nihms91072.pdf

Back Electron Transfer Suppresses the Periodic Length

Dependence of DNA-mediated Charge Transport Across Adenine

Tracts

Joseph C. Genereux

Katherine E. Augustyn

Molly L. Davis

Fangwei Shao

, and

Jacqueline

K. Barton

Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena,

California 91125

Abstract

DNA-mediated charge transport (CT) is exquisitely sensitive to the integrity of the bridging

-stack

and is characterized by a shallow distance dependence. These properties are obscured by poor

coupling between the donor/acceptor pair and the DNA bridge, or by convolution with other

processes. Previously, we found a surprising periodic length dependence for the rate of DNA-

mediated CT across adenine tracts monitored by 2-aminopurine fluorescence. Here we report a

similar periodicity by monitoring N

-cyclopropylguanosine decomposition by rhodium and

anthraquinone photooxidants. Furthermore, we find that this periodicity is attenuated by consequent

back electron transfer (BET), as observed by direct comparison between sequences that allow and

suppress BET. Thus, the periodicity can be controlled by engineering the extent of BET across the

bridge. The periodic length dependence is not consistent with a periodicity predicted by molecular

wire theory but is consistent with a model where multiples of four to five base pairs form an ideal

CT-active length of a bridging adenine domain.

INTRODUCTION

The DNA

-stack has the inherent ability to act as an efficient medium for charge transport

(CT).

Long range DNA-mediated CT is exquisitely sensitive both to the coupling of donors

and acceptors into the

-stack,

and to the presence of lesions, mismatches, protein-induced

distortions, and other defects in the integrity of base stacking.

This sensitivity has been

exploited in the development of novel classes of DNA-based sensing technologies

and might

be utilized

in vivo

by transcriptional activation and DNA repair pathways.

To realize fully

the potential of this technology, it is necessary to understand the mechanistic underpinnings

of DNA-mediated CT.

Recently, a periodic dependence on adenine tract length was observed for the fluorescence

quenching of photoexcited 2-aminopurine (Ap*) by DNA-mediated CT to guanine across the

adenine tract.

By standardizing to a system containing the redox-inactive base inosine, the

contribution to quenching solely due to CT between Ap* and guanine was isolated. The

amplitudes associated with this periodicity are substantial and greater than the observed

associated errors. Non-monotonicity of CT rate versus distance has since been observed

between gold and ferrocene across methyl-substituted oligophenyleneethynylene, but that

result was attributed to substantial torsional variations between polymers of different lengths,

an explanation that is not adaptable to these adenine tracts

. Instead, we interpreted our

E-mail: jkbarton@caltech.edu.

NIH Public Access

Author Manuscript

J Am Chem Soc

. Author manuscript; available in PMC 2009 November 12.

Published in final edited form as:

J Am Chem Soc

. 2008 November 12; 130(45): 15150–15156. doi:10.1021/ja8052738.

NIH-PA Author Manuscript

surprising result in the context of four or five base pairs being conducive to forming a CT-

active domain, leading to higher CT over an adenine tract that is an integer multiple of this

number. This interpretation is consistent with the conformationally gated character of DNA-

mediated CT over long distances,

with evidence for delocalization of the injected hole,

and

with evidence for a similar delocalization length in the formation of excimers along adenine

tracts.

A similar argument has been made to explain this result in the context of a polaron

hopping model,

and non-monotonicity was also observed in calculations that permitted

delocalization.

Importantly, Ap* fluorescence quenching is insensitive to processes that occur after the CT

event, including radical trapping, incoherent hopping or back electron transfer (BET). For hole

acceptors in DNA, product yields for different photooxidants scale inversely to the propensity

for BET,

and attenuating BET, both between the hole donor and the oxidized bridge and

between the hole donor and oxidized acceptor, extends the lifetime of the charge separated

state.

While other spectroscopic investigations of CT across adenine tracts have not revealed

a similar periodicity, these other studies have been performed on systems for which BET is

known to be substantial

15,16

or where slow trapping allows charge equilibration after the initial

CT step.

17,18

We have recently shown that for both hole and electron transport, CT efficiency

is dictated in the same manner by the dynamics and structure of the intervening DNA bases.

If the periodicity is the result of CT-active states that serve as more efficient pathways for

forward CT, then they will also mediate more efficient BET. Hence, we propose that

conformations that promote forward CT also promote BET, and this BET will serve to suppress

the apparent periodicity.

