Nitroazole universal bases in peptide nucleic acids

Article abstract of DOI:10.1021/ol990270q

(matrix presented). The syntheses of PNA oligomers containing potential ambiguous nucleobase analogues, namely 3-nitropyrrole and 5-nitroindole, have been accomplished. Hybridization properties of these PNAs with complementary oligodeoxynucleotides were evaluated by thermal denaturation experiments. Both novel residues exhibited little variation in T_m (≤1.5°C) when positioned against any of the four nucleoside bases. The capability to incorporate degenerate sites should further expand the utility of PNA in applications where precise sequence information is not available.

Full text of DOI:10.1021/ol990270q

ORGANIC
LETTERS
1999
Vol. 1, No. 10
1639-1641
Nitroazole Universal Bases in Peptide
Nucleic Acids
Hemavathi Challa, Melanie L. Styers, and Stephen A. Woski*
Department of Chemistry and Coalition for Biomolecular Products,
The UniVersity of Alabama, Tuscaloosa, Alabama 35487-0336
swoski@bama.ua.edu
Received September 3, 1999
ABSTRACT
The syntheses of PNA oligomers containing potential ambiguous nucleobase analogues, namely 3-nitropyrrole and 5-nitroindole, have been
accomplished. Hybridization properties of these PNAs with complementary oligodeoxynucleotides were evaluated by thermal denaturation
experiments. Both novel residues exhibited little variation in Tm (e1.5 °C) when positioned against any of the four nucleoside bases. The
capability to incorporate degenerate sites should further expand the utility of PNA in applications where precise sequence information is not
available.
With the pending completion of several genome projects, a
wealth of genetic information for several organisms is rapidly
becoming available. One key application of these data will
be the elucidation of the biological function of genes by
comparison of homologous sequences from different species.
Oligonucleotide primers based upon a reference sequence
from one organism can be used to identify orthologous
sequences from another.1 However, phenomena such as
sequence differences between species can complicate such
analyses by creating ambiguous sites for primer annealing.
Similarly, the degeneracy of the genetic code can introduce
numerous indeterminate base positions when designing
oligonucleotide probes from the amino acid sequence of a
protein (reverse genetics). To avoid the use of multiple probe
molecules to account for each possible sequence, nucleoside
analogues that lack base pairing specificity have been
incorporated.2 Ideally, such universal base residues will show
complete degeneracy without reducing the overall affinity
of the probe molecule for its complement. Such probes
should be valuable for primer extensions, in situ hybridiza-
tions, and the production of mutagenesis libraries. A number
of modified nucleosides have been studied as potential
universal residues. These include naturally occurring residues
such as inosine,3 nonnatural deoxyribofuranosides such as
3-nitropyrrole,4-6 5-nitroindole,7-10 and azole-4-carbox-
amides,11-13 and residues with both modified bases and
acyclic sugar portions.14
(3) Ohtsuka, E.; Matsuki, S.; Ikehara, M.; Takahashi, Y.; Matsubara, K.
J. Biol. Chem. 1985, 260, 2605-2608.
(4) Nichols, R.; Andrews, P. C.; Zhang, P.; Bergstrom, D. E. Nature
1994, 369, 492-493.
(5) Bergstrom, D. E.; Zhang, P.; Toma, P. H.; Andrews, P. C.; Nichols,
R. J. Am. Chem. Soc. 1995, 117, 1201-1209.
(6) Amosova, O.; George, J.; Fresco, J. R. Nucleic Acids Res. 1997, 25,
1930-1934.
(7) Loakes, D.; Brown, D. M. Nucleic Acids Res. 1994, 22, 4039-4043.
(8) Loakes, D.; Hill, F.; Linde, S.; Brown, D. M. Nucleosides Nucleotides
1995, 14, 1001-1003.
(9) Loakes, D.; Brown, D. M.; Linde, S.; Hill, F. Nucleic Acids Res.
1995, 23, 2361-2366.
(10) Loakes, D.; Hill, F.; Brown, D. M.; Salisbury, S. A. J. Mol. Biol.
1997, 270, 426-435.
(1) Hacia, J. G.; Makalowski, W.; Edgemon, K.; Erdos, M. R.; Robbins,
C. M.; Fodor, S. P.; Brody, L. C.; Collins, F. S. Nat. Genet. 1998, 18,
155-158.
(2) For a commentary, see: Burgess, K.; Welch, M. B. Chemtracts: Org.
Chem. 1995, 8, 187-190.
(11) Johnson, W. T.; Zhang, P.; Bergstrom, D. E. Nucleic Acids Res.
1997, 25, 559-567.
(12) Zhang, P.; Johnson, W. T.; Klewer, D.; Paul, N.; Hoops, G.;
Davisson, V. J.; Bergstrom, D. E. Nucleic Acids. Res. 1998, 26, 2208-
2215.
10.1021/ol990270q CCC: $18.00 © 1999 American Chemical Society
Published on Web 09/29/1999

