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Spectrophotometric analysis of nucleic acids: oxygenation-dependant

hyperchromism of DNA

Rupak Doshi1§*

, Philip J. R. Day1, Paolo Carampin

3, Ewan Blanch

2, Ian J. Stratford

4

and Nicola Tirelli3*

1School of Translational Medicine, Manchester Interdisciplinary Biocentre, The

University of Manchester, Manchester M1 7ND, UK

2Faculty of Life Sciences, Manchester Interdisciplinary Biocentre, The University of

Manchester, Manchester M1 7ND, UK

3Laboratory of Polymer and Biomaterials, School of Pharmacy & Pharmaceutical

Sciences, The University of Manchester, Manchester M13 9PL, UK

4 Experimental Oncology, School of Pharmacy & Pharmaceutical Sciences, The

University of Manchester, Manchester M13 9PL, UK

§ current address: Department of Pharmacology, School of Biological Sciences,

University of Cambridge, Cambridge CB2 1PD

Corresponding author:

Prof. Nicola Tirelli

Tel: +44 161 275 24 80

Fax: +44 161 275 23 96

Email: nicola.tirelli@manchester.ac.uk

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Abstract

The absorbance at 260 nm (A260) is ubiquitously used for nucleic acid quantification.

We show that following oxygenation, DNA solutions, experience alterations in both

spectral properties (hyperchromism in the UV region, λmax 260 nm), and DNA

conformation. The spectral changes caused by oxygen-DNA complexation are stable

for at least several weeks at room temperature or several hours at 37oC, but are also

reversible by purging with nitrogen. Our data indicate that DNA in working solutions

might already exist in the oxygen-complexed state, potentially confounding

spectrophotometric analyses. Further, the presence of these complexes does not

appear to impart cell toxicity in vitro or affect the biophysical functional behaviour

(e.g. hybridization) of DNA. Interestingly, our work also suggests that hybridization

could determine a release of bound oxygen, a phenomenon that could open the way to

the use of such systems as oxygen carriers.

Keywords: DNA analysis; DNA absorbance; charge-transfer complexes;

hyperchromism; oxygenation

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Introduction

Nucleic acids undergo oxidative chemical modifications and degradation through the

action of activated forms of oxygen, most typically singlet oxygen and reactive

oxygen species that are obtained through (multiple) single-electron reductive steps[1].

These are moderately produced during normal cell metabolism and more

conspicuously under a variety of pathological conditions[2], or can be

(photo)generated through the action of metal complexes[3], inorganic oxides[4, 5],

chemotherapic agents[6] or ionising or UV radiation[7, 8]. On the other hand,

molecular oxygen in its common triplet state per se is not known to introduce

chemical alterations to nucleic acids. The absence of chemical alteration, however,

does not preclude the occurrence of other forms of interactions: for example, nucleic

acids present the largest density of aromatic moieties among biomacromolecules and

aromatic compounds are well known to form complexes with oxygen[9].

As a consequence of the formation of complexes with oxygen, new features generally

appear in the UV-Vis spectra of organic molecules, corresponding to transitions from

ground state oxygen (

g

3 )-organic molecule complexes to charge-transfer complexes

[9, 10], which may further evolve to produce singlet oxygen ( g1 )[11] (Scheme 1).

Scheme 1: The intimate contact between oxygen and an organic (aromatic) system

enhances the UV absorption of the latter (possibly with a red-shift of the band), due to

the transition from a singlet contact complex state to a triplet, charge-transfer excited

state, which may eventually lead to the production of singlet oxygen. In these

reversible complexes the proposed stoichiometric ratio between organic molecules

and oxygen is 1:1 [12].

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It is noteworthy that the efficiency of the radiation-induced oxidative DNA damage is

strongly dependent on the presence of oxygen[1, 13, 14], which is converted into

DNA-damaging reactive oxygen species. This process may indeed require the

formation of transient complexes between nucleic acid and oxygen, which will then

facilitate oxygen activation. Such a pathway has already been proven to occur with

transition metal coordination compounds, where complexation of oxygen is a first

necessary step for its activation [15].

The intensity of the spectral band corresponding to the formation of charge transfer

complexes generally increases when organic solutions are purged with oxygen, and

this change can be reversed by purging them with nitrogen[16]. The hyperchromism is

particularly relevant for aromatic compounds, although common to most organic

compounds [17], and it is generally assumed to be proportional to the dissolved

oxygen concentration. It is interesting to note that generally the hyperchromism is not

accompanied by a sound change in the spectral location of the affected bands; the

little effect on the energetics of the transition may thus suggest a rather ancillary role

of oxygen in the transition.

