An Azo-based PNA monomer: Synthesis and spectroscopic study

Article abstract of DOI:10.1080/15257770.2011.604661

The full synthetic details and photospectroscopic characterization of a peptide nucleic acid (PNA) monomer suitable for Fmoc-based oligomerization chemistry that bears an azobenzene moiety as a base surrogate are reported. The monomer showed the ability t

Full text of DOI:10.1080/15257770.2011.604661

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An Azo-Based PNA Monomer: Synthesis
and Spectroscopic Study
Mohamed E. Moustafa a & Robert H. E. Hudson a
a Department of Chemistry , The University of Western Ontario ,
London, Ontario, Canada
Published online: 08 Sep 2011.
To cite this article: Mohamed E. Moustafa & Robert H. E. Hudson (2011) An Azo-Based PNA Monomer:
Synthesis and Spectroscopic Study, Nucleosides, Nucleotides and Nucleic Acids, 30:9, 740-751, DOI:
10.1080/15257770.2011.604661
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Nucleosides, Nucleotides and Nucleic Acids, 30:740–751, 2011
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2011.604661
AN AZO-BASED PNA MONOMER:
SYNTHESIS AND SPECTROSCOPIC STUDY
Mohamed E. Moustafa and Robert H. E. Hudson
Department of Chemistry, The University of Western Ontario, London, Ontario, Canada
The full synthetic details and photospectroscopic characterization of a peptide nucleic acid
2
(PNA) monomer suitable for Fmoc-based oligomerization chemistry that bears an azobenzene moiety
as a base surrogate are reported. The monomer showed the ability to quench the fluorescence emis-
sion of fluorescein and pyrene luminophores and proved to be a competent Fo¨ster resonance energy
transfer partner in a PNA-based molecular beacon.
Keywords Azobenzene; PNA; photoresponsive; quencher; fluorescence
INTRODUCTION
Peptide nucleic acid (PNA) is a synthetic DNA mimic based on an un-
natural polypeptide scaffold. It is a chemically and biologically robust design
that forms stable hybrids with both complementary DNA (cDNA) and RNA.
Due to the stability and selectivity of hybrid formation involving PNA, it has
widespread applications within genetic diagnostics.[1–3] In this vein, PNAs
have been employed in nucleic acid detection by various techniques such as
molecular beacons[4,5] and other types of reporter probes.[6,7]
Recent efforts have been directed toward the regulation of oligonu-
cleotide hybridization and recognition by proteins via external stimuli, es-
pecially light. Asanuma and colleagues[8–11] have synthesized deoxyoligonu-
cleotides bearing an azobenzene moiety and studied the effect of light irradi-
ation on duplex formation. It was found that the introduction of azobenzene
groups into oligonucleotides reversibly disrupts duplex formation upon ex-
posure to light of appropriate wavelength. This result was attributed to the
conformational change of azobenzene moiety in response to irradiation.
Azobenzene is well known to exist in two isomers, trans and cis, that undergo
Received 10 February 2011; accepted 8 July 2011.
The authors thank NSERC Canada and the School of Graduate and Postdoctoral Studies (Western)
for financial support of this work and the Egyptian Higher Education Ministry for scholarship support
for M. E. Moustafa.
Address correspondence to Robert H. E. Hudson, Department of Chemistry, The University of
Western Ontario, London, Ontario N6A 5B7, Canada. E-mail: rhhudson@uwo.ca
740

