γ-substituted peptide nucleic acids constructed from L-lysine are a versatile scaffold for multifunctional display

Article abstract of DOI:10.1002/anie.200603483

(Figure Presented) On display: This model shows DNA (pink) annealed to a peptide nucleic acid (PNA; orange) containing several L-lysine γ side chains (^LKγ-PNA) in the middle of the PNA oligomer. The ^LKγ-PNA side chains project away f

Full text of DOI:10.1002/anie.200603483

Communications
DOI: 10.1002/anie.200603483
Peptide Nucleic Acids
g-Substituted Peptide Nucleic Acids Constructed from
l-Lysine are a Versatile Scaffold for Multifunctional
Display**
Ethan A. Englund and Daniel H. Appella*
Angewandte
Chemie
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Peptide nucleic acids (PNAs), which contain an achiral,
uncharged aminoethylglycine (aeg) backbone, bind natural
nucleic acids through Watson–Crick base pairing with high
affinity and selectivity (Scheme 1a).[1–3] PNA oligomers are
not enzymatically degraded,[4] do not require high salt
concentration for binding,[2] and are useful in nucleic acid
detection systems[5] and as antisense molecules.[6]
means of conjugating functionality directly to PNA residues
has the potential to positively impact many applications that
employ aegPNA and stimulate development of new PNA-
based applications.
We initiated a program investigating the g position on the
aegPNA backbone for its potential as an effective conjugation
point (Scheme 1c).[16] In particular, l-lysine g-PNA (lKg-
PNA) is ideally suited to support a broad range of functional
groups without interfering with the binding to DNA or RNA.
Since our initial report, other researchers have similarly found
that side chains extending from the g position of aegPNA are
l
tolerated.[17] We herein present an extensive analysis of Kg-
PNA to demonstrate its improved binding affinity to DNA
and RNA compared with aegPNA, regardless of the group
that is conjugated to the amine of the lysine side chain. The
advantage of this approach is that the primary amine of the
Lys-derived side chain can be easily modified to display many
different groups. Furthermore, this basic strategy opens the
possibility of using PNAs as a scaffold for the multifunctional
display of groups in well-ordered, three-dimensional arrays.
Several moieties were attached to the g side chain to
gauge its stability when incorporated into aegPNA oligomers
(Scheme 2). Compared with an aegPNA oligomer, PNA
Scheme 1. aegPNA and backbone positions for side chains.
PNA research has focused on improving the favorable
binding properties of aegPNAs[7] while eliminating some of
the inherent weaknesses (e.g. solubility and bioavailability).[8]
Common strategies include conjugating moieties to PNA
termini,[9] utilizing PNA/DNA chimeras,[10] and synthesizing
PNA derivatives with backbone modifications.[11] Although
there are currently a large number of PNA derivatives and
variations, one challenging area of research has been the
strategic placement of side chains on the aegPNA scaffold. In
this regard, PNAs synthesized from chiral amino acids in
which the side chains extend from the a-carbon atom have
been examined (Scheme 1b).[12] The presence of positively
charged side chains at the a-carbon atom can result in
improvements in cellular uptake and duplex stability.[13]
However, neutral or negatively charged side chains from
the a position destabilize PNA/DNA duplexes, limiting their
utility. Some research has focused on appending functionality
onto these side chains, but in these cases, the modified PNA/
DNA duplexes are less stable than unmodified aegPNA/DNA
duplexes.[14] Recent studies also show that having multiple
a side chains positioned on adjacent PNA residues is detri-
mental to duplex stability, although such arrangements
improve the specificity of binding.[15] A strategy that improves
upon aegPNA binding properties while providing a facile
l
oligomers with Kg-PNA containing a primary amine on the
side chain (T1, C1, A1) show increased melting temperatures
[*] Dr. D. H. Appella
Laboratory of Bioorganic Chemistry
NIDDK, NIH, DHHS
Bethesda, MD 20892 (USA)
Fax: (+1)301-480-4977
E-mail: appellad@niddk.nih.gov
E. A. Englund
NIDDK, NIH, DHHS
Bethesda, MD 20892 (USA)
and
Northwestern University
Evanston, IL 60208 (USA)
Scheme 2. lKg-PNA monomers. T1, C1, A1, and T2 were synthesized by
using Cbz-protected side chains. T3, T4, T5, and T6 were synthesized by
using Fmoc-protected side chain amines, which were deprotected
during PNA synthesis on the solid support and coupled to carboxylic
acids. Cbz=benzyloxycarbonyl; Fmoc=9-fluorenylmethoxycarbonyl.
