((Title))

SERS and SERRS Detection of the DNA Lesion 8-Nitroguanine: a Self-Labeling Modification

Susan Dick,[a] Steven E. J. Bell*[a] Katie J. Alexander[b], Ian A. O’Neil[b] and Richard Cosstick*[b]

[a]S. Dick, Prof. S. E. J. Bell

Innovative Molecular Materials GroupSchool of Chemistry and Chemical EngineeringQueen’s University, Belfast, BT9 5AG, (UK)E-mail: S.Bell@qub.ac.uk

[b]K. J. Alexander, Dr. I. A. O’Neil, Prof. R. Cosstick

Department of Chemistry,

University of LiverpoolCrown Street, LiverpoolL69 7ZD, (UK)E-mail: rcosstic@liv.ac.uk

Supporting information for this article is given via a link at the end of the document.

www.chemsuschem.org

FULL PAPER

Abstract: Rapid and sensitive methods to detect DNA lesions are essential in order to understand their role in carcinogenesis and for potential diagnosis of cancers. The 8-nitroguanine DNA lesion, which is closely associated with inflammation-induced cancers, has been characterised for the first time by surface-enhanced Raman spectroscopy (SERS). This lesion has been studied as the free base, as well as part of a dinucleotide and oligodeoxynucleotides (ODNs) at 5 different excitation wavelengths in the range 785-488 nm. All nitrated samples produced distinctly different spectra from their control guanine counterparts, with nitro bands being assigned by DFT calculations. Additional resonance enhancement was observed at the shorter excitation wavelengths, these SERRS measurements allowed the detection of one nitrated guanine in over 1,300 bases. In addition, SER(R)S can be used to detect whether the unstable lesion is covalently attached to the ODN or has been released by hydrolytic depurination.

Introduction

The Development of simple, rapid methods to detect DNA lesions is important since such changes may be associated with disease. For example, it is estimated that about 20 % of human cancers are attributable to various types of chronic inflammation,[1] in which elevated levels of nitric oxide lead to the production of both reactive oxygen (ROS) and reactive nitrogen species (RNS), that are capable of chemically modifying DNA.[2] The resulting DNA lesions are often mutagenic and are closely associated with carcinogenesis.[3] The guanine base in DNA has the lowest oxidation potential and is therefore most susceptible to chemical modification[4] for example nitration by RNS, to form 8-nitro-2’-deoxyguanosine (8-nitro-dG). In this lesion introduction of the nitro substituent labilizes the glycosidic bond, releasing the 8-nitroguanine base and leaving behind an abasic site in the DNA.[5] In addition, 8-nitro-dG has been shown to cause G to T transversion mutations through miscoding with dA during DNA replication.[6,7] Studies have also demonstrated that 8-nitroguanine accumulates at the site of carcinogenesis in hamsters whilst analysis of surgical samples of human tumor tissues have shown that high levels of 8-nitroguanine are associated with a poor prognosis of the cancer.[1,8] Sensitive and quantitative methods for the detection of 8-nitro-dG are therefore important with regard to obtaining a more detailed understanding of the role this lesion plays in carcinogenesis and may provide new approaches for the early diagnosis of inflammation-related cancers.

The detection of 8-nitro-dG in DNA presents a particular problem due to the lability of the glycosidic bond which results in loss of the 8-nitroguanine base from DNA. This free modified base can be detected in tissues and sera by immunohistochemistry[9] and chromatographic methods,[10] but detecting the free nucleobase does not give information on the distribution and steady-state levels of this lesion in DNA. SERS has the potential to address this problem since chemical modification of nucleobases causes characteristic changes in their Raman spectra[11-13] allowing label-free DNA analysis in which the signals observed are those of the constituent bases rather than an extrinsic label.[14-16] For example, it has been shown that substitution of even a single base in 25-mer oligodeoxynucleotides (ODNs) can be detected using difference spectra.[15] Halas et al.[11] have shown that methylation of all 3 adenines in 12-mer ODNs gave detectable changes in their SERS spectra while Guerini et al. [16] detected methylation of adenine and cytosine with similar sensitivity. El-Sayed et al.[17] have monitored the effect of ROS on DNA and observed large spectral changes are associated with significant ds-DNA scission and oxidation. Additionally, SERS has the high sensitivity which will ultimately be required for intracellular measurements (detection limits of 10-8 and 10-9 mol dm-3 have been reported for ssDNA and ds-DNA, respectively)[14-16] and it requires only microliter samples.