An Azo-Based PNA Monomer
741
conformational change from the more thermally stable planar trans iso-
mer to the nonplanar cis isomer via illumination with ultraviolet (UV) light
(300 > λ > 400 nm). The trans form is recovered upon irradiation of the
cis isomer with visible light (λ > 400 nm) or via thermal relaxation. Azoben-
zenes possessing a ring substituted with an electron-donating substituent,
such as a dialkylamine, usually show a strong absorbance near 400 nm or
slightly longer wavelengths and display similar photophysical behavior.[12]
Herein, we report the synthesis of N,N -dimethylaminophenylaz-
obenzene moiety as an integral component of a PNA monomer. This
monomer is of interest because of its potential to photoregulate hy-
bridization and as a mimic of the universal quencher DABCYL (4-((4-
(dimethylamino)phenyl)azo)benzoic acid) for use in molecular beacons.
For the purposes of this report, we are interested in the fluorescence quench-
ing ability of an azo PNA monomer. We present results demonstrating its
ability to effectively act as a quencher in a molecular beacon construct.
RESULTS AND DISCUSSION
The synthesis of a PNA monomer bearing an azobenzene moiety
as a nucleobase replacement was accomplished via the standard series
of chemical transformations shown in Scheme 1. Diazotization of p-
aminophenylacetic acid was followed by coupling with N,N-dimethylaniline
to give 2-(4-((4ꢁ-(dimethylamino)phenyl)diazenyl)phenyl)acetic acid (1) in
80% yield. The azo-acetic acid derivative 1 was coupled with tert-butyl N -[2-
(Fmoc)aminoethyl]glycinate by dicyclohexylcarbodiimide (DCC) mediated
coupling to give the tert-butyl ester derivative 2 in 73% yield. Finally, the
tert-butyl group was removed by trifluoroacetic acid in dichloromethane
(50:50%) to afford the azobenzene PNA monomer 3 in 91% yield that was
suitable for oligomerization using Fmoc-based chemistry. It is worth not-
ing that silane quenchers should be avoided during the acidolysis of the
SCHEME 1 Synthesis of PNA monomer 3.

742
M. E. Moustafa and R. H. E. Hudson
FIGURE 1 UV-Vis spectral change of 3 in dichloromethane. Left: Under irradiation at 366 nm, moni-
tored at the time points shown. Right: Recovery of the spectrum by thermal relaxation monitored at 410
nm. The arrow indicates the direction of change of the peak in the spectra.
t-butyl ester, as reduction of the azo functional group was observed in their
presence.
Photoisomerization Studies and the Halochromic Effect
Figure 1 shows the UV/Vis absorption spectrum of PNA monomer 3,
which exhibited a characteristic broad absorption band centered at 414
nm (in dichloromethane) corresponding to π-π∗ transition of the trans-azo
moiety.[12] Upon illumination of 3 with near-UV light from a simple and
convenient handheld source (366 nm), a decrease in the intensity of π-π∗
band at 414 nm (trans) was observed corresponding to the light-driven trans
→ cis isomerization. Irradiation of the peak maximum was done to minimize
potential photochemical degradation of the dye.[13] The irradiation was also
necessarily done in the presence of baseto avoid confounding halochromic
effects due to photo-generated acid; however, the presence of base sup-
presses the photoisomerization.[14] The source of the acid is suspected to
be from photo-induced decomposition of the chlorinated solvent,[15] as the
same halochromic effects were observed for 2, which lacks a carboxylic acid
group, thus discounting the possibility of the carboxylic acid moiety of 3
being responsible. Recovery of trans-isomer occurred by thermal relaxation
in the dark (Figure 1, right). Incomplete recovery for the first cycle of the
photoisomerization has been observed previously but has not been ascribed
to any particular phenomenon such as photobleaching.[16]
The presence of the N,N -dimethylamino group imparts a susceptibility
to protonation that facilitates the study of pH effects on the azo-based PNA
monomer (halochromism). An ethanol solution of 3 was titrated with a
dilute solution of trifluoroacetic acid in ethanol (0.005 M). Upon addition
of the acid, a decrease in the π-π∗ absorption band for the nonprotonated
form at 409 nm with a concomitant increase in the absorption band for the

An Azo-Based PNA Monomer
743
FIGURE 2 Changes in the UV-Vis absorption spectrum of 3 in EtOH at 1.66 × 10−5 M during titration
with trifluoroacetic acid, from 0.3 to 2.4 equivalents (final TFA concentrations inset). The arrow indicates
the direction of change of the peak in the spectra.
protonated form at 524 nm was observed with an isosbestic point at 465 nm,
as shown in Figure 2. The spectral changes are accompanied by a dramatic
color change from yellow to deep red. This spectral change is explained with
the aid of Scheme 2. Although there are two basic sites in azo-PNA monomer
3, dimethylamino and azo groups,[17] protonation in EtOH favors the lower
energy, resonance-stabilized azonium ion over the ammonium form.
SCHEME 2 Two protonation states of the azo moiety: the ammonium and the resonance-stabilized
azonium.

