Received: 24 February 2023 Revised: 26 June 2023 Accepted: 5 July 2023

DOI: 10.1002/chir.23612

RESEARCH ARTICLE

Circular dichroism and UV–Vis detection of UV-induced

damage to nucleic acids

Reed C. Dowling 1 | Gregory T. Carroll 2 | David L. Kirschman 2 |

Mark B. Masthay 1 | Angela Mammana 1

1
Department of Chemistry, University of
Dayton, Dayton, Ohio, USA Abstract
2
Aerobiotix, Inc., Miamisburg, Ohio, USA In this report, we demonstrate that CD spectroscopy can be used as a tool to
detect changes to DNA upon irradiation with UV light. We follow the spectro-
Correspondence
scopic response of DNA samples irradiated at selected exposure times with both
Angela Mammana, University of Dayton,
Department of Chemistry, 300 College CD and UV–Vis spectroscopy. We analyzed four different nucleic acids to evaluate
Park Dr., Dayton, OH 45469, USA. the effect of the sequence on photodegradation. Only one polymer, calf thymus
Email: amammana1@udayton.edu
DNA, was a natural nucleic acid and contained all four nucleobases. The other
Funding information three were synthetic polymers and contained only one type of base pair: poly
University of Dayton (deoxyadenylic-deoxythymidylic) acid [poly (dA-dT)2] and poly (deoxyadenylic
acid)
poly (deoxythymidylic acid) [poly (dA)
poly (dT)], which contained only
adenine and thymine; poly (deoxyguanylic-deoxycytidylic) acid [poly (dG-dC)2],
which contained only guanine and cytosine. CD and UV–Vis spectra showed
sequence dependent changes. In particular, poly (dA)
poly (dT) undergoes
changes more rapidly than the other sequences investigated. The CD spectrum of
poly (dA)
poly (dT) gradually undergoes an inversion, suggesting a change in
helicity, before disappearing due to the unfolding of the double strand.

KEYWORDS
B-DNA, circular dichroism, germicide, nucleic acids, photodegradation, secondary structure

1 | INTRODUCTION their DNA and become “inactivated.”17,18 The main chemi-

UV germicidal irradiation technologies are used to
disinfect solid surfaces, air, and water.1,2 The Covid-19 pan-
demic has recently stirred increased interest in applying
UV light to surface and air disinfection.3–7 In operating
room settings, which are subject to infectious contami-
nants from many sources,8–13 UV germicide technology is
used to reduce the occurrence of surgical site
infections.14–16 In general, pathogens exposed to appropri-
ate dosages of UV light encounter problems in replicating
cal mechanism of disinfection involves photochemical
damage to nucleic acids, with thymine dimerization the
most often-cited photo-process that disrupts microbe repro-
duction.19,20 DNA has an absorption maximum at approxi-
mately 260 nm and absorbs UV light up to a wavelength of
roughly 300 nm, and hence traditional direct irradiation
requires a light source that emits in the UVC (200–
280 nm) and/or UVB (280–315 nm) regions. UVC is pre-
ferred as it covers the portion of the spectrum where DNA
has its highest extinction coefficients. Hg lamps are

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any
medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
© 2023 The Authors. Chirality published by Wiley Periodicals LLC.