Here we report the first application of SERS to studies of nitrative damage, specifically characterizing the 8-nitroguanine lesion. We have found that the high Raman scattering cross section of the nitro moiety makes it easier to detect nitration than other modifications using non-resonant SERS 785 nm excitation. More importantly, we also show that adding resonance enhancement of the nitrated base through the use of shorter excitation wavelengths to give surface-enhanced Resonance Raman scattering (SERRS), dramatically increases the signal of the nitrated guanine both in the free base and in ODNs. The combination of higher intrinsic scattering and resonance enhancement allows nitration to be detected at concentrations 2 orders of magnitude lower than other modifications, such as methylation. This is the first example of Raman monitoring of DNA modification at such low concentration and is a significant first step towards monitoring DNA nitration within living cells.

Results and Discussion

Initial studies were on the simple guanine and 8-nitroguanine nucleobases. The SERS spectra of both compounds obtained at 785 nm are shown in Figure 1. The spectrum of guanine (Figure 1(a)) is consistent with previous published spectra[18,19] and shows numerous modes associated with the purine ring and substituents. The 8-nitroguanine spectrum (Figure 1(c)) is distinctly different to that of unsubstituted guanine. The main difference is the appearance of strong new bands, at 1425, 1282, 1238 and 833 cm-1, which can be assigned to the nitro substituent by comparison with the nitro vibrations in the normal Raman spectra of nitrophenol[20] and DFT calculations (B3LYP/6-31G*, Supplementary Information) which predict νasym N-O (1406 cm-1), νsym C8-NO2 (1260 cm-1), νsym N-O (1240 cm-1) and δsym O-N-O (786 cm-1) bands, consistent with the experimental values. The atomic displacement vectors of these modes are shown in Figure 2. The calculations also predict some of the ring modes to shift by 10’s of cm-1 on substitution, but the general vibrational spectra remain recognizably similar. For example, the characteristic guanine ring breathing mode shifts +29 cm-1 in 8-nitroguanine (660→689 cm-1), in good agreement with the +34 cm-1 shift predicted by the DFT calculations.

Figure 1. SERS spectra of (a) guanine at 785 nm, (b) guanine at 532 nm, (c) 8-nitroguanine at 785 nm and (d) 8-nitroguanine at 532 nm. All measurements were taken at concentrations of 5x10-6 M. Spectra at 785 nm are shown on same absolute scale, (d) signal has been reduced in intensity by a factor of twenty to allow comparison with the other spectra.