744
M. E. Moustafa and R. H. E. Hudson
FIGURE 3 The effect of increasing amounts of 3 on the steady-state fluorescence emission spectra of
pyrene (left) and fluorescein (right), at 5 µM. Spectra were recorded in absolute EtOH and at room tem-
perature, λexcitation (pyrene) = 330 nm, λexcitation (fluorescein) = 480 nm. Final concentrations of 3 inset.
Fluorescence Quenching Ability of the Azo-Based PNA
Monomer 3
A preliminary qualitative study to assess the ability of the 4-(4-N,N -
dimethyl-aminophenyl)azobenzene PNA monomer to quench the emission
of fluorescein, pyrene, and sulforhodamine luminophores was made. PNA
monomer 3 is an analog of the well-established fluorescence quencher DAB-
CYL. PNA monomer 3 has a strong, broad absorption spanning the near-
UV and visible region centered at approximately 410 nm in EtOH. This
FIGURE 4 The effect of increasing amounts of 3 on the steady-state fluorescence emission spectra of
sulforhodamine 640 dye, λ
= 550 nm. Inset, expansion of the peak centered at ∼590 nm.
excitation

An Azo-Based PNA Monomer
745
FIGURE 5 Fluorescence emission spectra (λ
azobenzene-based PNA-MB at 2 µM in the following buffer: 100 mM NaCl, 10 mM Na2HPO4, 0.1 mM
EDTA, pH 7.0 PNA-MB + cDNA (——) and PNA-MB alone (– – –).
= 480 nm) of (4-(N,N -dimethylamino)phenyl)-
excitation
absorbance overlaps with the emission of several commonly used fluo-
rophores and, when in close proximity, may quench the fluorescence via
a Fo¨ster resonance energy transfer (FRET). The ability of 3 to effect in-
termolecular quenching of representative appropriate fluorophores was ob-
served for fluorescein (λexcitation = 480 nm; λemission = 524 nm) and pyrene
(λexcitation = 330 nm; λemission = 377, 395, and 418 nm) (Figure 3). The au-
thenticity of the quenching effect observed for fluorophores that matched
their emission to the absorption band of 3 was confirmed by using a dye that
is not a suitable FRET partner: sulforhodamine. The sulforhodamine dye is a
longer wavelength emitting dye (λexcitation = 550 nm; λemission = 590 nm) and
is not a suitable FRET partner for DABCYL. The results shown in Figure 4
also indicate that the PNA monomer 3 behaves in an analogous fashion as
free DABCYL and does not cause any significant fluorescence quenching of
sulforhodamine. The small (overall 7%) decrease in fluorescence intensity
is likely due to dilution or screening effects.
Application of 3 in a PNA Molecular Beacon
To assess the quenching ability of the azo-based PNA monomer in an
oligomer as a model molecular beacon, it was incorporated into a short 8-mer
PNA sequence: N-term-FctttttcAzK-C-term, where F, Az, and K represent fluo-
rescein, the azo-based monomer, and lysine, respectively. The N-terminus of
the oligomer was labeled with fluorescein while still bound to the resin via the
reaction with fluorescein isocyanate (FITC). The oligomer was purified by

746
M. E. Moustafa and R. H. E. Hudson
TABLE 1 Qualities of the absorption spectrum of 3 in various solvents
Solvent
(nm)
Benzene
DCM
CH3CN
Acetone
EtOH
DMF
DMSO
λ
407
25,300
411
33,400
412
29,600
407
34,200
409
15,500
414
29,300
421
39,300
max
ε (M−1 cm−1
)
reversed-phase high performance liquid chromatography (RP-HPLC) and
its identity was confirmed by mass spectrometry.
Similar to the results that have previously been reported in the litera-
ture, we found that a stemless molecular beacon was substantially quenched
in the single-stranded state, presumably as a result of FRET or collisional
deactivation due to the close proximity between the fluorophore and the
quencher.[18] Upon hybridization with a cDNA (5ꢁ-TCGAAAAAGCT), a
strong recovery of fluorescence from fluorescein was observed. As shown
in Figure 5, upon hybrid formation between the peptide nucleic acid molec-
ular beacon (PNA-MB) and the cDNA, both at 2 µM, the fluorescence signal
intensity increased by over 100-fold.
Effect of Solvent on the Absorption of 3
Compound 3 is soluble in a variety of solvents that span a wide range of
polarity and this provided the opportunity to screen for solvatochromism.
Both the wavelength of maximum absorbance in the UV-Vis spectrum (λmax
)
and the molar extinction coefficient (ε) vary with the nature of the solvent
(Table 1); however, no significant correlation to common polarity mea-
sures was found (Reichardt’s ETN, Kosower’s Z, or Taft’s π∗).[19–21] However,
these data still have use, such as to confirm incorporation of the modified
monomer into PNA oligomers and for the subsequent determination of the
oligomer concentration in solution.
CONCLUSION
We have described the synthesis of an Fmoc-PNA monomer possessing
an integral 4-(4-dimethylaminophenyl)azobenzene moiety as a nucleobase
surrogate. This PNA monomer showed the ability to undergo a light-driven
conformational change, which might be beneficial for photoregulation of
duplex stability between the PNA and complementary nucleic acids. The
azo-based PNA monomer also showed the ability to quench the fluorescence
of fluorescein and pyrene and would be compatible with the same panel
of dyes for which DABCYL is an effective quencher. The azo-based PNA
monomer was successfully incorporated into an oligomer using standard
Fmoc-based oligomerization in a molecular beacon design with fluorescein
as the optical label. As a result of hybridization of the azo-based PNA-MB to

