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Chemical Physics Letters 348 (2001) 255±262
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DNA/RNA nucleotides and nucleosides: direct measurement
of excited-state lifetimes by femtosecond
¯uorescence up-conversion
Jorge Peon, Ahmed H. Zewail *
Laboratory of Molecular Sciences, Arthur Amos Noyes Laboratory of Chemical Physics,
California Institute of Technology, Pasadena, CA 91125, USA
Received 20 August 2001
Abstract
Fluorescence decay times of the nucleosides: adenosine, guanosine, cytidine and thymidine, and of the corresponding
nucleotides, were determined using the technique of ¯uorescence up-conversion with femtosecond time resolution. The
excited-state lifetimes of these nucleic acid molecules all fall in the sub-picosecond time scale, con®rming the presence of
an ultrafast internal conversion channel for both the nucleotides and the nucleosides; the nucleotides lifetimes are
longer than those of the nucleosides by up to 20%. The ultrafast internal conversion is biologically relevant to the
stability of DNA, and our results support the sub-picosecond repopulation of the ground state, consistent with transient absorption studies on the femtosecond time scale. Ó 2001 Published by Elsevier Science B.V.
1. Introduction
The pathways of electronically excited nucleic
acid molecules are of considerable interest in
photochemistry and photobiology. Areas of potential relevance of these studies include: the
understanding of DNA and RNA photodamage
and repair [1,2], the use of intrinsic ¯uorescence
as a probe of DNA's molecular dynamics [3,4],
and the studies of energy transfer among the
bases in polynucleotides [5,6]. Our interest in this
subject arises from the on-going research on
*
Corresponding author. Fax: +1-626-792-8456.
E-mail address: [email protected] (A.H. Zewail).
DNA-mediated electron transfer and DNA's
characteristics when recognized by other ligands
[7±10].
Early steady-state measurements of DNA and
RNA bases, nucleosides and nucleotides demonstrated that these nucleic acid molecules exhibit
extremely low ¯uorescence quantum yields, on the
order of 10 4 at room temperature in aqueous
solutions (see Table 1) [11±15]. Also, several
studies have shown that the weak emission from
these compounds exhibits a large degree of anisotropy, even in the solution phase, indicating
that the ¯uorescent-state lifetimes are shorter than
their rotational reorientation times [11,16±18].
This set of observations strongly suggested that the
electronically excited-state dynamics of these
0009-2614/01/$ - see front matter Ó 2001 Published by Elsevier Science B.V.
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J. Peon, A.H. Zewail / Chemical Physics Letters 348 (2001) 255±262
Table 1
Measured lifetimes together with some photophysical parameters of the nucleosides and nucleotides
Nucleosides
a
Fluorescence maximum
(nm)
a
Fluorescence
quantum yield 10 4
b
Lifetime from transient absorption (fs)
c
Lifetime from ¯uorescence
up-conversion (fs)
Nucleotides
A
G
T
C
A
G
T
C
310
346
327
324
312
340
330
330
0.5
±
1.0
0.7
0.5
0.8
1.2
1.2
290
460
540
720
±
700
±
±
860 100
980 120
950 120
530 120 690 100 700 120 760 120 520 160
Reported values correspond to room temperature aqueous solutions.
kmax and /f for AMP, GMP, TMP and CMP were taken from [14, Table 1]; for Ado, Cyd and Thd from [15, Table 1]. kmax for Guo
was taken from [19] .
b
The lifetimes of the nucleosides from transient absorption are taken from [24,25]. For GMP, the value is taken from [26]. The
uncertainty in these values was reported as 40 fs.
a
molecules are dominated by a non-radiative decay
to a non-emitting state.
Using streak cameras [6,19,20], several studies
have attempted the resolution of these lifetimes, but
found that the decays were within the time resolution of the apparatus. The time resolution of the
streak-camera method is limited to a few picoseconds, and latter studies have indicated that the reported lifetimes can only be considered an upper
bound (vide infra). Pump-probe transient absorption studies have been made with sub-picosecond
time resolution and have obtained excited-state
lifetimes of the nucleobases on the order of 1 ps [21±
23]. Some of these studies were in¯uenced by multiphoton processes which lead to ionization in the
solvent and/or solute. As pointed out elsewhere
[24], for accurate results the experiments in the solution phase require careful consideration of the
pulse intensities as in many cases ionization of the
solvent can overwhelm the observed signal.
