Supporting Information for
UV-Induced Proton Transfer between DNA Strands
Yuyuan Zhang,1 Kimberly de La Harpe,2 Ashley A. Beckstead,1 Roberto Improta,3,* Bern
Kohler1,*
1
Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT
59717, United States
2
Department of Physics, United States Air Force Academy, USAF Academy, CO 80840,
United States
3
Consiglio Nazionale delle Ricerche, Istituto di Biostrutture e Bioimmagini, Via
Mezzocannone 16, 80136 Naples, Italy
*Corresponding Authors:
Bern Kohler: [email protected], Tel: +1 406-994-7931
Roberto Improta: [email protected], Tel: +39 081 2536614
Table of Contents
1. Equilibrium Constants for PT in Base Pair Radical Ions ……………………………… 2
2. Experimental Methods ………………………………………………………………… 3
3. Global Analysis ………………………………………………………………............... 5
4. Computational Details…………………………………………………………………. 6
5. Supporting Information References…………………………………………………....12
S1
1. Equilibrium Constants for PT in Base Pairs Radical Ions
The equilibrium constant, K, for the single proton transfer (SPT) reaction between a radical
ion nucleobase and its neutral Watson-Crick complement was estimated from the pKa values
in Table S1 using eq. 1,
−log10K = pKa(proton donor) – pKa(conjugate acid of the proton acceptor).
(1)
Table S1. pKa values for DNA base monomers (closed-shell species in black and radicals in
red) ordered from lowest to highest acidity. Values are from ref. 1 unless otherwise noted.
Acid / Base
pKa
Acid / Base
•
•–
2
G(+H6) / G
17.6 (calc.)
C(+H3)+ / C
H2O / OH–
15.7 a
A•+ / A(−H6)•
–
3
A / A(−H6)
>14
C•+ / C(−H4)•
C / C(−H4)–
>13 3
G•+ / G(−H1)•
•
•–
C(+H3) / C
>13
T•+ / T(−H3)•
•
•–
5
A(+H1) / A
12.1
A(+H1)+ / A
T / T(−H3)–
9.9 3
G(+H7)+ / G
–
3
G / G(−H1)
9.2 – 9.6
H3O+ / H2O
T(+H4)• / T•–
6.9
T(+H4)+ / T
a
-14
calculated as pKa = −log(Kw/[H2O]) = −log[10 / 55.56] = 15.7
b
calculated as pKa = −log[H2O] = −log[55.56] = −1.7
pKa
4.45 3
4.2 4
4.0
3.9
3.6
3.5
3.2 3
−1.7 b
−5 3
The predicted K values for SPT reactions of one-electron oxidized or reduced A·T
base pairs are as follows:
A•– + T → A(+H1)• + T(−H3)–
K = 10+12.1-9.9 = 10+2.2
(2)
A + T•+ → A(+H1)+ + T(−H3)•
K = 10+3.5-3.6 = 10-0.1
(3)
•–
–
•
•
+
+6.9-14
A + T → A(−H6) + T(+H4)
•+
K < 10
-5-4.2
A + T → A(−H6) + T(+H4)
K = 10
-7.1
= 10
-9.2
= 10
(4)
(5)
Only reaction 2 is predicted to have a significant driving force for proton transfer (PT) in
DNA with AT radical ion base pairs. Reactions 3 – 5 are not favored because T and T•– are
weak bases. A•– is proposed to form solely in the nonalternating (dA)18·(dT)18 duplex through
A→A intrastrand ET, which is not possible in the alternating d(AT)9·d(AT)9 duplex. The
above equilibrium constants thus provide a thermodynamic rationalization for interstrand PT
in the former, but not the latter duplex.
