotalus durissus, phosphodiesterase from calf spleen, and
alkaline phosphatase. The DNA fragmentation reaction was
analyzed by reverse-phase HPLC (Figure 2D) and revealed
besides the deoxynucleosides dC, dI (formed from dA by
contaminating adenosine deaminase activity), dG, T, and dAMe
a new compound eluting after 47.8 min. This new compound
was isolated and detected as positively charged ion at m/z 863.1
by electrospray ionization mass spectrometry (ESI-MS). The
observed mass is in good agreement with the calculated
molecular mass (863.4) of a protonated, with 3 modified 2′-
deoxyadenosine (dADans). This analysis demonstrates that the
fluorescent N-adenosylaziridine derivative 3 acts as a new
cofactor for M‚TaqI and can be used for fluorescence labeling
of DNA.
a
(a) 1,4-Diaminobutane, TEA, 99%. (b) 1. Trimethylsilyl chloride,
pyridine. 2. Dansyl chloride, 30%. (c) Mesyl chloride, TEA, DMAP, 43%.
(d) Formic acid, H2O (1:1), 55%. (e) Aziridine, EDIA, 36%.
Labeling of a Short Duplex Oligodeoxynucleotide. Fol-
lowing the binding study we tested whether the fluorescent
N-adenosylaziridine derivative 3 functions as a cofactor for the
DNA MTase M‚TaqI (Scheme 3). A short hemimethylated
duplex oligodeoxynucleotide (9‚10, 14 base pairs) containing
the 5′-TCGA-3′ recognition sequence of M‚TaqI was prepared
by annealing strand 9 with the methylated complementary strand
10 containing N6-methyl-2′-deoxyadenosine (AMe) instead of
2′-deoxyadenosine within the recognition sequence. Although
M‚TaqI also alkylates nonmethylated recognition sequences (see
labeling of plasmid DNA below) such a hemimethylated duplex
better resembles the natural hemimethylated substrate which is
formed after semiconservative DNA replication.
The duplex 9‚10 was analyzed by anion-exchange HPLC
(Figure 2A) and eluted after 23.2 min, and no additional peak
was observed, indicating that complete hybridization had
occurred. After incubation of the duplex 9‚10 with stoichiometric
amounts of M‚TaqI and excess of the fluorescent N-adenosyl-
aziridine derivative 3 the coupling reaction was monitored by
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Sequence-Specific Labeling of DNA
A R T I C L E S
Figure 2. Labeling of the short duplex oligodeoxynucleotide 9‚10 (10 µM) with the new fluorescent cofactor 3 (20 µM) and M‚TaqI (10 µM). (A) Anion-
exchange HPLC of the starting material 9‚10. (B) Analysis of the coupling reaction (analytical scale) after 15 h at 37 °C by anion-exchange HPLC (solid
line: UV-detection; dotted line: fluorescence detection, λex ) 330 nm, λem ) 551 nm). (C) Analysis of the coupling reaction (preparative scale) by anion-
exchange HPLC after additional proteinase K treatment (37 °C for 1 h; solid line: UV-detection, dotted line: fluorescence detection, λex ) 330 nm, λem
)
551 nm). (D) Reverse-phase HPLC analysis of the product duplex 9‚11 after enzymatic fragmentation with DNaseI, phosphodiesterase from Crotalus durissus,
phosphodiesterase from calf spleen and alkaline phosphatase (37 °C for 20 h). In addition to the nucleosides dC, dI, dG, T, and dAMe, the dansylated
nucleoside (dADans) was observed (dI was formed from dA by contaminating adenosine deaminase activity present in the commercial phosphodiesterase
from calf spleen).
Figure 3. Labeling of pUC19 plasmid DNA (28 nM, four M‚TaqI recognition sites) with the new fluorescent cofactor 3 (20 µM) and M‚TaqI (133 nM)
at 65 °C analyzed by anion-exchange HPLC. (A) UV-detection. (B) Detection of the dansyl fluorescence (λex ) 330 nm, λem ) 551 nm). Since fluorescence
detection was performed after UV-detection, a small delay of the retention time compared to A was observed.