Chirality. 2023;35:973–982. wileyonlinelibrary.com/journal/chir 973


974
typically used as germicides because their emission spec-
trum includes a strong line at 254 nm, which overlaps with
the DNA absorption band. Note that UVC irradiation can
also induce protein damage.21
The rich and dynamic nature of biological environ-
ments coupled with DNA's structural variations suggests
the potential for a complex mixture of products upon
exposing a micro-organism to UV light. When the
nucleobases of DNA absorb light, a number of photo-
products are possible.22 Both pyrimidines and purines
undergo photo-reactions, and the nature of the resulting
photo-products is dependent in part on the sequence and
flexibility of the DNA chains.23 It is generally assumed
that thymine (5,5-6,6) dimers formed between adjacent
deoxythymidylic acid nucleotides are the most important
photo-products in microbe inactivation.24 In fact, many
cursory discussions of the topic indicate this photoprod-
uct exclusively. However, additional photoproducts
containing and excluding thymine have been reported.
(6,4) thymine dimers are well-known.25 The spore photo-
product, 5-thyminyl-5,6-dihydrothymine, is another
example of an alternative thymine dimer.26 While
thymine dimerization is generally depicted as adjacent
thymine nucleobases reacting with each other, thymine
dimerization between non-adjacent nucleotides has been
reported.27 Additionally, thymine-cytosine and cytosine-
cytosine photo-products have been reported.22,28 Irradia-
tion of microbes with UV light is not limited to nucleic
acid damage, as UV-induced crosslinking between DNA
and proteins can occur.29 Given that photochemical reac-
tions often involve highly reactive intermediates and that
the interior of cells is chemically complex, it is expected
that a variety of products are generated, especially when
differences between pathogen species are considered.
Given the utility of UV disinfection and its growing
importance in inhibiting the spread of infectious agents,
enhancing our understanding of the effect of UV light on
DNA may lead to improved germicidal technologies
and/or the development of rapid protocols for screening
the effectiveness of irradiation devices. Investigating
molecular level changes involved in germicidal action,
including the exact mechanism of thymine dimerization,
is an ongoing challenge.1,30
Circular dichroism (CD) spectroscopy is a powerful
method for studying chiral materials including DNA.31
Understanding the chiroptical response of DNA to UV irra-
diation will further our understanding of UV-induced
DNA modification and its potential as a tool for the rapid
assessment of the efficacy of UV disinfection units.
Combined with UV–Vis and fluorescence spectroscopy,32
multiple channels can be used to detect changes in the pri-
mary and secondary structure of DNA.33 An advantage of
CD spectroscopy is that only chiral molecules give signals,
1520636x, 2023, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/chir.23612 by SEA ORCHID (Thailand), Wiley Online Library on [18/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
DOWLING ET AL.
which is particularly important in more complicated set-
tings where multiple chromophores can be present. Com-
bined with induced CD signals based on supramolecular
interactions, unique DNA sensors based on CD spectros-
copy that give rise to easily distinguishable signals in the
visible region have been developed.34 The utility of CD
spectra for characterizing a variety of systems including
solid substrates, metallic particles, supramolecular aggre-
gates, and small molecule interactions with proteins and
DNA, has been demonstrated.35–39 The efficiency of CD
spectroscopy in shedding light on the structure of molecu-
lar and biomolecular systems warrants its application to
the analysis of UV-irradiated DNA and may lead to the
development of new sensing strategies and technologies.
In this report, we demonstrate that CD spectroscopy
can be used as a tool to detect changes to DNA upon irradi-
ation with UV light (Figure 1). We follow the spectroscopic
response of DNA samples irradiated at selected exposure
times with both CD and UV–Vis spectroscopy. We analyze
four different nucleic acid sequences: calf thymus DNA,
poly (deoxyadenylic-deoxythymidylic) acid [poly (dA-dT)2],
poly (deoxyguanylic-deoxycytidylic) acid [poly (dG-dC)2],
and poly (deoxyadenylic acid)
poly (deoxythymidylic acid)
[poly (dA)
poly (dT)] as shown in Figure 1. In all cases,
both the CD and UV–Vis spectra undergo sequence depen-
dent changes. Poly (dA)
poly (dT), which contains thy-
mine nucleobases adjacent to each other on a single
homopolymeric strand and adenine nucleobases adjacent
to each other on the complementary homopolymeric
strand, undergoes changes more rapidly than the other
sequences investigated.
2 | MATERIALS AND METHODS
2.1 | General comments
Lyophilized natural and synthetic polynucleotides were
used. Calf thymus DNA was purchased from Sigma-Aldrich
and stored at 4 C. Poly (deoxyguanylic-deoxycytidylic) acid
sodium salt [poly (dG-dC)2], poly (deoxyadenylic-deoxythy-
midylic) acid sodium salt [poly (dA-dT)2], and poly (deox-
yadenylic)-poly (deoxythymidylic) acid sodium salt [poly
(dA)
poly (dT)] were purchased from Sigma-Aldrich and
stored at 20 C. Stock solutions of polynucleotides were
prepared in phosphate buffer (25 mM, pH 7).
2.2 | Instrumental measurements
CD spectroscopy was performed with a JASCO J-815 CD
spectropolarimeter. UV–Vis absorption spectroscopy was
performed with a JASCO V-630Bio UV–Vis