Since the UV/visible absorption spectrum of 8-nitroguanine shows a broad absorbance centered at 398 nm with an extinction coefficient of 4100 dm3mol-1cm-1, Raman spectra were recorded at 532 nm to look for indications of resonance or pre-resonance effects which would not be expected with 785 nm excitation.[21] These spectra (Figures 1 (b) and (d)) clearly show that the differences on nitration are indeed much more pronounced at the shorter excitation wavelength and the nitro group vibrations are even more dominant, which is strong evidence that significant resonance enhancement of the 8-nitroguanine occurs even at this wavelength, which is still > 100 nm away from the absorbance maximum. The spectra shown in Figure 1 have been normalized to allow easy comparison but the 785 nm spectra of both guanine and 8-nitroguanine are specifically shown on the same absolute intensity scale to highlight the similarity in the band intensities of both molecules, apart from the nitro bands, which are known to be somewhat better scatterers than most other groups.[20] In contrast, at 532 nm the 8-nitroguanine gives a much larger signal than guanine. Overall the absolute intensity of the 532 nm signals was lower than those at 785 nm, due to a combination of the instrument used and the fact that the colloid gave considerably less SERS enhancement at this wavelength (as discussed below). However, when this is corrected, as shown in Figure 1 (where the 532 nm spectra were rescaled to allow the guanine signals to be compared) it is obvious that the signal due to 8-nitroguanine is anomalously large, indeed the intensity needs to be reduced by a factor of 20 to bring it onto the same vertical axis. Closer examination of the 532 nm 8-nitroguanine spectrum also shows that the distinctive ring breathing mode at 689 cm-1 is barely detectable compared to the 833 cm-1 nitro bending vibration that it lies beside, while at 785 nm it was 2x larger than the nitro vibration, suggesting that the resonance enhancement is preferentially amplifying the vibrational modes of the nitro substituent. The change in the relative intensities of the two adjacent strong nitro bands at 1282 cm-1 and 1238 cm-1 in the 785 and 532 nm spectra is presumably due to different resonance enhancements of the two bands at the different wavelengths.

Figure 2. Atomic displacement vectors for the nitro group vibrations in 8-nitroguanine (DFT calculations using B3LYP functional and 6-31G(d) basis set).

Factors that could complicate the SERS spectra, such as the reduction of the pKa of NH-1,[7] in 8-nitroguanine and concentration dependence of SERS intensities were investigated. However, it was found that the spectra show relatively little variation with pH (see Supplementary Information). Similarly, we note that the SERS spectrum of 8-nitroguanine recorded under our experimental conditions is concentration-dependent. Increasing the concentration from 5x 10-6 mol dm-3 to much higher concentrations causes some bands to shift (see Supplementary Information), presumably due to change in the local environment around the nitro group on the surface as the concentration increases. However, this occurs at much higher concentrations than we would expect to use and in general it is important to emphasize that the spectra we recorded under a given set of concentration and excitation wavelength conditions were highly reproducible.

Studies on oligodeoxynucleotides containing 8-nitroguanine

The next step was to test if the 8-nitro guanine base could be observed in single-stranded (ss-DNA) by incorporating the lesion into ODNs. As noted above, 8-nitro-dG has a very labile glycosidic bond and rapidly undergoes depurination releasing the 8-nitroguanine base. It has previously been shown that stabilization of the glycosidic bond can be achieved by incorporating 8-nitroguanine into oligodeoxynucleotides as its 2´-O-methyl riboside.[7] Thus, 8-nitro-2’-O-methylguanosine (8-NO2-2’-O-Me-G) was used to prepare both 13-mer and 27-mer sequences (Figure 3), each containing a single 8-nitroguanine base, along with the 13-mer and 27-mer unmodified control sequences. In addition, since the SERS spectra of nucleosides and nucleotides can be quite different from the parent nucleobases, [24] we used 8-nitro-2’-O-methylguanosine dinucleotide (8-NO2-2’-O-Me-G dimer, Figure 3) as the best model system for nitrated guanine bases in DNA.

Figure 3. Structure of (a) 8-nitro-2’-O-methylguanosine dinucleotide, and 2’-O-methylguanosine dinucleotide, (b) 8-NO2-2’-O-methylguanosine ODNs and control ODN

The spectrum of the 8-nitro-2’-O-Me-G dimer at 785 nm (Figure 4(a)) is different to that of the 8-nitroguanine (Figure 1), in that it is dominated by a single stretching mode at 1238 cm-1, with the ring breathing mode at 689 cm-1 barely visible. The band at 1282 cm-1 which was assigned to NO2, appears to be suppressed in the dinucleotide at 785 nm. These changes are due to differences in the mode composition and frequency of the guanine ring vibrations caused by addition of the ribose at the N9 position rather than being additional vibrations arising from the 2’-methoxyribose substituent, because ribose sugars are known to have a very weak Raman spectra compared to that of aromatic nucleobases. [24]