[**] This work was supported in part by the Intramural Research
Program at NIDDK, NIH.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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(Tm) when complexed with the complementary antiparallel
interactions contribute to binding, the sequence specificity
was examined. When PNA 2 is annealed to DNAwith a single
base mismatch across from the g-PNA residue T1, a difference
in Tm was observed equal to or greater than aegPNA
mismatches (see the Supporting Information).
DNA (Table 1, PNA molecules 1–4). This increase in Tm was
consistent for different nucleobase monomers (thymine,
adenine, and cytosine) and demonstrated an additive effect
l
Several oligomers containing Kg-PNA were synthesized
Table 1: Thermal melting data[a] for PNA decamers bound to comple-
with a range of functional groups appended to their primary
amines to test whether bulky or nonionic groups could be
tolerated. When an oligomer containing an acetamide group
on the g-Lys side chain (Scheme 2, T3) was annealed to the
target DNA, the Tm was also higher than the analogous
aegPNA oligomer, further suggesting that the increase in
duplex stability is independent of positive charge on the side
chain. This increase in stability is consistent with results from
Dragulescu-Andrasi et al., who recently showed that g sub-
stitution with a serine-derived side chain in PNA monomers
enhances binding affinity by preorganization of the PNA
oligomer.[17a] The circular dichroism (CD) spectrum of PNA 8
suggests a definite helical conformation in the absence of
DNA (Figure 2). Considering the interest that PNA/peptide
conjugates have generated,[19] a small peptide chain (Ala, Ala,
Gly) was synthesized through Fmoc chemistry from the side
chain T4; this oligomer (Table 1, PNA 7) also showed a
consistent increase in Tm.
mentary antiparallel DNA.
PNA
Sequence[b]
Tm [8C]
1
2
3
4
5
6
7
8
9
10
11
12
13
H2N-G T A G A T C A C T-Lys
H2N-G T A G A T1 C A C T-Lys
H2N-G T A G A1 T C A C T-Lys
H2N-G T A G A T C1 A C T-Lys
H2N-G T A G A T2 C A C T-Lys
H2N-G T A G A T3 C A C T-Lys
H2N-G T A G A T4 C A C T-Lys
H2N-G T1 A G A T1 C A C T1-Lys
H2N-G A C T T T A C G A-Lys
H2N-G A C T5 T5 T5 A C G A-Lys
H2N-C C T C T T C C T C-Lys
H2N-C C T C T6 T C C T C-Lys
H2N-C C T C T6 T C C T6 C-Lys
49.7
51.4
53.1
51.8
36.4
51.2
51.7
54.5
42.4
49.3
34.5
44.6
46.4
[a] Solutions of DNA/PNA (1:1) were prepared in pH 7.0 buffer solution
consisting of 10 mm sodium phosphate, 0.1 mm EDTA, and 150 mm
NaCl. Strand concentrations were 5 mm in eachcomponent. Estimated
error is Æ0.58C. [b] Sequences are written from the N terminus to the
C terminus. PNA molecules 1–8 were bound to DNA 3’-CATCTAGTGA-5’,
9 and 10 were bound to DNA 3’-CTGAAATGCT-5’, and 11–13 were bound
to DNA 3’-GGAGAAGGAG-5’. Th elKg-PNA monomers are underlined
for clarity.
when multiple thymine monomers were incorporated
(Table 1, PNA 8). Because of the important role that chirality
and flexibility can have in PNA/DNA duplex formation, the
stereochemistry of lKg-PNA was examined. Oligomers incor-
porating d-lysine g-PNA (T2) showed greatly reduced binding
affinity with antiparallel DNA. Modeling based on the NMR-
solved structure of complexed PNA/DNA[18] suggests that the
side chains with S stereochemistry will orient along the
periphery of the duplex, whereas side chains with the
R stereochemistry are directed to the interior of the duplex
(Figure 1). To determine whether nonspecific charge–charge
Figure 2. CD spectrum of PNA 8 (25.0 mm). The spectrum was mea-
sured at 258C and represents an average of eight scans collected at
100 nmminÀ1
.