DOWLING ET AL.
F I G U R E 1 Well-known photo-
products of UV-irradiated DNA include
the thymine dimers shown (top).
Photochemical modification of B-DNA,
which has a characteristic CD spectrum,
is expected to induce conformational
and spectroscopic changes (middle).
Schematic representations of the four
types of DNA irradiated with UV light
and analyzed with CD spectroscopy are
shown (bottom).
spectrophotometer. Measurements were performed in a
quartz cuvette with a 1 cm pathlength. All solutions were
prepared in a phosphate buffer (25 mM, pH 7).
2.3 | Photochemical experiments
For UV irradiation, solutions in a quartz cuvette were
irradiated with a 100-watt Hg Arc lamp (Oriel Corp.)
with a maximum irradiance at 254 nm of approximately
60 mW/m2 at 0.5 m. UV–Vis and CD spectra were taken
immediately after irradiation for selected exposure times.
2.4 | Preparation of DNA solutions
All polynucleotide solutions were prepared in phosphate
buffer at concentrations that gave absorption maxima of
approximately 0.96 ± 0.02.
3 | R ES U L T S A N D D I S C U S S I O N
Calf thymus DNA and three synthetic polynucleotides
were irradiated with UV light (Hg-Lamp) and analyzed
1520636x, 2023, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/chir.23612 by SEA ORCHID (Thailand), Wiley Online Library on [18/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
975
with CD and UV–Vis spectroscopy in order to understand
how the presence and relative position of the nucleotides
affect spectroscopic changes. Calf thymus DNA, poly
(dA-dT)2, poly (dG-dC)2, and poly (dA)
poly (dT) were
all prepared in phosphate buffer at pH 7 and were each
irradiated and analyzed using the same procedure.
3.1 | Calf thymus DNA
Figure 2A shows the CD spectra for calf thymus DNA
in buffer before and after irradiation with UV light for
selected exposure times. The symmetric bisignate signal
shown indicates that the DNA is in the B form.40 The
spectrum contains a positive band at 276 nm and a neg-
ative band at 246 nm, both of which are plotted as func-
tions of irradiation time in Figure 2B (top). After
15 min of irradiation, significant reductions in the mag-
nitudes of the ellipticities (6.5 and 4.8 mDeg at 246 and
276 nm, respectively) are clearly evident. These magni-
tudes of attenuation correspond to a 43% reduction in
intensity of the negative signal and 41% reduction in
intensity of the positive signal. The CD signals are effec-
tively eliminated after 120 min of exposure to UV light,
with the signals at 246 and 276 nm converging to

976
F I G U R E 2 CD spectra of calf thymus DNA after irradiation with UV light for selected amounts of time (A). The CD signals at 246 and
276 nm are plotted as functions of irradiation time (B top). UV–Vis absorption spectra of calf thymus DNA after irradiation with UV light for
selected amounts of time (C). The absorption at 258 nm (left axis) and 310 nm (right axis) are plotted as functions of irradiation time
(B bottom).
approximately 0 mDeg from initial values of 14.7 and
+11.7 mDeg, respectively. The loss of the CD signal and
absence of the formation of new signals throughout the
irradiation process indicates an unfolding of the B form
upon irradiation.
The UV–Vis absorption spectra for calf thymus DNA
in phosphate buffer at pH 7 before and after irradiation
with UV light for selected exposure times are shown in
Figure 2C. The absorption maximum occurs at approxi-
mately 258 nm. Absorbance values at 258 and 310 nm are
plotted as functions of irradiation time in Figure 2B (bot-
tom). The signal at 258 nm decreases with exposure time
and drops from an initial value of approximately 0.962 to
0.388 after 150 min of exposure. A weak signal that shows
absorbance appearing above 300 nm, A > 300, grows with
irradiation time and reaches a maximum value at 310 nm
of approximately 0.065 after 60 min of exposure to UV
light. This signal gradually decays after 90 min of irradia-
tion. A fluorescence band with a maximum at approxi-
mately 402 nm and associated with the A > 300 signal was
recently reported.32 The fluorescence signal continues to
increase in intensity for up to 120 min of UV irradiation
and is stable for up to at least 150 min of irradiation, indi-
cating that multiple photoproducts might contribute to the
UV–Vis signal, not all of which are fluorescent. While the
absorption spectrum shows the growth of the A > 300 sig-
nal, the CD spectra do not show the growth of a new signal
in this region, suggesting either a lack of chirality in the
1520636x, 2023, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/chir.23612 by SEA ORCHID (Thailand), Wiley Online Library on [18/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
DOWLING ET AL.
photo-product or a lack of interaction of the photo-product
with chiral material.
The data reported in Figure 2 coupled with the recently
reported fluorescence spectra32 show that a combination of
CD, UV–Vis, and fluorescence techniques provides com-
plementary information regarding the photo-induced mod-
ification of DNA. The absorption spectra show both a
change in the DNA absorbance as well as the growth of a
weak signal at longer wavelengths. The CD spectra are
intimately related to the secondary structure of the DNA
and show a more compelling spectral change in a shorter
amount of exposure time as compared to UV–Vis and fluo-
rescence spectra, and provide two strong signals of opposite
sign to monitor. The previously reported fluorescence spec-
tra show a strong signal with an emission maximum at
402 nm corresponding to the growth of a new product.
Collectively, UV–Vis, CD, and fluorescence spectroscopy
show that DNA is modified by exposure to UV light under
the conditions employed.
3.2 | Poly (dA-dT)2
Figure 3A shows the CD spectra for poly (dA-dT)2 before
and after irradiation with UV light for selected exposure
times. Poly (dA-dT)2 shows a bisignate signal with a posi-
tive band at 264 nm and a negative band at 249 nm, both
of which are plotted as functions of irradiation time in