The most important comparison is of the SERS spectra of unmodified ODNs with their nitrated analogues. Figure 4 compares spectra of the 27-mers recorded using 785 nm excitation, where resonance enhancement of the nitro groups is not expected. Equivalent data for the 13-mers are shown in the Supplementary Information. Many of the bands have the same position and relative intensity in both spectra, which is unsurprising considering that only one of the bases within the sequence has been modified and all the others remain the same in both sequences. The spectra of the unmodified sequences are typical of those found for ss-DNA under these experimental conditions[14] in that they show numerous bands characteristic of the nucleobases which are present. The spectra are dominated by a strong band at 1333 cm-1, that is characteristic of deoxyadenosine, but also show bands at 1634 and 1576 cm-1 which are due to deoxycytidine and deoxyguanosine, respectively. Despite the overall similarity of the spectra, there is also a clear band in the 785 nm spectrum of the nitrated sequence at 1238 cm-1. This is the same position as the strongest band in the model 8-nitro-2’-O-Me-G dimer, It is impressive that the modification of a single base within this 27-mer can be easily observed by eye. This is possible because even under non-resonance conditions the vibrations of the nitro group in the 8-NO2-2’-O-Me-G ODNs have larger Raman scattering cross-sections than the ring modes in the base (as discussed above) and also because the strongest of these vibrations lies in a spectral region that is relatively free of interfering bands from the other nucleobases.

Figure 4. SERS spectra of (a) nitrated dinucleotide with (b) nitrated ODN (27-mer) and (c) control ODN (27-mer) at 785 nm. Bands previously assigned to individual nucleotides have been marked in (b) and (c) which are on the same vertical axis apart from being offset for clarity.

Importantly, with shorter wavelength 532 nm excitation the modified ODNs showed the same additional resonance (or pre-resonance) enhancement as the model nitro dimers (Figure 5). The control ODN gave a very low intensity spectrum, which is due to the low sensitivity of the spectrometer used and to the drop off in the SERS enhancement provided by the aggregated Ag colloid at this excitation wavelength. However, the nitrated 27-mer showed a strong SERRS spectrum, which was completely dominated by the vibrational modes of the single 8-nitroguanine base it contains, as shown by its similarity to the spectrum of the 8-NO2-2’-O-Me-G dimer, with barely detectable contributions from the other 26 bases in the sequence. This is very different from other SERS studies of single base substitution or modification experiments where the changes in the spectra were so small that could really only be clearly observed in difference spectra. Here the nitration dramatically changes both the overall intensity and bands in the spectra.

Figure 5. SERS spectra of (a) nitrated dinucleotide with (b) nitrated ODN (27-mer) and (c) control ODN (27-mer) at 532 nm. (b) and (c) are on the same vertical axis apart from being offset for clarity.

Since tuning the excitation wavelength to 532 nm selectively increased the SERRS enhancement of the nitrated guanine base it would be logical to attempt to further increase the resonance enhancement by shifting the excitation wavelength closer to λmax of the 8-nitroguanine absorption at ca. 400 nm. However, this also moves the excitation wavelength away from the surface plasmon of the aggregated colloid, which would be expected to decrease the surface enhancement. To test which of these effects was more important, spectra were recorded at a range of excitation wavelengths. The absolute sensitivities of the spectrometers used were normalized using toluene (which shows no SERS effect) and the relative SERS enhancement then measured using a standard SERS test molecule (thiophenol). The SERS enhancement factor of the test molecule (Figure 6) was found to peak at 633 nm and fall off at shorter wavelengths. Conversely, the resonance enhancement of the nitro- over the non-nitro samples (calculated from the relative intensities of the 1333 and 1234 cm-1 bands) maximized, as expected, at the shortest excitation wavelength. These data show that the optimum wavelength will be different depending on the circumstances of the measurement. 532 nm combines reasonable levels of SERS enhancement, which will give high overall signal levels, with reasonable resonance enhancement. However, for detection of small amounts of nitration the shortest wavelength is best, since this will give maximum enhancement of the nitro signals relative to the other bases.

