Synthesis of γ-substituted peptide nucleic acids: A new place to attach fluorophores without affecting DNA binding

Article abstract of DOI:10.1021/ol051143z

(Chemical Equation Presented) Molecular beacon strategies using PNA are currently restricted to fluorophore attachment to the ends of the PNA. We report the synthesis of PNA oligomers wherein fluorophores can be attached to the PNA backbone from novel γ-l

Full text of DOI:10.1021/ol051143z

ORGANIC
LETTERS
2005
Vol. 7, No. 16
3465-3467
Synthesis of
γ-Substituted Peptide
Nucleic Acids: A New Place to Attach
Fluorophores without Affecting DNA
Binding
Ethan A. Englund and Daniel H. Appella*
Laboratory of Bioorganic Chemistry, NIDDK, NIH, DHHS, Bethesda, Maryland
20892, and Northwestern UniVersity, EVanston, Illinois 60208
appellad@niddk.nih.goV
Received May 16, 2005
ABSTRACT
Molecular beacon strategies using PNA are currently restricted to fluorophore attachment to the ends of the PNA. We report the synthesis of
PNA oligomers wherein fluorophores can be attached to the PNA backbone from novel -lysine PNA monomers. Oligomers incorporating the
γ
modified PNA showed comparable thermal stability to the corresponding aegPNA oligomer with DNA. When the modified PNA oligomer was
annealed with complementary DNA, the fluorescence intensity increased 4-fold over the unbound PNA.
Sequence-specific nucleic acid detection is critical for many
medicinal and diagnostic applications. In this area, molecular
beacons (MBs) are popular tools for in ViVo nucleic acid
detection.1 In these systems, a nucleic acid probe exhibits a
fluorescent signal only in the presence of the target oligo-
nucleotide. MBs usually consist of a fluorophore and a
fluorescence-quencher attached at the termini of a nucleic
acid oligomer.2 When the termini are close to one another
(due to a stem-loop structure or hydrophobic interactions),
the fluorescence is quenched. Upon binding to the target
oligonucleotide, separation of the termini is accompanied by
an increase in fluorescence.
points in the oligonucleotide sequence, not just at the termini,
and allows incorporation of multiple fluorophores in prin-
ciple. Currently, such MBs rely on covalent attachment of
the fluorophore to a nucleobase.4
With the inception and continued study of peptide nucleic
acid (PNA),5 molecular beacon strategies incorporating this
nonnatural pseudopeptide have become increasingly popular.6
The use of “stemless” PNA molecular beacons (Figure 1) is
especially attractive due to its high binding affinity to natural
nucleic acids, high selectivity, and resistance to degradation.6b
MB strategies using PNA are currently restricted to fluoro-
phore attachment to the ends of the PNA6 or to the
Recently, quencher-free MBs have been synthesized from
DNA that utilize fluorophores quenched by nucleobases.3
This strategy allows the inclusion of fluorophores at various
(4) (a) Sando, S.; Abe, H.; Kool, E. T. J. Am. Chem. Soc. 2004, 126,
1081. (b) Fujimoto, K.; Shimizu, H.; Inouye, M. J. Org. Chem. 2004, 69,
3271.
(5) Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Science 1991,
254, 1497.
(1) (a) Paroo, Z.; Corey, D. R. J. Cell Biochem. 2003, 90, 437. (b)
Kaihatsu, K.; Janowski, B. A.; Corey, D. R. Chem. Biol. 2004, 11, 749. (c)
Tan, W.; Wang, K.; Drake, T. J. Curr. Opin. Chem. Biol. 2004, 8, 547.
(2) Tyagi, S.; Kramer, F. R. Nat. Biotechnol. 1996, 14, 303.
(3) Hwang, G. T.; Seo, Y. J.; Kim, B. H. J. Am. Chem. Soc. 2004, 126,
6528-6529.
(6) (a) Seitz, O.; Bergmann, F.; Heindl, D. Angew. Chem., Int. Ed. 1999,
38, 2203. (b) Svanvik, N.; Nygren, J.; Westman, G.; Kubista, M. J. Am.
Chem. Soc. 2001, 123, 803. (c) Kuhn, H.; Demidov, V. V.; Coull, J. M.;
Fiandaca, M. J.; Gildea, B. D.; Frank-Kamenetskii, M. D. J. Am. Chem.
Soc. 2002, 124, 1097. (d) Peterson, K.; Vogel, U.; Rockenbauer, E.; Nielsen,
K. V.; Kolvraa, S.; Bolund, L.; Nexo, B. Mol. Cell. Probes 2004, 18, 117.
10.1021/ol051143z This article not subject to U.S. Copyright. Published 2005 by the American Chemical Society
Published on Web 07/08/2005