An Azo-Based PNA Monomer
747
its complementary DNA, an increase of the fluorescence signal by 100-fold
was observed.
EXPERIMENTAL
All chemicals were obtained from commercial sources and were used
without further purification. Thin layer chromatography (TLC) was per-
formed on Merck Kieselgel 60 TLC plates. Flash column chromatography
(FCC) was performed using Merck Kieselgel 60, 230–400 mesh. NMR spectra
were recorded at 400 MHz for proton and at 100.6 MHz for carbon nuclei.
Chemical shifts are reported in parts per million (δ) and are reported rela-
tive to tetramethylsilane (0 ppm); they are referenced to the residual proton
in the following solvents: CDCl3 (7.26 ppm) and DMSO-d6 (2.49 ppm). 13
C
NMR spectra are referenced to the appropriate solvent signal: CDCl3 (77.0
ppm), DMSO-d6 (39.5 ppm), or CD2Cl2 (53.5 ppm). Multiplicities are de-
scribed as s (singlet), d (doublet), and m (multiplet). Coupling constants (J )
are reported in Hertz (Hz). Resonances due to restricted rotation around
the amide bond (rotamers) are reported as major (ma.) and minor (mi.).
High resolution mass spectra (HRMS) were obtained using electrospray ion-
ization time of flight (ESI-TOF) techniques. UV-Vis spectra were measured
with a Varian Cary 300 Bio spectrophotometer. Fluorescence spectra were
measured on a Photon Technologies International Quanta Master 7/2005
spectrophotometer.
2-(4-((4’-(Dimethylamino)phenyl)diazenyl)phenyl)acetic Acid, 1
To an ice-cooled stirred solution of 4-aminophenylacetic acid (1.0 g,
6.62 mmol) in 1 M HCl (30 mL), a cold solution of sodium nitrite (0.685 g,
9.93 mmol) in H2O (3 mL) was added dropwise. The mixture was stirred for
30 minutes and the resultant solution of diazonium salt was transferred to a
dropping funnel and then was added to a solution of N,N -dimethylaniline
(0.961 g, 7.9 mmol) in a mixture of glacial acetic acid and water (2:1 v/v,
30 mL). The mixture was stirred for 3 hours. A brown solid precipitated
after the pH was adjusted to 4 by use of an aqueous solution of 2 M sodium
acetate. The solid was collected by filtration and was washed sequentially with
cold water and ether and then dried to give product 1 in pure form (1.49 g,
1
80%), H NMR (400 MHz, CDCl3) δH: 7.78 (d, J = 8.9 Hz, 2H), 7.72 (d, J
= 8.2 Hz, 2H), 7.40 (d, J = 9.5 Hz, 2H), 6.76 (d, J = 8.6 Hz, 2H), 3.70 (s,
2H), 3.07 (s, 6H); 13C NMR (100 MHz, CDCl3) δC: 177.1, 152.3, 143.6, 134.4,
129.8, 124.9, 122.4, 111.5, 40.8, 40.3. HRMS (ESI): calcd. for C16H17N3O2
[M+] 283.1321, found 283.1323.