This effect is illustrated in Figure 7, which shows the spectra of mixtures of control and singly nitrated 27-mer ODNs recorded with 488 nm excitation. Although the overall signal levels were lower than at longer wavelengths there was still sufficient surface enhancement to give good quality spectra with 5x10-6 mol dm-3 samples. The spectra of the pure control and nitrated ODNs components are as expected from the measurements at other wavelengths, the unmodified sequence shows multiple bands due to the constituent bases with the strongest band at 1333 cm-1 due to adenine, whilst the nitrated ODN is dominated by bands due to the nitrated guanine base, notably the strong NO2 stretching vibration at 1248 cm-1. Since the absolute signal height of the nitrated ODN is significantly larger than that of the unmodified ODN, the nitrated component dominates the spectra of mixtures, even those which are predominantly composed of the unmodified ODN and it is only at lower relative concentrations of 8-NO2-2’-O-Me-G that the bands due to the other bases become visible. Most notably in Figure 6 the 1248 cm-1 nitro band is visible even in a mixture which is 98% unmodified ODN and 2% singly nitrated ODN. In this case the nitro band is visible without spectral subtraction, even though there is only one modified guanine for every 27*50 = 1350 nucleobases. This level of detection is exceptional, particularly in a rapid analysis which requires no labels and can be used at concentrations < 10-5 mol dm-3. Since similar signals are observed with single and double stranded DNA with the same base sequences14 we would expect similar detection limits for nitration in ds-DNA

.

Figure 6. Plot of SERS Activity and resonance enhancement vs excitation wavelength. Data was recorded at 488, 514, 532, 633 and 785 nm. Resonance enhancements were calculated by the relative enhancement of the nitrated ODN over the control ODN and SERS activity was calculated by the absolute intensity of thiophenol signal over that of toluene.

Figure 7. SERS Spectra of 27-mer ODNs, nitrated (8NO2) and non-nitrated (G), mixed in various concentrations, (a) 100% 8NO2, (b) 10% 8NO2, 90% G, (c) 5% 8NO2, 95% G, (d) 98% 8NO2, 2% G and (e) 100% G. All spectra were recorded at 488 nm and were normalised to reduce the much larger signals observed for samples with a higher proportion of the nitrated ODN to a level where they could be compared to the non-nitrated sample. Inset shows a 7 base section of the ODN with the nitrated position highlighted.

Since it is established that despite the stabilizing effect of the 2’-O-Me substituent the 8-NO2-2’-O-Me-G ODN is still susceptible to depurination,[7] experiments were performed to ensure that the nitrated ODN spectra were not due to 8-nitroguanine which was free in solution and thus able to preferentially attach to the enhancing surface. Figure 8 compares the spectrum of a sample of nitrated ODN with that of a sample designed to mimic the effect of depurination, prepared by mixing unmodified ODN with free 8-nitroguanine at a concentration equivalent to the amount of free base which would be present if all the 8-NO2-2’-O-Me-G in the modified ODN was released. Figure 8 shows that the spectrum of the model depurinated mixture is indeed dominated by the free 8-nitroguanine, but that this spectrum can easily be distinguished from that of the ODN in which the 8-NO2-2’-O-Me-G remains bound. For example in the mixture of control ODN with the nitrated base (Figure 8(b)) the relative intensities of the 1234 cm-1 and 1282 cm-1 bands are reversed compared to the nitrated ODN (Figure 8(a)). Additionally, free 8-nitroguanine gives a strong band at 834 cm-1, which is not observed in the spectra of the nitrated ODN.