To test this hypothesis and determine whether this periodicity is a general property of long

range DNA-mediated CT, in the present work we consider disparate donor-acceptor systems

with varying extents of BET (Figure 1). Previously, by measuring quantum yield of damage

at double guanine sites, we ranked a series of photooxidants by propensity for charge

recombination between the guanine cation radical and the reduced hole donor.

Two

photooxidants that are subject to only moderate BET are Rh(phi)

(bpy’)

(Rh) and

anthraquinone (AQ), while BET is highly efficient for Ap. Although these and other

photooxidants typically induce oxidation of native guanine sites to 8-oxoguanine and other

base-labile damage products,

18,20

facile BET between guanine cation radical and aminopurine

anion radical renders Ap photooxidation of guanine only observable with the

G trap.

Furthermore, to limit post-injection charge equilibration, we assay for arrival using N

cyclopropylguanine (

G) instead of guanine as a hole acceptor.

This fast

trap for cation

and anion radicals allows detection of pre-equilibrium CT processes that are obscured by the

slow trapping of guanine radical by water or oxygen. By modulating the extent of BET for a

series of

G-containing duplexes, we demonstrate that the periodic length dependence is

inherent to adenine tracts but is attenuated with increasing BET.

EXPERIMENTAL

Oligonucleotide Synthesis

DNA oligonucleotides were synthesized trityl-on using standard phosphoramidite chemistry

on an ABI DNA synthesizer with Glen Research reagents. 2-aminopurine was incorporated as

the N

-dimethylaminomethylidene protected phosphoramidite (Glen Research).

G-modified

oligonucleotides were prepared by incorporating the precursor base, 2-fluoroinosine-O

paraphenylethyl-2’-deoxyinosine (Glen Research), as a phosphoramidite at the desired

position. The resin was then reacted with 1 M diaza(1,3)bicyclo[5.4.0]undecane (DBU,

Aldrich) in acetonitrile to effectively remove the O

protecting group. The oligonucleotides

were subsequently incubated overnight in 6 M aqueous cyclopropylamine (Aldrich) at 60 °C

resulting in substitution, base deprotection, and simultaneous cleavage from the resin. The

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cleaved strands were dried

in vacuo

and purified by reversed-phase HPLC, detritylated by 80%

acetic acid for 15 min, and repurified by reversed-phase HPLC. Oligonucleotides were

characterized by MALDI-TOF mass spectrometry.

Rhodium-modified oligonucleotides were synthesized as described previously.

Briefly, the

detritylated resin-bound oligonucleotides were first modified with a nine carbon amine linker

by reaction with carbonyldiimidazole and diaminononane in dioxane. The amine-modified

strands were then reacted with [Rh(phi)

(bpy’)]Cl

(bpy’ = 4-(4’-methyl-2,2’-bipyridyl)

valerate) in 1:1:1 methanol:acetonitrile:isopropanol using O-(N-succinimidyl)-1,1,3,3-

tetramethyl uranium tetrafluoroborate (TSTU) as the coupling reagent. Cleavage from the resin

was accomplished by incubation in NH

OH at 60 °C for 6 hours. Strands were HPLC-purified

using a Varian C

reversed-phase column. The two diasteromeric conjugates, differing in

configuration at the metal center, have different retention times. However, both isomers were

collected together and used for subsequent experiments. MALDI-TOF mass spectrometry was

used to characterize the metallated DNA conjugates.

Anthraquinone (AQ)-tethered oligonucleotides were synthesized as described previously by

incorporating an anthraquinone phosphoramidite at the 5’-end of the oligonucleotides.

The

DNA was deprotected in NH

OH at 60 °C overnight. The resulting oligonucleotides were

purified once by reversed-phase HPLC and characterized by MALDI-TOF mass spectrometry.

All oligonucleotides were suspended in a buffer containing 50 mM NaCl, 20 mM or 5 mM

sodium phosphate, pH 7 and quantified using UV-visible spectroscopy. Duplexes were

prepared by heating equal concentrations of complementary strands to 90 °C for 5 min and

slow cooling to ambient temperature. Melting temperatures (T

) were obtained for all

duplexes. All duplexes melted between 50 – 60 °C at a 1.5 μM concentration in phosphate

buffer (PBS, 20 mM sodium phosphate, 50 mM NaCl, pH 7).