Labeling of pUC19 Plasmid DNA. We also tested whether
the new fluorescent cofactor 3 in combination with M‚TaqI can
be used to label long DNA molecules. The plasmid pUC19
which contains four recognition sites for M‚TaqI was incubated
with 3 and M‚TaqI and the labeling reaction followed by anion-
exchange HPLC (Figure 3). The plasmid eluted after 21.9 min
(UV-detection), and the integration of the corresponding signal
was constant over the incubation time of 8 h (Figure 3A). Only
a small broadening of the peak shape was observed during the
first 2 h. Most interestingly, detection of the dansyl fluorescence
revealed that the plasmid was fluorescently labeled with time,
and completion of the reaction was reached after 4 h (Figure
3B). Labeling of plasmid DNA was faster than labeling of the
short duplex 9‚10 because of the higher incubation temperature.
In a parallel experiment where M‚TaqI was omitted, labeling
of the plasmid was not observed (data not shown). This result
demonstrates that the labeling reaction is enzyme-catalyzed and
not a result of a direct reaction between 3 and DNA. To
investigate the specificity of the labeling reaction we performed
a control experiment in which pUC19 was first methylated using
M‚TaqI and the natural cofactor AdoMet (1), isolated, and then
incubated with the fluorescent cofactor 3 and M‚TaqI. Since
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A R T I C L E S
Pljevaljcic et al.
phosphodiesterase from Crotalus durissus and from calf spleen were
from Roche Diagnostics. Oligodeoxynucleotides 9 and 10 were
synthesized by MWG-Biotech, Ebersberg, Germany, and pUC19
plasmid DNA was purchased from New England Biolabs.
M‚TaqI catalyzes the methylation of each adenine residue within
the palindromic 5′-TCGA-3′ recognition sequence only once,
methylation of the recognition sequences in pUC19 should block
further alkylation by M‚TaqI and 3. Anion-exchange HPLC
analysis of this control experiment revealed no fluorescence
signal for the plasmid (data not shown). From this result we
conclude that the sequence specificity of M‚TaqI is not lowered
with the new fluorescent cofactor 3. Given the high sequence
specificity of M‚TaqI (the second-best found site is methylated
about 100000-fold slower than the canonical recognition site
by M‚TaqI; unpublished result), DNA labeling with M‚TaqI
should be highly specific.
General Procedures. All chemical reactions were carried out in
oven-dried glassware under an argon atmosphere, except for the
deprotection of the nucleoside 7. DC-alumina plates Sil-G60/UV254
(Merck) were used for TLC. Compounds were visualized by treatment
with a solution of ammonium molybdate (15 g) and cerium(IV) sulfate
(0.4 g) in 10% sulfuric acid (300 mL) and subsequent heating with a
heat gun. Flash chromatography was carried out using Merck silica
gel 60 (40-63 µm). HPLC was performed using a Beckman System
Gold equipped with a programmable solvent module 125, a diode array
detector module 168, an analogue interface module 406, and a Shimadzu
spectrofluorometric detector RF-551. NMR spectra were obtained using
Conclusions and Outlook
1
a Bruker AX 500 (500 and 125.7 MHz, for H and 13C, respectively).
This work illustrates the successful design of the new
fluorescent MTase cofactor 3 which is sequence-specifically
coupled with DNA using the DNA MTase M‚TaqI and provides
a novel method for sequence-specific labeling of large DNA
fragments. It is likely that derivatives of 3 containing other
reporter groups instead of the fluorescent dansyl group will also
function as cofactors for M‚TaqI and could be used for
sequence-specific labeling of DNA with a variety of reporter
groups. Furthermore, preliminary experiments in our laboratory
indicate that N-adenosylaziridine (2) is also a cofactor for N4-
and C5-cytosine DNA MTases. Thus, other DNA MTases with
different recognition sequences might also use the new fluo-
rescent cofactor 3, provided that, as in M‚TaqI, the 8-position
of the cofactor adenine ring is accessible from the solvent.