DOWLING ET AL.
F I G U R E 3 CD spectra of poly (dA-dT)2 after irradiation with UV light for selected amounts of time (A). The CD signals at 249 and
264 nm are plotted as functions of irradiation time (B top). UV–Vis absorption spectra of poly (dA-dT)2 after irradiation with UV light for
selected amounts of time (C). The absorption at 262 nm (left axis) and 310 nm (right axis) are plotted as functions of irradiation time
(B bottom).
Figure 3B (top). As for calf thymus DNA, the asymmetric
bisignate signal indicates that the DNA is in the B form.40
Significantly, the positive band has a shoulder at approxi-
mately 280 nm. After 15 min of irradiation, changes in
the secondary structure are easily detected. Reductions of
6.5 and 4.8 mDeg in the magnitudes of the 249 and
264 nm signals, respectively, are clear. The signal at
249 nm attenuates in magnitude by 6.0 mDeg, corre-
sponding to a 21% reduction in intensity of the negative
signal. Similarly, the signal at 264 nm attenuates by
3.1 mDeg, corresponding to a 22% reduction in intensity
of the positive signal. The CD signals are effectively
eliminated after 180 min of exposure to UV light, with
the signals at 246 and 276 nm converging to approxi-
mately 0 mDeg from initial values of 28.4 and
+14.1 mDeg, respectively. In agreement with the calf
thymus DNA results, the loss of the CD signal indicates a
loss in secondary structure upon extensive irradiation.
However, the rate of attenuation is slower than that of
calf thymus DNA.
Figure 3C shows the UV–Vis absorption spectra for
poly (dA-dT)2 before and after irradiation with UV light
for select exposure times. The absorbances at 262 and
310 nm are plotted as functions of irradiation time in
Figure 3B (bottom). Upon irradiation, the absorption
maximum at 262 nm decreases from an initial absor-
bance value of 0.931 to 0.308 after 210 min of exposure.
As found for the calf thymus DNA, the A > 300 signal
grows with irradiation time, although the rate of growth
1520636x, 2023, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/chir.23612 by SEA ORCHID (Thailand), Wiley Online Library on [18/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
977
is approximately four times slower. After 15 min of irradia-
tion, the signal at 310 nm increases by only approximately
0.009 absorption units compared to 0.035 units for the calf
thymus DNA. Regardless, both systems show relatively
low increases in absorbance, thus highlighting the limita-
tions of absorption spectroscopy without complementary
techniques for assessing DNA damage at shorter irradia-
tion times. The signal at 310 nm associated with this band
reaches a maximum value of approximately 0.056 after
120 min of exposure to UV light and gradually decays with
further irradiation. The relative reductions in the intensity
of the CD signals are greater in magnitude than those
observed in the absorption spectrum. The temporal behav-
ior of the UV–Vis signal is more complicated in that it first
increases slightly before decreasing. In fact, the UV–Vis
signal at 262 nm does not fall below its original value until
after 60 min of irradiation. While the UV–Vis spectrum
shows the growth of the A > 300 signal, the CD spectra do
not show the growth of a new signal in this region in
agreement with the calf thymus DNA results, again sug-
gesting either a lack of chirality in the photo-product or
lack of interaction with chiral material.
3.3 | Poly (dG-dC)2
Figure 4A shows the CD spectra for poly (dG-dC)2 in
buffer before and after irradiation with UV light for select
exposure times. Poly (dG-dC)2 shows an asymmetric