The predicted K values for SPT reactions of one-electron oxidized or reduced G·C
base pairs are as follows:
S2
G•– + C → G(+H6)• + C(−H4)–
K < 10+17.6-13 = 10+4.6
(6)
G + C•– → G(−H1)– + C(+H3)•
K > 10+13-9.6 = 10+3.4
(7)
+4.45-3.9
(8)
•+
•
+
+0.55
G + C → G(−H1) + C(+H3)
K = 10
G + C•+ → G(+H6)+ + C(−H4)•
K < 10+3.2-4.0 = 10-0.8
= 10
(9)
Note that the equilibrium constant in eq. 9 is an upper bound because protonation of G at O6,
the proton accepting site for C to G PT, should have a lower pKa than the experimental value
of +3.2 for G(+H7)+, the most stable form of protonated G in aqueous solution. The results in
equations 6 – 9 indicate that SPT is strongly favored in the base pair radical anions compared
to the base pair radical cations. Reactions 7 and 8 are more probable than reactions 6 and 9
for 265 nm excitation of the alternating d(GC)9d(GC)9 duplex because G→C ET is favored
over C→G on account of the lower ionization energy of G and the higher electron affinity of
C.
2. Experimental Methods
Transient absorption spectra at mid-IR wavelengths were acquired after exciting the duplexes
studied with 265-nm laser pulses. Solvent absorption prevents probing of O−H or N−H
stretching fundamentals, so time-resolved infrared (TRIR) spectra were recorded instead in
buffered D2O solution between 1400 and 1720 cm-1. The frequencies and intensities of the
modes in this double-bond stretching region are sensitive to proton shifts6 and one-electron
oxidation or reduction.7-9
The TRIR experimental apparatus has been described previously.10 Briefly, 800-nm
fundamental (3.5 W, 80 fs, 1 kHz) pulses were generated with a Ti:Sapphire regenerative
amplifier (Libra-HE, Coherent). 265-nm pump pulses were generated by an OPA (OPerA
Solo, Coherent) and used to excite the DNA duplexes. The UV-pump was attenuated to 3.0
μJ and focused to a beam diameter of approximately 500 μm (FWHM) at the sample. A
mechanical chopper (Newport) operating at 500 Hz (synchronized to the fundamental)
blocked every other pulse, allowing the calculation of the change in absorbance (ΔA)
measured with and without the UV pump pulse.
Broadband mid-IR probe pulses centered at 6150 nm and 6800 nm with ~200 nm
bandwidth were generated from a second OPA (TOPAS + nDFG, Coherent). A pair of
S3
transmissive CaF2 holographic wire-grid polarizers attenuated the mid-IR probe pulse to no
more than 200 μW at the sample and set the relative polarization between the electric field of
the pump and probe pulses to magic angle (54.7°). The mid-IR pulses were split into two
portions with approximately equal intensities. The “signal” portion was overlapped with the
pump beam at the sample, whereas the “reference” portion, displaced vertically from the
“signal” by 7.5 mm, travelled through the sample without interacting with the pump. Both
“signal” and “reference” were collimated after the sample, focused into a spectrograph (Triax,
Horiba), and dispersed onto a dual-row 64-element HgCdTe detector (Infrared Systems
Development).
The DNA sequences, with sodium counterions, were purchased from the Midland
Certified Reagent Company. During TRIR measurements, 2 mL DNA samples were
continuously circulated through a flow cell (Harrick Scientific Products, Inc.) with CaF2
windows (Photop Technologies, Inc). The linear flow rate of the sample was controlled by a
peristaltic pump (Cole Parmer) and set at approximately 0.1 m s-1 in the interaction region.
For the d(AT)9·d(AT)9 duplex, approximately 1050 nmol (amount of single strand)
d(AT)9 was dissolved in 2 mL of D2O buffer solution composed of 100 mM DPO42−/D2PO4−
phosphate buffer and 250 mM NaCl, giving approximately 9 mM sample concentration (per
nucleotide). Each sample was annealed in a water bath at 85 °C for 10 minutes and allowed
to cool down slowly to room temperature. Exciton-coupled circular dichroism (ECCD) and
UV-visible spectra were recorded as a function of temperature; FTIR spectra were also
recorded at the temperatures of the TRIR experiments.