Inspection of the known three-dimensional structures of other
DNA MTases, such as M‚HhaI,15 M‚PVuII,16 DpnM,17 M‚RsrI,18
and M‚MboII,19 in complex with AdoMet (1) or adenosine
derivatives reveals that this appears to be the case in most DNA
MTases. Thus, the new fluorescent cofactor 3 or derivatives
with other reporter groups could be useful for labeling a wide
variety of DNA sequences. This novel technique named
sequence-specific methyltransferase-induced labeling of DNA
(SMILing DNA) could find interesting applications in functional
studies of DNA modifying enzymes, molecular biology, DNA-
based medical diagnosis, and nanobiotechnology.
CDCl3 (δH ) 7.24 and δC ) 77.0) or [d6]DMSO (δH ) 2.49 and δC )
39.5) was used as solvent. Assignment of 13C signals are based on 1H,
13C-correlated 2D-NMR and on 13C-DEPT spectra. Electrospray ioniza-
tion mass spectra (ESI-MS) were obtained using a Finnigan LCQ
connected to a nano-electrospray ion source.22 Measurements were
carried out in the positive ion mode. The samples were dissolved in
CH3OH/water (1:1 v/v) containing 5% formic acid. UV-absorption
measurements were performed in water using a Varian Cary 3E
spectrometer. Fluorescence data were collected with a SLM-Aminco
AB2 spectrofluorometer. The excitation and emission bandwidths were
adjusted to 2 and 8 nm, respectively.
8-Amino[1′′-(4′-aminobutyl)]-2′,3′-O-isopropylideneadenosine (5).
To a solution of 8-bromo-2′,3′-O-isopropylideneadenosine (4) (628 mg,
1.6 mmol) in dry DMSO (10 mL) were added dry triethylamine (2.26
mL, 16.3 mmol) and 1,4-diaminobutane (0.82 mL, 8.1 mmol). The
solution was stirred at 110 °C and the reaction progress monitored by
TLC. After 4 h the solvent was removed under reduced pressure. The
residue was dissolved in water (50 mL) and the pH adjusted to 5.3
with acetic acid (0.1 M). The crude product was purified by cation-
exchange chromatography (Dowex 50 × 4, H+-form, 100 g, elution
with 600 mL of water followed by 1000 mL of 1 M potassium
hydroxide). Fractions containing the product were extracted with
chloroform. The organic layers were combined, and the solvent was
removed under reduced pressure to yield compound 5 (639 mg, 99%)
as a white solid (Rf 0.44, butanol/acetic acid/water 12:3:5): 1H NMR
(500 MHz, CDCl3): δ 1.33 (s, 3H; acetonide-H), 1.48-1.55 (m, 2H;
linker-H), 1.61 (s, 3H; acetonide-H), 1.64-1.70 (m, 2H; linker-H),
2.66-2.73 (m, 2H; linker-H), 3.33-3.42 (m, 2H; linker-H), 3.77-
Experimental Section
3
3.91 (m, 2H; 5′-H), 4.28-4.30 (m, 1H; 4′-H), 4.99 (dd, J ) 2.7, 6.3
3
Hz, 1H; 3′-H), 5.08 (dd, J ) 4.8, 6.3 Hz, 1H; 2′-H), 5.39 (s, br, 2H;
Materials. 8-Bromo-2′,3′-O-isopropylideneadenosine (4) and aziri-
dine were prepared according to literature procedures.13,20 (CAUTION:
Aziridine is hazardous and should be handled with care.) 1,4-Diamino-
butane was purchased from Aldrich, and dansyl chloride, trimethysilyl
chloride, mesyl chloride, and N-ethyldiisopropylamine were purchased
from Fluka. All reagents were of p.a. grade. Dry solvents were either
purchased or dried using common laboratory techniques.21 The DNA
MTase M‚TaqI was prepared as described before.12 Proteinase K was
obtained from Merck, and DNase I (grade II), alkaline phosphatase,
3
6-NH2), 6.15 (d, J ) 4.5 Hz, 1H; 1′-H), 6.55-6.60 (m, 1H; 8-NH),
8.10 (s, 1H; 2-H); 13C NMR (125.7 MHz, CDCl3): δ 25.30 (q;
acetonide-CH3), 25.73 (t; linker-C), 27.42 (q; acetonide-CH3), 29.60
(t; linker-C), 40.46 (t; linker-C), 42.69 (t; linker-C), 61.17 (t; 5′-C),
80.59 (d; 3′-C), 82.19 (d; 2′-C), 84.48 (d; 4′-C), 89.21 (d; 1′-C), 114.50
(s; acetonide-C(CH3)2), 117.68 (s; 5-C), 149.49 (d; 2-C), 149.95 (s;
8-C), 151.68 (s; 4-C), 151.72 (s; 6-C); ESI-MS m/z (relative inten-
sity): 394.3 (25) [M + H]+, 222.3 (100) [adenine + aminobutyl +
H]+.