978
F I G U R E 4 CD spectra of poly (dG-dC)2 after irradiation with UV light for selected amounts of time (A). The CD signals at 253 and
278 nm are plotted as functions of irradiation time (B top). UV–Vis absorption spectra of poly (dG-dC)2 after irradiation with UV light for
selected amounts of time (C). The absorption at 257 nm (left axis) and 310 nm (right axis) are plotted as functions of irradiation time
(B bottom).
bisignate signal that is consistent with the B form of
DNA.40 The absorption spectrum shows a positive band
at 278 nm and a negative band at 253 nm, both of which
are plotted as a function of irradiation time in Figure 4B
(top). In comparison with calf thymus DNA and poly
(dA-dT)2, the CD spectrum of poly (dG-dC)2 changes
more slowly. After 15 min of irradiation, the positive
band at 278 nm shows very little change. The negative
band at 253 nm reduces in magnitude by 2.3 mDeg, cor-
responding to an 8.7% reduction. Although the changes
in the spectrum are less impressive at 15 min compared
to calf thymus DNA and poly (dA-dT)2, UV-induced
changes in the structure of the DNA are clearly evident
as the CD signals continually attenuate with irradiation
time. The CD signals are effectively eliminated after
300 min of exposure to UV light, with the signals at
253 and 278 nm converging to approximately 0 mDeg
from initial values of 26.1 and +9.9 mDeg, respectively.
As for calf thymus DNA and poly (dA-dT)2, the gradual
loss of signal indicates an unfolding of the secondary
structure.
Figure 4C shows the UV–Vis absorption spectra for
poly (dG-dC)2 in buffer before and after irradiation with
UV light for select exposure times. The absorbances at
257 and 310 nm are plotted as functions of irradiation
time in Figure 4B (bottom). The absorption maximum at
approximately 257 nm attenuates upon irradiation,
decreasing from an initial value of approximately 0.969 to
0.218 after 300 min of exposure. As was shown for both
1520636x, 2023, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/chir.23612 by SEA ORCHID (Thailand), Wiley Online Library on [18/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
DOWLING ET AL.
calf thymus DNA and poly (dA-dT)2, the A > 300 signal
grows with irradiation time. After 15 min of irradiation,
the absorbance at 310 nm increases by only approxi-
mately 0.004 absorption units and reaches a maximum
value of approximately only 0.039 after 150 min of
exposure to UV light. The signal gradually decays after
180 min of exposure. The consistent presence of the
A > 300 signal regardless of nucleotide composition
(i.e., in both poly (dA-dT)2 and poly (dG-dC)2) indicates
that a mixture of products is generated in calf thymus
DNA, in which all four nucleobases are present. Interest-
ingly, the band at 257 nm undergoes a very rapid hypso-
chromic shift compared to calf thymus DNA and poly
(dA-dT)2. Additionally, it is worth noting that the inten-
sity of the band at 257 nm does not initially rise during
the first 30 min of irradiation as it does for poly (dA-dT)2.
Again, we do not see the growth of a band in the CD
spectrum that corresponds to the growth of the weak
A > 300 signal in the UV–Vis spectrum.
3.4 | Poly (dA)
poly (dT)
Figure 5A shows the CD spectra for poly (dA)
poly
(dT) before and after irradiation with UV light for select
exposure times. The spectrum for poly (dA)
poly
(dT) prior to irradiation is consistent with a B form
secondary structure.40 The spectrum includes two
positive bands at 283 and 261 nm and two negative bands
 1520636x, 2023, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/chir.23612 by SEA ORCHID (Thailand), Wiley Online Library on [18/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
DOWLING ET AL. 979

F I G U R E 5 CD spectra of poly (dA)
poly (dT) after irradiation with UV light for selected amounts of time (A). The inset shows the
initial B form signal (0 s), the inverted signal after 30 min of irradiation, and the loss of signal after 180 min of irradiation. The CD signals at
249 and 283 nm are plotted as functions of irradiation time (B top). UV–Vis absorption spectra of poly (dA)
poly (dT) after irradiation with
UV light for selected amounts of time (C). The absorption at 259 nm (left axis) and 310 nm (right axis) are plotted as functions of irradiation
time (B bottom).

at 249 and 269 nm. The CD signals at 283 and 249 nm
are plotted as a function of irradiation time in Figure 5B
(top). The CD spectrum changes much more rapidly for
the poly (dA)
poly (dT) compared to the other polynu-
cleotide samples analyzed, which is expected given that
all thymine nucleobases are adjacent to each other on a
single strand. This result is in agreement with the rapid
photo-degradation of poly (dA)
poly (dT) noted in our
UV–Vis results below. Interestingly, after 10 min, the
signal for the secondary structure of the polymer starts to
invert, reaching its maximum value after 30 min of
irradiation. Further irradiation of the sample results in
the complete disappearance of the CD signal, indicating a
loss of secondary structure. The CD signals are effectively
eliminated after 180 min of exposure to UV light, with
the signals at 249 and 283 nm converging (after inver-
sion) to approximately 0 mDeg from initial values of
54.5 and +6.9 mDeg, respectively.
Figure 5C shows the UV–Vis absorption spectra for
poly (dA)
poly (dT) in buffer before and after irradiation
with UV light for select exposure times. Absorbances at
259 and 310 nm are plotted as functions of irradiation
time in Figure 5B (bottom). The absorption maximum
at approximately 259 nm attenuates upon irradiation,
decreasing from an initial value of approximately 0.972 to
0.294 after 180 min of exposure. Similar to poly (dA-dT)2,
the early stages of irradiation are accompanied by a slight
increase in absorbance. After 25 min of irradiation, the
signal at 259 nm falls below its original value. The signal
continues to decrease with further irradiation and shows
no hypsochromic shift. As found for the calf thymus
DNA, poly (dA-dT)2 and poly (dG-dC)2, the A > 300 signal
grows with irradiation time. After 15 min of irradiation,
the absorbance at 310 nm increases by 0.084 absorption
units, which is the fastest rate of increase for the four
samples in this study and is perhaps expected, since it is
consistent with the rapid formation of thymine dimers
along a single DNA strand. After 30 min, the signal
reaches a maximum value of approximately 0.12 and
gradually decays with further irradiation.
The changes in the CD spectrum suggest a two-step
process. In the first step, there seems to be a transition of
the secondary structure of the polymer from a B-form
(right-handed helix) to what appears to be a Z-form (left-
handed helix) as the spectrum at 30 min resembles the
spectrum of the Z-form of Poly[d (AT)] reported by
Vorlickova et al.40 The transition is triggered by the
formation of the photoproduct. In the second step, the
full unfolding of the polymer occurs. The CD results
agree with the previously published fluorescence data, in
which the increase in emission was initially slow, and
then increased more rapidly after 30 min.32 We speculate
that in the first 30 min of irradiation, the predominant
photoproduct is the non-fluorescent thymine (5-5, 6-6)
dimer. Longer exposure times promote the accumulation
of the fluorescent thymine (6-4) dimer.