Photooxidation Experiments

Photooxidations of Rh-tethered oligonucleotides were carried out by irradiating 30 μL aliquots

of 10 μM duplex in PBS for 30 sec at 365 nm on a 1000 W Hg/Xe lamp equipped with a 320

nm long pass filter and monochromator. AQ-containing duplexes in PBS (30 μL, 10 μM) were

irradiated at 350 nm using the same apparatus for 5 min. Irradiation times were varied and the

decomposition was linear over the times used (supplementary information). Samples were

irradiated at various temperatures ranging from 20 to 80 °C. Ap-containing duplexes (30 μL,

10 μM) in PBS were irradiated as above at 325 nm without the long pass filter for 30 sec or

30 min.

To analyze for

G decomposition following irradiation, samples were digested to the

component nucleosides by phosphodiesterase I (USB) and alkaline phosphatase (Roche) at 37

°C, to completion. The resulting deoxynucleosides were analyzed by reversed-phase HPLC

using a Chemcobond 5-ODS-H, 4.6 mm × 100 mm column. The amount of

G per duplex

was determined by taking the ratio of the area of the HPLC peak for d

G to the area of the

peak for dT. For 30 minute irradiations, a small amount of thymine decomposition was

observed, as has been described previously.

Hence, redox-inactive inosine was used as the

internal standard for these experiments. The decomposition yield is taken as the percent loss

G between an irradiated sample and the dark control. Dark control HPLC traces were

confirmed to yield the correct relative amounts of dA, dC, dG, dI, dT, and d

G based on

duplex sequence. Irradiations were performed at least three times and the results averaged. Due

to the long irradiation times used for the Ap-I

G strands, actinometry was performed

using a 6 mM ferrioxalate standard

to allow comparison between experiments performed on

separate days. The given quantum yield is for the efficiency from the Ap* state to the ring-

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opened product. Fluorescence quenching for the Ap-I

was not expected to be observable

based on the quantum yield of

G damage, and hence was not explored.

Errors are presented at 90% standard error of the mean, using the Student’s t-distribution at

the appropriate degrees of freedom to determine confidence intervals.

RESULTS

Experimental Design

Figure 1 illustrates typical DNA-photooxidant assemblies. The Rh-A

, AQ-A

and Ap-A

series contain rhodium, anthraquinone, or 2-aminopurine separated from

G by a bridge

containing increasing numbers of adenines. For all Rh-modified assemblies there is a four base

pair segment surrounding the rhodium binding site to provide optimum intercalation of the

photooxidant. In Figure 1, the rhodium is shown intercalated two base pairs from the terminus,

but likely a mixture of binding sites (one and two bases in) are available to the diastereomers.

On the side distal to the hole trap, there is a constant three base sequence so that end effects

are minimized. Guanine can serve as a thermodynamic well if placed near the rhodium

intercalation site and, although the trapping rate is slow, BET to rhodium is comparably fast

at short distance.

Therefore, inosine was employed as a substitute for guanine near the

rhodium binding site to enhance

G decomposition.

9,19

Note that the first four adenine tract

sequences, Rh-A

through Rh-A

are composed of 20 base pairs, while that of Rh-A

’ through

Rh-A

are slightly longer, with 26 base pairs (supplementary information). Rh-A

and Rh-

’, both containing the 8 base pair long adenine tract but differing in length, yield equivalent

decomposition profiles with both time and temperature, and in subsequent results and figures,

the data from Rh-A

’ are presented. A series of HPLC traces from the time-course of AQ-A

degradation shows the well-resolved peaks corresponding to the six different natural and

unnatural nucleosides (Figure 2).

DNA-mediated Oxidative Decomposition of

G by Rh and AQ

Figure 3 shows the variation in the decomposition yield (Y) as a function of bridge length for

the Rh-A

and AQ-A

series. Notably, the same non-monotonic, apparently periodic decay is

observed for the Rh-A

series as was seen for the Ap* fluorescence quenching.

The apparent

period of about five base pairs is similar as well, as is the temperature dependence for the Rh-

sequences. Below the T

of the duplex, increasing temperature leads to increased

decomposition, but the amplitude of the periodicity is suppressed. Once the duplexes begin to

melt, unstacking the base pairs, the decomposition efficiencies sharply drop to zero

(supplementary information). This decrease in decomposition occurs between 50 – 60 °C.

Although the apparent periodicity is dampened, a similar profile is apparent with anthraquinone

as the pendant photooxidant (Figure 3). As with the Rh-A

series, photooxidation of the AQ-

assemblies show a shallow, non-monotonic periodic length dependence in yield. Decay

parameters and apparent period are comparable.