While the incorporation of universal bases into oligode-
oxynucleotides has been extensively studied, little work has
been performed on DNA analogues with completely modified
backbones. Among the most important analogues are the
peptide nucleic acids (PNAs).15-17 PNAs are surrogates for
natural oligonucleotides constructed on uncharged pseudopep-
tide backbones. Nucleobases are positioned on this backbone
such that they can hybridize with complementary sequences
of DNA and RNA to form double- and, depending upon
sequence, triple-helical complexes. Moreover, PNA can
readily hybridize with complementary DNA sequences with
concurrent displacement of the DNA complement (strand
invasion). Due to the uncharged nature of the backbone,
binding is usually very tight and largely independent of salt
concentration. PNA is generally more sensitive to the
presence of mismatches than DNA probes.16 Because of these
characteristics, PNA has been viewed primarily as a potential
antisense/antigene reagent.15,17 Significantly, PNAs have been
shown to specifically inhibit human telomerase in cells.18
PNAs are also being used in hybridization assays and
mutation analysis.17 Furthermore, recent work has shown that
PNAs19 and PNA-DNA chimeras20 can serve as primers for
some polymerases. While new applications are being devel-
oped for PNA, the incorporation of novel residues has not
received much attention. Described herein is the synthesis
of PNAs bearing the potential universal bases and an
examination of the hybridization properties of PNA oligo-
mers containing these residues.
Figure 1. A. Structures of modified PNA residues. B. Sequences
of PNA and DNA oligomers used in thermal denaturaion assays.
sigmoidal, indicating that double helix formation is coopera-
tive (Figure 2). PNA 1, which contains the 3-nitropyrrole
residue, showed Tm values ranging from 54.7 °C (opposite
Because of the success of the nitroazole deoxynucleoside
residues as universal bases,4-10 it was decided to examine
whether these heterocycles would behave similarly when
incorporated into PNA. The synthesis of the nitroazole PNA
monomers began with a two-step conversion of 3-nitropyrrole
and 5-nitroindole into their corresponding acetic acid deriva-
tives (synthetic details are provided in the Supporting
Information). Condensation of these carboxylic acids with
2-N-Fmoc-2-aminoethylglycine tert-butyl ester21 provided the
fully blocked monomers in good yields. Removal of the
C-terminal tert-butyl esters was accomplished by brief
treatment with trifluoroacetic acid, producing monomers for
PNA synthesis. PNA oligomers were made using the standard
automated solid-phase synthetic method22 except that the
novel residues were coupled. The PNAs were purified using
HPLC, and oligomers containing the novel residues produced
satisfactory MALDI-TOF mass spectra.23
Hybridization of the 15-mer PNA oligomers with oligode-
oxynucleotides was performed in PES buffer (10 mM
phosphate, 0.1 mM EDTA, 100 mM NaCl, pH 7), and the
stability of each of these complexes was evaluated by thermal
denaturation experiments using absorbance spectroscopy
(Figure 1). All of the absorbance vs temperature curves were
Figure 2. Thermal denaturation curves for PNA‚DNA double
helices. Absorbance was measured at 260 nm; the concentration
of each strand was 4 µM. A. PNA 1 (3-nitropyrrole) + comple-
mentary DNAs. B. PNA 2 (5-nitroindole) + complementary DNAs.
(13) Pochet, S.; Dugue´, L. Nucleosides Nucleotides 1998, 17, 2003-
2009.
(14) For example, see: Van Aerschot, A.; Rozenski, J.; Loakes, D.; Pillet,
N.; Schepers, G.; Herdewjin, P. Nucleic Acids Res. 1995, 23, 4363-4370.
(15) Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Science 1991,
254, 1497-1500.
(16) 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.
dG) to 56.2 °C (opposite dA) (Table 1). This range of Tm
values is smaller than that observed for oligodeoxynucleo-
tides containing the 3-nitropyrrole deoxynucleoside residue
1640
Org. Lett., Vol. 1, No. 10, 1999