8-Amino[1′′-(N′′-dansyl)-4′′-aminobutyl]-2′,3′-O-isopropylidene-
adenosine (6). To a solution of amine 5 (104 mg, 0,26 mmol) in dry
pyridine (7 mL) was added slowly trimethylsilyl chloride (0.07 mL,
0.53 mmol) at 0 °C, and the solution was stirred at room temperature
for 1 h. Subsequently, dansyl chloride (103.8 mg, 0.37 mmol, dissolved
in 3 mL of dry pyridine) was added, and the solution was stirred at
room temperature for 4 h. The progress of the reaction was monitored
by TLC. After complete conversion the solution was treated with water
(5 mL) at 0 °C, and the solvent was removed under reduced pressure.
(15) Klimasauskas, S.; Kumar, S.; Roberts, R. J.; Cheng, X. Cell 1994, 76, 357-
369.
(16) Gong, W.; O’Gara, M.; Blumenthal, R. M.; Cheng, X. Nucleic Acids Res.
1997, 25, 2702-2715.
(17) Tran, P. H.; Korszun, Z. R.; Cerritelli, S.; Springhorn, S. S.; Lacks, S. A.
Structure 1998, 6, 1563-1575.
(18) Scavetta, R. D.; Thomas, C. B.; Walsh, M. A.; Szegedi, S.; Joachimiak,
A.; Gumport, R. I.; Churchill, M. E. A. Nucleic Acids Res. 2000, 28, 3950-
3961.
(19) Osipiuk, J.; Walsh, M. A.; Joachimiak, A. Protein Data Bank, entry number
1G60.
(20) Gabriel, S. Chem. Ber. 1888, 21, 2664-2669. Gabriel, S.; Stelzner, R.
Chem. Ber. 1895, 28, 2929-2938.
(21) Perrin, D. D.; Armarego, W. L. F. Purification of Laboratory Chemicals,
3rd ed.; Pergamon Press: Oxford, 1988.
(22) Wilm, M.; Mann, M. Anal. Chem. 1996, 68, 1-8.
9
3490 J. AM. CHEM. SOC. VOL. 125, NO. 12, 2003

Sequence-Specific Labeling of DNA
A R T I C L E S
The crude product was purified by column chromatography (silica gel,
40 g, elution with 5% methanol in methylene chloride) to give
nucleoside 6 (50 mg, 30%) as a yellow fluorescent solid (Rf 0.54, 10%
methanol in methylene chloride): 1H NMR (500 MHz, [d6]DMSO):
δ 1.29 (s, 3H; acetonide-H), 1.39-1.43 (m, 2H; linker-H), 1.47-1.50
(m, 2H; linker-H), 1.53 (s, 3H; acetonide-H), 2.78-2.82 (m, 8H;
linker-H and N(CH3)2), 3.16-3.24 (m, 2H; linker-H), 3.50-3.58 (m,
H), 4.01-4.04 (m, 1H; 4′-H), 4.33-4.38 (m, 2H; 5′-H), 4.44-4.47
3