Recently, Pecourt et al. [24±26] made the ®rst
study of the excited-state dynamics of some nucleosides with femtosecond time resolution. In
these transient absorption experiments, sub-picosecond decay signals in the visible region were
observed and assigned to the lifetimes following
the probing of the S1 ! Sn absorption. Also, when
probing the UV spectral region near the red edge
of the ground state absorption, signals characteristic of the vibrationally excited ground state were
observed. These time dependent spectral features
indicated that the ultrafast internal conversion
gives rise to the electronic ground state with a large
excess energy (up to about 34 000 cm 1 ) which is
then transferred to the solvent on the time scale of
about 2±4 ps (vibrational cooling).
In order to obtain direct measurements of the
lifetimes of the S1 state, without contribution from
vibrational relaxation and solvation e€ects, direct
resolution of the ¯uorescence is needed on the
femtosecond time scale. In this Letter, we report a
direct determination of the excited-state lifetimes of
the eight nucleosides and nucleotides listed in
Scheme 1. This was accomplished by resolving the
emission decay using the technique of ¯uorescence
up-conversion in a non-linear optical crystal. The
sucient time resolution of this method provides
accurate values of the decay and allows us to observe the dependence on base structure. The
shortest lifetime measured in water solution is
520 160 fs and the longest is 980 120 fs. Clearly,
in all the nucleosides and nucleotides studied, these
molecules dissipate the energy very eciently.
2. Experimental
2.1. Up-conversion setup
The third harmonic of a 1 kHz chirped pulse
ampli®ed Ti:sapphire laser (k ˆ 270 nm, 0:5 lJ)
was used to excite the samples. Emission was col-
J. Peon, A.H. Zewail / Chemical Physics Letters 348 (2001) 255±262
257
Scheme 1. Molecular structures of the nucleosides and nucleotides studied here.
lected by a pair of parabolic focus mirrors and
crossed at an angle of about 5° with the gate beam
in the non-linear crystal. Fundamental pulses
…k ˆ 810 nm† were used to gate the ¯uorescence in
a 0.3 mm bBBO crystal (hcut ˆ 44°). The up-converted wavelength was in all cases near the maximum of the emission spectrum for each compound
(see Fig. 1). The UV signal that results from the
sum-frequency mixing was dispersed in a doublegrating monochromator and detected by a
photomultiplier tube. The instrument response
time of 360 fs was obtained from a di€erence frequency-mixing scheme of the pump and gate pulses using the same crystal.
The relative polarizations of the two beams
were adjusted at the magic angle (54.7°) to remove the contribution from rotational reorientation. The crystal's acceptance axis for the type-I
non-linear interaction was adjusted to be parallel
to the gate beam's polarization axis. Solutions
were studied in a 1 mm path-length rotating circular cell to avoid thermal and cumulative e€ects.
The integrity of the samples was veri®ed by
conventional absorption spectroscopy on the
Fig. 1. Steady-state absorption and ¯uorescence spectra of the
nucleotides studied here. The spectra were acquired in a 1 cm
cell at the concentration of 10 4 M in a 50 mM, pH ˆ 7 phosphate bu€er. The excitation wavelength for the ¯uorescence was
260 nm.
samples before and after the up-conversion experiments.
2.2. Samples
The following compounds were obtained from
Sigma and used without further puri®cation; RNA
nucleosides: adenosine (Ado), cytosine (Cyd) and
guanosine (Guo). DNA nucleoside: thymidine
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J. Peon, A.H. Zewail / Chemical Physics Letters 348 (2001) 255±262
(Thd). RNA nucleotides: adenosine 50 -monophosphate (AMP), cytosine 50 -monophosphate
(CMP) and guanosine 50 -monophosphate (GMP).
DNA nucleotide: thymidine 50 -monophosphate
(TMP). Unless indicated, aqueous solutions were
prepared in a 50 mM, pH ˆ 7 phosphate bu€er
using HPLC-spectrophotometric grade water
(Omnisolv, EM Science). The concentration of the
nucleosides and nucleotides in these studies was 3
mM with the exception of Guo; because of its reduced solubility in water, a concentration of 1.5
mM was used. Previous studies have veri®ed the
absence of association e€ects at similar concentrations for several nucleic acid monomers
[19,20,24]. Steady-state absorption measurements
were made in a Cary 500 spectrophotometer
(Varian). Static ¯uorescence spectra were taken in
a Fluormax2 spectro¯uorometer (Instruments
S.A.). All experiments were made at room temperature (294 1 K).