Figure 3. aegPNA derivative synthesized from orthogonally
protected (L)-Lysine.11
Figure 1. General principle of stemless PNA molecular beacons.
Fluorescence is observed upon duplex formation.
PNA monomers were synthesized from commercially avail-
able, orthogonally protected (L)-lysine. Fluorene was chosen
as the fluorophore in our initial examination of this system
because the fluorophore emission is, in general, effectively
quenched by thymine in numerous examples10 and in recent
MB studies.3 When unbound, the fluorene in this molecule
will presumably interact with the thymine in the PNA
oligomer due to compaction or hydrophobic interactions6c
so that the fluorescence is effectivly quenched. Once bound
to a complementary nucleic acid in a stable double helix,
the fluorene should emit increased fluorescence due to the
descreased interaction with the thymine residues. After
coupling Boc-γ-Lys(Fmoc)-T-OH to the PNA oligomer on
the solid support, the Fmoc was removed, and a MiniPEG
linker and 9-fluoreneacetic acid were coupled to the side
chain.11 PNA synthesis was then completed without incident.
nucleobases. Therefore, successful development of PNA-
based MBs requires that the termini of the PNA are in close
proximity to each other or to nucleobases in the absence of
target oligonucleotide (to ensure fluorescence quenching) or
that attachment of fluorophores directly to nucleobases does
not negatively impact oligonucleotide binding. Such require-
ments can make the design of PNA-based MBs challenging.
In this communication, we report a new strategy for
attaching side chains to the PNA backbone such that
fluorophores can be covalently attached. Our strategy should
facilitate the design of PNA-based MBs. While many PNA
modifications have deleterious effects on the binding,7 our
side chain does not compromise binding to DNA. As an
initial test of our strategy, we developed a rudimentary PNA-
based biosensor where fluorescent intensity increases upon
binding to fully complementary DNA (Figure 2).
Hybridization properties of the side chain-bearing PNA
oligomers with DNA (Figure 4) were examined using
Figure 2. General structure of DNA, aegPNA, and γ-substituted
PNA with a linker and fluorophore attached to it.
Figure 4. Synthesized PNA oligomers and DNA used in fluores-
cent and melting temperature (Tm) studies.
We designed a PNA monomer with an Fmoc-protected
lysine-derived side chain at the γ-carbon (Figure 3, Boc-γ-
Lys(Fmoc)-T-OH) to serve as the attachment point for a
fluorophore. On the basis of the NMR structure of PNA/
DNA duplexes, we deduced that the S stereochemistry at
the γ carbon would be ideal for side chain accommodation.8
Furthermore, inspection of the NMR structure indicated that
a side chain at the γ-position would be able to tolerate a
large flourophore without affecting duplex stability. While
PNA side chains are often attached to the R-carbon, we felt
that attachment of a large fluorophore at this position would
destabilize the PNA/DNA duplex.9 The side chain-bearing
variable-temperature UV and compared to unmodified
aegPNA (Table 1). The thermal denaturation studies showed
that oligomers incorporating the modified PNA residue
melted at a slightly higher temperature (Tm) than the
corresponding aegPNA oligomer with fully complementary
(9) (a) Dueholm, K. L.; Petersen, K. H.; Jensen, D. K.; Egholm, M.;
Nielsen, P. E.; Buchardt, O. Bioorg. Med. Chem. Lett. 1994, 4, 1077. (b)
Haaima, G.; Lohse, A.; Buchardt, O.; Nielsen, P. E. Angew. Chem., Int.
Ed. Engl. 1996, 35, 1939. (c) Zhou, P.; Wang, M.; Du, L.; Fisher, G. W.;
Waggoner, A.; Ly, D. H. J. Am. Chem. Soc. 2003, 125, 6878.
(10) (a) Telser, J.; Cruickshank, K. A.; Morrison, L. E.; Nextel, T. L. J.
Am. Chem. Soc. 1989, 111, 6966. (b) Mann, J. S.; Shibata, Y.; Meehan, T.
Bioconjugate Chem. 1992, 3, 554.
(7) Hyrup, B.; Egholm, M.; Nielsen, P. E.; Wittung, P.; Norden, B.;
Buchardt, O. J. Am. Chem. Soc. 1994, 116, 7964.
(8) Eriksson, M.; Nielsen, P. E. Nat. Struct. Biol. 1996, 3, 410.
(11) See Supporting Information for details.
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Org. Lett., Vol. 7, No. 16, 2005