(m, 1H; 3′-H), 5.08 (ddd ) q,
3J ) 5.5 Hz, 1H; 2′-H), 5.37 (d, J )
5.5 Hz, 1H; OH), 5.44 (d, 3J ) 5.5 Hz, 1H; OH), 5.72 (d, 3J ) 5.1 Hz,
3
1H; 1′-H), 6.48 (s, br, 2H; 6-NH2), 6.78 (t, J ) 5.3 Hz, 1H; 8-NH),
7.24 (d, 3J ) 7.8 Hz, 1H; arom-H), 7.57 (t, 3J ) 8.3 Hz, 1H; arom-H),
7.61 (t, 3J ) 7.8 Hz, 1H; arom-H), 7.88 (s, 1H; 2-H), 7.95 (t, 3J ) 5.7
Hz, 1H; NHSO2), 8.08 (d, 3J ) 6.9 Hz, 1H; arom-H), 8.28 (d, 3J ) 8.7
Hz, 1H; arom-H), 8.44 (d, 3J ) 8.7 Hz, 1H; arom-H); 13C NMR (125.7
MHz, [d6]DMSO): δ 27.24 (t; linker-C), 28.06 (t; linker-C), 37.91 (q;
SO2CH3), 43.07 (t; linker-C), 43.58 (t; linker-C), 46.41 (q; N(CH3)2),
71.21 (t; 5′-C), 71.45 (d; 3′-C), 71.61 (d; 2′-C), 82.20 (d; 4′-C), 88.63
(d; 1′-C), 116.44 (d; arom-C), 118.76 (s), 120.46 (d; arom-C), 124.98
(d; arom-C), 129.16 (d; arom-C), 129.54 (d; arom-C), 130.34 (s), 130.39
(d; arom-C), 130.68 (s), 137.35 (s), 149.95 (d; 2-C), 150.78 (s), 152.66
(s), 153.06 (s), 153.73 (s); ESI-MS m/z (relative intensity): 665.6 (85)
[M + H]+, 687.4 (100) [M + Na]+.
3
2H; 5′-H), 4.12-4.14 (m, 1H; 4′-H), 4.94 (dd, J ) 2.7, 6.1 Hz, 1H;
3
3′-H), 5.33 (dd, J ) 3.7, 6.1 Hz, 1H; 2′-H), 5.41-5.44 (m, 1H; 5′-
OH), 6.01 (d, 3J ) 3.5 Hz, 1H; 1′-H), 6.49 (s, br, 2H; 6-NH2), 6.85 (t,
3
3J ) 5.0 Hz, 1H; 8-NH), 7.22 (d, J ) 7.5 Hz, 1H; arom-H), 7.54-
7.61 (m, 2H; arom-H), 7.87-7.90 (m, 1H; NHSO2), 7.90 (s, 1H; 2-H),
8.08 (d, 3J ) 7.2 Hz, 1H; arom-H), 8.30 (d, 3J ) 8.5 Hz, 1H; arom-H),
8.43 (d, 3J ) 8.5 Hz, 1H; arom-H); 13C NMR (125.7 MHz, [d6]DMSO):
δ 25.42 (q; acetonide-CH3), 26.00 (t; linker-C), 26.96 (t; linker-C),
27.33 (q; acetonide-CH3), 41.92 (t; linker-C), 42.43 (t; linker-C), 45.21
(q; N(CH3)2), 61.40 (t; 5′-C), 81.14 (d; 3′-C), 81.50 (d; 2′-C), 85.29
(d; 4′-C), 87.85 (d; 1′-C), 113.38 (s), 115.24 (d; arom-C), 117.24 (s),
119.29 (d; arom-C), 123.72 (d; arom-C), 127.92 (d; arom-C), 128.31
(d; arom-C), 129.26 (s), 129.48 (d; arom-C), 136.27 (s), 148.89 (d;
2-C), 149.30 (s), 151.20 (s), 151.50 (s), 152.58 (s); ESI-MS m/z (relative
intensity): 627.1 (100) [M + H]+, 455.2 (8) [adenine + linker + dansyl
+ H]+.
