Nucleic acid-triggered fluorescent probe activation by the staudinger reaction

Article abstract of DOI:10.1021/ja0452626

Highly sequence specific and sensitive detection systems for DNA or RNA in vivo would be of great use in biology and medicine for monitoring gene expression and diagnosing diseased cells. Herein we describe a new nucleic acid-triggered fluorescent probe system that is activated by a biocompatible and chemoselective Staudinger reaction. Copyright

Full text of DOI:10.1021/ja0452626

Published on Web 11/23/2004
Nucleic Acid-Triggered Fluorescent Probe Activation by the Staudinger
Reaction
Jianfeng Cai, Xiaoxu Li, Xuan Yue, and John Stephen Taylor*
Department of Chemistry, Washington UniVersity, One Brookings DriVe, St. Louis, Missouri 63130
Received August 5, 2004; E-mail: taylor@wustl.edu
Scheme 1
Biological and biomedical research could be greatly aided by
probes that can detect specific RNA sequences in vivo with high
sensitivity and selectivity and with low background signal.1 Such
reagents coupled with highly sensitive detection methods2 could
be used to monitor gene expression in cells or whole organisms
and to detect and diagnose diseased or stressed cells. A number of
approaches to developing such probes have been described and
range from molecular beacons that fluoresce upon binding to a
target,3 to pairs of ODNs or analogues that fluoresce through a
Scheme 2a
fluorescent energy transfer mechanism,4 or through chemical release
of a fluorescence molecule5 or a quencher.6 For in vivo use, reagents
that are based on a chemical step require a biocompatible reaction
that can proceed with a high rate and efficiency in water at 37°
and pH 7. To this end, SN2 reactions6a-c and carboxylic acid ester5
and phosphodiester6d hydrolysis reactions have been explored. The
ability of a nucleic acid sequence to trigger the release a molecule
has also been proposed as a possible means of selectively releasing
drugs as well as probes in a target cell containing a unique or
uniquely overexpressed nucleic acid sequence (nucleic acid trig-
gered prodrug or probe activation, or NATPA).5,7 In this report,
we show that the Staudinger reaction can also be used for NATPA
to activate a fluorescent molecule in vitro.
The Staudinger reaction is the reaction between a phosphine and
an azide, which produces a reactive aza-ylide,8 that hydrolyzes
almost spontaneously to give a primary amine and the corresponding
phosphine. Bertozzi and co-workers have described a clever and
useful modification of the Staudinger reaction in which an ester
group on the phosphine captures the aza-ylide intermediate by
intramolecular cyclization, producing an amide efficiently and
selectively under physiological conditions.9 This Staudinger ligation
reaction is biocompatible and orthogonal to most biological
functionalities, and it has found extensive application to peptide
ligation and to both in vitro and in vivo bioconjugation.10 Bertozzi
and co-workers have also shown that a fluorescent dye can be
activated by the Staudinger reaction as a direct result of the change
in the oxidation state of phosphorus.11
We realized that the amide bond formation in the Staudinger
ligation reaction could also be used to activate a probe if one
replaces the methyl ester used in most applications with an ester
of a probe molecule (Scheme 1). To investigate this possibility we
examined the reaction between the monoalkylated fluorescein ester
of 2-carboxytriphenylphosphine 1 with R-azido acetic acid 2
(Scheme 2). Esters of monoalkylated fluoresceins are not fluores-
cent, but become fluorescent when hydrolyzed.12
a Reaction conditions: (i) DCC, DMAP, CH2Cl2, 12h, room temperature,
91%; (ii) 1:1 CF3COOH/CH2Cl2, 2h, 0 °C then room temperature, 93%;
(iii) N3CH2CO2H, 2:5 CH3CN/H2O, 10 mM phosphate buffer, pH ) 7.0,
room temperature, 2 h.
acid was added, a time and concentration dependent increase in
absorbance was observed (Figure 1). The apparent second-order
rate constant for this reaction was calculated to be 0.017 M-1 s-1
(1.0 × 10-6 µM-1 min-1), which is quite close to that reported for
related reactions.11,14
To test the suitability of the Staudinger ligation reaction for
NATPA we designed the three component system shown in Scheme
3 in which ester 1 was tethered to the amino terminus of one peptide
nucleic acid (PNA), and azidoacetic acid 2 was tethered to the
carboxy terminus of another PNA through the ꢀ-amino group of
lysine. A Staudinger ligation reaction between 6 and 7 would be
expected to produce PNA 9 and fluorescent PNA 10. PNA was
chosen because of its higher affinity toward complementary DNA
and RNA than DNA,15 its ability to invade regions of secondary
structure,16 its inability to cause degradation of the target RNA by
RNaseH,17 and its superior stability in serum and in cells.18
Additionally, we have found herein that PNA coupling and final
deprotection conditions are compatible with the ester being used.
PNA 7 was prepared by standard Fmoc-PNA synthesis with a
carboxy terminal Mtt-protected lysine, which was selectively
deprotected with 2% dichloroacetic acid and then coupled to
azidoacetic acid in the presence of HATU/2,6-lutidine/DIEA prior
to cleavage from the resin and complete deprotection with TFA/
Ester 1 was prepared from the tert-butyl ester of mono-O-
carboxymethylfluorescein
4 by coupling with 2-carboxytri-
phenylphosphine in the presence of DCC to form 5, followed by
deprotection with TFA/CH2Cl2 (Scheme 2). Azidoacetic acid was
synthesized by a published procedure.13 No significant increase in
absorbance at 454 nm indicative of 3 was observed for ester 1 when
incubated in aqueous acetonitrile at neutral pH, but when azidoacetic
9
16324
J. AM. CHEM. SOC. 2004, 126, 16324-16325
10.1021/ja0452626 CCC: $27.50 © 2004 American Chemical Society