To determine the viability of using g-PNA coupled to
bulky substituents that are located in close proximity, three
adjacent thymine g-Lys residues (Scheme 2, T5) were coupled
to hydrocinnamic acid (Table 1, PNA 10). The oligomer
showed a significant increase in Tm compared with the
aegPNA decamer (PNA 9). The corresponding single-base-
mismatch studies also showed slightly increased mismatch
l
discrimination for Kg-PNA 10 compared with its aeg ana-
logue (see the Supporting Information).
In some cases, backbone-modified PNAs can bind DNA
or RNA selectively.[20] Therefore, the binding of lKg-PNA to
RNA was examined. Two lKg-PNA oligomers, one composed
of T1 and the other containing multiple, bulky substituents T5,
were annealed to complementary RNA (Table 2). Both of the
lKg-PNA molecules showed increases in duplex stability
compared with aegPNAs that are very similar to the increase
Figure 1. A model of Lys g-PNA/DNA duplex (purple=base pairs,
orange=PNA backbone, fuscia=DNA backbone). The side chain at
the g position derived from l-(S)-lysine projecting away from the
duplex is shown in blue. The side chain at the g position derived from
d-(R)-lysine projecting into the minor groove is shown in yellow.
l
d
observed when Kg-PNA is annealed to DNA. A Kg-PNA
oligomer (containing T2) exhibited a large decrease in
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Table 2: Thermal melting data[a] for PNA decamers and complementary
antiparallel RNA.
rotation of thiazole orange must be restricted due to
intercalation[22b] or some other type of noncovalent associa-
tion. Because aegPNA lacks possible positions where further
functional groups can be easily appended, most aegPNA
oligomers only have one functional group attached per
PNA
Sequence[b]
Tm [8C]
1
2
5
9
10
H2N-G T A G A T C A C T-Lys
H2N-G T A G A T1 C A C T-Lys
H2N-G T A G A T2 C A C T-Lys
H2N-G A C T T T A C G A-Lys
H2N-G A C T5 T5 T5 A C G A-Lys
54.8
57.3
42.1
52.6
56.6
l
oligomer, usually to the N terminus. By using the Kg-PNA,
multiple fluorophores can be incorporated into a single-
molecular probe, increasing sensitivity of the probe without
negatively affecting the Tm of the PNA/DNA duplex. When
the oligomer containing two T6 residues (PNA 13) was
combined with the complementary DNA, a similar increase
in fluorescence intensity was observed. Although the effect of
having two thiazole orange molecules attached to PNA 13 did
not double the fluorescent intensity, there was a 30% larger
fluorescent intensity compared with the oligomer with one
thiazole orange fluorophore attached (Figure 3).
[a] Solutions of RNA/PNA (1:1) were prepared in pH 7.0 buffer consist-
ing of 10 mm sodium phosphate, 0.1 mm EDTA, and 150 mm NaCl.
Strand concentrations were 5 mm in eachcomponent. Estimated error is
Æ0.58C. [b] Sequences are written from the N terminus to the C termi-
nus. PNA molecules 1, 2, and 5 were bound to RNA 3’-CATCTAGTGA-5’;
PNA molecules 9 and 10 were bound to RNA 3’-CTGAAATGCT-5’. Th e
lKg-PNA monomers are underlined for clarity.
thermal stability when bound to complementary RNA. This
Herein, we have shown that lKg-PNA is a versatile
scaffold to attach a range of functional groups without
compromising the ability of PNAs to bind to complementary
nucleic acid sequences. In addition to this versatility, the
d
finding was also consistent with the binding affinity of Kg-
PNA oligomers to DNA. These results strongly indicate that
lKg-PNA binding properties are common to both DNA and
RNA.