In this study we investigate whether molecular oxygen can influence the spectral

properties of nucleic acids, following complex formation with their aromatic bases.

We have specifically tackled the following issues:

- Nucleic acid detection and quantification is overwhelmingly performed

spectroscopically using the UV absorbance at 260 nm (A260). This is a classic

method[18, 19], which, in particular for oligodeoxyribonucleotides, is well known

to show a dependence on the relative amounts of the different bases, as well as on

the secondary structure of the macromolecule [20, 21]. Spectral shifts that depend

on oxygen concentration may be an underlying and yet unrecognized Achilles’

heel of this ubiquitous method[22].

- If nucleic acids interact with oxygen in a reversible fashion, the resulting

complexes may be considered oxygen carriers. The biomedical interest in such

carriers would be in their possible use in hypoxia reversal, which is one of the most

promising strategies to fight cancer [23-26]. A number of current approaches

employ blood hyper-oxygenation [27-31], which, however, has a limited efficacy

since the constricted microvessels surrounding the tumour may block erythrocyte

passage [23], while additionally the oxygen carrying capacity of blood may be

already compromised due to tumour-associated anaemia [25, 27-29]. Synthetic

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oxygen carriers, such as fluorocarbons, have been used to overcome these

problems [32], however, novel biological systems with a “smart” behaviour

allowing responsive release, may be clearly advantageous.

Materials and Methods

Sample preparation

Sterile 1 x saline sodium citrate (SSC) buffer (150mM sodium chloride, 15mM,

sodium citrate tribasic, pH 7.5 + 0.2, Sigma Aldrich) was prepared using MQ water

(MilliQ Gradient A-10, Millipore, USA), and was used to dissolve ultrapure salmon

sperm DNA, (Invitrogen, USA) and calf thymus DNA (sodium salt, Type I fibres,

Sigma Aldrich), overnight at room temperature. Solutions were freshly prepared at

10µg/mL for all experiments, unless otherwise stated. Samples were protected from

light to prevent photodegradation of DNA solutions [33].

Oxygenation experiments

Three methods of oxygenation were used. The first and the third method employed

oxygen cylinders (puriss., > 99.998%, Sigma Aldrich), controlling the gas flow rate

through a pressure flow controlling valve, and a Gilmont® calibrated/correlated

flowmeter (size micro, Sigma Aldrich). Method 2 (“peaked” flow oxygenation)

method was based on the catalytic decomposition of hydrogen peroxide adapted from

previous reports [34]. Each oxygenation reaction was carried out by dissolving 1 g

ferric nitrate nonahydrate in 1 mL conc. nitric acid, diluting it to 5 mL with deionised

water, and adding 20 mL of a 30% hydrogen peroxide solution. In all methods, the

gas was directed via silicone tubing through the flowmeter, and bubbled into DNA

solutions using a sterile needle in screw-capped glass vials or microcentrifuge tubes.

UV analysis and dissolved oxygen measurements

UV spectra of DNA and oxygenated DNA samples were measured in a Lambda 25

UV/Vis Spectrophotometer with Peltier PT-6 temperature controller (Perkin Elmer,

USA), using 1 cm optical path quartz cuvettes. All absorbance spectra are displayed

as, A (OD units) = εlC, where ε (extinction coefficient), l (optical path length), C

(concentration). The purity and accuracy of DNA preparations were monitored using

A260 (UV absorbance at 260nm) and the A260/A280 ratio that a 50 µg/mL of double-

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stranded DNA solution should provide, respectively, 1.0 OD units and an average

ratio of 1.8 [35].

DO in solution, was measured using a SG6-SevenGo pro™ DO meter (Mettler

Toledo, USA). All DO measurements were recorded in real-time per minute at room

temperature (24.5oC + 0.2

oC), unless otherwise stated. The plots presented in the UV

and DO dose-response curves were obtained with the built-in sigmoid (Boltzmann)

fitting function of Origin Pro 8 (OriginLab, USA) signal(t) = A2 + (A1-

A2)/(1+exp((x-x0)/dx)), where signal is the DO conc. or A260, subject to fit-statistics.