For the dA18·dT18 duplex, approximately 500 A260 units of dA18 and 500 A260 units of
dT18 were mixed in the D2O buffer solution and annealed as described above. The ECCD and
FTIR spectra of the annealed sample were taken and carefully compared to the spectra
reported in the literature.11,12 Additional quantities of dA18 or dT18 were added to the solution
until both the CD and FTIR spectra compared well with reported spectra. Temperaturedependent CD and UV-Vis spectra of the samples used in the TRIR experiments are
displayed in Fig. S1.
For the d(GC)9·d(GC)9 duplex, approximately 1200 nmol (amount of single strand) of
d(GC)9 was dissolved in 2 mL of D2O buffer solution. With 250 mM NaCl, the
d(GC)9·d(GC)9 duplex is expected to adopt a stable B-form geometry. The final
concentration was approximately 10.8 mM (per nucleotide). ECCD and FTIR spectra of the
annealed samples were recorded at room temperature prior to the TRIR experiments. These
FTIR spectra are plotted in Figs. 2 and 3 in the main text in the double-bond stretching region.
S4
As can be seen in the melting curves in Fig. S1, d(GC)9·d(GC)9 does not melt in the
10 – 70 ˚C temperature range. TRIR experiments for this duplex were therefore performed at
room temperature. For the two A·T duplexes, which have melting points around 50 – 60 °C
at the salt concentrations used, samples were kept in a water bath set at 7°C during TRIR
measurements to ensure the integrity of the duplexes.
Fig. S1. Exciton-coupled circular dichroism (ECCD) spectra (A, D, G), UV-visible spectra (B,
E, H), and melting curves determined from the UV-visible spectra at 260 nm (C, F, I) for
d(AT)9·d(AT)9 (A – C), dA18·dT18 (D – F) and d(GC)9·d(GC)9 (G – I) in 100 mM phosphate
buffer (50 mM DPO42− + 50 mM D2PO4−) and 250 mM NaCl. CD signals (Δε) in panels A, D
and G were calculated using Δε = θCD / (32.98 deg · cL), where θCD is the CD signal in degree,
c is the concentration per nucleotide, and L is the optical path length (100 μm).
3. Global Analysis
Global analysis13 was performed using the Glotaran program described in ref 14. Lifetime
uncertainties reported here are twice the standard deviation (2σ). For d(GC)9·d(GC)9, two
time components were required to fit the time- and spectral-domain TRIR data. A third
component was added to model the constant offset at long times that arises from solvent
heating.15 For d(AT)9·d(AT)9 and dA18·dT18, the TRIR signals generally decay
biexponentially to a constant offset, but a fourth component improved the fit at very early
times (1 – 2 ps). The frequency-dependent amplitudes for each exponential term (component)
in the fitting function define the decay-associated difference spectra (DADS). These are
S5
shown in Fig. S2 along with the time constants determined by global fitting. The slowest
decay components (red traces in Fig. S2) are shown in Figs. 2 and 3 in the main text.
Fig. S2. Decay-associated difference spectra (DADS) for (A) d(AT)9·d(AT)9, (B) dA18·dT18,
and (C) d(GC)9·d(GC)9. The lifetimes from the global fits are also shown. Note that the red
trace in panel B has been multiplied by 3.
4. Computational Details
4a. Methods. The M052X functional16,17 was used as the reference functional for our
computational analysis. This functional provides an explicit dependence on the kinetic energy
density and has been parameterized with special reference to stacked systems. It has been
demonstrated that M052X is not biased by the traditional deficiencies of ‘standard’
functionals in describing charge transfer (CT) transitions.18 M052X has been successfully
applied to the study of excited states in several oligonucleotide systems, capturing well the
effects of stacking and hydrogen bonding interactions on excited state properties (see, for
example, the extensive comparison made in ref 19 with results of refs 20, 21). It was shown
previously that this functional accurately reproduces IR spectra of radical nucleobases in a
dinucleotide.9,22 Most of our calculations were performed using the cost-effective 6-31G(d)
basis set.