Figure 8. SERS spectra of (a) nitrated ODN with (b) control ODN mixed with 8-nitroguanine base and (c) free base of 8-nitroguanine. All spectra were recorded at 532 nm. (a) and (b) are on the same vertical axis apart from being offset for clarity.

Factors Determining the Ability of SERS to Detect DNA Modifications

There are some challenges with regard to using SERS to detect base modifications and base lesions in DNA. In particular, the observation that the absolute intensity of the SERS signals given by different nucleobases, although not identical, are typically quite similar to each other.[25] There is general agreement that adenine gives the strongest scattering, but even the relative intensities of cytosine, thymine and guanine, are the subject of debate and seems to vary with the enhancing medium.[24-27] Irrespective of the details, if the scattering cross-sections of the bases are broadly similar, modifying a small fraction of the bases will give only a small change in the overall signal for the DNA. For example, in previous work on detecting substitutions of single bases in 25-mer ss-DNA by SERS,[15] we found that spectra of the substituted 25-mers were almost indistinguishable by eye from the spectra of the original sequences and only SERS difference spectroscopy could detect the changes in the spectra associated with base substitution, which in turn placed high demands on the signal-to-noise ratios and reproducibility of the signals. Halas et al.[11] have investigated the SERS spectra of short 12-mer ODNs with all the bases of a given type modified e.g. with all 3 adenines methylated and under those favourable conditions did manage to observe differences in the spectra of the pure ODNs by simple comparison by eye. However, these differences became much harder to detect when the methylated 12-mer ODNs were diluted 50:50 with the unmodified 12-mers. In a related study, Guerrini et al.[16] showed that in a 20 base pair DNA duplex, methylation of 5 of the cytosines or 6 of the adenines gave differences that were just large enough to be observed without subtraction. In contrast, the introduction of resonance enhancement means modification at the levels discussed above (1 modified base per ca. 10-30 bases in the sequence) gives dramatic changes in the spectra (Figure 5), which instead of being barely visible by eye completely dominate the spectra.

As a final direct illustration of the increased sensitivity possible for guanine nitration over other previously studied modifications, Figure 9 presents the SERS spectrum of an unmodified 13-mer (Figure 9(b)) with that obtained with one of the guanine bases oxidized at the C8 position (to give 8-oxoguanine, Figure 9(c)) and the spectrum of the sequence where the same guanine was nitrated rather than oxidized. The SERS spectra of the nitrated sequence is dominated by the bands from the single nitrated base while that containing the oxidized guanine are virtually indistinguishable from the unmodified control, which is not surprising since under our experimental conditions the guanine signals are much lower in intensity than those of adenine and here we have made a small modification to just one of the 3 guanines in the 13-mer sequence.

Figure 9. SERS spectra of 13-mer ODNs (GCGTACXCATGCG) containing the following modifications (a) X = 8-NO2-2’-OMe-G, (b) 2’-OMe-G and (c) X = 8-oxoG. All spectra were recorded at 785 nm.

This comparison emphasizes the particular attraction of probing nitro species using SER(R)S, which is that the signal of the modified bases are significantly stronger than those of their unmodified counterparts, so they can be detected even against a background of signals from a much larger number of other bases with normal scattering cross sections. Moreover, as shown above, the relative size of nitro to unmodified base signals can be further enhanced by moving into resonance or pre-resonance with the absorption band of the 8-nitroguanine base. Under these pre-resonance conditions we move from the normal situation where the conventional methylation or oxidation of a single base in a sequence is barely detectable to one where the spectrum of the entire sequence is dominated by the enhanced signal of the single nitrated base.