Circular Dichroism (CD)

All CD spectra were measured with a Jasco J810 CD spectrometer with a Peltier

temperature controller set at 25oC, kept under a constant 5 L/min nitrogen

atmosphere, using a 0.1cm pathlength cuvette and parameters; scan range 210-300

nm, sensitivity 100 mdeg, bandwidth 1nm, scan speed 50 nm/min, response 1 sec, and

data pitch 0.2 nm. Every spectrum reported here, is an average of 4 accumulative

scans and has been displayed as CD signal (mdeg units) = (εL-εR)lC, where, εL-εR

(extinction coefficients for left and right circular polarized light), l (path length), and

C (concentration). DNA solutions for CD measurements were prepared in 0.5 x SSC

buffer to increase the signal-to-noise ratio.

Raman spectroscopy

All Raman spectra were collected using a ChiralRAMAN™ spectrometer (BioTools

Inc., USA) utilizing a 532 nm NdYVO4 laser and 7 cm-1

resolution, with 1.40 W laser

power, and a total data acquisition time of 1:02 minutes per spectrum. Every spectrum

is an average of 77 or 78 accumulations.

Oligonucleotide preparation and hybridization

Homo-15-mers of A/T/C (Metabion, Germany) were dissolved at 5 µg/mL in 1X SSC

buffer. Hybridization experiments were performed at room temperature (~24oC) as

100% complementarities were employed, and the formation of duplex DNA was

confirmed by DNA melting analyses against a control where 100% non-

complementarity was employed [35, 36].

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Cytotoxicity assay

The test employed a CellTiter 96®

One solution Cell Proliferation assay kit (Promega,

USA), based on the bio-reduction of MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-

carboxymethoxyphenyl)-2(4-sulfophenyl)-2H tetrazolium), into a brown formazan

product by metabolically active cells (mouse L-929 fibroblast cells (European

Collection of Cell Cultures (ECACC)). The cells (passage number below 6) were

seeded at a density of 8,000 cells/well in a 96 well plate, and incubated at 37ºC in 5%

CO2 atmosphere. After 24 hours, the medium in the wells was exchanged with fresh

medium containing buffer, and DNA or oxygenated-DNA, at concentrations ranging

200 µg/mL-25 µg/mL, filtered through a 0.22 µm sterile filter. After a further 24

hours, the medium was removed, and the wells were washed twice with PBS

(phosphate buffered saline), pH 7.4. Subsequently, a mixture containing culture

medium with MTS, but without FBS (foetal bovine serum) was added into each well.

After 3 hours, the absorbance at 490 nm was measured (µQuant, Biotek) and

expressed relatively to that of control wells.

Results and Discussion

DNA hyperchromism

In pilot experiments, DNA solutions showed a significant increase in UV absorbance

(hyperchromism, with p< 0.05 or 0.01) when purged with oxygen, and this effect was

reversed with a nitrogen flow. An example of this behaviour is reported in Figure 1,

where calf thymus DNA was exposed to a stream of nascent oxygen generated by

decomposition of hydrogen peroxide (method 2 in the experimental section).

Substantially identical effects were recorded also for salmon sperm DNA.

Interestingly, after purging nitrogen, the UV absorbance fell below that of the pristine

DNA solutions. Nitrogen purging causes a qualitatively similar effect also without

previous oxygenation (see Supplementary Information, SI Figure 1), indicating that

DNA solutions can present an oxygen-induced hyperchromism also when the

dissolved oxygen (DO) level is below saturation.

It is noteworthy that the hyperchromism is not only reversible, but also non-linearly

dependent on oxygen flow rate and dissolved oxygen concentration, with the largest

increase in absorbance corresponding to moderate increases in flow rate and DO

concentration.

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Figure 1. Left: UV spectra of calf thymus DNA solutions (10 µg/mL), oxygenated by

bubbling nascent oxygen (method 2) for 0, 5, 10, and 15 min; notably, the flow rate is

not constant (see Supplementary Information, SI Figure 2). Right: the spectrum of the

same solution after 15 min oxygenation with subsequent nitrogen purging for 5, 10

and 15 min. at ~0.1 bar. (Each spectral shift was confirmed as statistically significant

under a threshold of p<0.05 or 0.01)

Influence of the oxygenation method

The non-linear relationship between oxygen flow rate and DO concentration (and

amount of oxygen purged) on one hand and the increase in DNA absorbance on the

other, utilizing three different oxygenation methods was tested.

