Fluorescence and hybridization properties of peptide nucleic acid containing a substituted phenylpyrrolocytosine designed to engage guanine with an additional H-bond

Article abstract of DOI:10.1021/ja804233g

A new pyrrolocytosine derivative has been designed to selectively interact with guanine and has been evaluated in peptide nucleic acid where it imparts increased selective binding affinity for complementary oligonucleotides. The modified nucleobase also possesses an exceptionally high fluorescence quantum yield that is responsive to hybridization. Copyright

Full text of DOI:10.1021/ja804233g

Published on Web 08/30/2008
Fluorescence and Hybridization Properties of Peptide Nucleic Acid
Containing a Substituted Phenylpyrrolocytosine Designed to Engage Guanine
with an Additional H-Bond
Filip Wojciechowski and Robert H. E. Hudson*
Department of Chemistry, The UniVersity of Western Ontario, London, Ontario, Canada, N6A 5B7
Received June 11, 2008; E-mail: robert.hudson@uwo.ca
Over the past decade fluorescent DNA base analogues have
received considerable attention, and there continues to be a growing
interest in their design, study, and application.1 Fluorescent
nucleobase analogues that maintain fidelity of base-pairing, possess
a high fluorescence quantum yield, and undergo a significant change
in quantum yield upon hybridization are prized for use in the study
of nucleic acid conformation/dynamics and as hybridization probes.
The archetypal responsive fluorescent nucleobase is the adenine
analogue 2-aminopurine which retains complementarity to thymine/
uracil but also wobble pairs with cytosine. It has an exceptionally
high fluorescence quantum yield (Φf ) 0.68) that decreases when
incorporated into oligonucleotides owing to base-stacking interac-
tions2 and thus has found use as a probe of environmental changes.
There exists relatively few such fluorescent nucleobases comple-
mentary to other nucleosides; fewer yet have been incorporated
into oligonucleotides or analogues.3 With respect to the recognition
of guanine, the most studied examples are the tricyclic phenothiazine
analogue of cytosine (tC),4 its oxygen-containing homologue
phenoxazine (tCo),5 and 6-methylpyrrolocytosine (MepC).6 The tC
base has been studied in both DNA and peptide nucleic acid (PNA)
and its high fluorescence (Φf ) 0.20) has been reported to be
insensitive to the single or double stranded nature of the DNA and
the base sequence.7 The tCo analogue, when incorporated into DNA,
has an average quantum yield of 0.22 and has been referred to as
the brightest DNA-incorporated base analogue reported so far.5
Although pyrrolocytosine derivatives have been incorporated into
DNA and RNA and demonstrate responsiveness to hybridization,
these fluorophores are relatively inefficient (Φf ≈ 0.06).8
Figure 1. (a) Structures of nucleobases studied; cytosine, 6-phenylpyr-
rolocytosine (PhpC), [mono-o-(aminoethoxy)phenyl]pyrrolocytosine (moPh-
pC), and [bis-o-(aminoethoxy)phenyl]pyrrolocytosine (boPhpC). (b) Pro-
posed interaction of boPhpC with guanine.
Table 1. Tm Data (°C) for PNA:DNA and PNA:RNA Duplexes,
Duplex Stabilization (∆Tm) and Mismatch Discrimination for DNAa
match
mismatch (DNA target)
sequence
nucleobase
DNA
RNA
Z ) C
Z ) A
Z ) T
1
2
3
4
5
6
7
8
X ) cytosine 49.5
X ) PhpC
X ) moPhpC (+10.5) (+4.0)
X ) boPhpC (+9.5) (+3.0) (-15.0) (-16.0) (-16.0)
Y ) cytosine 51.0 63.0 (-15.5) (-14.0) (-15.0)
Y ) PhpC (+2.0) (+1.0) (-11.0)
Y ) moPhpC (+7.0) (+3.0) (-11.0) (-9.0) (-11.0)
Y ) boPhpC (+11.5) (+10.0) (-13.5) (-14.5)
57.0
(-7.5) (-4.5) (-13.5)
(+2.5) (-2.0) (-13.0) (-7.0) (-9.0)
b
b
b
b
b
b
a PNA sequences: GTA GAT XAC T-Lys and GTA GAT CYC
T-Lys.
mismatch base in DNA. b No observed cooperative
Z
)
transition. ∆Tm values in parentheses, positive values indicate duplex
stabilization.
on the phenyl ring serve the purpose of increasing the aqueous
solubility of the modified insert and providing a Hoogsteen
hydrogen bond to O(6) of guanine.13,14 For comparison, unsubsti-
tuted (PhpC) and monosubstituted (moPhpC) pyrrolocytosine-
containing PNAs were also prepared, Figure 1.
Oligonucleotide analogues capable of forming highly stable
complexes with their target nucleic acids can be productively
combined with fluorescent labels to construct specific, high affinity
probes as potential bioanalytical reagents.9 One such oligonucleotide
analogue is peptide nucleic acid (PNA).10 The increased affinity is
useful for stabilizing relatively short probes or to out compete other
ligands for the same target sequence. Herein, we describe an
exceptionally bright and environmentally sensitive pyrrolocytosine
nucleobase analogue designed for tight binding to guanine and its
hybridization performance in PNA.
The hybridization properties of sequences 1-812b were evaluated
with DNA and RNA and compared with the control oligomers (X
and Y ) cytosine), Table 1. Incorporation of PhpC into oligomers
resulted in a consistently modest increase in Tm (ca. +2.0 °C with
DNA) ascribed to increased π-stacking. Incorporation of boPhpC
resulted in a marked increase in binding affinity (+9.5 and +11.5 °C,
with DNA) presumably due, in large part, to an additional hydrogen
bond to guanine. Only 8, containing boPhpC, exhibited a significant
increase in affinity toward RNA. Such dramatic sequence effects are
also observed for other related cytosine nucleobase analogues.12
The formation of helical complexes was confirmed by circular
dichroism spectrophotometry. While single-stranded PNA (1-8)
characteristically showed no CD signal; complexes with cDNA for
all fully matched heteroduplexes (PNA:DNA) displayed a positive
Cotton effect at 260 nm and a shoulder at 280 nm and another positive
peak at ca. 220 nm. The similarity of spectral features for heterodu-
plexes formed whether possessing an unmodified cytosine, PhpC,
We have previously described the synthesis and fluorescence
emission properties of some para-substituted 6-phenylpyrrolocytosine
(PhpC) derivatives11 and report herein a new cytosine base analogue,
[bis-ortho-(aminoethoxy)phenyl]pyrrolocytosine (boPhpC), that is
designed to engage guanine with an additional hydrogen bond, Figure
1.12 The hybridization properties, fluorescence quantum yields, and
fluorescence response to cDNA and RNA are reported.
A domino Sonogashira cross-coupling/annulation reaction se-
quence with an appropriately derivatized terminal alkyne was
employed as the key step in the synthesis of the bicyclic nucleobase
luminophore (see Supporting Information). The dual appendages
9
12574 J. AM. CHEM. SOC. 2008, 130, 12574–12575
10.1021/ja804233g CCC: $40.75
2008 American Chemical Society