l
chemistry to introduce different side chains onto Kg-PNAs
PNA oligomers incorporating a fluorescent molecule,
such as thiazole orange,[21] have been used as detection
probes.[22] Expanding on our previous PNA-beacon strat-
egy,[16] a thiazole orange carboxylic acid derivative was
coupled to the g-Lys side chain (Scheme 2, T6) in a polypyr-
imidine PNA oligomer. Thiazole orange emits very low
fluorescence in its unbound state. However, when present in a
nucleic acid duplex, thiazole orange base stacking limits its
bond rotation and the molecule emits a fluorescent signal.[23]
When an oligomer containing the T6 residue (PNA 12) was
unbound or in the presence of noncomplementary DNA,
there was a very low background fluorescence (Figure 3).
However, when annealed to the complementary DNA,
PNA 12 gave a large increase in fluorescent intensity (about
two orders of magnitude). This increase was consistent with
the increase seen when thiazole orange was coupled to the
N terminus of the same oligomer (fluorescent intensity
approximately 42-times greater than at 530 nm).[24] Because
the lKg-PNA residue is located in the middle of the sequence,
base stacking on the end of the duplex is not possible (even
taking into account the length of the linkers). Therefore, bond
can be performed during solid-phase synthesis of the PNA
oligomer by judicious selection of orthogonal protecting
groups. We hope that these basic properties of lKg-PNAs can
be used to impact the areas of diagnostics and therapeutics
where aegPNA is currently used, and stimulate creative new
uses of PNAs as a scaffold for multifunctional display.
Experimental Section
lKg-PNA monomer synthesis and characterization is described either
in the Supporting Information or was described previously.[16]
Analytical TLC was carried out on Sorbent Technologies TLC
plates precoated with silica gel (layer thickness of 250 mm). Flash
column chromatography was performed by using a Biotage Flash12 +
apparatus on Biotage Si 25m columns. Fmoc-8-amino-3,6-dioctanoic
acid was purchased from Peptides International, Inc. BocLys(Cbz)-
OH (Boc = tert-butoxycarbonyl) was purchased from Advanced
ChemTech. Unless otherwise noted, all other commercially available
reagents and solvents were purchased from Aldrich and used without
further purification. Dichloromethane, THF, and N,N-dimethylfor-
mamide was passed through a column of activated alumina.[25] Unless
otherwise indicated, all reactions were performed under an inert
atmosphere of N2. Glassware was dried in an oven at 1808C for at
least 2 h prior to use. PNA oligomers were made by manual solid-
phase synthesis on 4-methylbenzhydrylamine (MBHA) resin as
previously described.[7] Oligomers were purified by reverse-phase
HPLC and characterized by electrospray or MALDI mass spectrom-
etry. The oligomers were quantified by using UVabsorbance at 808C.
Thermal denaturation studies were conducted by annealing
oligomer samples while monitoring the absorbance at l = 260 nm by
using a UV/Vis spectrophotometer. The buffered oligomer samples
were heated to 918C for 5 minutes and then cooled to 158C at a rate
of 1 degree per minute. The Tm was calculated as the maximum for the
first derivative of the melting curve. CD experiments were performed
on a Jasco J-175 spectropolarimeter. Samples in buffered solutions
were identical to the thermal denaturation studies.
Fluorescence studies were carried out on a Jobin Yvon Fluo-
romax 3, by using a duplex concentration of 0.5–2 mm. Samples were
prepared either by annealing oligomers under the same conditions as
were used for the thermal denaturation studies or without annealing.
Results were the same under both conditions. Samples were excited
l
Figure 3. Fluorescence intensity (If) of Kg-PNAs withthiazole orange
attached to the side chains: a) PNA 13 combined withthe complemen-
tary DNA; b) PNA 12 combined withthe complementary DNA;
c) PNA 13 by itself; and d) PNA 12 by itself.
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by using at a wavelength of 470 nm, and fluorescence was monitored
at 495–595 nm.
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Received: August 25, 2006
Published online: November 29, 2006
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Keywords: binding studies · chiral backbone · DNA structures ·
nucleic acids · peptide nucleic acids
.
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