- Method 1 (constant flow rate). Using two different flow rates (2.9 or 8.2 mL/min

+0.2 mL/min) the same plateau values of DO (~22 mg/L) and hyperchromism

(~1.5%) were achieved (see Supplementary Information, SI Figure 2). The final DO

level is considerably higher than the equilibrium saturation of oxygen at 1 bar (≈ 9

mg/L, from Henry’s Law), likely because of higher-than-atmospheric pressure of the

gas in the bubbles (due to Laplace pressure). As a consequence, the oxygenated

solutions are markedly super-saturated and therefore the plateau DO level is marked

as “super-saturation 1” in Figure 2.

DNA absorbance and DO level increased with comparable kinetics, and both more

rapidly with higher flow rate. The A260 increase, however, appeared to have a quicker

start; a comparison of DO level and A260 curves is provided in Supplementary

Information (see Supplementary Information, SI Figure 2). Although this may be due

to a time lag between th collection of DO and UV data, we are more inclined to

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ascribe it to transient super-saturation effects in the early stages of oxygenation (as

explained in the legend to SI. Figure 2).

Figure 2: Dissolved oxygen (DO) content (left) and A260 (right and, magnified, in the

insert) for salmon sperm DNA solutions (10 µg/mL) under different oxygenation

procedures. The freshly prepared solutions already contained an average of 6 mg/L

(+1 mg/L) DO at 25oC. Upon oxygenation the equilibrium saturation level was always

largely overcome and super-saturated solutions were obtained, where different levels

of super-saturation could be obtained. However, the super-saturation levels are not

proportional to the hyperchromic shifts. Sigmoid curves obtained by fitting the

experimental data with Boltzmann functions are visual guides.

- Methods 2 and 3 (pulsatile flow). In Method 2 nascent oxygen was generated

through the Fe(III)-mediated decomposition of hydrogen peroxide, producing a

peaked flow rate profile (see Supplementary Information, black circles in SI Figure

3). The maximum flow rate achieved with this method was close to the highest flow

rate in Method 1, but the two oxygenation methods differed in their time profiles

(peaked vs. constant flow). This may be explained by the partially pulsatile (bubbles)

nature of production of nascent oxygen, i.e. a quick alternation of slower and quicker

flow regimes. To complement this, in Method 3, regular and longer pulses were

generated by switching on and off the cylindered oxygen flow (10-sec pulses, every 1-

2 min, at 9-11 mL/min, with the A260 and A280 recorded after every pulse).

Both Methods 2 and 3, produced a similarly rapid increase in DO level (Figure. 2

left). Where Method 3 may wrongly appear faster, the flow rate in Method 2 has a

slow start and only the central part of the curve should be compared. The DO plateau

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level was appreciably larger than that achieved with method 1 and is indicated as

“super-saturation 2”. Since the flow rate or the total amount of oxygen purged are

similar or lower than in Method 1, this effect can be only ascribed to the nature of the

flow, i.e. short and intense pulses produced higher super-saturation than a constant

flow. In all three oxygenation methods A260 increased with increasing DO

concentration (Figure 2); however, Methods 2 and 3 provided much larger A260

increases for similar DO concentrations, for example ~47% hyperchromism at DO =

26 mg/L with method 2 vs. a ~2% increase at DO = 22 mg/L with method 1.

Stability of the hyperchromic effects

Irrespective of the amount of oxygenation-induced hyperchromism, the perturbation

was substantially maintained for at least three weeks at room temperature (Figure 3,

right) or 18 hours at 37oC (see Supplementary Information, SI Table 1). However, the

DO level decreased much faster than A260, with a decay to baseline over a period of

less than a week (Figure 3, left. See also Supplementary Information, SI Figure 4).

Figure 3: Stability of DO level (left) and A260 (right) of differently oxygenated DNA

solutions over time at room temperature, all stored in screw-capped glass vials under

air. The hyperchromism reaches already >50% of its asymptotic value within 90

seconds of oxygenation (SI. Figure 2), and this effect is very stable, while even the

asymptotic DO is not maintained for long.

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We noted that the stability of the spectral features was independent of the oxygenation

method (data not shown). Therefore, the DO level did not appear to influence either

the extent of oxygen-DNA interactions or their stability, since the same DO levels

corresponded to different hyperchromic values depending on the oxygenation method.