980
The two types of dimers are expected to have a differ-
ent effect on the stability of the double helix as shown in
Figure 6. Formation of the thymine (5-5, 6-6) dimer does
not alter its hydrogen bonding sites, allowing the DNA to
maintain a double-stranded secondary structure. More-
over, formation of the (5-5, 6-6) dimer affects the second-
ary structure in a manner that appears to invert the
helicity of the polynucleotide as suggested by the appear-
ance of a new bisignate CD spectrum containing opposite
signs compared to the original B form. In contrast, forma-
tion of the thymine (6-4) dimer results in the loss of
hydrogen bonding sites on one of the thymine nucleo-
bases, which is expected to weaken the stability of the
double strand and consequently of the helical structure.
The loss of hydrogen bonding likely results in a separa-
tion of the two strands, which is accompanied by a full
loss of secondary structure as the absence of CD signal
between 200 and 300 nm suggests.
1520636x, 2023, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/chir.23612 by SEA ORCHID (Thailand), Wiley Online Library on [18/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
DOWLING ET AL.
The results show that CD spectroscopy is a sensitive
technique for monitoring the photochemical modifica-
tion of DNA in combination with the absorption spectra
shown in this report and fluorescence data reported in a
previous article.32 Each technique provides unique infor-
mation. Together, these techniques provide rapid insights
into molecular level changes that occur when DNA is
irradiated with UV light with circular dichroism being
the most sensitive of the three: the CD spectra manifest
larger changes in magnitude in a smaller amount of time
as compared to both absorption and fluorescence spectra.
Our results indicate that while GC base pairs typically
receive less discussion regarding UV germicidal mecha-
nisms, they are clearly susceptible to UV damage. While
alternating AT base pairs are clearly damaged by UV
light, having thymine nucleobases adjacent to each other
on a single strand dramatically enhances the effective-
ness of the UV light. The rate enhancement for poly
F I G U R E 6 Proposed effects of
dimer formation on the secondary
structure of poly (dA)
poly (dT) are
shown. The CD spectra suggest that
formation of the (5,5-6,6) dimer during
the first stage of UV irradiation causes
an inversion of the helicity.
Accumulation of the (6,4) dimer causes
an unwinding of the helix and ultimate
loss of secondary structure as shown by
the complete loss of CD signal.