DNA-mediated Oxidative Decomposition of

G by Ap

To determine if periodicities could be observed in the presence of facile BET, we prepared the

series of duplexes ApA

. Figure 4 directly compares the CT yield for

G decomposition and

Ap* fluorescence quenching. Although oxidative damage to

G is observed,

G immediately

neighboring Ap does not allow a sufficiently long-lived charge-separated state, and BET

depletes the oxidized base faster than ring-opening.

This initial low yield for a single

intervening adenine, and much higher yield for three intervening adenines, is characteristic of

a system with rapid charge recombination.

14,15

Notably, although the length dependence

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G ring opening is comparable to the fluorescence quenching result, the corresponding

periodicity is completely suppressed.

For the Ap-I

sequences (Figure 5), there is substantially less damage, such that 30 min of

irradiation is necessary to achieve significant decomposition of

G. Nonetheless, BET is

suppressed, as only slightly more decomposition is observed for the Ap-I

sequence versus

the Ap-I

sequence. Importantly, the non-monotonicity is now recovered and is qualitatively

similar to that observed for the Ap* fluorescence quenching and Rh-A

systems.

DISCUSSION

Observation of Periodicities in Length Dependence of

G Decomposition

The dependence of

G oxidation by Rh or AQ on the length of the intervening adenine tract

is periodic. It is striking that this result is so similar to that seen with the Ap* fluorescence

quenching assay and that the periods are identical. The driving forces for photooxidation by

Ap*, Rh*, and AQ* vary over a range of 700 mV.

2,30,31

The fluorescence quenching assay

measures direct hole injection from Ap* into an orbital that includes the acceptor guanine,

while the

G assays directly measure the total CT yield to the hole acceptor, regardless of

mechanism. Nevertheless, despite these fundamental differences between the experiments, a

periodic length dependence is observed for all three cases and approximately the same apparent

period is observed. Importantly, when the slow, unmodified guanine trap is used, no periodicity

is observed, indicating the importance of assaying pre-equilibrium states in CT experiments.

Although the

G decomposition is a chemical event, the fast timescale of ring-opening defines

a fast clock where CT is rate-limiting, in contrast to biochemical experiments measuring

guanine decomposition.

For the Rh-A

series, with increasing temperature, the overall yield of CT increases, the length

dependence becomes shallower, and the periodicity is attenuated. For a direct CT event

between a donor and acceptor in contact, in which the donor and acceptor orbitals are already

aligned, higher temperatures are likely to decrease the probability that the orbitals will remain

aligned, and decreased CT results. In contrast, when the donor and acceptor are separated by

a dynamic bridge of base pairs, increasing the temperature allows a greater fraction of these

duplexes to access a CT-active domain, resulting in enhanced CT. Increased temperature has

a more prominent effect on CT through longer adenine bridges because there is a lower initial

probability of each bridging base being aligned in a CT-active conformation. This effect is

identical to that observed for Ap* fluorescence quenching.

Furthermore, for both cases, the

apparent periodicity is suppressed with increasing temperature, implying that the underlying

cause of the periodicity is the same. Periodicity is not as evident for the AQ-A

system as for

the Rh-A

sequences. This apparent decrease in amplitude could be because the AQ is separated

from the adenine tract by five bases, introducing dephasing processes. Furthermore, anionic

AQ radical can equilibrate between singlet and triplet states, the former being competent to

reduce oxygen,

generating a persistent hole in the DNA that can equilibrate over a long

timescale and damage

G independently of the bridging sequence; previous work

has,

however, shown only a modest effect of oxygen on

G ring-opening rates by AQ.

Nevertheless, there is clear deviation from monotonicity that is greater than experimental error,

and a period equivalent in length to that observed for the RhA

is evident.

In a sense, the examination of Ap-A

G sequences should represent an intermediate system

between studies of Ap* fluorescence quenching by guanine and assays of

G decomposition

by Rh photooxidants. The photooxidant is the same as in the fluorescence quenching study,

and

G decomposition is used as a proxy for charge separation, as with the Rh-(A)

and AQ-

(A)

series. Remarkably, the decay is monotonic (Figure 4), with a decreasing slope similar to

that observed in a system using stilbene as a photooxidant.

This could be due to a higher

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proportion of initial CT-active conformations for short lengths

or to changing distribution of

yield with length between superexchange, localized hopping, and delocalized hopping

mechanisms. Nevertheless, the only consistent difference between the Ap-A

system and the

other three is the presence of efficient BET. Clearly, we can control this non-monotonic effect

by changing the extent of BET.