These findings lead to the conclusion that the products are substantially not in a rapid

equilibrium (or not in equilibrium at all) with dissolved oxygen, being the result of

either a chemical modification or a strong complexation.

The nature of oxygen-DNA interactions

Chemical modification of nucleic acids during oxygenation would most likely mean

oxidation, with possible formation of (hydro)peroxides[37]. Any spectral feature

linked to the presence of peroxides would be expected to disappear by the addition of

a compound capable of decomposing them, such as TEMED (N,N,N',N'-tetramethyl

ethylene 1,2-diamine) [38, 39]; However, even high TEMED concentrations (Figure

4) did not affect the absorbance of oxygenated samples any differently than that of

untreated DNA or buffers, irrespective of the extent of the hyperchromism.

Figure 4: UV spectra of buffer, DNA, and DNA oxygenated with constant flow rate

(Method 1) at 2.9 mL/min, 6.3 mL/min, and 8.2 mL/min for 15mins each (DNA

concentration = 10µg/ml) as a function of the amount of TEMED (3x2µL increments

of pure TEMED). The increased absorbance at<260 nm is due to TEMED

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Raman spectroscopy, widely used for characterizing DNA structures [40, 41], showed

that oxygenation did not introduce new peaks (Figure 5), confirming the absence of

major chemical modifications. The bands in the range 600-800 cm-1

, assigned to

secondary structure-shifts independent of base concentrations [40], were largely

unchanged.

Figure 5. Raman spectra (buffer-subtracted and normalised) of salmon sperm DNA (1

mg/mL) solutions, before and after oxygenation with constant flow at 8.2 mL/min

(method 1) for 40 mins.

On the other hand, the Raman intensity ratio I790/1097, generally quantitatively

associated with A-helices, showed a ~50 % increase. However, since B↔A secondary

transitions did not occur (see later comments), this change could indicate a strong

modification of the spatial arrangement of the backbone relative to the bases [41].

Furthermore, the peaks in the range 1200-1600 cm-1

, which are known to be

hyperchromically sensitive to deformational vibration modes and stacking interactions

in the bases [40], showed marked intensity increases for oxygenated samples.

Substantially confirming the indications obtained from Raman spectra, Circular

Dichroism (CD) analysis showed all DNA preparations, irrespective of oxygenation,

to be in native B-type secondary conformation, with positive and negative bands at

275 nm and 246 nm, respectively (see Supplementary Information, SI Figure 5).

However, both bands were enhanced in intensity on oxygenation (Figure 6, see also

Supplementary Information, SI Figure 5).

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Figure 6. CD intensities of the bands peaked at 246 and 275 nm for salmon sperm

DNA at 24oC, 0.5 mg/mL in 0.5 x SSC buffer, as a function of oxygenation

conditions

These changes cannot be ascribed to the presence of A-, C- or Z-secondary structures

which have different characteristic CD profiles [42], and thus indicate non-secondary

conformational alterations. Interestingly, similar effects have been reported for DNA

as a function of the decrease in solution ionic content [43].

Influence of oxygenation on oligonucleotide hybridization

Having established that oxygenation did not introduce chemical modifications, but

produced minor, but substantially stable conformational changes, we engaged the

fundamental question whether complexation affected the functional behaviour of

nucleic acids, for example, hybridization.

Single-stranded 5 µg/mL homo-oligoT (15-mer) DNA solutions were oxygenated

through Method 3 up to a plateau in A260, corresponding to >250 mL of purged

oxygen, i.e. >25 minutes at a flow rate of 10 mL/min (see Supplementary information,

SI Figure 6, left). The post-hybridization melting profiles of both oxygenated and

native oligoT with homo-oligoA were virtually indistinguishable (Figure 7A), with an

identical melting temperature (Tm) extrapolated from the plot (~38oC) that was in

agreement with the theoretically predicted Tm [36]. The same result could also be

obtained by plotting the ratio A260/280 instead (see Supplementary Information, SI

Figure 6, right). It could therefore be concluded that oxygenation apparently did not

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affect the recognition capability of DNA strands, which is possibly the reason why the

“oxygenation effect” discussed here has remained unidentified.