Conclusions

The spectroscopic properties of the 8-nitroguanine: high scattering cross-section under resonance conditions, characteristic bands in an uncrowded spectral region, lack of interference with the biological processes of interest, and ability to discriminate between 8-NO2-2’-O-Me-G ODN and free 8-nitroguanine are precisely those which would be used in trying to select a good SERS or SERRS label. As such, this work can be regarded not so much an example of label-free SER(R)S as self-labeling SER(R)S, in that the process of interest generates its own SER(R)S label. The fact that this self-labeling occurs has the potential to transform studies of nitrative damage from being some of the most challenging areas of DNA modification due to their susceptibility for depurination into one of the areas which can be most easily characterized. This is therefore a significant first step towards extending the whole cell studies of the type recently published for oxidative damage in DNA,[17] to nitrative damage

Experimental Section

Reagents and Methods

Silver nitrate (99.9999%), hydroxylamine hydrochloride (99%), sodium hydroxide (≥97%), magnesium sulfate and guanine (98%) were all purchased from Sigma-Aldrich. Aqueous solutions and dilutions were prepared using distilled deionized (DDI, resistivity 18.2 M cm) water from a Barnstead NANOpure Diamond System. The hydroxylamine hydrochloride silver colloids were prepared according to the Leopold and Lendl method and had a max = 420 nm.28

The oligodeoxynucleotide sequence containing the 8-oxodeoxyguanosine modification was purchased from Integrated DNA Technologies (Belgium) and underwent HPLC purification by the vendor. Oligodeoxynucleotide sequences containing 8-nitro-2’-O-methylguanosine were prepared and purified as previously described, together with the corresponding 2’-O-methylguanosine control sequences.7 All oligodeoxynucleotides containing 8-nitroguanine showed the characteristic absorption at 395 nm.7 The synthesis of 8-nitroguanine and the 8-nitro-2’-O-methylguanosine dinucleotide are described in the Supplementary Information.

SERS Measurements

For SERS measurements, 50 L of colloidal solution was mixed with 50 L of analyte and then aggregated by 25 L 0.1 M magnesium sulfate. pH adjustments were made with dilute H2SO4 and NaOH and measured with universal indicator paper. Spectra were recorded on a number of instruments. Spectra at 785 nm excitation were recorded using an Avalon Instruments Ramanstation R1 which is fitted with a 785 nm diode laser and an echelle spectrograph. In this system the laser power at the sample was 160 mW and samples were held in 96 well polypropylene multiwell plates. A Horiba Scientific LabRAM HR 800 Raman microscope was used for 633 nm (HeNe) and 532 nm (Nd/YAG) excitation. The laser power at 633 nm was 9 mW (operating at 50% power, 4.1 mW) and at 533 nm, 101 mW. Finally, for excitation wavelengths of 514 nm and 488 nm, a Raman system which was built in-house was used. Briefly this comprises a Spectra-Physics 2020 Ar+ laser, a 180⁰ backscattering illumination/collection system, Jobin-Yon HR640 spectrograph and an Andor Technology CCD. Spectra were typically recorded with an exposure time of 6 x 10 s. SERS activity was calculated by comparing the ratio of absolute intensities of the spectra of toluene (normal Raman) and thiophenol (SERS) at each of the excitation wavelengths.

DFT Calculations

DFT calculations were carried out using the Gaussian 03 package using the B3LYP functional and 6-31G(d) basis set. Comparison of calculated and experimental spectra is tabulated in the Supplementary Information.

Acknowledgements

SD was supported by a Department for Employment and Learning studentship, Northern Ireland and KJA was funded by an Engineering and Physical Sciences Research Council studentship.

Keywords: SERS • SERRS • 8-nitroguanine • DNA • DNA lesion

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Inflamed DNA gets found out: The 8-nitroguanine DNA lesion, which is closely associated with chronic inflammation, has been characterised by surface-enhanced Raman (SERS) and resonance Raman (SERRS) spectroscopy. SERRS is exquisitely sensitive for this lesion and allows the detection of one 8-nitroguanine in over 1,300 bases.

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