We next considered the effect of eliminating BET while still assaying for ring-opening. The

timescale required for efficient charge injection is the nanosecond lifetime of Ap*, while BET

must compete with the faster ring-opening. Hence, we speculated that a bridge modification

that sufficiently decreased the rate of CT in both directions could eliminate BET while still

maintaining some efficiency for forward transfer.

14,34

Ap* does not oxidize inosine, and the

introduction of inosine into an adenine bridge substantially affects the CT yield. We introduced

three inosines between the aminopurine and the adenine tract (Figure 5). As expected, the total

CT efficiency dropped substantially, but the Ap-I

sequence has equivalent damage yield

to the Ap-I

sequence, indicating that BET has been mostly excluded from the system.

Importantly, the non-monotonicity is now restored, supporting the hypothesis that BET was

responsible for suppressing the periodicity.

These results are straightforward to reconcile with two recent studies on CT across adenine

tracts. In one system, transient absorption spectroscopy was used to measure the production of

NDI radical, with PTZ across an A tract participating as the hole acceptor.

No periodicity

was observed, but it was found that BET substantially depletes the charge separated state.

Similarly, another series of experiments considered CT across an adenine tract between two

capping stilbenes.

The length dependence found in this study is identical to that for Ap-

G, and no periodicity was observed. Furthermore, BET of the injected hole is rapid in

this system as well. Notably, although a recent theoretical treatment of three-adenine tracts

implied that the stiffness introduced by the bridging stilbene used in this study does not

profoundly influence local coupling constants

, this environment might well affect formation

of delocalized domains.

Conformational gating through delocalized CT-active domains

Previously, we interpreted the periodic length dependence in the context of a certain number

of bases being ideal for forming a CT-active domain.

When an integer number of CT-active

domains can readily form between the acceptor and donor, CT is accelerated, either coherently

through two mutually CT-active domains or incoherently by hopping between such domains.

For a non-integer number of domains, dephasing processes, such as domain drift, are required.

These processes are slower and decrease the probability of CT to the acceptor before charge

recombination. A similar argument has been made in the context of polaron hopping.

The

experiments described here do not distinguish between the two mechanistic arguments.

Nevertheless, the fact that BET suppresses the periodicity supports the notion that increased

CT across certain bridge lengths is the inherent source of the periodicity.

Since the conformationally gated domain hopping model ascribes the periodicity to the change

in A-tract length, it is interesting to compare distance dependences to a system in which the

A-tract length is fixed. This was accomplished by monitoring decomposition of

cyclopropyladenine (

A) serially substituted at each position within a 14 base pair adenine

tract.

In contrast to the

G trapping situation, there is no periodic variation of the yield

with

A position for a given A-tract length. This result is consistent with our domain hopping

model, as a given length A-tract will accommodate a similar domain structure regardless of

the placement of the trap.

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Other Theoretical Predictions of Periodic Distance Dependences

There have been theoretical predictions of a periodic length dependence of CT. In particular,

when the energies of the donor, bridge, and acceptor are similar, on-resonance CT has been

calculated to have a periodic length dependence.

36–38

In these theoretical studies of molecular

wires, though an exponential distance dependence was found for off-resonance CT, smooth,

bounded periodicities were predicted for on-resonance coupling; energetic inhomogeneities

along the bridge could attenuate the periodicities.

Although these studies modeled the wire

between metals, the same analyses could apply to a sufficiently gated charge transfer system,

such that the donor can be excited independently of the bridge. It is possible that DNA fulfills

that requirement based on the apparent conformational gating. A separate novel approach to

determine the coupling across a molecular bridge formulated the lengthening of the bridge as

iterative perturbations. Here, too, a non-monotonicity was predicted for on-resonant transfer,

but was aperiodic and unstable with respect to the coupling parameters.

Interestingly, Renger and Marcus have calculated a periodic length dependence for CT across

an A-tract DNA bridge using a model that allowed delocalization of the electron hole over

several bases.

These periodicities were eliminated by incorporation of a static disorder term.

The periodic length dependence found here does not appear to be related to on-resonance CT.

The periods are the same for the different photooxidants, Ap, Rh, AQ, with different oxidation

potentials; this similarity argues that the periodicity is not electronic in nature. More

importantly, these theoretical periodicities are all with regard to donor-acceptor separation, not

adenine tract length. Only the CT-active domain model predicts that serially inserting a

trap along a constant A-tract will eliminate the periodicity; a quantum or symmetry effect would

be, if anything, more pronounced in such a system.