748
M. E. Moustafa and R. H. E. Hudson
tert-Butyl-2-(N-(2-(((9H-fluoren-9-
yl)methoxy)carbonylamino)ethyl)-2-(4-((4’-
(dimethylamino)phenyl)diazenyl)phenyl)acetamido)
Acetate, 2
To a solution of 2-(4-((4-(dimethylamino)phenyl)diazenyl)phenyl)acetic
acid (0.5 g, 1.76 mmol) in dry N, N -dimethylformamide (DMF)
(7 mL), dicyclohexylcarbodiimide (DCC) (0.366 g, 1.76 mmol) and
N-hydroxybenzotriazole (HOBt) (0.238 g, 1.76 mmol) were added.The reac-
tion mixture was degased for 15 minutes and was then stirred for 1 hour fol-
lowed by the dropwise addition of a solution of the Fmoc-backbone ester, tert-
butyl 2-(2-(((9H-fluoren-9-yl)methoxy)carbonylamino)ethylamino)acetate
(0.635 g, 1.6 mmol) in 5 mL of dichloromethane. The mixture was stirred
at room temperature for 36 hours. The dicyclohexylurea byproduct was re-
moved by filtration and the filtrate was poured into ice water with stirring,
which produced a brown precipitate. The solids were collected by filtra-
tion and then subjected to column chromatography using hexanes/CH2Cl2
(70:30 v/v) as the eluent. Yield of 2: 0.852 g, 73%. Compound 2 exists in
solution as a pair of slowly exchanging rotamers; signals attributed to ma-
1
jor (ma.) and minor (min.) rotamers are designated: H NMR (400 MHz,
CDCl3) δH: 7.82 (d, J = 8.2 Hz, 2H), 7.79–7.74 (m, 4H), 7.58 (d, J = 7.4
Hz, 2H), 7.38–7.27 (m, 6H), 6.74 (d, J = 8.2 Hz, 2H), 4.38 (d, J = 7.1
Hz, 2H), 4.22 (m, 1H), 3.95 (s, 0.6H, mi.), 3.91 (s, 1.4H, ma.), 3.75 (s,
1.3H, ma.), 3.67 (s, 0.7H, mi.), 3.58–3.48 (m, 2H), 3.39–3.31 (m, 2H), 3.07
(s, 6H), 1.48 (s, 9H). 13C NMR (100 MHz, CDCl3) δC: 156.5, 152.4, 143.7,
141.2, 129.5, 127.6, 127.1, 125.0, 119.9, 112.6, 83.1, 82.3, 49.9, 47.1, 40.8,
28.0. HRMS (electrospray ionization (ESI)): calcd. for C19H43N5O5 [M+]
661.3264, found [M+H] 662.3342.
2-(N-(2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)ethyl)-2-(4-
((4’-(dimethylamino)phenyl)diazenyl)phenyl)acetamido)acetic
Acid, 3
To an ice-cooled suspension of compound 2 (0.5 g, 0.75 mmol) in
dichloromethane (2 mL), trifluoroacetic acid (TFA) (2 mL) was slowly added
and the reaction mixture was stirred for 1 hour in an ice bath and for 3 hours
at room temperature. The mixture was evaporated in vacuo to dryness and
the remaining residue was coevaporated with dichloromethane (3 × 10 mL),
dissolved in dichloromethane (2 mL), and precipitated with the addition of
diethyl ether (30 mL). The resultant mixture was cooled to 0◦C for 12 hours,
filtered, washed with cold ether, and dried to yield the title compound as a
red solid (0.416 g, 91%). 1H NMR (400 MHz, dimethyl sulfoxide (DMSO)-
d6) δH: 8.11 (s, 1H), 7.89 (d, J = 7.4 Hz, 2H), 7.77 (d, J = 8.6 Hz, 2H),