DOWLING ET AL.
(dA)
poly (dT) is likely a result of the adjacent thymine
units being positioned in a manner that facilitates
dimerization.
The methodologies described above can be used to
test the effectiveness of specific irradiation equipment.
Additionally, the efficacy for photochemically modifying
specific sequences of DNA can be tested. The protocols
described herein can be applied to assess photochemical
damage to proteins and RNA as well.
A C K N O WL E D G M E N T S
Reed C. Dowling acknowledges an ISE Corps award
(University of Dayton).
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are avail-
able from the corresponding author upon reasonable
request.
ORCID
Angela Mammana https://orcid.org/0000-0002-6237-
8784
R EF E RE N C E S
1. Kowalski W. Ultraviolet germicidal irradiation handbook: UVGI
for air and surface disinfection. Springer Science & Business
Media; 2010. doi:10.1007/978-3-642-01999-9
2. Reed NG. The history of ultraviolet germicidal irradiation for
air disinfection. Public Health Rep. 2010;125(1):15-27. doi:10.
1177/003335491012500105
3. Barnewall RE, Bischoff WE. Removal of SARS-CoV-2
bioaerosols using ultraviolet air filtration. Infect Control Hosp
Epidemiol. 2021;12(8):1-8. doi:10.1017/ice.2021.103
4. Raeiszadeh M, Adeli B. A critical review on ultraviolet
disinfection systems against Covid-19 outbreak: applicability,
validation and safety considerations. ACS Photon. 2020;7(11):
2941-2951. doi:10.1021/acsphotonics.0c01245
5. Kumar A, Sagdeo A, Sagdeo PR. Possibility of using ultraviolet
radiation for disinfecting the novel COVID-19. Photodiagnosis
Photodyn Ther. 2021;34:102234. doi:10.1016/j.pdpdt.2021.
102234
6. Nardell EA. Air disinfection for airborne infection control with
a focus on COVID-19: why germicidal UV is essential. Photo-
chem Photobiol. 2021;97(3):493-497. doi:10.1111/php.13421
7. Lo CW, Matsuura R, Iimura K, et al. UVC disinfects
SARS-CoV-2 by induction of viral genome damage without
apparent effects on viral morphology and proteins. Sci Rep.
2021;11(1):1-1. doi:10.1038/s41598-021-93231-7
8. Bischoff WE, Russell G. Correlation of the air–surface nexus
of bacterial burden during routine patient care. Infect
Control Hosp Epidemiol. 2020;41(11):1353-1354. doi:10.1017/
ice.2020.436
9. Carroll GT, Kirschman DL, Mammana A. Increased CO2 levels
in the operating room correlate with the number of healthcare
workers present: an imperative for intentional crowd control.
Patient Saf Surg. 2022;16(1):35. doi:10.1186/s13037-022-00343-8
1520636x, 2023, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/chir.23612 by SEA ORCHID (Thailand), Wiley Online Library on [18/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
981
10. Carroll GT, Kirschman DL. Catalytic surgical smoke filtration
unit reduces formaldehyde levels in a simulated operating
room environment. ACS Chem Health Saf. 2023;30(1):21-28.
doi:10.1021/acs.chas.2c00071
11. Carroll GT, Kirschman DL. Discrete room pressure drops pre-
dict door openings and contamination levels in the operating
room setting. Perioper Care Oper Room Manag. 2022;29:100291.
doi:10.1016/j.pcorm.2022.100291
12. Chisari E, Largoza G, Clarkson S, Krueger CA, Kirschman D,
Parvizi J. Many common pathogens are present in the
operative room air during surgery. J Arthroplasty. 2022;37(12):
2427-2430. doi:10.1016/j.arth.2022.07.007
13. Kowalski WJ. Aerobiological engineering handbook: airborne dis-
ease and control technologies. McGraw Hill Professional; 2005.
14. Curtis GL, Faour M, Jawad M, Klika AK, Barsoum WK,
Higuera CA. Reduction of particles in the operating room using
ultraviolet air disinfection and recirculation units. J Arthro-
plasty. 2018;33(7):S196-S200. doi:10.1016/j.arth.2017.11.052
15. Bischoff W, Russell G, Willard E, Stehle J Jr. Impact of a novel
mobile high-efficiency particulate air–ultraviolet air recircula-
tion system on the bacterial air burden during routine care.
Am J Infect Control. 2019;47(8):1025-1027. doi:10.1016/j.ajic.
2018.12.019
16. Cook TM, Piatt CJ, Barnes S, Edmiston CE Jr. The impact of
supplemental intraoperative air decontamination on the out-
come of total joint arthroplasty: a pilot analysis. J Arthroplasty.
2019;34(3):549-553. doi:10.1016/j.arth.2018.11.041
17. Burby PE, Simmons LA. Regulation of cell division in bacteria
by monitoring genome integrity and DNA replication status.
J Bacteriol. 2020;202(2): 10-1128. doi:10.1128/JB.00408-19
18. Cutler TD, Zimmerman JJ. Ultraviolet irradiation and the
mechanisms underlying its inactivation of infectious
agents. Anim Health Res Rev. 2011;12(1):15-23. doi:10.1017/
S1466252311000016
19. Beukers R, Berends W. Isolation and identification of the
irradiation product of thymine. Biochim Biophys Acta. 1960;
41(3):550-551. doi:10.1016/0006-3002(60)90063-9
20. Setlow RB, Swenson PA, Carrier WL. Thymine dimers and
inhibition of DNA synthesis by ultraviolet irradiation of cells.
Science. 1963;142(3598):1464-1466. doi:10.1126/science.142.
3598.1464
21. Chan HL, Gaffney PR, Waterfield MD, et al. Proteomic analysis
of UVC irradiation-induced damage of plasma proteins:
serum amyloid P component as a major target of photolysis.
FEBS Lett. 2006;580(13):3229-3236. doi:10.1016/j.febslet.2006.
05.002
22. Douki T, Cadet J. Individual determination of the yield of the
Main UV-induced dimeric pyrimidine photoproducts in DNA
suggests a high mutagenicity of CC Photolesions. Biochemistry.
2001;40(8):2495-2501. doi:10.1021/bi0022543
23. Becker MM, Wang Z. Origin of ultraviolet damage in DNA.
J Mol Biol. 1989;210(3):429-438. doi:10.1016/0022-2836(89)
90120-4
24. McAteer K, Jing Y, Kao J, Taylor JS, Kennedy MA. Solution-
state structure of a DNA dodecamer duplex containing a
cis-syn thymine cyclobutane dimer, the major UV photoprod-
uct of DNA. J Mol Biol. 1998;282(5):1013-1032. doi:10.1006/
jmbi.1998.2062
25. Franklin WA, Haseltine WA. The role of the (6-4)
photoproduct in ultraviolet light-induced transition mutations