It is remarkable that we are able to observe these periodicities in DNA CT using disparate

assays so long as the experiments probe events on a fast timescale and isolate convoluting

processes such as BET and trapping events. The observations here underscore the utility of

applying cyclopropylamine-modified bases as fast traps for CT. More importantly, it is clear

that engineering differing extents of BET allows control over the extent of length-dependent

periodic behavior.

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

Acknowledgment

We are grateful to the NIH (GM49216) for their support. We thank also the Caltech SURF program for a summer

undergraduate fellowship (M.L.D.). In addition, we are grateful for the helpful comments provided by our reviewers.

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22. The rate of ring-opening for

G has not been measured directly, although indirect results from

experiments in DNA suggest a rate of

≥

−

. BET from guanine cation radical to thionine anion

radical bound non-covalently to DNA has been measured as subpicosecond,

although photolysis

of thionine bound to DNA yields no detectable base-labile guanine damage despite clear evidence

by transient absorption spectroscopy that photooxidation occurs.

In contrast,

G is facilely

decomposed by DNA-tethered thionine, indicating that the

G ring-opening rate is at least

nanosecond. Similar results are obtained with Ap photooxidation, where forward transport to guanine

is 200 ps over a three adenine tract,

and no damage is observed to guanine due to facile BET,

but

G ring-opening is nonetheless observed.

Thus

G ring-opening appears to be much faster

than charge trapping at unmodified guanine. Model studies have been less illuminating.

A neutral

N-alkylcyclopropylaminyl radical

25a

was observed to ring-open with a rate of at least 7.2 × 10

−

. However, this rate is most likely accelerated by phenyl substitution on the cyclopropyl group,

though attenuated by virtue of being the neutral, rather than cation radical. A model system closer

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31. The currently accepted oxidation potential for guanosine is ~1.29 V

, although it is notable that this

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Figure 1.

Photooxidants, modified bases, and assemblies used to probe CT events in DNA. At top are

the structures of the rhodium and anthraquinone complexes utilized, and structures of

aminopurine, inosine, and

G. The rhodium complex is tethered to the 5’ end of amino

modified DNA by a nine carbon linker as represented in the center left, and the anthraquinone

is capped on the 5’ end through the phosphate. Representative assemblies, indicating position

of photooxidants, are shown on the bottom. Duplex length is conserved across individual series

by removing base-pairs distal to the hole trap (see text and supplemental information).

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Figure 2.

Overlaid HPLC traces at 260 nm for digested nucleosides from AQA

irradiated at 350 nm for

0, 1, 2, 3, 5, 7, 10, and 15 min. Traces are normalized to the height of the dT peak, and the inset

demonstrates that the peak corresponding to d

G steadily degrades with respect to increased

irradiation time. Conditions are as described in Experimental.

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Figure 3.

CT yields (Y) as a function of bridge length for the Rh-A

series and AQ-A

series. Results at

three temperatures are shown for the Rh-A

series: 20 °C (red circles), 30 °C (blue triangles),

and 40 °C (green x’s); AQ-A

experiments are at ambient temperature. Duplexes (10 μM) were

irradiated at 365 nm in 20 mM sodium phosphate, 50 mM NaCl, pH 7.0 as described in the

text. The bridge length is defined as the number of adenines between the photooxidant and the

trap. The experiments were repeated at least three times, the results averaged, and the error is

expressed as 90% confidence intervals of the mean.

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Figure 4.

CT yields (y) as a function of bridge length for the Ap-A

series (red, open circles), as

determined by ring-opening of

G. Duplexes (10 μM) were irradiated at ambient temperature

for 30 sec at 325 nm in 5 mM sodium phosphate, 50 mM NaCl, pH 7.0 as described in the text.

The experiments were repeated at least three times, the results averaged, and the error is

expressed as 90% confidence intervals of the mean. On the same plot, fluorescence quenching

from reference (6) is shown for comparison (blue, closed circles).

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Figure 5.

CT quantum yields (

) as a function of bridge length for the Ap-I

series, as determined by

ring-opening of

G. Duplexes (10 μM) were irradiated at ambient temperature for 30 min at

325 nm in 5 mM sodium phosphate, 50 mM NaCl, pH 7.0 as described in the text. The

experiments were repeated at least eight times, the results averaged, and the error is expressed

as 90% confidence intervals of the mean. Quantum yields were determined using actinometry

on 6 mM ferrioxalate.

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