Solvent effects were accounted for by the polarizable continuum model (PCM),23 and
explicit water molecules were not included. Although water molecules bind extensively to
DNA strands in aqueous solution, their inclusion in the calculations is less important than for
base monomers because at least some of the H-bonding sites are involved in base pairing.
Furthermore, the large system size makes it impractical to include the large number of
explicit solvent molecules that would be needed to reproduce solvation effects in an unbiased
way. When the number of explicit water molecules is too small, unrealistic coordination
S6
geometries can be found that overestimate the solute-solvent interaction energy and affect the
calculation of the IR spectra.
The calculated vibrational frequencies at the PCM/M052X/6-31G(d) level were
scaled by 0.95 for comparison with experiment. The vibrational spectra for the biradicals
generated by interstrand H-atom transfer, calculated at the PCM/PBE0/6-31G(d) level, were
scaled by a factor of 0.955. In some figures below, calculated frequencies were broadened by
a Gaussian function of 10 cm-1 and 20 cm-1 FWHM for the A·T and G·C duplexes,
respectively. The calculations yield absolute integrated absorption coefficients for the various
vibrational transitions. These values were scaled by a constant factor to give the best
agreement with experimental FTIR spectra.
4b. Model Systems Used in the Calculations. The lack of analytical second derivatives for
PCM/TD-M052X calculations made calculating IR spectra of the excited base tetramers
prohibitively expensive. For this reason, the vibrational spectra of the CT excited states
shown in column B of Fig. S3 were modeled as the sum of ground-state spectra calculated
separately for the two constituent base pair radical ions. Specifically, IR spectra were
calculated for the singly-charged tetramers shown in column C of Fig. S3 in the harmonic
approximation, following full geometry optimization of each in its electronic ground state.
This approach is an extension to duplex DNA structures of one used to successfully calculate
IR spectra of CT states of single-stranded dinucleotides by combining ground-state spectra of
the constituent radical ions.9,22
S7
Fig. S3. Tetramer units used in the DFT calculations. (A) Ground state, (B) CT states without
PT (1, 2 and 4) and CT states with PT in the base pair radical anion (3 and 5), (C) tetramer
radical cations and anions used in the ground-state calculations to estimate the IR spectra of
CT states 1, 3, and 5 in panel B.
4c. Calculated Vibrational Spectra. The ground-state vibrational spectra of the two A·T
sequence isomers calculated in the harmonic approximation are shown by the black,
uppermost traces in panels B and D of Fig. S4. The scaled frequencies are in excellent
agreement with the experimental FTIR bands (gray dashed lines), establishing the reliability
of the theoretical approach. Note that the spectrum for PT in the anionic pair is not calculated
for the AT/AT dimer base step because it is stable against PT according to estimates from
experimental pKa values (Section 1). Furthermore, the negative charge on the anion of
AA/TT is always located on the T residue during geometry optimization; A•− is not possible
in these ground-state calculations.
S8
Fig. S4. Top: long-lived DADS for (A) d(AT)9·d(AT)9 and (B) dA18·dT18 in D2O solution.
Bottom: calculated vibrational spectra for (C) AT/AT and (D) AA/TT as neutrals (black
traces; the FTIR spectra of the corresponding duplexes are shown by the gray dot-dashed
curves for comparison) and as the tetramer radical cations and radical anions with the
structures shown to the right of each spectrum.