Table 1. Thermal Melting Temperature (Tm) of Modified PNA
with Complementary and Mismatched Antiparallel DNAa
Tm (°C)
DNA 2
DNA 3b
DNA 4b
DNA 5b
aegPNA
PNA 1
PNA 6
48.9
50.5
50.3
36.0 (12.9)
30.0 (20.5)
34.0 (14.9)
29.9 (20.6)
32.0 (16.9)
34.9 (15.6)
a Solutions of 1:1 oligonucleotide/PNA were prepared in pH 7.0 buffer
consisting of 10 mM sodium phosphate, 0.1 mM EDTA, and 150 mM NaCl.
Strand concentrations were 5 µM in each component. Estimated error is
(0.5 °C. b ∆Tm (°C) in parentheses represents the difference in Tm between
fully complementary duplex and duplex with a single mismatch.
Figure 6. (a) Emission spectra (in arbitrary units) of PNA 6
complexed with DNA 2 with conditions matching those used in
Figure 5. (b) PNA 6 alone.
DNA (DNA 2). Discrimination of single-base mismatches
(DNAs 3-5) was also similar to, or higher than, that of the
corresponding aegPNA.
On the basis of our results, we sought to determine if the
modification was general or site specific, both in binding
characteristics and fluorescent properties. We predicted that
the fluorene would remain quenched even if not directly
incorporated on a thymine PNA residue. Fluorescent studies
of PNA 6 (Figure 6) showed a similar increase (∼4-fold) in
fluorescent intensity when bound to the complementary DNA
(2).
We investigated the fluorescent intensity changes of our
PNAs when bound to fully complementary and singly
mismatched single-stranded DNA. PNA 1 was weakly
fluorescent (λmax ) 425) in the absence of DNA. When PNA
1 was annealed with complementary DNA (2), the fluores-
cence intensity increased 4-fold over that of the unbound
PNA (Figure 5). When combined with DNA 4 or 5,
In conclusion, we have developed a method to modify the
PNA backbone with side chains that can support a fluorescent
group. Our modification shows no deleterious effects on
DNA binding. Although the fluorene was very efficiently
quenched when the oligomers were unbound, the modest
increase in fluorescent intensity suggests that the fluorene
still interacts with the thymine residues. This could be due
to intercalation of the fluorene residue into the duplex or
aggregation effects. While the fluorescence increase is not
sufficient for most sensor applications, because of the
versatility of our strategy we expect to be able to install a
variety of different fluorophores at multiple positions to
optimize for highly efficient biosensors.
Figure 5. (a) Emission spectra (in arbitrary units) recorded at 23
°C of PNA 1 bound to DNA 2 (5.0 µM). Fluorescent spectra were
recorded using an excitation wavelength of 340 nm. Buffer
conditions are the same as those described in Table 1. (b) PNA 1
bound to DNA 3. (c) PNA 1 bound to DNA 4. (d) PNA 1 bound
to DNA 5. (e) PNA 1 alone.
Acknowledgment. Funding was provided by Northwest-
ern University and NIDDK, NIH, DHHS. We gratefully
acknowledge Fred Lewis for assistance with the fluorescent
studies.
containing a single-base mismatch opposite the modified
PNA segment, the fluorescence remained near baseline
levels. Annealing with DNA 3 (TT mismatch) gave a 2.5-
fold increase in fluorescence intensity, which is still con-
siderably less than that exhibited by the fully complementary
DNA (2).
Supporting Information Available: Synthesis of the
γ-Lysine(Fmoc) PNA monomer, along with the solid-phase
synthesis of PNAs 1 and 6. This material is available free
of charge via the Internet at http://pubs.acs.org.
OL051143Z
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