C O M M U N I C A T I O N S
purification and handling. The reaction also showed good mismatch
discrimination, dropping in initial rate by 37- and 31-fold opposite
templates with the mismatch opposite the azido (8b) and fluorogenic
(8c) components, respectively (Figure 2).
Further work will be needed to see how efficient the activation
of fluorescence can be made by adjusting the length and confor-
mational properties of the linkers to the azide and ester. For in
vivo use, additional substitution of the fluorogenic component may
be necessary to increase the stability of the phosphine to oxidation
and to minimize fluorescence activation by enzymatic hydrolysis.5b
The PNAs will also have to be rendered membrane-permeable and
may also have to be lengthened to bind efficiently to the low
concentration of mRNAs present inside human cells.19
Figure 1. Rate of formation of 3 from 100 µM 1 as a function of the
concentration of 2 in 2:5 CH3CN/H2O, 10 mM phosphate, pH 7, at room
temperature. The amount of 3 was monitored by its absorbance at 454 nm.
Acknowledgment. Dedicated to Peter Dervan on the occasion
of his 60th birthday. Supported by NIH CA92477 with assistance
of the Washington University Mass Spectrometry Resource with
support from Grant No. P41RR0954.
Supporting Information Available: Experimental procedures and
analytical data for the synthesis of compounds 1, 2, 4-7, and analysis
of the kinetics. This material is available free of charge via the Internet
at http://pubs.acs.org.
References
(1) For reviews, see: (a) Dirks, R. W.; Molenaar, C.; Tanke, H. J. Histochem.
Cell Biol. 2001, 115, 3-11. (b) Paroo, Z.; Corey, D. R. J. Cell Biochem.
2003, 90, 437-442.
(2) For recent examples of single molecule mRNA detection, see: (a) Yao,
G.; Fang, X.; Yokota, H.; Yanagida, T.; Tan, W. Chemistry 2003, 9, 5686-
5692. (b) Knemeyer, J. P.; Herten, D. P.; Sauer, M. Anal. Chem. 2003,
75, 2147-2153.
(3) (a) Tyagi, S.; Kramer, F. R. Nat. Biotechnol. 1996, 14, 303-308. (b)
Sokol, D. L.; Zhang, X.; Lu, P.; Gewirtz, A. M. Proc. Natl. Acad. Sci.
U.S.A. 1998, 95, 11538-11543. (c) Fang, X.; Mi, Y.; Li, J. J.; Beck, T.;
Schuster, S.; Tan, W. Cell Biochem. Biophys. 2002, 37, 71-81. (d) Xi,
C.; Balberg, M.; Boppart, S. A.; Raskin, L. Appl. EnViron. Microbiol.
2003, 69, 5673-5678.
Figure 2. Rate of formation of 10 from 2 µM 6 in 10 mM phosphate, pH
7, at room temperature in the presence or absence of 5 µM 7 and 5 µM 8a,
b, or c. The amount of 10 was monitored by its fluorescence at 514 nm
(454 nm excitation). See SI for expanded plots of the data.
Scheme 3
(4) (a) Cardullo, R. A.; Agrawal, S.; Flores, C.; Zamecnik, P. C.; Wolf, D.
E. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 8790-8794. (b) Tsuji, A.;
Koshimoto, H.; Sato, Y.; Hirano, M.; Sei-Iida, Y.; Kondo, S.; Ishibashi,
K. Biophys. J. 2000, 78, 3260-3274. (c) Tsuji, A.; Sato, Y.; Hirano, M.;
Suga, T.; Koshimoto, H.; Taguchi, T.; Ohsuka, S. Biophys. J. 2001, 81,
501-515. (d) Ishibashi, K.; Tsuji, A. Anal. Chem. 2003, 75, 2715-2723.