3. Results and discussion
The steady-state absorption and ¯uorescence
spectra of the nucleotides studied are presented in
Fig. 1. The ¯uorescence spectra have been multiplied by a relative response function to correct for
the wavelength dependence of the spectro¯uorometer. The spectra of the nucleosides are very
similar to those of the corresponding nucleotides.
The maximum emission wavelength and the
overall shape of the spectra are consistent with the
room temperature spectra available in the literature [4,6,11,13,14,19]. In all cases, the integrated
¯uorescence is very weak due to the extremely
Fig. 2. Femtosecond ¯uorescence up-conversion transients obtained as a function of pump-gate delay time. Aqueous solutions of 3
mM concentration of the nucleosides or nucleotides were made in a 50 mM, pH ˆ 7 phosphate bu€er.
J. Peon, A.H. Zewail / Chemical Physics Letters 348 (2001) 255±262
259
Fig. 3. Femtosecond ¯uorescence up-conversion transients
obtained as a function of pump-gate delay time. Aqueous solutions of 3 mM Thd (®lled circles) or TMP (open circles) were
made in a 50 mM, pH ˆ 7 phosphate bu€er.
small ¯uorescence quantum yield. This however is
not necessarily a direct forecaster of the up-conversion signal magnitude since up-conversion resolves the emission in a very small time segment.
Figs. 2 and 3 show ¯uorescence up-conversion
signals from 3 mM solutions of the nucleotides
and nucleosides in the pH ˆ 7 phosphate bu€er.
The up-converted ¯uorescence wavelength was 330
nm for Cyd, CMP, Thd and TMP; 320 nm for Ado
and AMP; and 340 nm for Guo and GMP. These
wavelengths are near the maximum of the respective ¯uorescence spectra. In all cases, the signal
corresponds to an instantaneous rise followed by a
single exponential decay to the baseline. The decay
times of these signals have been obtained from
convoluted non-linear least-square ®ts to the data
using the gaussian instrument response function.
The results have been compiled with other relevant
data in Table 1. It should be noticed, particularly
for Ado and AMP, that the time resolution of
these experiments is close to the sub-picosecond
decay and the ®ts have a slight systematic deviation from the data on the decay side of the traces.
We veri®ed that no up-conversion signal could
be detected from the bu€ered water free of the
solute, under identical alignment conditions. Fig. 3
shows the ¯uorescence up-conversion scans for
Thd and TMP on the same plot. The presence of
the phosphate group in the deoxyribose sugar re-
Fig. 4. Femtosecond ¯uorescence up-conversion transients
obtained for a series of ¯uorescence wavelengths as a function
of pump-gate delay time. The aqueous solution of 3 mM TMP
was made in a 50 mM, pH ˆ 7 phosphate bu€er.
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J. Peon, A.H. Zewail / Chemical Physics Letters 348 (2001) 255±262
sults in a slight increase in the lifetime of the ¯uorescent state, which changes from 700 to 980 fs.
Similarly, the lifetime increases from 690 to 860 fs
in the Guo/GMP case and from 760 to 950 fs in the
Cyd/CMP case.
To test for contributions from solvation, Raman scattering and others, we made a series of
experiments in which the up-converted ¯uorescence was varied from 310 to 350 nm. As shown
for TMP, for all wavelengths, the same single exponential decay to the baseline was observed with
only a small di€erence in the decay times as indicated in Fig. 4; the scan for kfluor ˆ 310 nm where
the S=N was only 4, is less reliable. The slightly
shorter lifetimes observed in the red side of the
¯uorescence spectrum of TMP, might be a signature of some solvation and vibrational relaxation
processes occurring in the S1 state on this time
scale. In fact, Callis has indicated that for the
nucleobase thymine, vibrational relaxation most
likely occurs on a time scale shorter than the excited-state lifetime. This idea was based on the
observation that both the ¯uorescence spectrum
and the anisotropy remain unchanged upon varying the excitation wavelength from near the 0±0
transition at 290 nm to an excess energy of 6000
cm 1 [15,16].
In Table 1, we also include the excited-state
decay times obtained from pump-probe experiments by Pecourt et al. As can be seen, the lifetimes we report here are somewhat longer than
those obtained from transient absorption studies.
However, the ordering of the lifetimes reported
here for the nucleosides is consistent with that
from the transient absorption study: Ado <
Guo < Thd < Cyd. The discrepancies between the
transient absorption and the up-conversion measurements may be due to the fact that transient
absorption, at certain wavelengths, could show a
signi®cant contribution from solvation/vibrational
relaxation. In ¯uorescence up-conversion, the observed decay at di€erent wavelengths of the emission band separates the decay of unrelaxed and
relaxed population.