982
in E. coli. Mutat Res. 1986;165(1):1-7. doi:10.1016/0167-8817
(86)90002-7
26. Setlow P, Li L. Photochemistry and photobiology of the spore
photoproduct: a 50-year journey. Photochem Photobiol. 2015;
91(6):1263-1290. doi:10.1111/php.12506
27. Love JD, Minton KW. Ultraviolet induced dimerization of
non-adjacent pyrimidines in poly[d(A-T)]. J Biol Chem. 1992;
267(35):24953-24959. doi:10.1016/S0021-9258(19)73990-8
28. Palmeira L, Guegen L, Lobry J. UV-targeted dinucleotides are
not depleted in light-exposed prokaryotic genomes. Mol Biol
Evol. 2006;23(11):2214-2219. doi:10.1093/molbev/msl096
29. Smith KC, Hodgkins B, O'Leary ME. The biological importance
of ultraviolet light induced DNA-protein crosslinks in
Escherichia coli 15 TAU. Biochim Biophys Acta, Nucleic Acids
Protein Synth. 1966;114(1):1-15. doi:10.1016/0005-2787(66)
90248-6
30. Nagpal A, Dhankhar D, Cesario TC, Li R, Chen J, Rentzepis PM.
Thymine dissociation and dimer formation: a Raman and syn-
chronous fluorescence spectroscopic study. Proc Nat Acad Sci.
2021;118(6):e2025263118. doi:10.1073/pnas.2025263118
31. Mammana A, Carroll GT, Feringa BL. Circular Dichroism of
Dynamic Systems: Switching Molecular and Supramolecular
Chirality. In: Comprehensive Chiroptical spectroscopy: applica-
tions in Stereochemical analysis of synthetic compounds, natural
products, and biomolecules. John Wiley & Sons, Inc.; Vol. 2;
2012:289-316.
32. Carroll GT, Dowling R, Kirschman DL, Masthay MB,
Mammana A. Intrinsic fluorescence of UV irradiated DNA.
J Photochem Photobiol A. 2023;437:114484. doi:10.1016/j.
jphotochem.2022.114484
33. D'Urso A, Nardis S, Pomarico G, Fragalà ME, Paolesse R,
Purrello R. Interaction of Tricationic Corroles with
single/double helix of Homopolymeric nucleic acids and DNA.
J am Chem Soc. 2013;135(23):8632-8638. doi:10.1021/ja4023539
1520636x, 2023, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/chir.23612 by SEA ORCHID (Thailand), Wiley Online Library on [18/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
DOWLING ET AL.
34. D'urso A, Mammana A, Balaz M, et al. Interactions of a
tetraanionic porphyrin with DNA: from a Z-DNA sensor to a
versatile supramolecular device. J am Chem Soc. 2009;131(6):
2046-2047. doi:10.1021/ja808099u
35. Carroll GT, Jongejan MGM, Pijper D, Feringa BL. Spontaneous
generation and patterning of chiral polymeric surface toroids.
Chem Sci. 2010;1(4):469-472. doi:10.1039/c0sc00159g
36. Carroll GT, Pollard MM, van Delden R, Feringa BL. Controlled
rotary motion of light-driven molecular motors assembled on a
gold film. Chem Sci. 2010;1(1):97-101. doi:10.1039/c0sc00162g
37. Thorpe S, Mammana A. Spectroscopic study of porphyrin self-
assembly: role of pH, time, and chiral template. Chirality. 2020;
32(1):5-16. doi:10.1002/chir.23140
38. Fitz S, Mammana A. Spectroscopic study of the pH dependent
interaction of an achiral molecular photo-switch with poly-
glutamic acid. J Photochem Photobiol. 2020;388:112146. doi:10.
1016/j.jphotochem.2019.112146
39. Mammana A, Carroll GT, Areephong J, Feringa BL. A
Chiroptical Photoswitchable DNA complex. J Phys Chem B.
2011;115(40):11581-11587. doi:10.1021/jp205893y
40. Vorlickova M, Kejnovska I, Bednarova K, Renciuk D, Kypr J.
Circular dichroism spectroscopy of DNA: from duplexes to
Quadruplexes. Chirality. 2012;24(9):691-698. doi:10.1002/chir.
22064
How to cite this article: Dowling RC, Carroll GT,
Kirschman DL, Masthay MB, Mammana A.
Circular dichroism and UV–Vis detection of
UV-induced damage to nucleic acids. Chirality.
2023;35(12):973‐982. doi:10.1002/chir.23612