8-Amino[1′′-(N′′-dansyl)-4′′-aminobutyl]-5′-(1-aziridinyl)-5′-deoxy-
adenosine (3). Nucleoside 8 (20 mg, 30 µmol) was dissolved in dry
aziridine (1 mL) and N-ethyldiisopropylamine (350 µL) and the solution
stirred at room temperature for 3 d. The reaction was monitored by
analytical reverse-phase HPLC (Hypersil-ODS, 5 µm, 120 Å, 250 mm
× 4.6 mm, Bischoff, Leonberg, Germany). Compounds were eluted
with acetonitrile (0% for 5 min, followed by a linear gradient to 35%
in 30 min and to 70% in 10 min) in triethylammonium acetate buffer
(0.1 M, pH 7.0) and a flow rate of 1 mL/min. After the reaction was
complete, the solvent was removed under reduced pressure, and the
crude product was purified by column chromatography (silica gel, 2 g,
elution with 10% methanol in methylene chloride) to give nucleoside
3 (6.7 mg, 36%) as a yellow fluorescent solid (Rf 0.23, 10% methanol
8-Amino[1′′-(N′′-dansyl)-4′′-aminobutyl]-2′,3′-O-isopropylidene-
5′-O-mesyladenosine (7). To a solution of nucleoside 6 (181 mg, 0.32
mmol) and (dimethylamino)pyridine (40 mg, 0.32 mmol) in dry
methylene chloride (20 mL) was added dry triethylamine (1.1 mL, 8.0
mmol) and the solution cooled to 0 °C. Mesyl chloride (200 µL, 2.6
mmol) was added, and the solution was stirred for 30 min. The reaction
was quenched with a cold, saturated sodium hydrogencarbonate solution
(5 mL). The solution was extracted three times with cold chloroform
(10 mL), the organic phases were combined, and the solvent was
removed under reduced pressure. The crude product was purified by
column chromatography (silica gel, 40 g, elution with 3% methanol in
methylene chloride) to give nucleoside 7 (96 mg, 43%) as a yellow
1
in methylene chloride); H NMR (500 MHz, [d6]DMSO): δ 1.19-
1.22 (m, 2H; aziridine-H), 1.32-1.34 (m, 2H; linker-H), 1.37-1.39
(m, 2H; linker-H), 1.59-1.61 (m, 2H; aziridine-H), 1.94 (dd, 3J ) 3.2
2
Hz, J ) 13.5 Hz, 1H; 5′-Ha), 2.74-2.79 (m, 2H; linker-H), 2.81 (s,
6H; N(CH3)2), 2.91-2.95 (m, 1H; 5′-Hb), 3.07-3.16 (m, 2H; linker-
H), 3.94-3.96 (m, 1H; 4′-H), 4.19-4.21 (m, 1H; 3′-H), 4.63-4.67
1
fluorescent solid (Rf 0.55, 10% methanol in methylene chloride); H
3
3
(m, 1H; 2′-H), 5.20 (d, J ) 4.1 Hz, 1H; OH), 5.30 (d, J ) 6.8 Hz,
NMR (500 MHz, CDCl3): δ 1.37 (s, 3H; acetonide-H), 1.45-1.48 (m,
2H; linker-H), 1.59-1.61 (m, 5H; linker-H and acetonide-H), 2.85 (s,
6H; N(CH3)2), 2.96 (s, 3H; SO2CH3), 2.98-3.02 (m, 2H; linker-H),
3.32-3.36 (m, 2H; linker-H), 4.33-4.43 (m, 3H; 5′-H and 4′-H), 5.03
(m, 1H; 3′-H), 5.52 (dd, 3J ) 2.5, 6.5 Hz, 1H; 2′-H), 6.04 (d, 3J ) 2.5
Hz, 1H; 1′-H), 6.13 (s, br, 2H; 6-NH2), 6.91 (t, 3J ) 5.8 Hz, 1H; 8-NH),
7.13 (d, 3J ) 7.3 Hz, 1H; arom-H), 7.43 (t, 3J ) 8.2 Hz, 1H; arom-H),
7.50 (t, 3J ) 7.9 Hz, 1H; arom-H), 8.10 (s, 1H; 2-H), 8.23 (d, 3J ) 7.0
Hz, 1H; arom-H), 8.37 (d, 3J ) 8.5 Hz, 1H; arom-H), 8.51 (d, 3J ) 8.6
Hz, 1H; arom-H); 13C NMR (125.7 MHz, CDCl3): δ 24.62 (q;
acetonide-CH3), 25.30 (t; linker-C), 26.89 (t; linker-C), 27.04 (q;
acetonide-CH3), 37.50 (q; SO2CH3), 41.58 (t; linker-C), 42.70 (t; linker-
C), 45.44 (q; N(CH3)2), 68.38 (t; 5′-C), 80.10 (d; 3′-C), 82.11 (d; 2′-
C), 83.29 (d; 4′-C), 88.63 (d; 1′-C), 115.16 (d; arom-C), 118.94 (d;
arom-C), 123.23 (d; arom-C), 128.20 (d; arom-C), 129.70 (d; arom-
C), 130.37 (d; arom-C), 149.78 (d; 2-C), 151.84 (s), 152.41 (s); ESI-
MS m/z (relative intensity): 705.3 (70) [M + H]+, 609.7 (100)
[cyclonucleoside + H]+.