An Azo-Based PNA Monomer
749
7.69–7.66 (m, 4H), 7.40–7.30 (m, 6H), 6.83 (d, J = 8.6 Hz, 2H), 4.31 (m,
1H), 4.20 (d, J = 6.6 Hz, 2H), 3.85 (s, 0.5H, ma.), 3.75 (s, 1.5H, mi.), 3.63 (s,
2H), 3.40 (m, 2H), 3.19 (m, 2H), 3.05 (s, 6H); 13C NMR (100 MHz, CD2Cl2)
δC: 161.1, 157.4, 144.4, 141.7, 136.5, 131.1, 128.2, 127.6, 125.6, 120.4, 119.9,
115.8, 76.3, 67.5, 47.6, 42.1, 40.0. HRMS (ESI): calcd. for C35H35N5O5 [M+]
605.2638, found [M+H] 606.2716.
Photoisomerization of 3
A solution of 3 (2.06 × 10−5 M) in dichloromethane with 7.08 × 10−2
M of NEt3 was irradiated in a quartz cuvette at room temperature using
a UVGL-58 hand-held lamp (6 W, 366 nm) at a distance of approximately
4 cm. At given time points, a UV-Vis spectrum was collected. The time course
for spontaneous reversion was monitored at the wavelength of the maximum
change (410 nm).
Luminescence Studies on 3
The quenching ability of the 4-(N,N -dimethylamino)phenylazobenzene-
containing PNA monomer 3 was tested against three luminophores: fluores-
cein, pyrene, and sulforhodamine. Separate solutions of the luminophores
and 3 were prepared at 5 µM in spectrophotometric grade absolute ethanol.
Steady-state fluorescence emission spectra were collected at room temper-
ature in the appropriate spectral window by excitation at λexcit. = 480, 330,
and 550 nm for fluorescein, pyrene, and sulforhodamine, respectively. To
1-mL solutions of the luminophore, small aliquots of 3 were added to give
final PNA monomer concentrations in the range from 1 × 10−8 to 4 ×
10−7 M; changes in volume were neglected. For sulforhodamine, a second ex-
periment, for which the results are presented in Figure 4, was done at higher
PNA concentrations to confirm its inability to be an effective quencher.
Aliquots of a stock solution of PNA at 33 mM were used, resulting in final
concentrations of 3 in the range from 6.6 × 10−5 to 2.7 × 10−4 M.
PNA-MB Synthesis, Isolation, and Purification
A PNA 8-mer was synthesized on a 5-µmol scale using the standard
FastMoc module on an Applied Biosystems 433A synthesizer using NovaSyn
TGR (Rink amide) resin. The only modification to the synthesis protocol
was extension of coupling times to 30 minutes. After the PNA synthesis was
completed, including N-terminal deprotection, the resin was transferred to a
manual peptide synthesis vessel and was washed several times with each of dry
CH2Cl2 and DMF. In a minimal amount of DMF, fluorescein isothiocyanate
(FITC) (6 equiv.) and DIEA (10 equiv.) were added and the reaction mixture
was shaken in dark for 6 hours. The resin was washed with CH2Cl2 and DMF,
and completion of FITC coupling was confirmed by Kaiser test.

750
M. E. Moustafa and R. H. E. Hudson
The PNA oligomer was cleaved from the resin by treatment with neat TFA
(1 mL) for 90 minutes. Afterward, the resin was removed by filtration and
the filtrate was evaporated under a nitrogen stream. The residue was treated
with cold anhydrous diethyl ether (1 mL) to precipitate the crude PNA.
Purification of the crude PNA was done by reversed-phase high pressure
liquid chromatography (RP-HPLC) using a 250 mm × 4.6 mm, C18-bonded
˚
phase, 100-A pore, 5-µm particle size column. The elution conditions were
a binary gradient elution at 1 mL/minute with the solvent system of TFA in
H2O (Solvent A, 0.05%, v/v) and TFA in CH3CN (Solvent B, 0.05%, v/v).
Details for the gradient: 0–5 minutes, 100% A; 5–20 minutes, 100% to 60%
A; 20–30 minutes, 60% to 0% A; 30–40 minutes 100% B.
The PNA isolated by RP-HPLC was characterized by ultra performance
liquid chromatography (UPLC)-MS (ESI-TOF; for C122 H148 N39O34S: found
2735.1440 [M+]-[3H]+, calcd. 2735.0772). PNA stock solutions were pre-
pared in deionized water and stored at 4◦C. Concentrations of PNA
oligomer were determined by measuring UV absorbance at 260 nm at
80◦C and the extinction coefficients were calculated by summing the val-
ues for the individual nucleobases, FITC (70,000 M−1 cm−1), and 4-(N,N -
dimethylaminophenyl)azobenzene (32,600 M−1 cm−1).
Luminescence Studies on PNA-MB
The steady-state fluorescence intensity of PNA-MB (N-FctttttcAzK-C,
where F = fluorescein, Az = azo-based PNA monomer, K = lysine) at 2
µM in an aqueous buffer consisting of 100 mM NaCl, 10 mM Na2HPO4, and
0.1 mM EDTA at pH 7.0 was measured by excitation at 480 nm. The hybrid
was formed by adding cDNA (5’-TCGAAAAAGCT-3’) at 2 µM concentration
and heating to 90◦C for 3 minutes, followed by annealing by slow cooling at
room temperature over the course of an hour.
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