Figure 7. A: Hybridization of oxygenated oligoT/untreated oligoT with oligoA, and

resulting melting curves (insert-negative hybridization control, oligoA-C), B: Re-

oxygenation of the hybridized duplex. Oxygenated oligoT and oligoA, shown for

comparison. (Plots adjusted for inter-oligo extinction coefficient differences)

Furthermore, the hybridized A-oxyT duplex was re-oxygenated, and it was found that

the plateau was obtained at an amount of oxygen (400-500 mL) nearly double than

that used for single stranded oligoT or oligoA (Figure 7B). This seemed to indicate

that, a) the oxygen was somehow released during the hybridization process, and b) the

oxygenation capacity was not affected by hybridization. The latter finding is

reasonable considering that complexation to oxygen would not be expected to involve

H-bonding groups, but essentially only electron clouds of the aromatic rings.

However, oxygen complexation and hybridization may be somehow intertwined, with

the conformation rearrangements following hybridization possibly resulting in a

recognition-responsive release of oxygen.

Implications for DNA quantification

DNA quantification using A260 is a commonly adopted global practice [35]. Our

results, however, show clear evidence that this value is vulnerable to differences in

oxygen concentration, likely causing significant laboratory-to-laboratory variations,

due to protocol differences in the DNA source, preparation, handling, and storage, i.e.

overall history of the sample. Worth mentioning is that due to the similar reasons it is

virtually impossible to propose a universal correction factor to account for complexed

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oxygen. We therefore suggest that in a series of comparative measurements

demanding accurate quantification, a parallel nitrogenation control should be

performed. From our investigations on single-stranded oligodeoxyribonucleotides, we

presume that RNA solution spectra would also show the same oxygenation-led

perturbations.

Can oxygenated DNA be an oxygen carrier?

Following oxygenation, DNA appears to retain oxygen under physiological conditions

for long periods (Figure 3 right, and SI. Table 1), and possibly releases it upon

hybridization (in a responsive fashion) (Figure 7). This could open the way for the use

of oxygenated DNA as an oxygen carrier, provided that the construct shows negligible

toxicity on mammalian cells. Indeed the standard MTS cytotoxicity assay on mouse

L-929 fibroblasts showed that identical to non-oxygenated DNA, oxygenated DNA

solutions did not have any deleterious effects on cell viability, for concentrations

ranging 25µg/mL- 200µg/mL (see Supplementary Information, SI Figure 7).

Recent studies suggest that micromolar concentrations of oxygen, delivered to the

tumour, can show noticeable results in hypoxia reversal-based chemo- or

radiotherapeutic intervention [23-25]. Both earlier studies on the spatial arrangements

of oxygen complexed with aromatic compounds [44] and later reports [12] have

indicated that the complexation could occur at 1:1 molar ratio. If the same assumption

is applied to DNA, then, hypothetically, the highest DNA concentration used, i.e. 200

g/mL, corresponding to ~0.6 mM of subunits, could potentially complex 0.6 mM

oxygen.

Considering that the assumed molar concentration is markedly below what has been

tested for in vivo non-viral gene therapy [45, 46], nucleic acids may resemble

potential oxygen-carriers for hypoxia reversal. Further, the stability of oxygen

complexation, coupled to its reversibility through hybridization, could allow for the

release of oxygen to potentially occur simultaneously to gene/antisense RNA and

RNAi etc. therapies.

Conclusions

The influence of oxygenation on the UV absorbance of DNA solutions is a finding of

great significance for biological analysis and its consequences may be far-reaching,

Page 16 of 19

since it implies that precise and reliable results could be obtained only by controlling

the level of oxygenation of the DNA solutions.

More work should be done to characterise the (responsive) reversibility of oxygen-

DNA interactions. This phenomenon is potentially advantageous for boosting dual

therapy, but controlling the localisation of the oxygen release (packaging the

complexes into appropriate vehicles) is also essential. It is worth mentioning that

although the use of DNA as an oxygen-carrier offers the opportunity to club gene

therapy, aromatic chemotherapeutic agents may also be used to reversibly complex

oxygen. However, we do not suggest these complexes to have a therapeutic effect per

se, since oxygen can only assist clinical results, possibly allowing a lower dosage of

radiation or drugs.

Acknowledgements

We would like to thank Mr. Sanjay Nilapwar for his help with CD measurements. NT

gratefully acknowledges financial support from EPSRC (grant No. EP/C543564/1 and

Advanced Research Fellowship). RD thanks AstraZeneca and CRUK for a generous

bursary award.

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