(5) (a) Ma, Z.; Taylor, J. S. Bioconjugate Chem. 2003, 14, 679-683. (b)
Ma, Z.; Taylor, J. S. Bioorg. Med. Chem. 2001, 9, 2501-2510.
(6) (a) Sando, S.; Kool, E. T. J. Am. Chem. Soc. 2002, 124, 9686-9687. (b)
Sando, S.; Kool, E. T. J. Am. Chem. Soc. 2002, 124, 2096-2097. (c)
Sando, S.; Abe, H.; Kool, E. T. J. Am. Chem. Soc. 2004, 126, 1081-
1087. (d) Sando, S.; Sasaki, T.; Kanatani, K.; Aoyama, Y. J. Am. Chem.
Soc. 2003, 125, 15720-15721.
(7) Ma, Z.; Taylor, J.-S. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 11159-
11163.
(8) (a) Staudinger, H.; Meyer, J. HelV. Chim. Acta 1919, 2, 635-646. (b)
Gololobov, Y. G.; Kasukhin, L. F. Tetrahedron 1992, 48, 1353-1406.
(9) Saxon, E.; Bertozzi, C. R. Science 2000, 287, 2007-2010.
(10) For a recent review, see: Kohn, M.; Breinbauer, R. Angew. Chem., Int.
Ed. 2004, 43, 3106-3116.
m-cresol. PNA 6 was prepared by coupling the ester 1 to the amino
terminal amine prior to cleavage and deprotection.
(11) Lemieux, G. A.; De Graffenried, C. L.; Bertozzi, C. R. J. Am. Chem.
Soc. 2003, 125, 4708-4709.
(12) Li, X.; Taylor, J. S. Bioorg. Med. Chem. 2004, 12, 545-552.
(13) Dyke, J. M.; Groves, A. P.; Morris, A.; Ogden, J. S.; Dias, A. A.; Oliveira,
A. M. S.; Costa, M. L.; Barros, M. T.; Cabral, M. H.; Moutinho, A. M.
C. J. Am. Chem. Soc. 1997, 119, 6883-6887.
The ester linkage of PNA 6 is very stable in aqueous solution at
pH 7, as indicated by the lack of increase in fluorescence of a 2
µM solution of 6 over a period of 2 h (Figure 2). When 2 µM 6
and 5 µM 7 were incubated together under the same conditions,
10 formed with an initial rate of 8 × 10-5 µM‚s-1, which is 8
times that calculated for the reaction of 2 µM 1 and 5 µM 2, and
may be due to association of the PNA molecules. When 2 µM 6
and 5 µM 7 were incubated in the presence of 5 µM of the fully
complementary strand 8a, 10 formed with an initial rate of 0.015
µM‚s-1, which corresponds to a 188-fold increase in rate over the
reaction in the absence of DNA template. The reaction appeared
to approach only 50% completion, presumably due to the presence
of some oxidized phosphine resulting from exposure to air during
(14) Leffler, J. E.; Temple, R. D. J. Am. Chem. Soc. 1967, 89, 5235-5246.
(15) Egholm, M.; Buchardt, O.; Christensen, L.; Behrens, C.; Freier, S. M.;
Driver, D. A.; Berg, R. H.; Kim, S. K.; Norden, B.; Nielsen, P. E. Nature
1993, 365, 566-568.
(16) Peffer, N. J.; Hanvey, J. C.; Bisi, J. E.; Thomson, S. A.; Hassman, C. F.;
Noble, S. A.; Babiss, L. E. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 10648-
10652.
(17) Uhlmann, E. Biol. Chem. 1998, 379, 1045-1052.
(18) Demidov, V. V.; Potaman, V. N.; Frank-Kamenetskii, M. D.; Egholm,
M.; Buchard, O.; Sonnichsen, S. H.; Nielsen, P. E. Biochem. Pharmacol.
1994, 48, 1310-1313.
(19) The copy number for human mRNAs ranges from 1 to 10 000/cell,
corresponding to 0.001-10 nM for a cell with a volume of 1 pL.
JA0452626
9
J. AM. CHEM. SOC. VOL. 126, NO. 50, 2004 16325