We further made studies of the e€ect of pH on
the excited-state decay characteristics; these measurements were helpful in elucidating the power of
up-conversion in resolving, with high sensitivity,
the decay of protonated and unprotonated species.
We made an up-conversion study of a solution of
Guo at pH ˆ 3. Fujiwara et al. [19] performed
streak-camera measurements of the ¯uorescence of
these solutions and were able to isolate the weak,
fast decaying ¯uorescence of neutral guanosine
molecules from the more prominent, long lived
¯uorescence of guanosine molecules protonated at
position 7 …GuoH‡ †. The ¯uorescence spectrum of
the protonated form is centered at 382 nm and
overlaps the ¯uorescence from Guo, whose maximum occurs at 346 nm. As Fujiwara et al. showed,
in the pH regime where protonated and un-protonated species coexist, the time resolved emission
shows a fast decaying feature due to Guo and a
much longer component from GuoH‡ .
The results of our up-conversion experiments
are presented in Fig. 5. The ¯uorescence wavelength for this experiment was 340 nm; the pH of
the solution was adjusted by adding a few milliliters of an HCl solution to the Guo solution. At
this pH the Guo=GuoH‡ ratio is approximately
6.3 (pKa …GuoH‡ † ˆ 2:2) [19]. The traces in Fig. 5
are described by the sum of three exponential
terms with s1 ˆ 660 fs (67%), s2 ˆ 3:4 ps (18%)
and s3 ˆ 209 ps (15%). The long lifetime we observed is consistent with the 197 ps component
resolved in the streak-camera experiment and
corresponds to the ¯uorescence lifetime of
Fig. 5. Femtosecond ¯uorescence up-conversion transients
obtained as a function of pump-gate delay time. Aqueous solutions of 3 mM Guo at pH ˆ 3 were used.
J. Peon, A.H. Zewail / Chemical Physics Letters 348 (2001) 255±262
GuoH‡ . The sub-picosecond component we resolved for the pH ˆ 3 solution is the same as the
¯uorescence lifetime we measured at pH ˆ 7 for
Guo. The 3.4 ps component is most likely due to
relaxation processes in the S1 state of GuoH‡ at
the probing wavelength.
In the streak-camera experiment, the ultrafast
component was reported to have a 5 1 ps time
constant. When we sum the contributions from
our two observed ultrafast decays of 660 fs and 3.4
ps, a 85% total amplitude is obtained, in agreement with the relative amplitude of 90% of the 5 ps
component measured with the streak-camera.
Thus, with our up-conversion sensitivity we are
able to resolve the true ultrafast dynamics of the
species present. This case-study demonstrates the
suitability and improved accuracy of the up-conversion experiments for the characterization of the
ultrafast decay even in the presence of longer-lived
¯uorescent species.
The mechanism for the rapid electronic deactivation of the nucleic acid molecules by internal
conversion is clear, but the pathways for energy
disposal need further elucidation. Theoretical
studies have pointed out that the ultrafast
quenching is probably related to the vibronic
coupling between nearby np and pp states, which
occurs in heterocyclic and aromatic-carbonyl
molecules (proximity e€ect or pseudo Jahn±Teller
distortion) [27±29]. Also, conical intersections
have been proposed to be responsible for ultrafast
internal conversion processes in several molecules
[30±32], including nucleic acid components [24]. A
complete understanding of the femtosecond deactivation mechanism should account for several
observed phenomena, including the ordering of the
excited-state lifetimes of the four nucleotides, the
dramatic change upon modi®cations of the molecular structure, and the in¯uence of the phosphate binding (nucleosides vs nucleotides) on the
observed lifetimes.
4. Conclusion
Our results provide direct measurements of the
femtosecond decay of the electronically excited
states of eight nucleosides and nucleotides.
261
Because we monitor the population loss to the
ground state, these measurements provide accurate
description of the non-radiative decay by internal
conversion. The range of lifetimes measured is
from 520 to 980 fs. Vibrational relaxation and
solvation occur on a time scale less than or comparable to those observed here. On such a time
scale, the conversion of electronic energy to vibrational dissipation is biologically relevant as it
ensures a highly photostable molecular structure in
the DNA and RNA assemblies.
Acknowledgements
This work was supported by a grant from the
National Science Foundation. We wish to thank
Dr. Dongping Zhong for his help with the initial
setup of the experiments.
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