8-Amino[1′′-(N′′-dansyl)-4′′-aminobutyl]-5′-O-mesyladenosine (8).
Nucleoside 7 (96.2 mg, 0.14 mmol) was dissolved in aqueous formic
acid (50%, 10 mL), and the resulting solution was stirred at room
temperature for 4 d. After complete conversion the solvent was removed
under reduced pressure, and remaining solvent was coevaporated with
a mixture of water and methanol (1:1, 3 × 5 mL). The crude product
was purified by column chromatography (silica gel, 15 g, elution with
10% methanol in methylene chloride) to give nucleoside 8 (49.2 mg,
55%) as a yellow fluorescent solid (Rf 0.23, 10% methanol in methylene
chloride); 1H NMR (500 MHz, [d6]DMSO): δ 1.36-1.42 (m, 2H;
linker-H), 1.47-1.53 (m, 2H; linker-H), 2.77-2.79 (m, 2H; linker-H),
2.81 (s, 6H; N(CH3)2), 3.07 (s, 3H; SO2CH3), 3.17-3.20 (m, 2H; linker-
3
1H; OH), 5.90 (d, J ) 7.2 Hz, 1H; 1′-H), 6.42 (s, br, 2H; 6-NH2),
7.23 (d, J ) 7.2 Hz, 1H; arom-H), 7.55-7.61 (m, 3H; arom-H and
8-NH), 7.87 (s, 1H; 2-H), 7.95 (t, J ) 5.6 Hz, 1H; NHSO2), 8.08 (d,
3
3
3J ) 7.2 Hz, 1H; arom-H), 8.28 (d, 3J ) 8.6 Hz, 1H; arom-H), 8.43 (d,
3J ) 8.6 Hz, 1H; arom-H); 13C NMR (125.7 MHz, [d6]DMSO): δ
26.92 (t; aziridine-C), 27.43 (t; linker-C), 28.01 (t; linker-C), 30.02 (t;
aziridine-C), 43.02 (t; linker-C), 43.65 (t; linker-C), 46.41 (q; N(CH3)2),
62.96 (t; 5′-C), 71.14 (d; 2′-C), 72.29 (d; 3′-C), 85.31 (d; 4′-C), 87.11
(d; 1′-C), 116.45 (d; arom-C), 118.20 (s), 120.45 (d; arom-C), 124.96
(d; arom-C), 129.16 (d; arom-C), 129.57 (d; arom-C), 130.00 (s), 130.36
(d; arom-C), 130.68 (s), 137.37 (s), 149.86 (d; 2-C), 151.49 (s), 152.42
(s), 152.66 (s), 153.43 (s); ESI-MS m/z (relative intensity): 612.7 (100)
[M + H]+.
Fluorescence Titration. To a solution of cofactor 3 (1 µM, based
on a molecular extinction coefficient ꢀ335 ) 4600 cm-1 M-1 for the
dansyl group) in buffer A (20 mM Tris acetate, pH 7.9, 50 mM
potassium acetate, 10 mM magnesium acetate, 1 mM DTT and 0.01%
Triton X-100) were added increasing amounts of M‚TaqI in buffer A
containing cofactor 3 (1 µM) at 25 °C, and the dansyl fluorescence
intensity (λex ) 330 nm, λem ) 551 nm) was monitored after each
addition. Fitting the real solution of the quadratic equation for one
binding site14 to the titration data was performed with the data analysis
program GraFit.23
Fluorescence Labeling of Duplex Oligodeoynucleotide 9‚10 and
Analysis of the Product Duplex 9‚11. Molecular extinction coefficients
at 260 nm of oligodeoynucleotides 9 and 10 were calculated according
(23) Leatherbarrow, R. J. 1992, GraFit, Version 3.0; Erithacus Software Ltd.:
Staines, U.K.