The thermodynamic parameters for formation of PNA‚
DNA double helices containing nitroazole bases (Table 1)
show similar trends to the analogous DNA‚DNA system.26
In both a large drop in ∆H from the unsubstituted system is
seen, presumably arising from the loss of hydrogen bond-
ing.26,27 In both cases, this loss is partially counterbalanced
by an increase in ∆S. This entropic effect has been attributed
to the greater conformational freedom seen by the nucleobase
positioned opposite to the non-hydrogen bonding universal
base25 or to solvation/desolvation effects.26 Future work is
needed to fully explore this phenomenon. Nevertheless,
incorporation of the nitroazole universal base residues into
both PNA‚DNA and DNA‚DNA double helices appears to
behave in a qualitatively similar fashion. However, as would
be expected given the greater mismatch sensitivity of PNA
probes, substitution of PNA with the nitroazole residues is
more destabilizing than in DNA probes.
Table 1. Thermodynamic Parameters for the Hybridization of
Nitroazole-PNAs with Complementary DNAsa
Y
Tm (°C)
∆H (kJ /mol)
∆S (kJ /mol‚K)
X ) Np
X ) Ni
X ) T
A
G
C
T
A
G
C
T
56.2
54.7
55.9
55.1
58.4
58.9
59.7
59.3
68.5b
-317
-318
-309
-306
-338
-344
-343
-350
-446b
-0.85
-0.86
-0.83
-0.82
-0.91
-0.93
-0.97
-0.94
-1.20b
A
a Absorbance vs temperature curves were measured at 260 nm in PES
buffer. Tm values were determined by multiplying the maxima of the
derivative plots (Tmax) by 0.971. Thermodynamic parameters were calculated
by the method of Gralla and Crothers.24 b Data from Egholm et al. (ref
16).
Incorporation of the universal bases 3-nitropyrrole and
5-nitroindole into PNA oligomers can be accomplished easily
and produces sites that are completely degenerate with
respect to base-pairing preferences. The substitution of these
residues in the PNA strand of a PNA‚DNA double helix is
slightly destabilizing; however, both of these complexes show
significantly higher Tm values than the corresponding DNA‚
DNA double helix containing an A-T base pair (Tm ) 53.3
°C).16 To test the generality of these substitutions, experi-
ments are currently underway to examine the effects of
sequence context on hybridization. Overall, the ability to
introduce completely degenerate sites into PNA probes
should greatly enhance their utility as tools for molecular
biology.
(1.5 °C versus 3 °C).4,5,7 The substitution of a natural base
with 3-nitropyrrole is mildly destabilizing. A PNA‚DNA
duplex with the same sequence studied here but containing
a normal A-T pair in the variable position was found by
Egholm and co-workers16 to have a Tm of 68.5 °C. Duplexes
with nitropyrrole-nucleobase pairs have approximately
equivalent stabilities when compared to mismatched duplexes
containing a thymine residue in the PNA strand against dG,
dC, or T in the DNA strand.16
The incorporation of the 5-nitroindole residue into the PNA
strand produces a molecule (PNA 2) with improved hybrid-
ization characteristics (Table 1). The range of Tm values for
PNA 2 was narrower than that of PNA 1 (∆Tm ) 1.3 °C).
More significantly, incorporation of the nitroindole residue
was less destabilizing, only showing a drop in Tm of
approximately 9 °C versus the matched duplex. These results
correspond well with those seen in oligodeoxynucleotides
with nitropyrrole and nitroindole bases.4,5,7 Oligonucleotides
with either of these residues showed little variation in Tm
when positioned against any of the four natural bases (∆Tm
) 3 °C),4,5,7 but duplexes containing the 5-nitroindole
residues were more stable.7 Presumably, this stabilization is
due to the improved stacking ability of the indole moiety as
compared to that of the smaller pyrrole.10 The higher
enthalpies calculated for duplexes with nitroindole versus
duplexes with nitropyrrole also support the role of improved
stacking (Table 1). Because PNA‚DNA double helices
maintain base stacking arrangements similar to those seen
in DNA‚DNA duplexes,25 it is not surprising that the
5-nitroindole residue is less destabilizing.
Acknowledgment. M.L.S. was supported by an REU
grant from the NSF (CHE-9531496). The authors are grateful
to Dr. Joseph Hacia for helpful discussions and to
Greg Miller for measurement of high-resolution FAB mass
spectra.
Supporting Information Available: Synthetic details for
3-nitropyrrole and 5-nitroindole PNA monomers and PNA
oligomers, experimental details for thermal denaturation
experiments, and discussion of calculations used to determine
thermodynamic parameters. This material is available free
of charge via the Internet at http://pubs.acs.org.
OL990270Q
(21) Thomson, S. A.; Josey, J. A.; Cadilla, R.; Gaul, M. D.; Hassman,
C. F.; Luzzio, M. J.; Pipe, A. J.; Reed, K. L.; Ricca, D. J.; Wiethe, R. W.;
Noble, S. A. Tetrahedron 1995, 51, 6179-6194.
(22) PNA Chemistry for the Expedite Nucleic Acid Synthesis System;
PerSeptive Biosystems, Inc.: Framingham, MA, 1998.
(23) PNA 1 (H-TGT ACG Np CAC AAC TA-NH2) m/z 4033.5 [M +
H]+, calcd 4033.6. PNA 2 (H-TGT ACG Ni CAC AAC TA-NH2) m/z
4083.9 [M + H]+, calcd 4084.3.
(24) Gralla, J.; Crothers, D. M. J. Mol. Biol. 1973, 78, 301-319.
(25) Leijon, M.; Graeslund, A.; Nielsen, P. E.; Buchardt, O.; Norden,
B.; Kristensen, S. M.; Eriksson, M. Biochemistry 1994, 33, 9820-9825.
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1997, 25, 1935-1942.
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Acta 1996, 79, 488-498.
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1997, 21, 19-31; Uhlmann, E.; Peyman, A.; Breipohl, G.; Will, D. W.
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Chem. Biol. 1999, 6, 343-351.
(19) Lutz, M. J.; Benner, S. A.; Hein, S.; Breipohl, G.; Uhlmann, E. J.
Am. Chem. Soc. 1997, 119, 3177-3178.
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