C O M M U N I C A T I O N S
Table 2. Photophysical Data of boPhpCa
Exc, λmax (nm)
Em, λmax (nm)
Φfb
dioxane
1-octanol
acetone
378
379
378
372
365
455
461
456
461
468
0.75
0.74
0.62
0.61
0.32
ethanol
aqueous bufferc
a Referring to Figure 1, fluorophore structure
R
)
CH2CO2Et.
b Quantum yield determination detailed in ESI. c Consists of 100 mM
NaCl, 10 mM Na2PO4, 0.1 mM EDTA, pH 7.
Figure 2. Fluorescence emission spectra and quantum yield of sequence
4 (left) and sequence 8 (right), each containing the boPhpC modification.
moPhpC, or boPhpC suggests similar global conformations. Impor-
tantly, the CD spectra clearly showed the formation of duplexes
possessing a mismatched basepair with an approximately 15 nm
bathochromic shift of the 260 nm band. All PNA also formed helical
complexes with complementary RNA (see Supporting Information).
To establish whether the increased affinity toward cDNA or RNA
was due to the additional hydrogen bond to guanine or electrostatic
interactions, moPhpC was synthesized. If electrostatic interactions were
the major source of increased duplex stability, moPhpC possessing
only one amino group should hybridize with lower affinity than
boPhpC. However, this is not the case, as moPhpC yields a hybrid
with similar thermal stability (cf. 3 and 4) suggesting that the increased
affinity is not solely due to electrostatic interactions but due to specific
and discriminating bonding interactions to guanine.
binding to guanine with excellent base recognition specificity. We
anticipate that this modification will find use as a valuable reporter
group in PNA-based hybridization probes and in potential antisense/
antigene applications.
Acknowledgment. We thank NSERC Canada for support of
this work. Mr. Andrew Wallman is thanked for early contributions
to this work.
Supporting Information Available: Characterization data, synthetic
procedures, and experimental details. This material is available free of
charge via the Internet at http://pubs.acs.org.
The excellent base discrimination ability of boPhpC is illustrated
by using singly mismatched DNA (Table 1). In both PNA sequences
a mismatch was detrimental to duplex stability as indicated by the
decrease in Tm (∆T m g -13.5 °C). This discrimination is equivalent
to or better than cytosine itself.
Next, we investigated the fluorescence properties of boPhpC
because of its demonstrated excellent binding properties and
increased PNA solubility. The photophysical study of boPhpC
revealed an exceptional quantum yield which was sensitive to the
molecular environment. Although the quantum yield is strongly
influenced by the environment and decreases with increasing the
polarity of the medium (Table 2), it remains remarkably high in
aqueous solution. The value of Φf ) 0.32 (ꢀ365 ) 6650) for the
boPhpC luminophore is comparable to the best fluorescent cytosine
analogues yet reported.
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