9
J. AM. CHEM. SOC. VOL. 125, NO. 12, 2003 3491

A R T I C L E S
Pljevaljcic et al.
to the nearest-neighbor method,24 and concentrations were determined
by UV spectroscopy. Annealing of the complementary strands 9 and
10 was performed by heating an equimolar solution to 95 °C for 2 min
followed by slow cooling to room temperature.
A solution (500 µL) of duplex 9‚10 (10 µM), cofactor 3 (20 µM),
and M‚TaqI (10 µM) in buffer B (20 mM Tris acetate, pH 6.0, 50 mM
potassium acetate, 10 mM magnesium acetate and 0.01% Triton X-100)
was incubated at 37 °C. After 15 h an aliquot was injected onto an
anion-exchange HPLC column (Poros 10 HQ, 10 µm, 4.6 mm × 100
mm, Applied Biosystems), and compounds were eluted with potassium
chloride (0.2 M for 5 min followed by linear gradients to 0.5 M in 5
min and then to 1.0 M in 30 min) in Tris hydrochloride buffer (10
mM, pH 7.0) at a flow rate of 4 mL/min.
The pH of the reaction solution was adjusted to 8.0 with a potassium
hydroxide solution (10 M). Then, a solution (4 µL) of proteinase K
(31 µg/µL) in Tris hydrochloride buffer (50 mM, pH 8.0) and calcium
chloride (1 mM) was added and the mixture incubated at 37 °C for 1
h. The proteolytic fragmentation was monitored by anion-exchange
HPLC as described above. For further characterization the duplex 9‚
11 was isolated by reverse-phase HPLC (conditions see synthesis of
cofactor 3, retention time: 25.0 min).
phosphatase (13.7 µL, 13.7 U). After incubation at 37 °C for 20 h an
aliquot (100 µL) was injected onto the analytical reverse-phase HPLC
column (see synthesis of cofactor 3), and the products were eluted with
an acetonitrile gradient (0 to 10.5% in 30 min followed by 10.5 to
28% in 10 min and 28 to 70% in 15 min) in triethylammonium acetate
buffer (0.1 M, pH 7.0) at a flow rate of 1 mL/min.
Fluorescence Labeling of pUC19 Plasmid DNA. A solution of
pUC19 DNA (28 nM, four M‚TaqI recognition sites), cofactor 3 (20
µM), and M‚TaqI (133 nM) in buffer B was incubated at 65 °C.
Aliquots were withdrawn after different incubation times and injected
onto an anion-exchange HPLC column (Nucleogen 4000-7 DEAE, 7
µm, 125 mm × 6 mm, Macherey-Nagel, Du¨ren, Germany), and
compounds were eluted with potassium chloride (0.3 M for 5 min
followed by a linear gradient to 1.5 M in 30 min) in potassium
phosphate buffer (30 mM, pH 6.5) containing acetonitrile (20%) at a
flow rate of 1.5 mL/min.
Methylation of pUC19 Plasmid DNA. A solution of pUC19 DNA
(66 nM), 1 (80 µM), and M‚TaqI (7.5 nM) in buffer A containing
bovine serum albumin (1 mg/mL) was incubated at 65 °C for 1 h. The
methylated DNA was purified using the Qiagen PCR Purification Kit.
Enzymatic fragmentation of the purified duplex 9‚11 (0.57 OD at
260 nm) in buffer (228 µL, 10 mM potassium phosphate, pH 7.0, 10
mM magnesium chloride) was carried out by adding DNase I (13.7
µL, 2.7 U), phosphodiesterase from Crotalus durissus (13.7 µL, 41
mU), phosphodiesterase from calf spleen (13.7 µL, 55 mU), and alkaline
Acknowledgment. We thank Nathalie Bleimling for the
preparation of M‚TaqI and Roger Goody for his con-
tinuous support. This work was supported by the Deutsche
Forschungsgemeinschaft.
(24) Cantor, C. R.; Warshaw, M. M.; Shapiro, H. Biopolymers 1970, 9, 1059-
1077.
JA021106S
9
3492 J. AM. CHEM. SOC. VOL. 125, NO. 12, 2003