The band assignments presented below (and in the main text) are based on the
dominant character of each normal mode. For d(AT)9·d(AT)9, the strong absorption band
observed at 1630 cm-1 (bands a and b) has contributions from both the C2=O stretch of T•−
and the C4=O stretch of T that is base paired with A•+. From panel B of Fig. S4 we see that
only these two bands can lead to vibrational activity in this region. PT in the cationic tetramer
predicts a strong absorption between ground-state bands 1 and 2 that is absent in our
experimental spectrum and can therefore be ruled out. The prominent positive band seen near
1575 cm-1 (band c in Fig. 2C) is assigned to the symmetric carbonyl stretch of T•−, consistent
with previous observations in the d(AT) dinucleotide7 and the dT18 single strand.24 Our
calculations predict a fundamental at 1599 cm-1 with C4O stretching character for T•− in a
duplex. A ring mode of A•+ calculated at 1571 cm-1 may also contribute.
For d(A)18·d(T)18, only PT in the anionic base pair results in vibrational activity
between the two highest-energy ground state bands 1 and 2, corresponding to band aʹ in Fig.
S9
S4B. Furthermore, only this process predicts strong vibrational activity in the lower
frequency region of band c. The twin bands at 1595 cm-1 and 1549 cm-1, assigned to ring inplane vibrations of A(+D1)• and C4O stretching of T(−D3)−, respectively, are thought to be
responsible for the broad absorption band c observed in the experiment. Other possibilities do
not produce strong absorption in these regions.
Fig. S5. (A) Long-lived DADS for d(GC)9·d(GC)9 in D2O solution. (B) Calculated
vibrational spectra of the neutral GC/GC tetramer (black trace; the FTIR spectrum of the
d(GC)9·d(GC)9 duplex is shown by the gray dot-dashed curve for comparison) and for the
GC/CC tetramer radical cations and radical anions with the structures shown to the right of
each spectrum.
The calculated spectra for the GC/GC model tetramer are shown in Fig. S5. Only PT in
the anionic tetramer, producing G(−D1)−, results in a strong absorption band c on the blue
side of band 3. Band c is assigned to the C−O stretch coupled with a ring vibration of
G(−D1)−. Band b at ~1625 cm-1 is assigned to C(+D3)•, but the cationic tetramer may also
contribute to this feature. The possibility of PT in the base pair radical cation was also
considered. In this case, a proton would move away from the ‘hole’ created by intrastrand ET.
Although our calculations predict that PT in the cationic base pair (reaction 8) produces
S10
vibrational activity at 1700 cm-1 due to carbonyl stretching of the closed-shell C(+D3)+, the
marker band for G(−D1)• at 1550 cm-1 is not observed (the 1550 cm-1 band, band e, is shaded
gray in Fig. S5). The band observed at ~1700 cm-1 (band a) was assigned to G•+ based on
previous experiments as discussed in the main text.
We considered the possibility that photoexcitation transfers an electron and proton (i.e.
transfer of a hydrogen atom) between bases in the same base pair, an excited state identified
in previous computational studies.20,25,26 Excited state calculations performed on the 9meA·1me-T single base pair (methyl groups at the N9 and N1 positions of A and T, respectively
were substituted for the 2’-deoxyribose groups), shown in panel C of Fig. S6, indicate that
this mechanism fails to reproduce the positive feature observed at 1630 cm-1 (bands a and b
in panel A). Furthermore, the calculations predict a positive signal between bands 1 and 2,
which is not seen experimentally. Similarly, H-atom transfer from 9me-G to 1me-C in a
single base pair does not lead to a strong vibrational band at ~1595 cm-1 (band c in panel D of
Fig. S6). These calculations confirm that H-atom transfer does not account for the long-lived
signals.
Fig. S6. Long-lived DADS for (A) d(AT)9·d(AT)9 and (B) d(GC)9·d(GC)9 following 265 nm
excitation. Difference spectra for the biradical single base pairs (C) 9-meA(−D6)•·1meT(+D4)• and (D) 9me-G(−D6)•1me-C(+D4)• calculated at the PCM/PBE0/6-31G(d) level.
The difference spectra were obtained by subtracting the ground state spectrum from that of
the excited state.
S11
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